FungaI BiotechnoIogy

in Agricultural, Food,
and Environmental
Ap pIications
edited by
Ddip K. Arora
National Bureau of Agriculturally Important Microorganisms
New Delhi, lndiu

Associate Editors
Paul D. Bridge
British Antarctic Survey
Cambridge, United Kingdom

Deepak Bhatnagar
U S . Department of Agriculture
New Orleans, Louisiana, U.S.A.

m
MARCEL

DEKKER
MARCEL
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NEWYORK BASEL
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 MYCOLOGY SERIES

Editor
J. W. Bennett
Professor
Department of Cell and Molecular Biology
Tulane University
New Orleans, Louisiana

Founding Editor
Paul A. Lemke

1. Viruses and Plasmids in Fungi, edited by Paul A. Lemke

2. The Fungal Community: Its Organization and Role in the Ecosystem, edited by Donald T. Wicklow and
George C. Carroll
3. Fungi Pathogenic for Humans and Animals (in three parts), edited by Dexter H. Howard
4. Fungal Differentiation:A Contemporary Synthesis, edited by John E. Smith
5. Secondary Metabolism and Differentiationin Fungi, edited by Joan W. Bennett and Alex Ciegler
6. Fungal Protoplasts, edited by John F. Peberdy and Lajos Ferenczy
7. Viruses of Fungi and Simple Eukaryotes, edited by Yigal Koltin and Michael J. Leibowitz
8. Molecular lndustrial Mycology: Systems and Applications for Filamentous Fungi, edited by Sally A.
Leong and Randy M. Berka
9. The Fungal Community; Its Organization and Role in the Ecosystem, Second Edition, edited by George
C. Carroll and Donald T. Wicklow
10. Stress Tolerance of Fungi, edited by D. H. Jennings
11. Metal lons in Fungi, edited by Gunther Winkelmann and Dennis R. Winge
12. Anaerobic Fungi: Biology, Ecology, and Function, edited by Douglas 0. Mountfort and Colin G. Orpin
13. Fungal Genetics:Principles and Practice, edited by Cees J. Bos
14. Fungal Pathogenesis: Principles and Clinical Applications, edited by Riclhard A. Calderone and Ronald
L. Cihlar
15. Molecular Biology of Fungal Development,edited by Heinz D. Osiewacz
16. Pathogenic Fungi in Humans and Animals: Second Edition, edited by Dexter H. Howard
17. Fungi in Ecosystem Processes, John Dighton
18. Genomics ofPlants and Fungi, edited by Rolf A. Prade and Hans J. Bohriert
I9. Clavicipitalean Fungi: Evolutionary Biology, Chemistry, Biocontrol, and Cultural Impacts, edited by
James F. White Jr., Charles W. Bacon, Nigel L. Hywel-Jones, and Joseph W. Spatafora
20. Handbook of Fungal Biotechnology, Second Edition, edited by Dilip K. Arora
21. Fungal Biotechnology in Agricultural, Food, and EnvironmentalApplications, edited by Dilip K. Arora

Additional Volumes in Preparation

Handbook of Industrial Mycology, edited by Zhiqiang An

Preface

The study of fungal biotechnology is proceeding at an unprecedented rate with an array of new tools to generate a
wealth of disciplines and subdisciplines. By means of modern biotechnology, fungi have justified their practical
application to varied domains of human enterprise, and thus promise considerable potential in the agricultural, food, and
environmental spheres. The successful application of fungal biotechnological processes in these areas requires the
integration of a number of scientific disciplines and technologies. These may include subjects as diverse as agronomy,
chemistry, genetic manipulation, and process engineering. The practical use of newer techniques such as genetic
recombination, bioinformatics, and robotics has revolutionized modern biotechnology-based agri-food industries, and
created the enormous range of possible applications of fungi.
Tremendous biodiversity of agriculturally important fungi exists—the benefit of which is not fully harnessed. The level
of technology required to take full advantage may range from the simple introduction of a single fungus in biocontrol
processes to the extensive manipulation of the organism that facilitates overproduction of a particular enzyme or metabolite.
In modern agri-industry, fungi offer many established beneficial roles, particularly as biofertilizers, mycorrhizae, and
biocontrol agents of pathogens, pests, and weeds. As pathogens, fungi represent a heavy negative impact on human health,
agriculture, and environment. In agriculture, annual crop losses by phytopathogenic fungi in the field and also during post-
harvest exceed 200 billion Euros, and in the United States alone, over $600 million are spent annually on agricultural
fungicides. The balance of beneficial and detrimental effects is reflected in many other areas of agriculture and horticulture.
Fungi that inhabit tropical or temperate soils, as mycorrhiza, endophytes, phytopathogens, entomopathogens, or simple
saprophytes, are significant resources in transformation of biological matter, and they offer many bioproducts including
secondary metabolites, antibiotics, and catabolic enzymes of enormous potential.
The world has won a crucial battle in the area of food security, but the war is still on. A total of 800 million people—that
is, one of every six persons in the developing world—do not have access to food. One-third of all pre – school-age children
in the developing countries face food insecurity. In the food and feed arena, fungi are historically important as mushrooms
and fermented foods and in baking and brewing. Such roles are supplemented by the provision of fungi to offer food
processing enzymes and additives, and more recently the development of protein-based foodstuffs from filamentous fungi.
On the detrimental side, fungi cause extensive spoilage of stored and processed foodstuff. Through direct pathogenesis and
biodeterioration of foods and other agricultural commodities, fungi cause considerable economic consequences as well. In
these cases, techniques developed from biochemistry and molecular biology can be deployed to analyze the relevant
processes, and to evolve tools for the detection, characterization, and tracking of the organisms involved. Although such
endeavors may seem rather far removed from the traditional definition of fungal biotechnology, the information derived can
be pivotal in understanding the underlying intricate processes, and arriving at suitable control measures.
The utilization of fungi in the environment is a more recent development, and can have particular association with both
food and agriculture, with fungal remediation of land having implications for biofertilizers, mycorrhizae, and food crop

iii
iv Preface

development, among many other considerations. The degradative activities of fungi have also been harnessed in programs
related to bioremediation of contaminated land, treatment of industrial wastes, and biotransformation of specific
compounds. Many of the applications of fungal biotechnology in these areas rely not on identifying new activities but in
harnessing and expanding roles that the fungi undertake normally in the environment.
Several books on the role of fungi in agricultural, food, and environmental applications have appeared since the 1990s.
However, subjects relating to these areas are so broad that no single book can provide all the available information.
Consequently, this book complements the others by providing valuable information that is not available elsewhere. The
book encompasses a broad range of information on biotechnological potential of entomopathogenic fungi, ergot alkaloids,
fungi in disease control, the development of mycoherbicides, control of nematodes, control of plant disease, strategies for
controlling vegetable and fruit crops, mycotoxigenic fungi, development of biofungicides, production of edible fungi,
fermented foods, and high-value products such as mycoprotein, yeasts in the wine industry, the role of fungi in the dairy
industry, molecular detection of fungi in food and feeds, antifungal food additives, the importance of fungi in forest and arid
ecosystems, the role of fungi in the biomineralization of heavy metals, bioconversion of distillery waste, decoloration of
industrial waste, and fungal degradation of cellulose, hydrocarbons, dye water, and explosives.
Together with its companion publication, the Handbook of Fungal Biotechnology, Second Edition (Marcel Dekker,
2004), this incomparable book reigns as the top source on the role of fungi in agriculture, food technology, and
environmental applications. The book will be useful for teachers and students, in both undergraduate and graduate studies,
in departments of agricultural microbiology, food science, food technology, food engineering, microbiology, environmental
sciences, botany, bioengineering, plant pathology, mycology, and, of course, biotechnology. In addition, the book will be
useful for agri-food producers, research establishments, and government and academic units.
No work of this magnitude can be accomplished without the support and contributions of many individuals. I am deeply
indebted to my colleagues and associate editors who have assisted me throughout the production of this book. I appreciate
the hard work of authors for their up-to-date discussions on various topics and immense persistent cooperation. My
gratitude is expressed to my teacher J. L. Lockwood. I thank Ms. Sandra Beberman (Vice President) and Ms. Dana Bigelow
(Production Editor) at Marcel Dekker, Inc., for their dedicated assistance and advice in editorial structuring at all stages of
the production of this book.

Dilip K. Arora
Contents

Preface iii
Contributors ix

I AGRICULTURAL BIOTECHNOLOGY

1 Biotechnological Approaches in Plant Protection: Achievements, New Initiatives, and Prospects 1

Sally Ann Leong
2 Chemical Identification of Fungi: Metabolite Profiling and Metabolomics 19
Kristian Fog Nielsen, Jørn Smedsgaard, Thomas Ostenfeld Larsen, Flemming Lund,
Ulf Thrane, and Jens Christian Frisvad
3 Isozyme Analysis in Fungal Taxonomy, Genetics, and Population Biology 37
Stephen B. Goodwin
4 Molecular Methods for Identification of Plant Pathogenic Fungi 49
Maren A. Klich and Edward J. Mullaney
5 The Application of Molecular Markers in the Epidemiology of Plant Pathogenic Fungi 57
Paul D. Bridge, Tanuja Singh, and Dilip K. Arora
6 Molecular Biology for Control of Mycotoxigenic Fungi 69
Robert L. Brown, Deepak Bhatnagar, Thomas E. Cleveland, and Zhi-Yuan Chen
7 Biotechnological Potential of Entomopathogenic Fungi 79
Travis R. Glare
8 Biotechnological Potential of Ergot Alkaloids 91
M. Flieger, P. Mehta, and A. Mehta
9 Fungi as Plant Growth Promoter and Disease Suppressor 101
M. Hyakumachi and M. Kubota
10 Challenges and Strategies for Development of Mycoherbicides 111
Susan M. Boyetchko and Gary Peng

v
vi Contents

11 Biofungicides 123
Beom Seok Kim and Byung Kook Hwang
12 Molecular Biology of Biocontrol Trichoderma 135
Christian P. Kubicek
13 The Biological Control Agent Trichoderma from Fundamentals to Applications 147
A. Herrera-Estrella and I. Chet
14 Biological Control of Fungal Diseases on Vegetable Crops with Fungi and Yeasts 157
Zamir K. Punja and Raj S. Utkhede
15 Control of Postharvest Diseases of Fruits Using Microbes 173
Wojciech J. Janisiewicz
16 Arbuscular Mycorrhizal Fungi in Plant Disease Control 183
Lisette J. C. Xavier and Susan M. Boyetchko
17 Commercialization of Arbuscular Mycorrhizal Biofertilizer 195
Pragati Tiwari, Anil Prakash, and Alok Adholeya
18 Control of Nematodes by Fungi 205
Hans-B€orje Jansson and Luiz V. Lopez-Llorca

II FOOD AND FEEDS

19 Fungi in Food Technology: An Overview 217

George G. Khachatourians
20 Fungi and Fermented Food 223
T. B. Ng
21 Production of Edible Fungi 233
R. D. Rai
22 Mycoprotein and Related Microbial Protein Products 247
Juan Ignacio Castrillo and Unai Ugalde
23 Genetic Variability of Yeast in Wine Fermentation 257
Amparo Querol, M. Teresa Fern
andez-Espinar, and Eladio Barrio
24 Yeast in the Dairy Industry 269
T. K. Hansen and M. Jakobsen
25 Flavors and Aromas 281
Renu Agrawal
26 Antifungal Food Additives 291
Purbita Ray and Michael B. Liewen
27 Molecular Detection of Fungi in Foods and Feeds 299
J
anos Varga
28 The Role of Spoilage Fungi in Seed Deterioration 311
Naresh Magan, Vicente Sanchis, and David Aldred
29 Mycotoxins 325
Fun S. Chu and Deepak Bhatnagar
Contents vii

30 Genetics and Biochemistry of Mycotoxin Synthesis 343

Jiujiang Yu

III ENVIRONMENTAL BIOTECHNOLOGY

31 Cellulose Degradation by Fungi 363

Justine M. Niamke and Nam Sun Wang
32 The Importance of Wood-Decay Fungi in Forest Ecosystems 375
Nia A. White
33 The Biodegradation of Lignocellulose by White Rot Fungi 393
Gary Ward, Yitzhak Hadar, and Carlos G. Dosoretz
34 Biomineralization of Heavy Metals 409
T. C. Crusberg, S. S. Mark, and A. Dilorio
35 Decoloration of Industrial Wastes and Degradation of Dye Water 419
Kirsten Schliephake, Warren L. Baker, and Greg T. Lonergan
36 Bioconversion of Distillery Waste 431
Jozefa Friedrich
37 Degradation of Hydrocarbons by Yeasts and Filamentous Fungi 443
John B. Sutherland
38 Biodegradation of Azo Dyes by Fungi 457
John A. Bumpus
39 Fungal Degradation of Explosives 471
J. L. Faull, S. C. Baker, S. Wilkinson, and S. Nicklin
40 Restoration of Mycorrhizae in Disturbed Arid Ecosystems 481
Season R. Snyder and Michael F. Allen

Index 493
Contributors

Alok Adholeya The Energy and Resources Institute, New Delhi, India

Renu Agrawal Central Food Technological Research Institute, Mysore, India

David Aldred Cranfield University, Bedford, United Kingdom

Michael F. Allen Center for Conservation Biology, University of California, Riverside, California, USA

Dilip K. Arora Banaras Hindu University, Varanasi, India

S. C. Baker School of Biological and Chemical Sciences, Birkbeck College, London, United Kingdom

Warren L. Baker Environment and Biotechnology Centre, Swinburne University of Technology, Melbourne,
Australia

Eladio Barrio University of València, València, Spain, Instituto de Agroquı́mica y Tecnologı́a de Alimentos,
CSIC, València, Spain

Deepak Bhatnagar U.S. Department of Agriculture– Agricultural Research Service,

New Orleans, Louisiana, USA

Susan M. Boyetchko Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada

Susan M. Boyetchko Saskatoon Research Centre, Agriculture and Agri-Food Canada, Saskatoon,
Saskatchewan, Canada

Paul D. Bridge* Birkbeck College, University of London, London, and Royal Botanic Gardens Kew, Surrey,
United Kingdom

Robert L. Brown U.S. Department of Agriculture–Agricultural Research Service,

New Orleans, Lousiana, USA

John A. Bumpus University of Northern Iowa, Cedar Falls, Iowa, USA

Juan Ignacio Castrillo University of Manchester, Manchester, United Kingdom

*Present affiliation: British Antarctic Survey, Cambridge, United Kingdom

ix
x Contributors

Zhi-Yuan Chen Louisiana State University, Baton Rouge, Lousiana, USA

I. Chet The Weizmann Institute of Science, Rehovot, Israel

Fun S. Chu University of Wisconsin, Madison, Wisconsin, USA

Thomas E. Cleveland U.S. Department of Agriculture– Agricultural Research Service, New Orleans, Lousiana,
USA

T. C. Crusberg Worcester Polytechnic Institute, Worcester, Massachusetts, USA

A. Dilorio Worcester Polytechnic Institute, Worcester, Massachusetts, USA

J.L. Faull School of Biological and Chemical Sciences, Birkbeck College, London, United Kingdom

M. Teresa Fernández-Espinar University of València, València, Spain, Instituto de Agroquı́mica y Tecnologı́a

de Alimentos, CSIC, València, Spain

M. Flieger Dr. H.S. Gour University, Saugor, India; Institute of Microbiology, Czech Academy of Sciences,
Prague, Czech Republic

Jozefa Friedrich National Institute of Chemistry, Ljubljana, Slovenia

Jens Christian Frisvad BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark

Travis R. Glare AgResearch, Lincoln, New Zealand

Stephen B. Goodwin U.S. Department of Agriculture—Agricultural Research Service, and Purdue University,
West Lafayette, Indiana, USA

Yitzhak Hadar The Hebrew University of Jerusalem, Rehovot, Israel

T. K. Hansen The Royal Veterinary and Agricultural University, Frederiksberg, Denmark

A. Herrera-Estrella Centro de Investigación y Estudios Avanzados, Unidad Irapuato, Irapuato, México

Byung Kook Hwang College of Life and Environmental Sciences, Korea University, Seoul, South Korea

M. Hyakumachi Gifu University, Yanagido Gifu, Japan

M. Jakobsen The Royal Veterinary and Agricultural University, Frederiksberg, Denmark

Wojciech J. Janisiewicz Appalachian Fruit Research Station, U.S. Department of Agriculture –Agricultural
Research Service, Kearneysville, West Virginia, USA

Hans-Börje Jansson Universidad de Alicante, Alicante, Spain

George G. Khachatourians College of Agriculture, University of Saskatchewan, Saskatoon, Canada

Beom Seok Kim Institute for Structural Biology and Drug Discovery, Medical College of Virginia, Virginia
Commonwealth University, Richmond, Virginia, USA

Maren A. Klich U.S. Department of Agriculture –Agricultural Research Service, New Orleans, Louisiana, USA

Christian P. Kubicek Institute of Chemical Engineering, Vienna, Austria

M. Kubota Gifu University, Yanagido Gifu, Japan

Thomas Ostenfeld Larsen BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark

Contributors xi

Sally Ann Leong U.S. Department of Agriculture –Agricultural Research Service, and University of Wisconsin,
Madison, Wisconsin, USA

Michael B. Liewen General Mills, Inc., Minneapolis, Minnesota, USA

Greg T. Lonergan Environment and Biotechnology Centre, Swinburne University of Technology, Melbourne,
Australia

Luis V. Lopez-Llorca Universidad de Alicante, Alicante, Spain

Flemming Lund BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark

Naresh Magan Cranfield University, Bedford, United Kingdom

S. S. Mark* Worcester Polytechnic Institute, Worcester, Massachusetts, USA

A. Mehta Dr. H.S. Gour University, Saugor, India; Institute of Microbiology, Czech Academy of Sciences,
Prague, Czech Republic

P. Mehta Dr. H.S. Gour University, Saugor, India; Institute of Microbiology, Czech Academy of Sciences,
Prague, Czech Republic

Edward J. Mullaney U.S. Department of Agriculture–Agricultural Research Service, New Orleans, Louisiana,
USA

T. B. Ng The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

Justine N. Niamke University of Maryland, College Park, Maryland, USA

S. Nicklin DSTL, Sevenoaks, Kent, United Kingdom

Kristian Fog Nielsen BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark

Gary Peng Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada

Anil Prakash Barkatullah University, Bhopal, Madhya Pradesh, India

Zamir K. Punja Simon Fraser University, Burnaby, British Columbia, Canada

Amparo Querol University of València, València, Spain, Instituto de Agroquı́mica y Tecnologı́a de Alimentos,
CSIC, València, Spain

R. D. Rai National Research Centre for Mushroom, Solan, Himachal Pradesh, India

Purbita Ray General Mills, Inc., Minneapolis, Minnesota, USA

Vicente Sanchis Universitat de Lleida, Lleida, Spain

Kirsten Schliephake Environment and Biotechnology Centre, Swinburne University of Technology,

Melbourne, Australia

Tanuja Singh Banaras Hindu University, Varanasi, India

Jørn Smedsgaard BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark

Season R. Snyder Sapphos Environmental, Inc., Pasadena, California, USA

*Present affiliation: Cornell University, Ithaca, New York, USA

xii Contributors

John B. Sutherland National Center for Toxicological Research, U.S. Food and Drug Administration,
Jefferson, Arkansas, USA

Ulf Thrane BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark

Pragati Tiwari The Energy and Resources Institute, New Delhi, India

Unai Ugalde University of the Basque Country, San Sebastián, Spain

Raj S. Utkhede Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Agassiz, British
Columbia, Canada

János Varga University of Szeged, Szeged, Hungary

Nam Sun Wang University of Maryland, College Park, Maryland, USA

Gary Ward MIGAL-Galilee Technology Center, Kiryat Shmona, Israel

Nia A. White University of Abertay, Dundee, Scotland, United Kingdom

S. Wilkinson DSTL, Sevenoaks, Kent, United Kingdom

Lisette J.C. Xavier Saskatoon Research Centre, Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan,
Canada

Jiujiang Yu U.S. Department of Agriculture –Agricultural Research Service, New Orleans, Louisiana, USA

Naresh Magan Cranfield University, Bedford, United Kingdom

Carlos G. Dosoretz Technion –Israel Institute of Technology, Haifa, Israel

1
Biotechnological Approaches in Plant Protection:
Achievements, New Initiatives, and Prospects

Sally Ann Leong U.S. Department of Agriculture – Agricultural Research Service, and University of
Wisconsin, Madison, Wisconsin, USA

1 CHALLENGES FOR FOOD SECURITY IN In the last century, the Green Revolution addressed the food
THIS CENTURY AND BEYOND needs of the human population through the development of
high yielding and early maturing varieties that performed
Today we face many critical issues in agriculture: (a) an under favorable conditions of nutrition and moisture (Khush
exponentially growing human population; (b) recurrent 2001). Prior to this time, increased production was dependent
on expansion of land area for crop production. In recent years,
famine; (c) the destruction of natural landscapes such as
yield gain through breeding has not kept pace with population
tropical rain forests to extend agriculture to previously unused
growth (Serageldin 1999). Furthermore, genotype is not the
lands; (d) the exodus of human civilization from rural
only factor limiting productivity. Abiotic and biotic stress
communities to cities; (e) the destruction of environmental
factors also contribute to losses in yield both pre and
quality resulting from exposure to agrochemicals, erosion of
postharvest. For example, in Asia these technical constraints
soils and salinization of soils as well as exhaustion and
on rice production may reduce production by 23% (Evanson
contamination of fresh water resources; (f) the loss of bio-
et al. 1996). Socioeconomic constraints also contribute to
diversity through monocropping and the destruction of natural practices that affect yield. Ninety percent of the world’s rice is
habitats; (g) the reliance of agricultural production, transport, grown in Asia on small farms with limited resources. Thus,
and storage systems on fossil fuel; (h) the acquisition and decisions are made based on economics rather than achieving
concentration of agricultural wealth by multinational corpor- technically optimum yields. Despite much research on the
ations; and (i) an issuant lack of knowledge by a growing application of biotechnology to solve these technical
proportion of human civilization on how to cultivate, prepare, production constraints, biotechnology has had limited impact
and preserve food. The United Nations Food and Agriculture to date on rice production in Asia (Houssain 1997). This has
Organization predicts that agricultural productivity in the been due in part to the reluctance to adopt the use of genetically
world will be able to sustain the growing human population by modified organisms (GMOs) in most countries of Asia. China
2030 but hundreds of millions of people in developing is the only Asian country that has embraced biotechnology on
countries will remain hungry and environmental problems any notable scale as a solution to technical production
caused by agriculture will remain serious (FAO 2002). By constraints in agriculture (Huang et al. 2002; Pray et al. 2002).
2025,83% of the expected global population of 8.5.2 billion For example, over four million smallholders have been able to
will be in the developing world (United Nations 2002). The increase yield and reduce pesticide costs and adverse health
social consequences are obvious. Food is a basic human need effects of applying pesticides by using transgenic Bt-cotton
and right. How can we sustain the food needs of the earth’s (Pray et al. 2002). Another constraint relates to the complex-
biotic community in the 21st century and beyond while ities of ownership rights that can delay and discourage the
preserving environmental quality and the diversity and quality scientific application of biotechnology discoveries and their
of life on earth (Time, August 26, 2002)? What solutions can transfer to the market place (Kowalski et al. 2002). Finally,
biotechnology provide to address these problems (Khush public concern about the safety of consuming GMOs has left
and Bar 2001)? tons of food aid containing transgenic corn untouched in

1
2 Leong

Zimbabwe and Zambia despite the prediction that 13 million maps were developed for key fungal pathogens such as
people are now at risk of famine due to severe drought in Magnaporthe grisea (Farman and Leong 1995; Nitta et al.
Southern Africa (Paarlberg 2002). Organic growers worldwide 1997; Skinner et al. 1993; Sweigard et al. 1993), Phytophtora
have rejected GMOs as an allied technology to disease and pest infestans (van der Lee 2001), and Leptosphaeria maculans
management. Late blight in potato is still very difficult to (Pongam et al. 1988). These studies also began to reveal the
control in these farming systems (Mader et al. 2002). complexities of these genomes in terms of repeated DNAs,
Biotechnology as applied to plant protection against fungal their function as transposable elements (Goff et al. 2002;
pathogens has seen three phases of development over the last Hamer et al. 1989; Kachroo et al. 1997), their distribution
20 years: (a) the application of molecular markers to marker- within the genome (Goff et al. 2002; McCouch et al. 1988;
assisted breeding and the map-based cloning of genes Nitta et al. 1997; Yu et al. 2002), and their role in genome
associated with disease resistance and the plant defense evolution and host recognition (Farman 2002; Farman et al.
response as well as to study fungal pathogenesis and host 2002; Kang et al. 2001; Song et al. 1997; 1998). Comparative
recognition; (b) the development of routine methods for stable maps were generated in plants by mapping markers across
and transient transformation of plants and fungi with foreign genera and showed considerable synteny within families of
genes; and (c) the application of New Biology approaches to plants (Ahn and Tanksley 1993; Bennetzen and Freeling
study plant growth and development and mechanisms of plant 1993; Chen et al. 1997; Dunford et al. 1995; Gale and Devos
response to abiotic and biotic stresses and fungal pathogen- 1998; Hulbert et al. 1990; Saghai Maroof et al. 1996; Tanksley
esis. The purpose of this short review is to provide a critical et al. 1992). The mapping of phenotypic markers, both native
analysis of these recent biotechnological approaches to plant and induced by mutation, followed closely behind and yielded
protection with emphasis on fungal pathogens. precise information on the chromosomal location of genes
important to plant defense (Ronald et al. 1992; Wang et al.
1995) and fungal host specificity (Dioh et al. 2000; Smith and
2 TESTED STRATEGIES Leong 1994; Sweigard et al. 1993) and led to their cloning by
chromosome walking (Cao et al. 1997; Farman and Leong
2.1 Marker-Assisted Breeding and Map-Based 1998; Martin et al. 1993; Orbach et al. 2000; Song et al. 1995;
Cloning of Genes Sweigard et al. 1995). The cloning of a plethora of disease
2.1.1 Molecular Maps resistance genes from many plant species has shown that they
belong to a small number of structural classes (Brueggeman
With the advent of recombinant DNA technology came the et al. 2002; Chauhan and Leong 2002; Dangl and Jones 2001;
application of cloned DNAs as probes to genomic DNA of the Meyers et al. 1999; Xiao et al. 2001) (Figure 2). By contrast,
source organism and the revelation that different alleles could the predicted structures of fungal cultivar specificity genes are
be detected between individuals based on restriction fragment quite diverse (Bohnert et al. 2001; De Wit and Joosten 1999;
length polymorphisms (Helentjaris et al. 1985) (Figure 1). Orbach et al. 2000; Sweigard et al. 1995).
Genetic maps based on these and other types of molecular These studies have been complemented by the mapping
markers (Table 1) were developed for many organisms of candidate genes such as the PR (pathogenesis-related)
including crop plants such as rice (McCouch et al. 1988; proteins in plants that were discovered from differential
http://rgp.dna.affrc.go.jp/publicdata/geneticmap2000/index. expression of RNA and protein during plant infection
html), lettuce (Kesseli et al. 1994), tomato (Tanksley et al. (Muthukrishnan et al. 2001) or resistance gene analogs based
1992), alfalfa (Brouwer and Osborn 1999), and Brassica on the conserved structural features of disease resistance
spp. (Kole et al. 2002), among others. Likewise molecular genes (Boyko et al. 2002; Chauhan et al. 2002; Faris et al.

Table 1 Molecular markers used in mapping of traits

Marker
RFLP Restriction fragment length polymorphism
RAPD Random amplified polymorphic DNA
APD Amplified polymorphic DNA
CAP Cleaved amplified polymorphism
AFLP Amplified fragment length polymorphism
Figure 1 Cosegregation of a RFLP marker (R-23 16) with
Microsatellite Polymorphism based on different numbers
Pi-CO39(t) locus in homozygous F2 susceptible progenies.
on mono, di, tri, or
Genomic DNA of CO39 (R, resistant), 51583 (S, susceptible) and
tetranucleotide repeats
F2 progenies was digested with Dra1, blotted and probed with
CDNA-AFLP cDNA amplified restriction fragment length
R-2316. Recombinant progenies show DNA fragments from both
polymorphism
parents. Phosphoimage of Southern blot is shown.
Biotechnological Approaches in Plant Protection 3

Figure 2 Different classes (A – G) of plant disease resistance genes [reviewed in Chauhan and Leong (2002)): Genes in classes A and B
are cytoplasmic proteins differing only in their N-terminal domains; Class C genes encode putative transmembrane molecules with an
extracellular LRR domain; Xi21 is a transmembrane protein with an extracellular LRR domain; Pto is a cytoplasmic Ser/Thr kinase;
RPW8 contains a putative N-terminal TM domain and a CC domain; Hm1 is a unique enzyme that detoxifies a fungal toxin; Abbreviations
for domains: TIR, Drosophila Toll/Human Interleukin-lreceptor; CC, Coiled-coil; NBS, Nucleotide binding site; LRR, Leucine-rich
repeat; TM, Transmembrane; Ser/Thr, Serine/threonine kinase.

1999; Gebhardt and Valkonen 2001; Huang and Gill 2001; Li and 93-11 has revealed numerous NBS-LRR-containing
et al. 1999; Shen et al. 1998; Speulman et al. 1998). This sequences as well as sequences potentially encoding other
analysis has been particularly well advanced in wheat and its minor classes of R genes and Arabidopsis genes known to
relative Aegilops tauschii. Resistance and defense response control defense response signal transduction (Goff et al. 2002;
genes in A. tauschii are localized in clusters primarily in Yu 2002). Preliminary studies based on conservation of
distal/telomeric regions of the genome (Boyko et al. 2002) RGAs in comparative maps of the grasses have shown
while in Chinese spring wheat defense response genes are evidence for some conservation but also redistribution of this
localized in clusters and/or at distal regions of chromosomes class of genes among the grasses (Leister et al. 1998).
(Li et al. 1999). In many cases, these genes or gene homologs Likewise a detailed comparison of a syntenic region between
have been correlated with loci that affect quantitative or barley and rice did not reveal any candidate resistance genes
single gene resistance in the respective plants. For example, in rice that could be the ortholog of Rpgl in barley (Han et al.
QTLs with large effects in wheat were shown to contain 1999; Kilian et al. 1997).
RGAs or clusters of defense response genes such as catalase,
chitinase, thaumatins, and an ion channel regulator (Faris
et al. 1999). Similar results are emerging in the genomes of 2.1.2 Differential cDNA-AFLP Screens
potato (Gebhardt and Valkonen 2001), Arabidopsis (Speul-
man et al. 1998) and pepper (Pflieger et al. 2001). Differential cDNA-APLP screening has been done to isolate
Efforts are underway to functionally characterize 179 hypersensitive response (HR)-specific genes to the Clade-
NBS-LRR-encoding genes that may encode disease resist- sporium fulvum elicitor Avr4 in tomato and has led to the
ance genes in the Arabidopsis genome (Figure 2). These isolation of a previously known and corresponding disease
have been organized into subclasses and their distribution resistance gene cluster Cf-4 as well as numerous new
mapped to the chromosomal sequence (Michelmore 2002; candidate genes involved in the HR response (De Wit et al.
www.nibh-rs.ucdavis.edu). A publicly available, draft 2002; Takken et al. 2001). This method is a robust and
ordered sequence of the rice genome is anticipated by the inexpensive way to identify differentially expressed genes
end of 2002 (http://rgp.dna.affrc.go.jp/cgi-bin/statusdb/ involving the digestion of cDNAs with two different
seqcollab.pl) and will allow comparisons to be done across restriction enzymes and the amplification of the resulting
syntenic regions of grass genomes (http://www.gramene.org!). products after ligation to adapters for these enzymes. The
The unordered draft sequence of rice varieties Nipponbare sizes of the resulting amplicons are measured by gel
4 Leong

electrophoresis and resulting fragments can be excised and such as Arabidopsis have large LD structures on the order of
sequenced. Comparison of this method with differential 250 kb or 1 cM (Nordborg et al. 2002) while outbreeding
display has shown the cDNA-AFLP method to be superior plants like maize have very small LD structures in the order of
(Jones and Harrower 1998). Using the cDNA-AFLP kbs (Buckler and Thornsberry 2002). Using this approach,
technique, Durrant et al. (2000) found a strong coincidence Thornsberry et al. (2001) were able to associate poly-
between the expression of genes involved in race-specific morphisms found in the Dwarf8 gene of maize with variation
resistance and the wound response in tobacco cell cultures. seen in flowering time.
Collectively, these candidate genes will provide additional In Saccharomyces cereviseae whose entire genome
markers for studies of disease resistance traits in the potato sequence is known, a QTL for high temperature growth
and tomato genomes. Infection-specific cDNA-AFLPs have (Htg) commonly found in clinical isolates was rigorously
also been identified in Arabidopsis thaliana inoculated analyzed to identify the responsible genes (Steinmetz et al.
with Peronospora parasitica (van der Biezen et al. 2000). 2002). Using reciprocal hemizygosity tests, involving
Interestingly, most fragments were derived from the fungal selective gene disruption of candidate genes in both parental
pathogen showing the power of this method to study genes genomes and then forming diploid hybrids among these
expressed in the pathogen, which in many cases may strains, three genes were found to contribute to the phenotype
represent a minor component of the mass of the tissue studied. in the QTL interval. However, the alleles of two genes came
Previous genetic studies by Valent et al. (1991) have from one parent strain while that of the third came from the
shown that infection of some grass hosts by M. grisea is a other parent. By contrast attempts to employ natural sequence
quantitatively inherited trait. The cDNA-AFLP approach variation or mRNA expression levels determined from several
would allow for the identification a unique set of cDNA- natural isolates of yeast did not provide a clue to which
AFLPs in each progeny showing varying degrees of gene(s) in the interval contributed to the phenotype. Thus
pathogenicity and in some cases segregating with patho- employing LD and association by decent to accelerate gene
genicity. This approach has been recently used to create identification in an interval may not always succeed and
genome-wide transcription maps of Arabidopsis and potato genetic studies will be required to study inheritance and create
and study inheritance of the cDNA-AFLPs in segregating reciprocal hemizygotes in targeted regions of the genome.
populations (Brugmans et al. 2002). Thus phenotypes can be The use of allele-specific gene silencing methods (see below)
directly associated with molecular genotypes and candidate may allow this to be done in the F1 generation of plants while
gene fragments can be excised from gels for further analysis. targeted gene disruption methods can be used in fungi such as
We are using this approach in an attempt to identify major and Ustilago maydis that have a stable diploid phase and facile
minor genes controlling resistance to blast and drought gene knockout system. Transformation of haploid fungi with
tolerance in Eleusine coracana. wild type and disrupted, endogenous or alternative alleles of
Computational methods for relating the size of the AFLP candidate genes might be a useful strategy for those fungi that
restriction fragment products to the predicted restriction cannot form stable diploids. In fact these strategies have been
fragment products from sequenced cDNA libraries have been used to unravel the complex functions of the east and west
developed and used to identify putative, infection stage- alleles of b mating type locus of U. maydis (Gillissen et al.
specific, pathogenicity factors from the plant pathogenic 1992; Kamper et al. 1995). Gene silencing has been used in
nematode Globoderu rostochiensis without need for sequen- fungi such as P. infestans in which silencing of the fungal
cing the gel fragments (Qin et al. 2001). For those organisms elicitin INF1 increased virulence on Nicotiana benthamiana
having fully sequenced, annotated full length cDNA libraries (Kamoun et al. 1998).
such as Arabidopsis (Seki et al. 2002), this approach provides
for the rapid functional classification of the cDNA-AFLPs. 2.1.5 Candidate Gene Validation

2.1.5 Identification of QTL-Associated Genes It should be emphasized that candidate genes are simply
“candidate” genes and that confirmation of a gene’s function
Very few QTL studies in plants have led to the cloning of a with a genetically and/or expression-defined phenotype must
single gene within a QTL that is responsible for the variation be done by transformation and complementation tests
seen [reviewed in Buckler and Thornsberry (2002)]. These (Farman and Leong 1998; Orbach et al. 2000; Song et al.
few examples represent QTLs that had major effects on 1995; Wang et al. 1999; Yoshimura et al. 1998). Recently
variation. Buckler and Thornsberry (2002) have proposed that gene silencing has also been successfully applied to study
association approaches should be also considered to provide gene function in several plants (Azevedo et al. 2002;
improved resolution and to reduce the time of analysis as Baulcomb et al. 2002; Peart et al. 2002; Wesley et al. 2001).
mapping populations are not needed since natural variation in This method involves the cloning of a small fragment
a population is investigated instead. The resolution of (, 200 nucleotides) of a gene into an expression vector and
association that can be obtained depends on the linkage transforming the plant [reviewed in Baulcomb et al. (2002)].
disequilibrium (LD) structure of the population of organism The resulting small RNA is made double stranded and then is
being studied and some insight on candidate gene(s) to target. digested into small dsRNA fragments (siRNA, small
Studies on LD structure have shown that inbreeding plants interfering RNA), which are thought to guide RNAse to the
Biotechnological Approaches in Plant Protection 5

nascent wild-type gene transcript and causes it to be degraded dispensable chromosome controls production of a host-
thus leading to a net loss in the gene’s expression. Direct specific toxin (Hatta et al. 2002). Likewise Han et al. (2001)
bombardment of plant cells with dsRNA is also possible found that genes required for pathogenicity on pea are present
(Schweizer et al. 2000). In the examples described earlier, the on a dispensable chromosome of Nectria haematococca.
specific genes RAR1 in barley, and Rx, N , Pto, and EDS1 in Exploitation of the multitude of novel genotypes found in
N. benthamiana implicated in disease resistance signaling plant germplasm now available in gene banks will often
were silenced and found to be essential for the signaling require the methods of molecular mapping and candidate gene
process. This approach is being extended to the high isolation described above to identify the genes that contribute
throughput analysis of the HR candidate genes from tomato to these unique phenotypes (Fulton et al. 1997; Tanksley and
noted earlier (De Wit et al. 2002) as well as in a normalized McCouch 1997; Xiao et al. 1998). This is true even for plant
cDNA library from N. benthamiana (Baulcomb et al. 2002). genomes for which the entire genome sequence is known,
Interestingly, many genes in N. benthamiana were found in thus allowing candidate genes to be isolated in related
the preliminary round of analysis to affect the HR while not genomes. For example, the short stature mutation sg1 found in
affecting pathogen growth while some genes were affected in green revolution rice variety IR8 encodes a mutant
both phenotypes when silenced. Moreover almost 1% of the biosynthetic gene for gibberellin while the semidwarf
genome appears to affect the HR response. phenotype in green revolution varieties of wheat is conferred
by mutations in the gibberellin signaling pathway (Sasaki
2.1.5 Integration of Molecular Biology with et al. 2002). In addition, many previously unidentified genes
Classical Breeding have been found in every genome that has been sequenced
(Goff et al. 2002; Yu et al. 2002). In the case of disease
The application of molecular markers to traditional breeding resistance genes, the functions of only a few are known in
has provided a powerful method to accelerate breeding as each sequenced genome and not all LRR-containing coding
phenotypic tests are not essential and rapid DNA isolation sequences are likely to function in disease resistance. For
methods using hole punch-sized pieces of leaf tissue are example, a LRR receptorlike transmembrane protein kinase
possible in young seedlings (Huang et al. 1997). Thus tightly gene, that is gibberellin-induced and specifically expressed
cosegregating or gene(allele)-specific markers can be in growing tissues of deep-water rice, may function in
followed and confirmation of phenotype can be done on a hormone signaling (van der Knaap 1999). The genetic
selected set of plants within a population that are destined for location of disease resistance genes must be determined in
further crossing. Phenotypic validation is essential as the genome that contains the functional gene if the
recombination, gene conversion or other confounding events reference genome sequence lacks a functional copy. These
can take place, even within the gene being studied, leading to tenets also apply to the identification of fungal genes
an inaccurate scoring based on markers alone. As we learn involved in plant recognition. Examples exist for the
more about the function of plant genes and specific alleles of complete absence of a recessive gene in fungal strains that
these genes in disease resistance through mapping and have lost cultivar specificity (van den Ackerveken et al.
functional tests, we can anticipate the increased application of 1992; Farman et al. 2002). Furthermore, the recently
molecular markers and gene chips (see later) to the breeding released genomic DNA sequence of M. grisea having 7X
of disease resistance in plants. In particular, it will be coverage (http://www-genome.wi.mit.edu/annotation/fungi/
interesting to know what contribution pathogenesis-related magnaporthe/) does not contain the AVRI-CO39 cultivar
proteins, which are generically present in all plant genomes, specificity gene (RS Chauhan, D Lazaro, and SA Leong,
make to quantitative resistance. Is expression more efficient unpublished data). This genome sequence is thus useless
in some genomes than others because of the gene’s placement without precise genetic mapping data for this AVR gene that
in clusters and/or their specific transcription regulatory can be used to identify in the reference, sequenced genome,
elements and/or their duplication or absence in some genomes a contiguous sequence spanning the genome between these
and/or the efficacy of specific alleles? Likewise, what is the markers. This sequence can then be used to develop new
genetic and molecular basis of host specialization in the genetic markers and to probe libraries of a strain that does
fungi? The location of many disease resistance and defense carry AVR1-CO39. In fact this AVR gene was originally
genes at the ends of chromosomes in wheat may affect their cloned using a more laborious chromosome walking
stability through recombination and chromosome breakage strategy in the genome of a strain carrying the functional
as well as their expression through unique chromatin gene (Farman and Leong 1998).
organization (Faris et al. 2000). The telomeric location of
AVR1-PITA in M. grisea has been shown to contribute to its
instability leading to strains with increased virulence (Orbach
et al. 2000). The BUF1 gene of M. grisea appears to be readily 2.2 Transgenic Plants As a Tool for Plant
deleted in one parental chromosome by intrachromosomal Protection
recombination of repeated DNA flanking the locus as a result
of mispairing of homologous chromosomes during meiosis Following clues from the molecular biology of plant-microbe
(Farman 2002). In Alternaria alternata a conditionally interactions, many genes involved in disease resistance and
6 Leong

the defense response have been cloned from a variety of achievements have been made in the last few years and many
plants (Dangl and Jones 2001). A number of these genes have major resistance genes associated with fungal disease
been tested for their ability to control fungal pathogens in resistance have been cloned or at least tagged with molecular
transgenic plants grown in the laboratory and to a more markers (Brueggeman et al. 2002; Chauhan and Leong 2002;
limited extent in the field. In addition, natural or synthetic Gebhardt and Valkonen 2001). Thus, we can expect many
antimicrobial peptides and genes for resistance to pathogen- new developments with regard to this field in the next
derived toxins have been introduced into plants. This work is decade. Furthermore, despite public concern about transgenic
thoroughly reviewed by Bent and Yu (1999); Rommens and crops, the global adoption of transgenic crops continues
Kishore (2000), and Melchers and Stuiver (2000). Only to increase, particularly in the United States, where
more recent studies providing significant new findings or 74.8 million acres were planted in transgenic crops including
extensions of this work will be considered here. corn, soybean, cotton, and canola in 2000 (Transgenic Crops
Current efforts have continued to focus on introduction of 2002). This represented about 50% of the total soybean and
disease resistance genes, natural and synthetic antimicrobial cotton acreage planted in that year. The International Service
peptides as well as selected enzymes such as chitinase and for the Acquisition of Agri-Biotech Applications predicts that
b-glucanase into several crop plants and the laboratory and the world market for genetically engineered plants will be $8
field evaluation of these plants for disease resistance. Little billion in 2005 and $25 billion by 2010 (http://nature.biotech.
has been reported in the literature on the field performance of com) (Figure 3). Plant pathogens cause $30 –50 billion dollars
these transgenic plants, although about 5% of all permits for of loss annually in crop productivity (Baker et al. 1997) thus
field testing of transgenic plants in the United States over the justifying this investment in biotechnological approaches to
last decade have been for transgenic plants having fungal crop protection. The reduction in use of agrochemicals for
resistance (Information Systems for Biotechnology 2002). disease control is another important incentive for this
Moreover, most permits have been issued to companies and technology. Japanese growers spend more than $600 million
this proprietary work is not yet in the public domain. No a year to control diseases on rice (Bonman 1998). Already, the
transgenic plants having fungal resistance have been reduction in insecticide use in China through use of Bt
approved for use as food and/or feed (AgBios 2002). transgenic crops has impacted farmer income and health
Despite this seemingly limited progress, some promising (Huang et al. 2002; Pray et al. 2002).

Figure 3 Impact of genomics on the world economy.

Biotechnological Approaches in Plant Protection 7

2.2.1 Defense Pathways for resistance in rice to M. grisea carrying the AVR1-CO39
gene (Farman and Leong 1998) will be useful in other grass
Despite many projections made in the reviews listed earlier, a species such as perennial rye grass as functional copies of the
flurry of published reports has not followed. In many cases, AVRl-CO39 gene are found in the grey leaf spot pathogen
this can be attributed to the observation being made first in (ML Farman personal communication).
Arabidopsis and not in a crop plant. However, this lag is being Coexpression of the C. fulvum Avr9 and tomato Cf-9 genes
addressed with Arabidopsis genes to assess function in crop in Brassica napus was investigated as a method for inducing
plants and through the identification of homologs of broad-spectrum resistance to fungal pathogens (Hennin et al.
Arabidopsis genes. For example, the Arabidopsis NPR1 2001). Induction of PR1, PR2, and Cxc750 was detected
gene (Cao et al. 1998) when overexpressed in rice caused following injection of the Avr9 peptide obtained from
enhanced resistance to the rice pathogen Xanthomonas oryzae intercellular fluids of B. napus transgenic plants expressing
pv. oryzae (Chern et al. 2001) and the investigators were able Avr9 into B. napus expressing the tomato Cf-9 gene. F1 plants
to retrieve, in a two hybrid screen, a bZIP family of interactors and progeny from a cross of the Avr9 and Cf-9 plants were
showing that a similar pathway of signaling is likely present evaluated for resistance to fungi. Disease development was
in rice as Arabidopsis (Zhang et al. 1999). In addition, delayed at the site of infection of L. maculans and Erysiphe
Yoshioka et al. (2001) have shown that Arabidopsis will polygoni but enhanced at the site of infection of Sclerotinia
respond to the rice fungicide probenazole by induction of PR sclerotiorum. Thus, heterologous expression of AVR-R gene
genes and show enhanced resistance to Pseudomonas pairs may be a useful strategy for control of fungal disease in a
syringae pv. tomato DC3000 and P. parasitica Emco5. This variety of plants. However, the finding that Arabidopsis
response was dependent on a functional NPR1 gene and was resistance gene RPM1 requires another plant gene RIN4 in
compromised in NahG transgenic plants further supporting order to accumulate and interact with avrRpm1 or AvrB
the connection of this pathway with generalized resistance to (Mackey et al. 2002) as well as the inability to show direct
pathogens in both Arabidopsis and rice. Six NPR1 homologs interaction of the products of Avr9 with Cf-9 (van der Hoorn
are reported in the recently released Nipponbare genome et al. 2002; Luderer et al. 2001) suggests that this strategy
sequence (Goff et al. 2002). It will be interesting to see how must be used cautiously. This may explain the inability of van
overexpression and silencing of these genes affects resistance der Hoorn et al. (2002) to see a necrotic response in the
of rice to key fungal pathogens of rice such as Rhizoctonia nonsolanaceous plant lettuce with this gene combination.
solani and M. grisea. More recent reports on the use of the coexpression strategy for
Overexpression of the Arabidopsis ACD2 (accelerated cell plant protection against fungi in solanaceous plants have not
death) gene leads to tolerance of susceptible Arabidopsis been made (Melchers and Stuiver 2000), however the
plants to P. syringae infection by reducing disease symptoms coexpression of Avr9 and Cf-9 under control of nematode
associated with cell death such as ion leakage, while allowing inducible promoters in tobacco has been studied (Bertioli et al.
the bacteria to grow to similar levels as in susceptible plants 2001). Surprisingly these plants underwent spontaneous
(Mach et al. 2001). Fungal pathogens were not tested. necrosis in the absence of the nematode. Evidence for activity
Broader testing of other genes that have shown wide- of the genes was found both in aerial and root tissue.
spectrum disease resistance to bacterial, fungal, and viral The cloning of MLO locus of barley (Shirasu et al. 1999),
pathogens when overexpressed such as Prf (Oldroyd and which confers nonrace-specific resistance to Blumeria
Staskawicz 1998) and Pto (Tang et al. 1999) in tomato has not graminis f. sp. hordei, has been followed with investigations
been reported. Nor have further reports been made on of its potential use for control of different fungal pathogens.
constitutively active variants of Pto (Rathjen et al. 1999). Jarosch et al. (1999) found that in contrast to increased
Presumably, this approach can be used in other crop plants. resistance conferred by recessive alleles of MLO to powdery
Introduction of the bacterial blight resistance gene Xa21 mildew, these barley plants have increased susceptibility to
into elite rice cultivars has lead to the expected resistance penetration by M. grisea despite showing similar ability to
phenotype when inoculated with X. oryzae pv. oryzae; wild type plants to respond to M. grisea elicitor. Likewise
however, strains that are virulent on Xa21 were not tested nor Kumar et al. (2001) showed that mlo plants were more
were other pathogens (Tu et al. 1998). Performance of these susceptible to the necotrophic pathogen Bipolaris sorokini-
lines was tested under natural field conditions without any ana. These reports reveal the complexity of various fungal
apparent loss of yield performance (Tu et al. 2000). interactions with the host and the difficulty of using a single
Disease resistance genes from one crop plant have now strategy to control multiple pathogens.
been successfully used in other crop species. For example, the Recent work on MLO has shown that it is a novel
Bs2 resistance gene from pepper confers resistance to calmodulin-binding protein that is responsive to both abiotic
X. campestris pv. vesicatoria in tomato in the laboratory as and biotic stresses through down regulating the oxidative
well as in preliminary field tests (Staskawicz et al. 2002; Tai burst and cell death response (Kim et al. 2002a,b; Piffanelli
et al. 1999). Work from my laboratory in conjunction with et al. 2002). Binding to calmodulin is essential to full function
studies from the laboratories of Mark Farman at the of MLO (Kim et al. 2002a). A rice homolog of MLO that also
University of Kentucky and Yukio Tosa at Kobe University interacts with calmodulin was isolated (Piffanelli et al. 2002).
has suggested that the Pi-CO39 (t) gene (Chauhan et al. 2002) It will be interesting to see how silencing of MLO in rice
8 Leong

plants affects interaction with biotrophic and necotrophic have shown that the synthetic antimicrobial peptide DE41 is
pathogens. active at the micromolar range against many important
bacterial and fungal plant pathogens. Crude protein extracts
2.2.2 Antifungal Proteins and Peptides from transgenic tobacco plants constitutively expressing
D4E1 showed ability to reduce growth of A. flavus and
Several new reports have appeared on the use of antifungal V. dahliae while control plant extracts did not (Cary et al.
proteins such as Ag-AFP from Aspergillus giganteus, 2000). Furthermore, the transgenic plants showed increased
chitinase, b-glucanase and Ribozome-Inactivating Proteins resistance to Colletotrichum destructivum. The D4El gene has
(RIP) (Chareonpornwattana et al. 1999; Datta et al. 2002; been introduced into cotton and was shown to be present in
Oldach et al. 2001), thaumatinlike protein (PR-5) (Datta et al. cottonseed. Reduction of aflatoxin in cottonseed oil is a
1999), and human lysozyme (Takaichi and Oeda 2000) in desired outcome. Dow AgroSciences LLC has licensed the
plants for protection against fungal disease and show technology and is collaborating with USDA-ARS scientists
different levels of promise for these approaches. Chitinase who developed the technology to further evaluate the efficacy
and AFP appear to increase resistance in wheat, however, of the transgenic plants (SeedQuest 2002). The effects of this
the results were not corroborated with levels of these peptide on plant growth or other nontarget organisms have not
proteins in transgenic plants (Oldach et al. 2001). Other been reported. Finally, the antimicrobial peptide MS1-99, an
efforts to introduce chitinases in wheat have led to gene analog of magainin 2, a defense peptide secreted from the skin
silencing (Chareonpornwattana et al. 1999). The recent of the African clawed frog, was expressed from the
isolation of cDNA clones for novel acidic chitinases and chloroplast genome of tobacco and showed significant ability
b-1,3-glucanases from wheat spikes infected by Fusarium to control many phytopathogenic bacteria and fungi (DeGray
graminearum (Li et al. 2001) is exciting as these enzymes et al. 2001). Trangenic plant homogenates inhibited the fungi
may be more effective in control of this pathogen in this A. flavus, F. monilzjbrme and V. dahliae and anthracnose
tissue. Introduction of infection-related chitinase and rice lesions were absent in transgenic plant infected with
thaumatinlike protein into rice has led to moderate control of C. destructivum. Transformation of the chloroplast genome
sheath blight caused by R. solani (Datta et al. 2002). Field is an innovative approach to control the spread of the
evaluation of these plants is underway. Finally, studies on transgene as pollen will not carry the transgenic chloroplast.
carrot transformed with human lysozyme, which can cleave Evidence for pollen transfer of transgenes at the commercial
b-1,4 glycosidic bonds of peptidoglycan in bacterial cell field level is now available (Reiger et al. 2002).
walls and chitin in fungal cell walls, suggest that this While the use of peptides has shown significant promise,
approach may have promise for control of E. heraclei and thorough testing of the toxicity of plants producing these
A. dauci (Takaichi and Oeda 2000). peptides will be important. Their potential ability to inhibit
Natural and synthetic peptides have been evaluated for microflora that are essential to plant health as well as health of
control of pre and postharvest damage by fungi. Ali and animals, humans, and birds needs careful evaluation. Feeding
Reddy (2000) studied four cationic peptides for antimicrobial raw potatoes containing a cecropin-melittin cationic peptide
activity in vitro and in plants. All were shown to have chimera to mice was not a convincing test of the toxicity as
significant activity in the micromolar range against the animals do not normally eat this food and lost weight on
P. infestans and A. solani completely inhibiting growth of this diet until it was supplemented with normal feed (Osusky
the fungi on potato tissues. Alfalfa antifungal peptide defensin et al. 2000). More realistic tests are needed. The use of cooked
from seeds of Medicago sativa was shown by Gao et al. potatoes should be tested. Furthermore, only short-term
(2000) to have significant activity against Verticillium dahliae effects were studied. The survival and biological impact of
in vitro, and transgenic potato plants expressing the peptide large quantities of these peptides in the environment resulting
showed a reduced area under the disease progress curve from crop plant decay also needs to be evaluated. These issues
compared to vector control plants. Moreover, resistance was are only beginning to be addressed in a multitrophic context
correlated with the levels of peptide found in root samples. for insect resistant transgenic plants (Groot and Dicke 2002).
Similar results were obtained for transgenic potato plants These issues need to be critically addressed at the time of risk
expressing a N terminus-modified cecropin-melittin cationic assessment. Public concern has been spurred largely by a lack
peptide chimera (Osusky et al. 2000). The efficacy of the on confidence in transgene technology because of the lag time
peptide against Phytophthora cactorum and F. solani in responding to the large-scale effects that agrochemicals
infection was demonstrated in variety Desiree while not are having on human and environmental health despite early
affecting plant growth or tuber morphology or size. Tubers warnings by Carson (1962) many decades ago.
remained resistant for more than one year and the peptide
could be detected in this tissue. By contrast transgenic Russet 2.2.3 Phytotoxin Detoxification
Burbank plants showed significant morphological alterations
and resembled lesion mimic plants, produced very small In planta studies of the hydroxylation and glycosylation of
tubers, and showed less resistance to P. cactorum. Trangenic destructin B, a phytotoxin produced by A. brassicae, to a
raw tubers were fed to mice without significant growth effects nontoxic product have shown a correlation between plant
relative to untransformed tubers. Rajasekaran et al. (2001) resistance with phytoalexin production and the efficiency of
Biotechnological Approaches in Plant Protection 9

these modifications of the toxin in Brassica spp. (Pedras et al. source fungus to create a disruption of the sequence in the
2001). These data suggest that improved resistance can be genome by gene replacement. This method has been adapted
engineered in or transferred within Brassica hosts of to a high throughput scheme and the resulting mutants are
A. brassicae by enhancing hydroxylation of destruxin B. then tested for phenotypic alterations using a battery of tests.
The analysis of the biosynthetic pathway of saponins, The success of this scheme is not dependent on access to the
antimicrobial metabolites of plants, may allow the transfer of full genome sequence of a fungus.
these genes to other plants. Mutants defective in the saponin Agrobacterium tumefaciens T-DNA is also being used to
avenacin in oat were studied and shown to define seven loci directly mutagenize the genome of a number of fungi (De
and to be compromised in their ability to resist fungal attack Groot et al. 1998; Mullins et al. 2001; Rho et al. 2001) as well
(Haralampidis et al. 2001). The sad1 gene was shown to as plants such as rice (An et al. 2001) and Arabidopsis (see the
encode b-amyrin synthase. following: Krysan et al. 1999). M. grisea and F. oxysporum
mutants altered in virulence have been identified (Mullins
et al. 2001; Rho et al. 2001). The randomness of insertion in
3 NEW INITIATIVES AND PROSPECTS: THE the target genome and the possibility that some mutants
NEW BIOLOGY may be caused by secondary events needs to be thoroughly
evaluated in fungi, nevertheless, this method provides a
3.1 From Genome Sequence to Gene to Mutant to straightforward way of tagging genes in fungi and appears to
Function be superior to restriction enzyme-mediated insertion (REMI)
mutagenesis in which the transforming DNA containing a
Research in the last five years has been increasingly driven by selectable marker is linearized and transformed with a
the availability of whole genome sequences and cDNA restriction enzyme to promote insertion at restricted sites in
libraries from many plants and microorganisms (National the genome (Balhadere et al. 1999; Bolker et al. 1995; Lu et al.
Center for Biotechnology Information, http://www.ncbi.nlm. 1994; Sweigard et al. 1998). This method did not generate
nih.gov). The Arabidopsis genome sequence was released in certain kinds of expected mutants presumably due to the lack
2001 (Arabidopsis Genome Initiative 2001) and the draft of sites for this enzyme in the genes or the inaccessibility of
DNA sequence of two subspecies of rice was released this the genes to enzyme because of their lack of expression under
year (Goff et al. 2002; Yu et al. 2002). Likewise, several the conditions of growth and/or transformation. Mutants
fungal genome sequences are now available for model lacking a DNA insertion in the mutated gene have also been
organisms as well as plant pathogens (http://www-genome. documented using this method. Thus reisolation of the mutant
wi.mit.edu/annotation/fungi/magnaporthe/; http://www- gene is not simple.
genome.wi.mit.edu/annotation/fungi/neurospora/; Friedrich
et al. 2001; Turgeon and Yoder 2002). Consortia of scientists
have been established to bring together fungal genomic 3.3 Transient Expression of Fungal Genes in
resources for a research community. These include M. grisea Plants
(http://www.riceblast.org/) and Phytophthora (http://www.
ncgr.org/pgc; Torto et al. 2002). Sequenced libraries of cDNAs from Phytophthora are being
Comparative analysis of several fungal genome sequences systematically tested for their ability to induce resistance or
including that of C. heterostrophus, F. graminearum, and disease symptoms in tobacco and potato by transient
Botrytis cinerea, with those of Neurospora crassa and yeast expression using potato virus X (PVX) and A. tumefuciens
has led to the identification of putative common essential T-DNA vectors as delivery systems (Takken et al. 2000;
genes, candidate fungal-specific genes, and candidate Torto et al. 2002). Agroinfiltration has been used in several
pathogenicity genes (Turgeon and Yoder 2002). Systematic plant systems to study the interaction phenotype of products
analysis of these candidate pathogenicity genes through of avirulence genes and specific resistance genes (van der
knock out studies is underway. In addition, genes for Hoorn et al. 2000; Tai et al. 1999). Often plants are scored
polyketide synthases and nonribosomal peptide synthetases simply for formation of a HR or necrosis. Whether this is truly
are being identified and systematically disrupted since many representative of the natural response to infection is unclear. It
secondary products from fungi are known to have functions as will be interesting to see what kinds of the candidate genes are
host-specific toxins. found and whether their disruption or silencing corroborates
the heterologous expression tests described here.

3.2 Random Mutagenesis with Transposons

3.4 Microarrays
Another strategy for identification of pathogenicity genes has
been to introduce transposons in fungal genomic DNA cloned With the recent availability of massive amounts of DNA
into cosmid vectors (Hamer et al. 2001). The insertion site of sequence data for many organisms, new technologies have
the transposon is sequenced to assess what function has been emerged to study this information in a high through put and
disrupted. Each tagged insert is then transformed into the cost effective manner. Microarrays based on spotting of
10 Leong

thousands of cDNAs on glass surfaces or synthesis of gene- sequence tag) database of M. truncatulu, and good matches
specific oligonucleotide microarray (OMA) on glass surfaces were found with calreticulin, nonseed lectins and an ion
have allowed the parallel analysis of expression of large channel. These early results are promising, as a homolog of
numbers of genes (Brown and Botstein 1999; Fodor 1997; the nonseed lectins is known to be part of the pea nodule
Lashkari et al. 1997). For example, new understanding of: (a) where it is thought to function as a storage protein. A similar
stage-specific gene expression during sporulation in yeast relationship at this location of the root might exist between
(Chu et al. 1998); (b) the functional role of a set of yeast the plant and fungus at the arbuscular membrane.
mutants each carrying a deletion in one of 5,916 open reading
frames (96.5% of total) (Giaever et al. 2002); (c) the
transcriptional hierarchy of metabolic genes in yeast (DeRisi 3.6 Plant Model Systems
et al. 1997); and (d) gene expression during meiosis (Primig
et al. 2000) has resulted from the use of OMAs. Microarrays Considerably more progress has been made toward our
are currently being used to study global gene expression in understanding of plant genes to date because of the higher
Arabidopsis (Zhu et al. 2001a; see later) and rice (Zhu et al. investment that has been made in plant biology. From
2001b). 1985 – 1995, the Rockefeller Foundation sponsored an
One caution in using these arrays is that they only provide international research program in rice biotechnology that
information on gene expression (RNA abundance) while led to the generation of rice molecular maps, transformation
many cellular processes are regulated at the translational or of rice, and the characterization of many basic biochemical
posttranslational levels. Giaever et al. (2002) found that many pathways for abiotic stress and disease and insect resistance in
genes required for fitness through gene knock out did not rice. The United States National Science Foundation
show increased expression using OMAs. Moreover, many (NSF)-Arabidopsis 2010 and Plant Genome Initiatives have
genes that showed increased expression were not required for also had a major impact on plant science research in the last
fitness. In addition, Primig et al. (2000) found very different decade (Ausubel 2002). Many of the projects deal with how
expression profiles when they compared RNA from different Arabidopsis responds to and resists pathogens such as: (a) the
yeast strains undergoing meiosis. The overlapping gene set Arabidopsis RPM1 disease resistance signaling network; (b)
could be correlated with genes previously known to have a expression profiling of plant disease resistance pathways; (c)
role in meiosis. This result emphasizes the need to use functional and comparative genomics of NBS-LRR-encoding
different isolates to find the core set of genes that are needed genes; (d) Functional genomics of quantitative traits.
for a biological process that shows differential gene Expression level polymorphisms (ELPs) of QTLs affecting
expression. disease resistance pathways in Arabidopsis; and (e) the
endgame for research genetics. Isolation and distribution of a
knockout mutant for every gene in Arabidopsis. More
3.5 Proteomics information can be obtained at the web sites of these projects
(Ausubel 2002). National Science Foundation Plant
Proteomics is the study of all proteins from a living organism. Genome has also funded allied work in other crops plants
The most advanced approach is the use of mass spectrometry (httn://www.nsf.gov). This research represents the cutting
to study whole cell/tissue protein content and modifications edge of plant science using the latest tools: microarrays to
(Haynes and Yates 2000). The method has also been used to study gene expression, T-DNA insertion mutants, large scale
analyze proteins in biological complexes (Link et al. 1999). mutant hunts, molecular mapping, and structure-function
Although this technology is very new and requires analysis of genes at the whole genome level.
sophisticated equipment and technical expertise, it offers a High through put methods for creating mutants using gene
more realistic view of the physiology of the cell at a given silencing that allow gene libraries or cDNA collections to be
point in time. Many studies that have traced RNA expression cloned in a silencing vector using an in vitro recombinase
as a means to identify important genes in a biological process should facilitate systematic functional analysis of plant
have led to disappointing results when the gene(s) is disrupted genes in plant defense (Wesley et al. 2001). Conventional
(Basse et al. 2000; Giaever et al. 2002; Timberlake and mutagenesis of plants carrying a construct such as a luciferase
Marshall 1988). Furthermore, redundancy in gene function or GUS (b-glucuronidase) reporter gene under the control of a
can confound this type of study. Analysis of the proteome can defense gene promoter are yielding new and interesting
distinguish which family member is made and modifications classes of mutants with constitutive broad-spectrum disease
that it may have undergone. resistance (Maleck et al. 2002). These mutants showed strong
Initial studies on the identification of membrane proteins resistance to P. parasitica isolates Noco2 and Emco5 and
from arbuscular mycorrhizas formed between M. truncatula variable resistance to Elysiphe cichoracearum. In addition to
and Glomus versiforme led to the identification several T-DNA, endogenous and heterologous plant transposable
protein from microsomes fractionated from mycorrhizal elements are being used to systematically mutate the rice
roots using 2D polyacrylamide gel electrophoresis and genome and isolate promoter elements via enhancer trapping
MALDI-TOF-MS (Mussa et al. 2002). Spectra of the four elements (Greco et al. 2001; Hirochika et al. 2001). The
proteins identified were queried against the EST (expressed transcriptome of Arabidopsis has been characterized during
Biotechnological Approaches in Plant Protection 11

systemic acquired resistance (Maleck et al. 2000). Analysis of from the Rockefeller Foundation, the Mcknight Foundation,
402 putative transcription factors from Arabidopsis using USDA-ARS and the Graduate School of the University of
OMAs allowed the identification of transcription factors that Wisconsin to SAL and a fellowship from the Department of
may be involved in regulation of various pathways responsive Biotechnology, Government of India to RSC.
to environmental stress and bacterial infection (Chen et al.
2002). These systems level approaches to analysis of model
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2
Chemical Identification of Fungi: Metabolite Profiling and
Metabolomics

Kristian Fog Nielsen / Jørn Smedsgaard / Thomas Ostenfeld Larsen / Flemming Lund /
Ulf Thrane / Jens Christian Frisvad BioCentrum-DTU, Technical University of Denmark, Lyngby,
Denmark

1 INTRODUCTION Commonly used macro- and micromorphological char-

acters (rough conidia, fluffy mycelia, color, etc.) can be
The identification of filamentous fungi has always been difficult to determine unequivocally and are difficult to link to
considered difficult and many misunderstandings and gene sequences. The production of secondary metabolites can
misidentifications can bee found in the literature (Frisvad typically be linked to a particular gene sequence or gene
1989; Mantle 1987). Phenotypic characters, e.g., morphology cluster and is most likely to be regulated as a response to
and growth on selected media have traditionally formed the growth factors. However, there are far more metabolites than
basis for fungal taxonomy (Domsch et al. 1980; Mantle 1987; genes as demonstrated by modern metabolomics. Schwab
Pitt 1979; Raper and Fennell 1977; Raper and Thom 1949). (2002) estimates that there might be as many as 10– 100
Advancements in the developments of analytical method- metabolites for each gene in higher organisms. Therefore, a
ology have allowed the use of “secondary” metabolite metabolite profile can provide an indirect method of detecting
profiling for fungal identification and been used to revise the a large set of metabolite coding genes which are expressed at
taxonomy within genera of Penicillium, Aspergillus, the same time. Metabolite profiling also allows detection of a
Fusarium, Alternaria, and their perfect states. The success specific gene cluster through the identification of different
of metabolite profiling in the classification of filamentous compounds from that pathway. Turner (1971) and Turner and
fungi relies on the fact that a major part of the fungal growth is Aldridge (1983) suggested subdividing secondary metabolites
expressed by the production of numerous diverse metabolites, according to their biosynthetic origin. In this way a selected
most of which are excreted into the media. The extracellular metabolite originating from a pathway, e.g., polyketide,
metabolites have been termed the exome, a subgroup of the terpene, diketopiperazine, and cyclopeptide is treated as
metabolome (all metabolites), and these are related to the representative of that particular pathway. This is important, as
genome as illustrated in Figure 1. The reasons why most only a limited number of members of a biosynthetic pathway
filamentous fungi produce such a diverse profile of are expressed under a given set of conditions, e.g., external
secondary metabolites are still unclear, but they are probably stimuli and growth conditions (Section 2.2). Mantle (1987)
produced as a result of stimuli and are directed against, or has reviewed secondary metabolite production by Penicillium
support actions on, receptor systems (Christophersen 1996) or species based on biosynthetic pathways. He suggested a
as outward directed (extrovert) differentiation products. renaming of original isolates according to new taxonomic
Possible others functions, include chemical signaling systems and emphasized that frequent misidentifications have
between organisms (Christophersen 1996; Frisvad 1994a). lead to errors, especially for isolates no longer available for
Williams et al. (1989) described their functions as “. . . serve the scientific community. Frisvad and Filtenborg (1983)
the producing organisms by improving their survival first demonstrated the advantage of secondary metabolite
fitness . . . .” profiling in fungal taxonomy with the genus Penicillium,

19
20 Nielsen et al.

2 MYCOLOGY AND BIOSYSTEMATICS

Biosystematics can be divided into taxonomy (¼the theory of

classification), nomenclature, identification and phylogeny.
The species is the central concept in biosystematics. It is
important to note that classification deals with the natural
nonoverlapping hierarchical grouping of isolates into species,
species into genera, etc, whereas identification deals with
allocating new isolates to already existing classes in an
effective, nonequivocal, and practical way.
Figure 1 The profile of extra-cellular metabolites or the exome
is linked to genomics through a mostly one-way connection from 2.1 Classical Identification
the genome. Far more metabolites than genes are found in most
cells, and metabolite profiles are phenotypic characters as are
The characters most often used in identification keys for
most other characters used in classical mycology.
filamentous fungi are based on arrangements, forms, sizes,
and ornamentations of mature asexual and sexual structures.
using a simple agar-plug-TLC technique. They later also Another kind of latent characters used in identification keys
included HPLC methods in their studies. Efficient identifi- are responses to abiotic factors such as macromorphology and
cation based on metabolite profiling relies on combining this colony growth rate or diameter at different temperatures,
information with more classical tools and á priori knowledge. water activities, pH, redox potential, etc. (Pitt 1973). The
For general use it is important to know at least the genus of the problem with all these different tests is the potential large
fungus being studied and which growth media to use. The number needed (economy) and the need for standardization.
analytical methodology depends on the group/genus being Morphological features occasionally overlap or can be
studied, but can vary from the simple TLC approach to difficult to record precisely (Frisvad et al. 2000), as they
hyphenated LC-MS-MS. This combined approach is illus- may be dependent on the media used (for example different
trated in Figure 2. It is important to note that efficient brands) and is particularly pronounced for colony diameters
identification of fungi will, in most cases, require the use of and colors. Standardization of incubation conditions (Pitt
profiles of metabolites from crude extracts, rather than single 1973) and the use of chemically defined media have been
or selected metabolites. However, a limited number of proposed to avoid these problems. Image analysis has also
species-specific metabolites can be used efficiently as markers been explored in Penicillium (Dörge et al. 2000), here the
for particular species in particular cases. This chapter focuses information found in fungal colonies such a colony texture,
on the practical considerations in the use of metabolite diameter and color from very accurately recorded and
profiles in identification. The text follows the general calibrated images could identify terverticillate penicillia, and
approach illustrated in Figure 2, and four different cases of has recently been used for clones recognition (Hansen et al.
various techniques are given. 2003).
In order to identify filamentous fungi it is necessary to
identify the fungi to genus level before using traditional keys
or chemotaxonomic methods. Filamentous fungi can be
identified to genus level by the use of keys [e.g., Samson et al.
(2000)]. Once the genus is known references to keys and
taxonomic treatments can be found, but as new species are
described each year, it may be difficult to obtain good and up-
to-date keys for large genera. Knowledge of the associated
funga (mycobiota) of different habitats can be a major help in
identifying the most common species (Filtenborg et al. 1996).
For example the only Penicillia that can grow on citrus fruits
are Penicillium italicum, P. ulaiense, and P. digitatum, so
identification of greenish mould growth on citrus fruits will be
relatively easy.

2.2 Cultivation and Media for Metabolite

Figure 2 Efficient identification of filamentous fungi requires a Profiling
synthesis of mycology, analytical chemistry and informatics
although the metabolite profile can sometime give the full Most fungi have evolved on solid matrices, and hence solid media
answer. are generally better than liquid media in terms of quantity and
Chemical Identification of Fungi 21

number of metabolites produced. On agar media, any section, profiles of metabolites rather than single metabolites
contamination is usually visible, and an agar plug technique can should be used for identification. It is very important to note
be used to sample different parts of the fungal colony and its that metabolite profiles are strongly influenced by the
surroundings. analytical scheme used, thus full metabolite profiles can
In general, agar media for optimal secondary metabolite only be compared if they are produced by the same analytical
and mycotoxin production have been based on media protocol. Over the years, many specific techniques have been
containing yeast extract. Yeast extract sucrose (YES) broth developed to determine a few selected metabolites, mostly
was introduced as a “semisynthetic” broth medium for known mycotoxins, from cultures and from complex samples
aflatoxin production by Davis et al. (1966). It was later shown such as food and feed. Some of these methods can be
to be a very effective general secondary metabolite expanded to include a broad spectrum of metabolites, but
production medium when used with a crude yeast extract dedicated profiling methods are required to efficiently obtain
(DIFCO or SIGMA) and formulated as an agar medium (YES the best possible metabolite profiles and allow reliable
agar) by Frisvad (1981); Frisvad and Filtenborg (1983), and identification.
has been used for Penicillium, Aspergillus, Fusarium, Developments in chromatography and mass spectrometry
Alternaria, and many other fungal genera (Andersen et al. (MS) have greatly increased their resolution, sensitivity and
2002; Thrane 2001). Other media including Czapek yeast productivity in analytical chemistry. There is, therefore, a set
autolysate (CYA) agar, Potato dextrose (PD) agar can be used of tools available that allows fast metabolite screening from a
to supplement YES agar, depending on the genus being very small amount of sample. As a result, a broad range of
considered, as seen in Table 1. Some of these semisynthetic metabolites can now be determined in one analysis with high
agar media can occasionally give problems as certain brands selectivity. Furthermore, several of the newer techniques have
of yeast extract, malt extract, potato extract, agar, peptone, or shown their potential as general rapid profiling methodology
tryptone, etc. may differ significantly in composition, working directly on raw samples or extracts. These include
although this may be diminished by adding trace elements MS, nuclear magnetic resonance, and FT-IR (including NIR),
and magnesium sulphate (Filtenborg et al. 1990). which eliminate a time consuming chromatographic step.
However, currently it is not wise to fully eliminate
chromatography and one should rather use systems based
on complementary methods. In planning an identification
3 TECHNIQUES FOR METABOLITE from an analytical approach the following considerations
PROFILING must be made: (a) Á priori knowledge about metabolites
produced by the genus, (b) whether the general chemical
Metabolite profiling for identification in its simplest form classes are alkaloids, acids, neutrals, volatiles, as well as large
consists of three elements: getting hold of the metabolites or small molecules, (c) sample matrix or growth medium
(e.g., extraction), determining the compounds (the profile) composition and interference, (d) number of samples to
(e.g., analysis), and data processing (e.g., chemometrics). handle, (e) expected biological variation, and if large or small
This will include all relevant metabolites needed for reliable chemical diversity is expected, (f) sensitivity of analytical
identification of a fungus, and as discussed in the previous instrumentation, and (g) cost.

Table 1 Recommended media for microscopy and chemotaxonomy of important fungal genera

Metabolite
Genus Teleomorphic state Microscopy profiling
Penicillium subgenus: Furcatum, Eupenicillium MEA YES, CYA
Penicillium, Aspergilloides
Penicillium subgenus Talaromyces MEA OAT, MEA, YES
Biverticillium
Aspergillus Eurotium, Emericella, Neocarpenteles, Neosartorya, MEA YES, CYA
Petromyces, Neopetromyces, Chaetosartorya, Sclerocleista,
Hemicarpenteles, Fennellia
Paecilomyces Byssochlamys MEA YES, PD
Stachybotrys Melanopsamma CMA PD, ALK
Trichoderma Hypocrea OAT, CMA YES, PD
Fusarium Gibberella, Nectria, Cosmospora, Haematonectria SNA YES, PD
Alternaria Lewia GAK DRYES
Media recipes, see text or our website http://www.biocentrum.dtu.dk/mycology/analysis/.
22 Nielsen et al.

Figure 3 The agar plug extraction method is a simple scheme by which extracts for metabolite profiling can be prepared rapidly using
only a minute amount of solvent (Smedsgaard 1997a).

3.1 Extraction Nearly all sample preparations start with either an

extraction based on distribution of the analyte between two
The first step in metabolite profiling for fungal identification immiscible phases or by a gas phase sampling. One phase is
is to obtain the metabolites produced by the fungi growing on the fungus (biomass) and/or growth medium, the other is
some sort of substrate, see Section 2.2 and Table 1. the solvent or a purge gas. Some physical (thermal or
Depending on the group of metabolites of interest, two mechanical) assistance may be needed to help the rapid
main schemes can be used to collect metabolites for a profile: distribution of metabolites between the two phases. There
(a) an extraction approach is used for the nonvolatile may be some overlap in the compounds determined by the
metabolites, as illustrated in Figure 3, in combination with two different approaches, e.g., some of the terpenes can be
HPLC and/or MS and (b) a headspace approach is used for the found in both extracts and by headspace analysis. The key
volatile metabolites as illustrated in Figure 4, in combination point is to select extraction solvents with a high affinity for
with GC or GC –MS. metabolites of interest. Adjusting pH can enhance the
solubility of metabolites in the extraction solvent.
Furthermore, choice of extraction solvent can also be used
to favor extraction of a particular subgroup, and a selection
of several solvents can be used to extend the range of
metabolites.
Many extraction protocols use grams of material and many
milliliters of solvents, however these procedures can be
miniaturized due to the very high sensitivity and selectivity of
modern instrumentation. A simple method is the plug
extraction method (Smedsgaard 1997a) illustrated in
Figure 3, where a few 6 mm plugs (0.5– 1 cm2 area) are cut
from plates of the fungus and extracted with about 500 ml of
solvent. The extraction is performed ultrasonically to improve
extraction efficiency and speed. If the raw extract is
compatible with the subsequent analysis it can be injected
directly, otherwise the solvent have to be evaporated and the
sample redissolved in an appropriate solvent (Figure 3). Other
analytical applications requires a more elaborate sample
preparation particularly if GC –MS analysis is required.
Figure 4 Collection of volatile metabolites using adsorption If fungi are to be identified in complex natural samples,
techniques. A stainless steel petri dish lid with a standard 1/400 e.g., foods or building materials a much more elaborate
Swagelocke fitting in the centre is used to hold the different sample preparation protocol is needed. It will nearly always
adsorption tubes or with a septum to be used with a SPME be necessary to remove interfering matrix compounds, by
syringe. Sampling is done by placing an adsorption tube, open liquid–liquid extraction or by passing the crude extract
only in the lid end, in the fitting and leaving the lit on the cultures through a disposable mini-column (solid phase extraction,
for a few hours. The tube or syringe is then analyzed. SPE).
Chemical Identification of Fungi 23

Sampling the volatile metabolites from fungi growing in wavelength. The chromatogram [e.g., plot of absorbance vs.
culture can be done by two approaches: either by one of three time] shows peaks representing the compounds eluting from
different dynamic headspace (HS) methods using direct the column. This technique requires that the compounds have
collection of volatiles present in the gas phase above the a chromophore that absorbs light at the selected wavelength
growing fungus or by extraction of volatiles present in the and that the solvent is transparent (or at least does not have
fungal biomass (or sample). Figure 4 illustrates the simple any significant absorption). Fluorescence can be measured
method to collect volatile metabolites by diffusive sampling with a selected pair of excitation/emission wavelengths in a
described by Larsen and Frisvad (1994; 1995c). The volatile similar fashion, if the compounds have a fluorophore. Several
metabolites are collected by adsorption on an adsorbent, e.g., mycotoxins have distinct fluorophores, whereas others can be
activated carbon black, a synthetic polymer like Tenax TA, or derivatized prior to detection.
recently a coated fiber (Solid Phase Microscale Extraction, Identification of fungi can in some cases be achieved by
SPME). Carbon black tubes are normally desorbed using identifying a set of specific metabolites produced, regardless
solvents, e.g., dichloromethane or diethylether. Tenax tubes of the analytical method. This requires that relevant
and SPME fibers are analyzed by thermal desorption, the metabolites to be identified with high certainty, and usually
SPME fibers in a split/splitless injection port, the tubes using requires either use of an authentic standard and/or several
thermal desorption equipment. spectral and chromatographic techniques.
Spectral detection by a UV-spectrometer (DAD or PDA) is
commonly used as these techniques greatly enhance the
3.2 HPLC information available by providing structural information
about each peak (compound). In HPLC – UV detection, a
HPLC is by far the most common analytical technique used UV-spectrum is collected at regular intervals in such a way
for metabolite profiling due to its versatility, relative ease of that between 10 –20 spectra are collected across a chromato-
operation, and the broad spectrum of metabolites that can be graphic peak. The resulting data file is illustrated in Figure 5
determined directly. Basically, HPLC is an integration of a and it is important to understand the structure of these files to
separation and detection system working with separation in a fully exploit the data of a full HPLC –UV chromatographic
liquid phase. matrix. The chromatographic UV-traces at selected wave-
Metabolite profiling using HPLC is almost always done lengths are present at the time axis and a UV spectrum is
using gradient elution on reversed phase material (C8 –C18 present at a specific retention time. The full data matrix can be
phase or similar) with polar mobile phase. The most widely visualized as a chemical image of the sample and can also be
used mobile phases are water-acetonitrile or water-methanol used for fungal identification without identifying the
containing some modifier, e.g., tri-fluoroacetic acid (TFA). metabolites (Nielsen et al. 1998; 1999; Thrane et al. 2001).
The flow rates used for metabolite profiling depend on the Figure 5 shows that quite different looking chromatograms
columns, but in general analytical columns with diameters can be obtained from the same sample by selecting different
between 2 and 4 mm are used at flow rates from 0.2 to wavelengths. This feature can be used to enhance the
1 ml/minute. Smaller diameter columns will give a better chromatographic resolution and to find specific metabolites,
separation at the cost of lower absolute sensitivity, as a e.g., viomellein is at 18.69 minutes on the 400 nm trace in
smaller amount of sample can be injected. As described in the Figure 5 and overlaps with puberuline at 18.40 minutes on the
introduction to this section identification of fungi from 220 nm trace. These traces are all profiles of the sample and as
metabolite profiles can be done either by detection of specific such can be used alone or in combination, or to aid specific
metabolites or by using the full chromatogram as a profile. In detection. As quantitative detection is based on Beers law it is
both cases it is important to keep the analytical conditions as necessary to use the same wavelength for quantifying
constant as possible. In the first case, the identification of standards and unknowns for each compound but it is not
metabolites can be determined from the retention times used necessary to use the same wavelength for different
together with standards. Possible spectral detection is compounds.
described later. The effect of small, unavoidable shift, in Identification or characterization of compounds can be
retention time over time can be reduced by using a series of achieved from UV spectra if the compound has a characteristic
alkylphenones to calculate a retention index for each peak in chromophore structure (bond structure typical containing p-
the chromatograms (Frisvad and Thrane 1987). If the full electrons systems), e.g., xanthomegnin, which is two
profiles are to be used constant analytical conditions will anthraquinone systems, has a chromophore giving an
reduce the alignment needed (Nielsen et al. 1998; 1999). In absorption maximum around 410 nm. This is fortunately the
cases of metabolite profiling, detection of compounds eluting case for many fungal metabolites but not for all (Cole and Cox
from the HPLC column is mostly done by UV detection, 1981). The combination of retention time or preferably the
fluorescence detection (FLD), or MS. The UV detection retention index and a characteristic UV spectrum is quite
accounts for the majority of the applications, however there is reliable for identification of many fungal metabolites (Frisvad
currently considerable growth in the use of MS (see section and Thrane 1987; 1993), however standards are needed for
3.4). In classical HPLC, compounds are detected by confirmation. The use of UV-spectra in combination with
measuring the absorbance of the column eluent at a specific other detectors, e.g., FLD can greatly enhance the specificity.
24 Nielsen et al.

Figure 5 The HPLC-UV data matrix showing both information about composition in form of chromatographic traces and structural
information in form of UV spectra. Analysis of a plug extract from Penicillium cyclopium, IBT 16932, grown on CYA collecting approx.
one spectrum/sec, with a resolution of 4 nm.

For identifying fungi, it is not necessary to know the complex, the two ochratoxin producing species A. niger (only
identity of all of the components in a chromatographic profile. 6% of the isolates produces ochratoxins) and A. carbonarius
If a peak has unique features in terms of retention time /index produce many interfering components that elute across the
and chromophore it can be designated with a code and used whole chromatogram (Figure 6) and obscures ochratoxin
along with the metabolites known from a particular species. detection. The FLD gives a very high specificity and
The most efficient identification can be done if metabolites sensitivity (1 –20 pg on column), with the possibility of
are grouped in chromophore families (known and unknown obtaining full scan fluorescence spectra. This is illustrated in
compounds), as compounds with similar UV spectra often Figure 6 where an A. niger isolate has been analyzed by
belong to the same bio-synthetic pathway. Therefore, if just HPLC with simultaneous UV and FLD, ochratoxins A and B
one member of a chromophore family is present it can be used are hidden in the UV chromatograms under peaks of
as indication of that particular pathway is active in a fungus. tetracyclic components. The simultaneous detection of all
ochratoxin analogues (a, b, A and B) serves as an extra
3.2.1 Case I. Ochratoxin A Determination in confirmation.
Aspergillus niger
Ochratoxins can easily be detected in the two penicillia, 3.3 Gas Chromatography and GC – MS
P. nordicum and P. verrucosum as well as in A. ochraceus and
Petromyces alliaceus where they are good species markers. Gas chromatography (GC) can be used to analyze volatiles
They can be detected using HPLC with UV detection with full directly and numerous semivolatile components after
scan UV spectral confirmation. However, in the A. niger derivatization, e.g., trichothecenes, amino acids, sugars,
Chemical Identification of Fungi 25

Figure 6 Extract from an Aspergillus niger isolate analysed by HPLC-DAD-FLD. Ochratoxin A, B, a, and b, are easily seen in the
lower fluorescence trace (ex. 230 nm, em. 450 nm) with the fluorescence spectra of ochratoxin A and a inserted. In the upper UV trace
(210 nm), several tetracyclic compounds (UV spectra inserted) obscure the detection of Ochratoxin A, B, and a.

lipids, and sterols. The advantage of GC is its very high information). Both TOF and MS–MS instrumentation greatly
separation power in comparison to HPLC and its relative ease increases the capability of the instruments, and the metabolite
of operation. Furthermore, GC is easily interfaced to MS profiles that can be obtained from these instruments are
(GC– MS) or other spectral detectors, forming a powerful tool generally not needed for fungal identification and they are
that can both deliver high separation power and give therefore not discussed further.
structural information in one run. For metabolite profiling, the most important detection
The most important step in GC is the injection, which if method is MS which in many cases will give a molecular ion
performed poorly can have a severe effect on the separation (and thereby molecular mass) and a characteristic fragmenta-
power. The most commonly used techniques for liquids are tion pattern—the mass spectrum—from each compound
split, splitless, on-column injection, thermal adsorption of eluting from the column. The limitations of MS in the
trapped volatiles, and headspace (Grob 1993). Currently fused identification of unknowns can be an insufficient information
silica columns for gas –liquid chromatography are used due to content of the mass spectrum to stereo and positional isomers
the high resolution power which is needed to separate complex in aromatic systems (Ramaswami et al. 1986). A great
mixtures of volatiles such as mono- and sesquiterpenes. potential for MS is the ability to scan for a selected number of
Different stationary phases as well as film thickness can be characteristic ions—selected ion recording (SIR or SIM)
chosen depending on the polarity and volatility of the which can improve detection sensitivity from the ng level to
compounds to be separated. More volatile compounds require the pg levels.
a thicker film column, whereas high separation power is best Flame ionization detection (FID) is an important and
obtained by thin film columns (Grob 1993). robust GC detector, which basically detects carbon atoms in
The mass spectrometer can be used to: (a) provide the sample with high sensitivity and over a large dynamic
structural information from fragmentation in electron impact range, but with no structural information. If metabolite
ionisation (EI) easily searched in databases, (b) accurate mass profiles are compared on two different columns of
using the modern time-of-flight (TOF) instrument, or (c) using substantially different polarity, similarity in the retention
ion-traps (or multistage MS –MS instruments) as very high times of metabolites on these two columns can be used for
selectivity detectors (or to get very detailed fragmentation their indirect identification (Davies 1990).
26 Nielsen et al.

Fourier transform infrared spectrometry (FTIR), can be used 3.3.1 Case II: Volatile Metabolites for Identification
to detect compounds eluting from GC columns. Most Of Penicillia
functional groups, in particular the carbonyl group (CvO),
have a unique absorption (fingerprint) from vibration energy. Volatile metabolites are believed to play an important role in
The FTIR will therefore give an unambiguous identification of chemical interactions between fungi and other organisms.
functionality of the functional groups of compounds and it is Recently the total volatile profile of the endophytic fungus
possible to identify these by comparing spectra to those of Muscodor albus was demonstrated to effectively inhibit or
known authentic compounds. The method is nondestructive kill a number of other fungi and bacteria (Strobel et al. 2001).
and can therefore be used in combination with either FID or In general volatile production is correlated with spore
MS. GC–FTIR will often allow discrimination between formation fitting well with a chemical ecological point of
structural- and stereo-isomers making the method a very view. The insects, often adapted to the toxic metabolites
powerful supplement to GC–MS. The major drawback of GC– produced by the fungi, can act as vectors for fungal spores for
FTIR is a lower sensitivity compared to GC-FID and GC-MS. their further spread.

Figure 7 Chromatograms showing profiles of volatile sesquiterpenes produced by Penicillium roqueforti (IBT 16403) and P. carneum
(IBT 6884). The two compounds produced in largest amounts by P. roqueforti are b-elemene and (þ)-aristolochene, whereas the three
largest peaks in the P. carneum chromatogram represents geosmin, an unknown sesquiterpene and dodecanoic acid methyl ester.
Chemical Identification of Fungi 27

Some important studies have shown that the production of compounds, and a lot of spectral information can also be
some possibly species-specific sesquiterpenes could be found in the atlas of Joulian and König (1998). A good review
related to the production of important mycotoxins such as of methods for the identification of sesquiterpenes can be
aflatoxins (Zeringue et al. 1993) and trichothecenes (Jelen found in König et al. (1999).
et al. 1995). Often fungal metabolites are only present in As mentioned in Section 3, the sensitivity can be greatly
particular compartments of the organism such as in the enhanced by the use of SIR, and the method has been used
conidia (or sclerotia), and these may have a role in protecting together with SPME to investigate how early volatile
them against being eaten by other organisms. Microfungi such metabolites can be detected (Larsen 1997). The SIR of four
as the penicillia usually produce a species-specific set of to seven of the most characteristic ions of mainly
volatile metabolites (Larsen 1998; Larsen and Frisvad 1995a). sesquiterpenes from cheese-associated fungi allowed the
Often even very closely species such as P. roqueforti and identification to species level within two days, at which time
P. carneum (Figures 7 and 8) can easily be differentiated by they had not started to sporulate and were only white mycelia.
the volatiles they produce. Volatiles from a mixed culture of P. roqueforti and
Fungal volatile metabolites include small ketones, P. commune, inoculated in a ratio of 1000:1, could be used
alcohols, esters, and hydrocarbons such as small alkenes, to detect both fungi within three days, showing the possibility
mono- and sesquiterpenes. The latter are by far the most of checking starter cultures for cross-contamination.
relevant metabolites for identification purposes or for use as
biomarkers (Larsen and Frisvad 1995b). The major
sesquiterpenes produced by P. roqueforti are b-elemene, 3.4 Atmospheric Pressure Ionization MS
selenine, and patchuline (Larsen and Frisvad 1995b)
together with aristolochene (Figure 8), recently reported The last decade has seen a tremendous development in
by Demyttenaere et al. (2002). biological MS, and it is currently one of the fastest growing
In general P. carneum produces relatively lower amounts analytical techniques in biotechnology.
of sesquiterpenes than P. roqueforti, however, the species has The MS is the determination of the mass to charge ratio of
a much more pronounced moldy odor than P. roqueforti due charged species of molecules or highly specific fragments of
to the production of the moldy smelling compound geosmin these (as in GC –MS). These can be either positively charged
together with large amounts of isopentanol (not shown in or negatively charged. In the atmospheric pressure ionization
Figure 8). (API) LC–MS techniques, ions are formed at atmospheric
It should be emphasized that the production of some pressure and transferred into the vacuum of the mass analyzer.
volatile compounds is strongly related to medium compo- There are two predominant ionization techniques: Electro-
sition, e.g., lipid degradation of fat rich media leads to the spray ionization (ESI); and atmospheric pressure chemical
production of ketones and secondary alcohols, as seen in ionization (APCI). In ESI the eluant from the column is
Camembert and especially Blue cheese production. The NIST sprayed though a narrow bore capillary to which a high
and Wiley MS databases contains many spectra of mono- and voltage is applied (around 3 kV). This will produce a spray of
sesquiterpenes (generated a 70 eV) for identification of single highly charged droplets. The solvent is evaporated from the
droplets by a heated gas, leading to shrinkage and
disintegration to charged species through a complex process.
The ions are formed either in the solvent before spraying or
during the spray droplet shrinkage and the key parameters
influencing the ion production are: solvent composition
(surface tension, volatility, modifiers, pH, ion strength),
source parameters (temperature, drying gas flow, potential),
and interaction between analytes in the sample (Berkel 2000).
The charged species are then sampled into the vacuum of the
mass analyzer.
In APCI the eluant from the HPLC column is sprayed
through a co-axial capillary with a heated gas to evaporate the
solvent. Evaporated solvent molecules are ionized by a
corona discharge from a needle that is usually placed across
the sampling orifice. Analyte molecules are ionized by
chemical reaction in the gas phase at atmospheric pressure
through a process much like the classical chemical ionization.
Figure 8 Structures of the main fungal terpene metabolites The ions are sampled into the mass analyzer by a process
obtained from the headspaces of Pencillium roqueforti and similar to ESI (see Table 2).
geosmin produced by P. carneum. 1) Limonene 2) b-elemene In general, ESI is the most versatile technique for a very
3) g-patchulene 4) b-caryopyllene 5) b-myrcene 6) (þ )- broad range of bio-molecules and also the easiest to use,
aristolochene 7) a-selinene 8) geosmin. therefore ESI is also the most frequently used technique.
28 Nielsen et al.

Table 2 Comparing some of the characteristics of the two major ionization techniques in LC-MS

ESI APCI
Type of ions positive M þ Hþ, M þ Naþ, solvent adducts Mþ, M þ Hþ
Negative ions (M 2 H)2 M2, (M 2 H)2
Ionization in Solvent (solvent gas interface) Gas phase
Solvents Some ions required Apolar solvent possible
Fragments None or few Common
Multiple charged ions Yes No
Clusters and complexes Many Few
Mass range High (multiple charged ions) As the mass analyser
Typical flow rate with HPLC 5 – 500 ml/min 300 ml/min– 1.5 ml/min

Not all molecules are ionized (usually protonated) in the charge determination. The performance of two common mass
positive mode, and are detected much better as negative ions. analyzers is shown in Figure 9. The TOF analyzer raw data
Gas phase chemistry is however not like solvent chemistry, (often called a continuum spectrum) show a resolution of
and many carboxylic acids are much more efficiently approx 7500 (half height) and the quadrupole approx 900
protonated in gas phase by positive ESI than determined as (half height). Mass spectra are normally used as centroid
anions in negative ESI. Negative ESI will give many fewer (stick) spectra for mass determination where the stick is
adducts and clusters than positive ESI, see Table 3, and so it is placed at the center of the continuum peak. This also reduces
therefore easier to interpret spectra. However, a higher the disk space needed to store the spectra.
sensitivity can be obtained for some classes of compounds in Formula can be calculated from the mass, and with
APCI than in ESI. sufficient mass accuracy the number of possible structures
The charge to mass ratio is determined using a mass is limited if sensible limits for composition are applied
analyzer, which is either: Quadrupol, time of flight (TOF), (Table 5). Combining a mass spectrometer with HPLC will
ion-trap, sector (electric and magnetic) or an ion-cyclotron allow the recording of mass spectra as peaks are eluted
(ICR). These analyzers can be grouped into scanning from the column. As was the case collecting UV-spectra,
analyzers (mass filters) where ions of just one mass to charge mass spectra are collected continuously with about 10 –20
ratio can pass at a time and nonscanning analyzers where all spectra across a peak to produce a data matrix containing
ions entering the analyzer are detected (Table 4). Mass both chromatographic and mass spectral information see
resolution and accuracy are the two most important factors for Figure 10.
the identification of compounds whereas sensitivity and scan Ion traces are highly specific chromatographic profiles of
speed are of most chromatographic importance. the samples which depend on the mass accuracy. Figure 10
Accurate mass determination relies on both sufficient shows an example of high accuracy ion traces corresponding
resolution to separate isotopes and a very stable mass to to the protonated mass of puberuline (444.2287 Da) and
xanthomegnin (575.1187 Da) both using a window width of
15 ppm (6.7/8.7 mDa) showing only one peak in each;
Table 3 Quasi-molecular ions are often seen along with compare this to Figure 5. For each peak a mass spectrum can
adducts in a low mass positive electrospray mass spectrum. This be retrieved, given structural information about the sample. In
is highly solvent and parameter dependent. Very few adducts and this case using an accurate TOF mass spectrometer also gives
clusters are seen in negative ESI an estimate of peak formula. Comparing the HPLC-MS image
in Figure 10 to the HPLC-UV image in Figure 5 there are
Adducts significantly more details and higher specificity in the former
þ þ þ of these two. The data shown on Figures 5 and 10 were
Charging by H , Na , K
Solvent adducts H2O, CH3OH, CH3CN acquired during the same run using a nondestructive UV
Dimers 2M þ Hþ, 2M þ H3Oþ, 2M þ Naþ detector in series with the MS to give the maximum
Multiple charged Double charged is rarely seen information.
below mass 800 – 1000 Da/e There are some restrictions on the use of HPLC-MS which
Fragments – H2O, depend on the ionization techniques: the eluants must be
Others Fe, Zn, and other metal volatile including modifiers (e.g., acids), the flow rate must be
complexes are sometime seen e.g. suitable for the interface and modifiers/ion strength must
(M 2 Hþ) þ Fe3þ þ H3CCOO2 match the ionization technique (ESI þ /2 or APCI þ /2 ). In
þ general, most reversed phase chromatographic solvent
M ions can be seen from
some types of compounds systems can be used, e.g., water, acetonitrile, and methanol
with the modifiers acetic acid, formic acid, and ammonium
Chemical Identification of Fungi 29

Table 4 Typical performance of the different mass analyzer although this may vary due to special designs. The sensitivity is relative to
the other analyzers and dependent on the analytical approach

Resolution Accuracy Accuracy at 1000 Da Sensitivity Scan speed Relative price

Quadrupol 1000 – 2000 100 – 300 ppm 0.1– 0.3 Da medium high low
Iontrap 1000 100 – 300 ppm 0.1– 0.3 Da medium high low
TOF . 10.000 2 – 5 ppma 2 – 5 mDaa high Very high Medium
Sector . 50.000 1 – 5 ppma 1 – 5 mDaa high low High
FT-ICR-MS . 100.000 0.05 – 0.1 ppma 0.05– 0.1 mDaa high Very high Very high
a
Using internal mass correction.

Figure 9 Comparing mass resolution and accuracy of the puberuline M þ Hþ ion C27H30O3N3 mass 444.2287 Da/e) from analysis of a
crude plug extract see the case below. A peak width at half height of about 0.06 Da and a precision of approx. 1 mDa (centroid data) is
found by the TOF instrument (top) and a peak width of about 0.5 Da and a precision of approx. 30 mDa by the quadrupole instrument
(bottom).

acetate. However, trifluoroacetic acid should particularly be ion traces corresponding to these can easily be drawn. It is
avoided in APCI (both positive and negative), whereas it can then a simple matter to interpret. In most cases it is not
be used in low concentration in positive ESI. It is very necessary to use high resolution/accurate mass spectrometers
difficult to run ESI in pure organic solvent due to volatility, for fungal identification. In practical identification, a
and the limit seems to be around 90–95% acetonitrile-water, combination of several metabolites with different retention
however ACPI works well in a pure organic solvent. time is used as a mark for each species thereby limiting the
Metabolite profiling by HPLC –MS (ESI or APCI) are very number of misidentifications. However it may be necessary to
efficient tools for identification and classification of fungi. As use more than one ionization technique, as some components
the specificity is very high, rapid chromatographic methods are difficult to ionize in positive ESI.
can be used. If a list of expected ions can be made, then the If the goal is to get a full profile of all metabolites
produced under specific conditions for metabolomics, then
high resolution/accurate mass determination is a major
advantage, as it also provides the molecular composition of
Table 5 Number of structures possible for mass 444.230 Da
the ions.
with different mass accuracies

Number of
Accuracy structures Composition limits
3.4.1 Case III: Direct Infusion MS

1 ppm (0.44 mDa) 1 C , 100 H , 500 a. Metabolite Profiling in Taxonomy Of Penicillium Series
5 ppm (2.2 mDa) 7 O , 12 Viridicata. An advantage of ESI mass spectrometry is that
50 ppm (22 mDa) 61 N , 10 the analytical conditions can be optimized to limit
DBE , 50 fragmentation and cluster formation. In the ideal case, only
protonated molecules are observed from each compound in a
30 Nielsen et al.

Figure 10 The HPLC-ESIMS data matrix showing both information about composition in form of chromatographic traces and mass
information in form of mass spectra. Analysis of a plug extract from Penicillium cyclopium, IBT 16932, grown on CYA collecting
approx. One spectrum/sec. With a mass resolution of 6000 and an accuracy , 5 ppm (Micromass LCT with lockspray).
Table 6 Production of secondary metabolites by cereal associated Penicillia

Speciesa

Metabolite I II III IV V VI VII VIII IX

Xanthomegnin 0 þ þ 0 0 þ þ 0b
0
Viomellein 0 þ þ 0 0 þ þ 0b 0
Aurantiamine þ þ 0 0 0 0 0 0 0
Viridamine 0 0 0 0 0 þ 0 0 0
3-methoxyviridicatin 0 þ 0 þ þ 0 þ 0 0
Brevianamide A 0 0 0 0 0 þ 0 0 0
Ochratoxin A 0 0 0 0 0 0 0 0 þ
Citrinin 0 0 0 0 0 0 0 0 þ
Penitrem A 0 0 0 0 0 0 0 þ 0
Oxaline 0 0 0 0 0 0 0 þ 0
Terrestric acid þ 0 þ 0 0 0 0 0 0
Penicillic acid þ þ 0 þ þ þ þ þ 0
Verrucosidin þ 0 0 þ 0 0 0 þ 0
a
I: Penicillium aurantiogriseum; II: P. freii; III: P. tricolor; IV: P. polonicum; V: P. aurantiocandidum; VI: P. viridicatum; VII: P. cyclopium; VIII:
P. melanoconidium; IX: P. verrucosum and detected by TLC.
b
The metabolites can be detected by HPLC.
Chemical Identification of Fungi 31

sample, thus injecting a mixture of compound will give a resolution using the correlation coefficient and UPGMA
mass profile of the sample. This approach was used by linkage.
Smedsgaard in a study of the terverticilllate penicillia As it can be seen from Figure 12 most species cluster
(Smedsgaard 1997b; Smedsgaard and Frisvad 1997). In that together, however P. aurantiocandium is found in the
study, crude plug extracts from cultures were injected directly P. cyclopium and P. tricolor clusters. One P. viridicatum is
into the mass spectrometer in positive ESI mode with solvent grouped with P. aurantiogriseum and another is an outlier.
and other parameters optimized to minimize fragmentation The ions (metabolites) that are important to the
and cluster. Samples were used at low concentration to reduce segregation of the species can be found using Principle
matrix effects, thus avoiding “the winner takes it all effect.” Component Analysis (PCA) as the PCA loadings (Figure
About 10– 15 samples can be analyzed per hour by this 13). If we consider loadings above 0.04 or below 2 0.04, 47
approach, which have recently been used to study other ions are found in the plot from the first three principal
organisms such as bacteria (Vaidyanathan et al. 2002) and components. Of these, 15 ions correspond to the protonated
actinomycetes (Higgs et al. 2001). ions from 15 of the most important metabolites produced by
Nine of the major species in the Penicillium series these species (out of about 28 metabolites). Furthermore
Viridicata (The Penicillium aurantiogriseum complex) were five ions correspond to the 13C isotope of major
analyzed by direct injection on a quadrupole mass metabolites. Five to six characteristic metabolites are not
spectrometer (Smedsgaard and Frisvad 1996). Figure 11 found as they are not produced on these media, however
shows crude extract spectra from three different species they can be seen in the mass spectra from some of the
cultivated on CYA (Samson et al. 2000). The spectra show known producers. Four significant ions of unknown
significant difference between the species and ions corre- structure are found from the loadings plot at mass 205,
sponding to the protonated mass of major known metabolites. 235, 243, and 274 Da.
These spectra can be stored and searched in a spectral
database using the software included with most instruments,
although this software in general is designed for EI spectra
(Smedsgaard 1997b). To prove the concept in more detail a 3.5 A Combined Approach: The Agar Plug-TLC
cluster analysis of the full centroid mass spectra (normalized) Method
from 45 isolates of the nine major Penicillium series
Viridicata species (Figure 12) was performed. This cluster The agar plug method was first introduced as a taxonomic tool
analysis is done directly on the centroid spectra with 1 Da for Penicillium in 1983 (Frisvad and Filtenborg 1983).

Figure 11 ESIþ mass spectra from injection of crude extracts of three Penicillium isolates.
32 Nielsen et al.

Figure 12 Cluster analysis of 45 mass spectra from direct infusion ESI þ MS of crude culture extracts of Penicillium species.
Calculated using correlation coefficient and UPGMA linkage.

The method uses a combined extraction—TLC analysis, plate by gently pressing the wetted side down on the plate.
where the extraction is done by adding a drop of solvent on top The advantage of the method is that it is simple, cheap
of a small plug cut from a colony. After a few seconds, the equipment is required, sample throughput is high, and with
solvent remaining on top of the colony is applied to a TLC many groups of fungi can be identified as illustrated on
Penicillium series Viridicata (the P. aurantiogriseum
complex) later. In the general Penicillium procedure plugs
from CYA media are examined for intracellular metabolites,
whereas the plugs from YES are examined for extra-cellular
metabolites. A normal 20 by 20 cm plate accommodate 21
lanes from each side, and so 10 isolates can be analyzed
(Figure 14).
The TLC plates are eluted in saturated chambers, dried and
examined in daylight and under UV light (366 nm and 254 nm)
and spots appearing are noted (color, shape, etc.). The spots are
marked gently with a pencil (or the plate is photographed). This
procedure is repeated after: the whole plate is sprayed with
AlCl3 and heated at 1308C for 8 minutes; the CAP side is
sprayed with Ce(SO4)2; the TEF side is sprayed with ANIS and
heated at 130 8C for 8 minutes.
Figure 13 Loadings from the first three principal components On our web site http://www.biocentrum.dtu.dk/mycology/
from PCA analysis of direct infusion mass spectra collected from analysis/tlc/ houses a collection of pictures of TLC plates
45 isolates of Penicllium species from the Viridicata series. from 18 of the cost common Penicillium species.
Chemical Identification of Fungi 33

Figure 14 The TLC-agar plug method. A 3 mm plug is cut from a colony and is placed directly on the TLC plate with the agar side down
for the extra cellular metabolites or a drop of solvent is added to the mycelium side and the plug is placed with the wetted side down. The
plate is eluted in two systems and the dried plate is examined under UV light before and after spraying.

3.5.1 Case IV: Identification of Cereal Borne vianamide A, viridamine, ochratoxin A, and citrinin are visible
Penicillia by TLC as colored spots under UV light (365 nm), usually in highest
quantities on CYA. Aurantiamine and viridamine are both
Cereals represent a habitat with a limited associated funga, seen as blue spots, however viridamine is more light blue and
and it is relatively easy to determine Penicillium species has a lower Rf than aurantiamine. Viridamine will identify the
when kernels are placed on DG18 (Dichloran 18% Glycerol fungus as P. viridicatum, and can be confirmed by the
agar), DRYES (Dichloran Rose Bengal Yeast Extract agar) presence of brevianamide A as a yellow spot. Aurantiamine
or DYSG (Dichloran Yeast Extract 18% Glycerol agar). can be produced by two species, but simultaneously detection
When the Penicillium species are isolated and inoculated in of 3-methoxyviridicatin as a blue spot under 255 nm light, and
3-points on the identification media CYA, MEA (Malt xanthomegnin and viomellein identifies P. freii.
extract agar), YES, and CREA (Creatine Sucrose agar) P. verrucosum is identified by a tailing yellow spot of
(Samson et al. 2000) it is relatively easy to observe that on citrinin and a blue green spot of ochratoxin A. The TLC
MEA, most isolates have two stage branched rough plate should then be sprayed with AlCl3 and then heated at
stipes (terverticillate) and smooth conidia (excluding 1308C for 8 minutes. Penitrem A is the visible as a bluish
P. hordei), and that growth is inhibited on CREA. black spot in daylight and oxaline as a yellow brown spot
At this stage it is not possible to make a definite identification very close to the application point. If both metabolites are
as these criteria fit with the following nine Penicillium present on CYA the fungus is P. melanoconidium. The
species found on cereals: P. aurantiogriseum, P. freii, TLC plate should then be spayed with ANIS and heated to
P. tricolor, P. polonicum, P. aurantiocandidum, 1308C for 8 minutes. In cultures from YES yellow tailing
P. viridicatum, P. cyclopium, P. melanoconidium spots in daylight identify terrestric acid that in combination
(Penicillium series Viridicata), and P. verrucosum with aurantiamine definitely identify the isolate as
(Penicillium series Verrucosa). Agar plugs from twenty P. aurantiogriseum. A yellow brown spot under UV light
isolates on CYA and YES can be applied on a TLC plate on CYA detects verrucosidin, which in combination with
(20 £ 20 cm) within 30 min, and the plate developed in TEF 3-methoxyviridicatin identifies P. polonicum. P. tricolor is
(Figure 14). identified as producer of terrestric acid, xanthomegnin and
The two secondary metabolites xanthomegnin and viomel- viomellein. Except P. tricolor and P. verrucosum all these
lein are always seen simultaneously as two brown spots under species produce penicillic acid seen as a bluish red spot
visible light in samples from CYA, and reduces the number of under UV light after ANIS spray. In summary eight of
possible species to four, i.e. only four cereal-borne Penicillium nine species can be identified solely based on TLC results
species (Table 6). Aurantiamine, 3-methoxyviridicatin, bre- however the species, P. aurantiocandidum cannot be
34 Nielsen et al.

identified using TLC results alone, and must be combined morphology, growth and metabolite production. Mycologia
with the poor sporulation on CYA. 94:392 –403.
Berkel GJV (2000). Electrolytical deposition of metals on the high-
voltage contact in an electrospray emitter: implications for
gas-phase ion formation. J Mass Spectrom 35:773 –783.
3.6 Data Handling, Processing, and Christophersen C (1996). Theory of the origin, function, and
Chemometrics evolution of secondary metabolites. In: Atta-ur-Rahman ed.
Studies in Natural products chemistry 18. Stereoselective
Multivariate statistical methods (chemometrics, taxometrics) synthesis (part K). Amsterdam: Elsevier. pp 677 – 737.
Cole RJ and Cox RH (1981). Handbook of Toxic Fungal Metabolites.
are ideal for evaluating chemotaxonomic data. Some of these
London: Academic Press.
methods are best used for unsupervised classification Davies NW (1990). Gas chromatographic retention indices of
approaches such as cluster analysis, multidimensional monoterpenes and sesquiterpenes on methyl silicone and
scaling, correspondence analysis, and PCA, whereas other Carbowax 20M phases. J Chromatogr 503:1– 24.
methods such as Partial Least Squares Discriminant (PLS-D) Davis ND, Diener UL, and Eldridge DW (1966). Production of
analysis and soft independent modeling of class analogy aflatoxins B1 and G1 by Aspergillus flavus on a semisynthetic
(SIMCA) are more suited for discriminant analysis and medium. Appl Microbiol 14:378.
identification (Frisvad 1994b; Söderström and Frisvad 1984). Demyttenaere JC, Adams A, Van Belleghem K, De Kimpe N, Konig
The immense quantities of data collected by modern WA, and Tkachev AV (2002). De novo production of
analytic instruments dictates some form of automatic data (þ )-aristolochene by sporulated surface cultures of Penicillium
handling and analysis (Nielsen et al. 1999) although problems roqueforti. Phytochemistry 59:597 –602.
Domsch KH, Gams W, and Anderson T-H (1980). Compendium of
such as handling of simultaneous UV, MS, and nuclear
soil fungi. London: Academic Press.
magnetic resonance data without component identification
Dörge T, Carstensen JM, and Frisvad JC (2000). Direct identification
needs to be solved, as well as issues relating to the storage in of pure Penicillium species using image analysis. J Microbiol
searchable databases, and how data can be combined with Methods 41:121– 133.
other biodiversity information. Filtenborg O, Frisvad JC, and Thrane U (1990). The significance of
yeast extract composition on metabolite production in
Penicillium. In: Samson RA, Pitt JI eds. Modern Concepts in
4 CONCLUSION AND SUGGESTIONS Penicillium and Aspergillus Classification. New York: Plenum
Press. pp 433 – 441.
Filtenborg O, Frisvad JC, and Thrane U (1996). Moulds in food
Profiles of secondary metabolites provide powerful tools for spoilage. Int J Food Microbiol 33:85 –102.
fungal identification and can give insight in very large parts of Frisvad JC (1981). Physiological criteria and mycotoxin production
the fungal genome, in addition of being functional and as aids in identification of common asymmetric penicillia. Appl
ecological characters. Metabolite profiling is currently being Environ Microbiol 41:568– 579.
revolutionized by developments in MS as well as chemo- Frisvad JC (1989). The connection between the penicillia and
metrics and data handling which is necessary to cope with the aspergilli and mycotoxins with special emphasis on mis-
immense quantities of data collected (@1 Gb/day). Chemical identified isolates. Arch Environ Contam Toxicol 18:452 –467.
characters can be used directly in synoptic keys. They are Frisvad JC (1994a). Classification of organisms by secondary
metabolites. In: Hawksworth DL ed. The identification and
also very suitable for databases as they can be accurately
characterization of pest organisms. Wallingford: CAB
recorded using specific chemical methods. Natural classi- International. pp 303 – 320.
fications are often based on a polyphasic approach, where Frisvad JC (1994b). Correspondence, principal coordinate, and
ideally many different ecologically relevant characters redundancy analysis used on mixed chemotaxonomical
should be used. qualitative and quantitative data. Chemom Intell Lab Syst
23:213 –229.
Frisvad JC and Filtenborg O (1983). Classification of Terverticillate
Penicilia based on profile of Mycotoxins and other Secondary
ACKNOWLEDGEMENTS
Metabolites. Appl Environ Microbiol 46:1301 – 1310.
Frisvad JC and Thrane U (1987). Standardised High-Performance
This project was supported by The Danish Technical Liquid Chromatography of 182 mycotoxins and other fungal
Research Council under the project “Functional biodiversity metabolites based on alkylphenone retention indices and
in Penicillium and Aspergillus” (grant no. 9901295) and UV-VIS spectra (Diode Array Detection). J Chromatogr
Center for Advanced Food Studies (LMC). 404:195 – 214.
Frisvad JC and Thrane U (1993). Liquid column chromatography of
mycotoxins. In: Betina V ed. Chromatography of mycotoxins:
Techniques and applications. Journal of Chromatography
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3
Isozyme Analysis in Fungal Taxonomy, Genetics,
and Population Biology

Stephen B. Goodwin U.S. Department of Agriculture—Agricultural Research Service, and

Purdue University, West Lafayette, Indiana, USA

1 INTRODUCTION examples of isozyme analysis for the major groups of fungi

will be discussed to illustrate the many potential applications
Isozyme analysis is a powerful technique for assaying genetic of this extremely useful and powerful technique.
variation within and among populations. This versatile
approach has been used on bacteria, fungi, plants, and most
2 WHAT IS AN ISOZYME?
major groups of animals, from insects and other invertebrates
to fish and mammals. The appeal of isozyme analysis stems
from its ease of use, low cost, and speed. DNA-based Isozymes are different forms of a single enzyme that perform
technologies can provide a higher level of polymorphism with the same or similar function. Each form has some small
concomitantly greater resolution. However, when sufficient change that allows it to be distinguished from other forms
variation is present, isozymes provide the fastest, easiest (isozymes) of the same enzyme. The changes usually result
answers to biological questions at a significantly lower cost from point mutations in the DNA that cause single amino-acid
compared to other available techniques. substitutions into the protein making up the enzyme. Changes
Isozyme analysis has a long history in the fungi. that affect the charge or size of the final enzyme may alter its
Differences among general protein patterns of species of mobility in an electric field. Such changes often can be
Neurospora and Pythium were first noted during the early detected by electrophoresis, and this forms the basis for
1960s (Chang et al. 1962; Clare 1963). This approach isozyme analysis.
developed into isozyme analysis with the application of The terms “isozyme” and “allozyme” often are used
methods for detection of specific enzyme activities. Since the interchangeably and can be a source of great confusion.
mid 1960s, the technique has been applied to questions of However, the terms are not identical. Isozymes that have been
fungal taxonomy, genetics, population biology, and species or analyzed genetically and are known to be alleles at a single
strain identification (Micales et al. 1986; 1992) but it is only genetic locus can be called allozymes. Therefore, all
during the past 15 years that the true potential of the technique allozymes are also isozymes, but not all isozymes are
has been realized. allozymes. Isozyme is a more general term and should be used
The purpose of this chapter is to introduce the concept of unless the genetic basis of the different enzyme forms is
isozyme analysis in fungi. Strategies for developing new known for certain.
isozyme systems and for addressing particular questions
about fungal taxonomy, genetics, and population biology
will be discussed. Particular emphasis will be placed on 3 ISOZYME TECHNIQUES FOR FUNGI AND
identifying common pitfalls and barriers to successful OOMYCETES
implementation of new isozyme systems, especially inter-
pretation of banding patterns, preparation of tissue samples, Many methods have been developed for separating enzyme
and methods and approaches to data analysis. Specific variants and visualizing isozyme variation. The basic

37
38 Goodwin

approach is to obtain crude extracts of total soluble proteins, a small amount of cross contamination will not affect the
separate the proteins by electrophoresis through some type of results due to the relatively low sensitivity of the technique.
solid matrix (usually a gel), then visualize the isozymes by To maintain enzyme activity, all samples should be kept on
use of chemicals that produce a visual reaction in response to ice whenever possible. Tissue extracts also can be frozen at
specific enzyme activity. General methods of isozyme 2808C for several weeks if necessary with little or no enzyme
analysis have been described elsewhere (Selander et al. degradation. However, this should be tested for each system
1971; Shaw and Prasad 1970; Soltis et al. 1983) and will not specifically before used on a large number of samples.
be repeated here. However, aspects of isozyme analysis that
are specific to fungi will be discussed briefly. Additional
information can be found in previous reviews of isozyme 3.2 Choice of Separation Matrix
analysis and compilations of recipes specifically adapted for
fungi (Micales et al. 1986). Gels of 12% hydrolyzed potato starch have been the mainstay
of isozyme analysis for decades, Advantages are cost and
throughput. A single starch gel can yield 3 –4 horizontal slices
3.1 Types of Tissue and Preparation of Samples (possibly more), each of which can be stained for a different
for Isozyme Analysis of Fungi enzyme. Depending on the size of the filter-paper wicks used
for sample loading and the width of the gel, 20– 50 or more
Fortunately, virtually any type of fungal tissue can be used for samples can be loaded per gel. Thus, more than 200 data
isozyme analysis. This could include sections of sporocarps points (50 individuals times four enzymes) can be obtained
collected in the field or produced in culture, mycelia from per gel once the system is optimized, and an average of 80 or
plates or liquid cultures, or even conidia washed from lesions more is common. This high sample capacity is excellent for
or cultures. The main requirement is that the tissue must be population genetics analyses that require large sample sizes
living so the enzymes are functional. Old cultures that are for meaningful conclusions. Starch gels can be photographed
dead or dying will not give satisfactory results. This precludes easily for a permanent record.
dried herbarium material as the activity of most enzymes will A newer approach is to use precast cellulose-acetate
be destroyed during the drying process. However, living plates obtained from commercial suppliers. This technique
material that has been lyophilized and stored at 2808C will has been used for many years in medical diagnostics but has
maintain enzyme activity for many months and possibly for only been applied to isozyme analysis in general since the mid
years. Lyophilization also may be a good option to save 1980s (Hebert and Beaton 1993). Advantages of cellulose
material until a sufficient quantity is available for analysis if acetate are that the pre-cast gels can be purchased ahead of
only limited amounts of material can be collected at a time. time and stored dry until needed, so there is no time, effort, or
Regardless of source or amount, the fungal material must skill involved in pouring the gels; specially designed gel
be macerated to release the enzymes. Lyophilized material boxes and sample applicators can be purchased so a
can be frozen quickly with a small quantity of liquid nitrogen laboratory can be set up quickly; and the complete analysis
and ground in a mortar after the nitrogen has evaporated. can be run in an hour from start to finish, so many runs can be
Small amounts of tissue can be ground in microcentrifuge completed in the same day. Very small volumes are needed so
tubes using a glass rod as a pestle. Disposable plastic pestles extremely small amounts of tissue can be analyzed. The
designed specifically for use in microcentrifuge tubes also can stained gels can be photographed or the gels themselves dried
be used. Fresh tissue can be crushed with a disposable plastic and saved for a permanent record.
pestle attached to a variable-speed electric drill. Adhering Isozymes also can be separated on many other types of
liquid and tissue should be wiped from the pestle between solid matrix and approach, such as polyacrylamide gels or
samples, but there is no need to clean the pestle completely as using isoelectric focusing. Polyacrylamide in particular is
often used in an attempt to gain increased resolution.
Although these systems often do reveal additional bands, the
banding patterns can be difficult to interpret genetically.
Furthermore, throughput and ease of use are lower, and costs
often are higher compared to starch and cellulose acetate.

3.3 Enzyme Activity Staining

Once the proteins have been separated on a gel, a set of

staining protocols must be followed to reveal the isozymes.
Figure 1 Isozymes of glucose-6-phosphate isomerase in 12 Specific enzyme activity staining is what makes isozyme
isolates of the barley scald pathogen, Rhynchosporium secalis. analysis possible. Recipes and techniques for staining
This haploid fungus yields a single band in each lane hundreds of enzyme activities are provided in numerous
corresponding to one of the three putative alleles 87, 100, or 114. compilations (Micales et al. 1986; Selander et al. 1971; Shaw
Isozyme Analysis of Fungi 39

and Prasad 1970; Soltis et al. 1983) and are beyond the often give complicated banding patterns that are difficult or
purview of this chapter. However, a brief description of the impossible to interpret without a genetic analysis. Unless
major types of staining systems is provided here. Three crosses can be made easily those systems are best avoided.
staining systems are used commonly for isozyme analysis:
positive, negative, and fluorescent. The vast majority of
isozyme visualization systems are based on positive staining. 4.1 Interpretation of Isozyme Banding Patterns
The general idea is that activity of a specific enzyme in Haploids
generates a colored precipitate at zones of enzyme activity.
These isozymes are visualized as dark bands against a light For haploid fungi, each individual ideally will produce a
background (Figure 1). Negatively stained isozymes are single band on a gel. Such isozymes can be scored easily by
visible as light-colored bands against a dark gel. Common assuming that each band corresponds to an allele at a single
negative staining systems include those for the enzymes genetic locus. Each allele can be named by indicating its
superoxide dismutase and catalase. A third class of enzyme relative migration distance on the gel. The easiest approach is
staining system works by generating bands that fluoresce to designate the most common allele as number 100. Then all
when exposed to ultraviolet light. Commonly used other alleles can be indicated by their migration distance
fluorescent staining systems include those for arylesterase relative to the 100 allele. For example, an allele migrating
and b-glucosidase. 14% faster than allele 100 would be allele 114, and one
migrating 13% slower would be allele 87 (Figure 1).
More than one band per haploid individual may indicate
4 INTERPRETATION OF ISOZYME DATA multiple loci. If alleles at each locus migrate within a limited,
defined region of the gel then it may be possible to score each
The best guide for interpreting isozyme banding patterns is to locus separately. Alleles at each locus can be named as
perform a thorough genetic analysis. Unfortunately, for many indicated previously but with a locus designation, e.g., Gpi-1
fungi this may be difficult or impossible. The purpose of this 100 and Gpi-2 100 to indicate the 100 alleles at two loci
section is to provide enough information for accurate coding for the enzyme glucose-6-phosphate isomerase.
interpretation of isozyme banding patterns even without a Enzymes giving more complicated banding patterns in
genetic analysis. Accurate interpretation of isozyme banding haploids should be avoided as accurate interpretation is
patterns requires knowledge of the ploidy of the organism and almost impossible without thorough genetic analysis. “Null”
the number of subunits required to form the active enzyme. alleles—those in which no enzyme activity is detected—
Most enzymes should give a relatively simple banding pattern should be regarded with suspicion as it is difficult to
with only one to three bands per individual (Figure 1). distinguish a null allele from a null caused by faulty
Enzymes with relatively nonspecific stains (e.g., esterases) technique. Furthermore, it is difficult to imagine a haploid
being truly null for important enzyme activities. All potential
null alleles should be verified thoroughly—most probably
will turn out to be errors caused by poor enzyme extraction
from particular individuals or other problems with technique.

4.2 Interpretation of Isozyme Banding Patterns

in Diploids, Dikaryons, and Polyploids

Interpretation of isozyme banding patterns in diploids and

dikaryons is much more complicated, and is aided by
knowing whether a particular enzyme is mono- or multimeric.
For monomeric enzymes, homozygous diploid individuals
should produce a single band on a gel as described for
haploids. Diploid individuals that are heterozygous at a locus
for a monomeric enzyme will give two bands in a properly
stained isozyme gel following electrophoresis. Each band can
be interpreted easily as a different allele at a single genetic
Figure 2 Subunits with 2 2 and 2 3 charges combine at locus. Banding patterns are more complicated for enzymes
random to form an active dimeric enzyme. A, In this example, composed of two or more subunits. In these cases the subunits
random combination of subunits yields three dimers with total must join together to form the active enzyme. Because
charges of 24, 2 5, and 26 in a ratio of 1:2:1. B, The pattern that dimeric enzymes (those composed of two subunits) are the
results on a gel after electrophoresis and staining. Direction of most common, they will be the subject of this discussion.
migration is indicated by the arrow. However, the concepts covered here are directly applicable to
40 Goodwin

enzymes composed of three or more subunits. To correctly oomycete Phytophthora infestans at the Gpi locus, and was
interpret isozyme banding patterns produced by dimeric confirmed by thorough genetic analyses (Goodwin et al. 1992).
enzymes, it is necessary to understand how the subunits Other banding patterns can be produced by individuals
combine to form the active enzyme. In heterozygous diploid containing three or more alleles separated by unequal charges.
individuals, the subunits produced by the two alleles combine For example, an individual heterozygous for three alleles in
at random in the cytoplasm of the cell to form the active uneven steps, e.g., 2 2, 23, and 25, would yield a six-
molecule. For the hypothetical case of a diploid with two banded pattern in a 1:2:1:2:2:1 ratio. Band-intensity ratios in
alleles, one coding for a subunit with a 2 2 charge, the other polyploids also can be affected by the number of copies of
with a 2 3 charge, there are four possible ways the subunits particular alleles at a locus. For example, an individual with
can be joined to form the active dimer (Figure 2A). Three three alleles, two of which are identical, e.g., 2 2, 24, and
types of dimer can result: two homodimers (when identical 24, should give rise to a three-banded pattern in a 1:4:4 ratio.
subunits combine) and a heterodimer (composed of two This has been documented in the US-1 genotype of
different subunits). In Figure 2A, each 2 2 subunit can join P. infestans which has two copies of the 100 and one copy
with another 2 2 subunit (to form a 2 2/2 2 homodimer) or of the 86 allele at the Gpi locus (Goodwin et al. 1992). Even
with a 2 3 subunit (for a 2 2/23 heterodimer). Similarly, half more complicated banding patterns are produced by
of the 23 subunits will pair with a 2 2 subunit and half with a tetrameric enzymes and by multiple loci that share alleles.
23 subunit. Notice that for every 2 2/2 2 homodimer Interpretation of these patterns was covered in detail
(total charge ¼ 2 4) there are two 2 2/2 3 heterodimers (total elsewhere (Micales et al. 1992). Fortunately, most enzymes
charge ¼ 2 5) and one 2 3/2 3 homodimer (total are dimeric so the principles discussed previously will apply
charge ¼ 2 6), for a ratio of 1:2:1. directly. An understanding of these basic principles combined
When these molecules are separated according to charge in with genetic analyses should allow unambiguous interpret-
an electric field, they will migrate to three regions on the gel, ation of virtually any isozyme pattern.
which after staining will be visualized as three distinct bands
(Figure 2B). Notice that the middle (heterodimer) band is
approximately twice as intense as either homodimer band, 4.3 Choosing Among the Available Enzyme
reflecting the 1:2:1 ratio in the numbers of each molecule. Systems
This pattern is unmistakable on isozyme gels (Figure 3).
The pattern is slightly more complicated for trisomic or Every isozyme project begins with a choice of enzymes.
polyploid individuals possessing three alleles. In this case, the Although it is not possible to predict which enzymes will
subunits pair at random as before, only now there are nine
possibilities (Figure 4A). If the different alleles vary by
single-step charge differences, for example with charges of
22, 2 3, and 24, then the 23/23 homodimer will have the
same 26 charge as the 2 2/24 heterodimers and will migrate
to the same place on the gel during electrophoresis. This will
produce a five-banded phenotype in which the intensities of
the bands should be in a ratio of approximately 1:2:3:2: 1
(Figure 4B). This pattern is seen in the US-8 genotype of the

Figure 3 Banding patterns of glucose-6-phosphate isomerase

in a diploid. Heterodimers are seen as more intensely staining
bands between two other bands. The gel shows interspecific Figure 4 Formation of dimers in a triploid. A, The subunits can
hybrids between the oomycetes Phytophthora infestans and its combine nine ways to form dimers with five possible charges. B,
close relative P. mirabilis. The P. infestans parent was Separating these dimers on a gel and staining gives five bands in a
heterozygous 86/122and the P. mirabilis parent was homozygous ratio of intensities of 1:2:3:2: 1. The most intense band results
l08/108. Interspecific hybrids are 86/108 (lanes 1,2,4,7, and 8) or from co-migration of 2 2/24 and 2 3/2 3 heterodimers. All six
108/122 (lanes 3,5,6, and 9). Lanes 10 (homozygous 108/108) bands could be visible if the differences in charge among alleles
and 11 (86/122) indicate self fertilization of the P. mirabilis and were uneven. Direction of migration is indicated by the
P. infestans parents, respectively. arrow.
Isozyme Analysis of Fungi 41

work with a particular fungal species, some guidance can be be performed with NTSYS, but it is available with some of the
obtained from previous research. The best approach is to other computer programs as well as the program WinBoot
screen a few isolates on a large number of enzyme systems, (Yap and Nelson 1996).
then choose those enzymes which give well resolved, easily Specific applications of isozyme analysis to taxonomic
storable banding patterns. However, choosing which enzymes questions include identifying strains of Trichoderma harzia-
to try first can be daunting. Enzyme testing results from 27 num (Zamir and Chet 1985), varieties of Verticicladiella
published surveys plus three unpublished surveys are wageneri (Otrosina and Cobb 1987), or anastomosis groups
summarized in Table 1. Among the 82 enzymes listed, 32 within Rhizoctonia (Damaj et al. 1993). The technique also
never worked and another 20 were successful in less than 50% can be used to identify fungal cultures to species (Six and
of the surveys. The most promising enzymes to try in an initial Paine 1997). Isozyme analyses have confirmed a high level of
survey would be the 20 enzymes that were tried four or more genetic differentiation among host-associated varieties of
times and provided useful information in 50% or more of the Leptographium wageneri (Zambino and Harrington 1989)
studies surveyed (indicated in bold in Table 1). and have revealed previously unknown genetic subdivision
within various species of Phytophthora (Mchau and Coffey
1994; 1995). In the rust genus Puccinia, isozyme variation
5 APPLICATIONS OF ISOZYME ANALYSIS can distinguish among species and also among formae
speciales on different hosts (Burdon and Marshall 1981).
Isozyme analysis can be applied to address a wide variety of The most common use of isozyme analysis in fungal
biological questions in the fungi. These markers usually are taxonomy is to divide isolates into species and to test how
considered to be selectively neutral so are ideal for looking at well biochemical species identification corresponds to
sources of inoculum for pathogenic fungi or gene flow among classical taxonomy. Usually, species groups identified by
populations. The markers can indicate whether specific isozyme analysis correspond quite closely with those
tissues are haploid or diploid, can aid strain or species identified morphologically (Hsiau and Harrington 1997;
identification, and can be used to identify hybridization either Oudemans and Coffey 1991a,b; St. Leger et al. 1992; Surve-
in the laboratory or in nature. Iyer et al. 1995). However, sometimes two or more taxa are
found to be the same genetically (Oudemans and Coffey
1991b; Yoon et al. 1990) and are combined into a single
5.1 Taxonomy and Species Identification species. The opposite also is a common result of isozyme
analysis: single species frequently can be divided into two or
Isozyme analysis was first applied to fungal taxonomy during more species based on previously hidden genetic differen-
the 1960s (Clare 1963; Hall 1967; Meyer et al. 1964; Peberdy tiation uncovered by isozyme analyses (Altomare et al. 1997;
and Turner 1968). Since then it has been applied at many St. Leger et al. 1992).
taxonomic levels from typing individual strains to delimita-
tion and identification of species. The best enzymes for
taxonomic purposes are those that are monomorphic within 5.2 Genetics
but different among taxa. A caveat is that isozymes cannot be
used to infer phylogenetic relationships. This is because bands In addition to simple Mendelian genetics (Bonde et al. 1988;
with the same migration rate on a gel in fact may not be Burdon et al. 1986; Hellman and Christ 1991; Shattock et al.
identical. Furthermore, it is not possible to infer which allele 1986a; Spielman et al. 1990) and linkage analysis (May and
is ancestral or to estimate the number of mutations that cause Royse 1982b), isozymes can be used to distinguish hybrid
the isozymes to migrate differently; alleles of similar size may from nonhybrid progeny in both intra- (May and Royse
be more different evolutionarily than those with larger 1982a; Shattock et al. 1986b) and inter-specific crosses
migration distances on a gel. Therefore, clustering algorithms (Goodwin and Fry 1994), and to infer the ploidy level of
such as neighbor joining (Saitou and Nei 1987) that allow for vegetative hyphae (Goodwin et al. 1994; Shattock et al.
unequal rates of evolution on branches are not appropriate for 1986b). Dimeric enzymes are ideal for this kind of analysis
isozyme data. Instead, isozyme data are analyzed usually by (Figure 3). Isozymes also can be used to analyze parasexual
calculating a simple distance coefficient and drawing clusters genetics in fungi. In addition to laboratory genetics, isozyme
with the Unweighted Pair Group Method with Arithmetic analysis implicated somatic hybridization as the probable
mean (UPGMA) (Michener and Sokal 1957). These analyses origin of a new forma specialis of cereal rust in Australia
can be performed with several computer programs such as (Burdon et al. 1981) and identified naturally occurring hybrids
NTSYSpc (Rohlf 1998), POPGENE (http://www.ualberta.ca/ among field isolates of Phytophthora species (Man in ‘t Veld
-fyeh/index.htm), PHYLIP (http://evolution.genetics. et al. 1998). Estimates of relatedness based on isozyme
washington.edu/phylip.html), or PAUP* (http://paup.csit. analysis among strains of Agaricus brunnescens were used to
fsu.edu/index.html). Cluster analyses always should be aid the choice of parents in a mushroom breeding program
accompanied by bootstrap analysis or some alternative (Royse and May 1982b). Most studies have shown normal
method of indicating the level of statistical support for Mendelian segregation of isozyme alleles (i.e., the isozymes
particular groupings. Unfortunately, bootstrap analysis cannot can be considered allozymes), although instances of aberrant
Table 1 Isozyme systems tested and success rates in various groups of fungi and oomycetes. The 20 enzymes that were tested four or more times and were useful in 50% or
42
more of the surveys are indicated in bold
No. times useful/no. times testeda
Enzyme E.C. no.b Ascoc Basidd Deute Oomf Percent useful Avg. no. of alleles
Acid phosphatase 3.1.3.2 3/4 6/6 1/3 77 1.6
Aconitate hydratase 4.2.1.3 5/9 3/7 3/5 2/2 56 1.9
Adenosine deaminase 3.5.4.2 0/1 1/1 50 2.0
Adenylate kinase 2.7.4.3 0/3 1/4 0/2 1/1 20 1.0
Alanine aminopeptidase 3.4.11.12 1/3 33 3.0
Alanine dehydrogenase 1.4.1.1 1/1 0/1 0/1 33 1.0
Alcohol dehydrogenase 1.1.1.1 0/4 2/6 0/4 0/1 13 5.0
Aldehyde oxidase 1.2.3.1 0/3 0/2 0
Alkaline phosphatase 3.1.3.1 2/6 1/5 0/3 0/1 20 2.0
a-Amylase 3.2.1.1 0/1 0
Aryl (fluorescent) esterase 3.1.1.2 1/1 3/3 1/2 1/1 86 1.8
L -Ascorbate oxidase 1.10.3.3 0/1 0
Aspartate aminotransferase (glutamate oxalate transaminase) 2.6.1.1 4/7 8/10 3/5 0/1 65 2.3
Aspartate oxidase 1.4.3.16 0/1 0
Carboxylesterase 3.1.1.1 3/7 4/5 1/4 50 2.6
Catalase 1.11.1.6 1/1 6/7 2/2 90 1.7
Catechol oxidase (tyrosinase) 1.10.3.1 0/1 0
Creatine kinase 2.7.3.2 0/1 0
Cytosol (leucine) aminopeptidase 3.4.11.1 7/10 9/14 5/7 1/2 67 2.3
Cytosol nonspecific dipeptidase 3.4.12.18 1/1 1/2 3/5 4/4 75 1.9
Dihydrolipoamide dehydrogenase (diaphorase) 1.8.1.4 2/4 4/8 1/3 1/2 47 2.1
Dipeptidyl carboxypeptidase I 3.4.15.1 0/1 0
Enolase 4.2.1.1 0/1 1/1 50 3.0
Formate dehydrogenase 1.2.1.2 0/1 0
Fructose-bisphosphatase 3.1.3.11 0/2 0/2 1/3 1/1 25 2.0
Fructose-bisphosphate aldolase 4.1.2.13 2/2 1/6 0/3 1/2 31 2.5
Fumarate hydratase 4.2.1.2 3/6 2/4 2/5 1/3 44 1.7
Galactose dehydrogenase 1.1.1.48 0/1 0
b-Galactosidase 3.2.1.23 0/2 0/1 0/1 0
Glucokinase 2.7.1.2 0/1 0
Glucose dehydrogenase 1.1.1.47 0/4 0/1 0/1 0/1 0
Glucose-6-phosphate dehydrogenase 1.1.1.49 7/8 1/5 5/6 2/2 71 1.5
Glucose-6-phosphate isomerase 5.3.1.9 10/10 10/12 5/5 4/4 94 2.3
b-Glucosidase 3.2.1.21 0/1 0/1 2/4 0/1 29 3.5
Glucosyl transferase 2.4.1.11 0/1 0
b-Glucuronidase 3.2.1.31 0/1 0/1 0
Glutamate dehydrogenase 1.4.1.2 3/3 5/11 1/3 1/1 56 1.8
Glutamate dehydrogenase NADP 1.4.1.3 1/1 100 1.0
Glutamate pyruvate transaminase 2.6.1.2 1/2 2/3 60 2.0
Glutathione reductase 1.6.4.2 0/1 1/2 0/1 25 2.0
Glyceraldehyde-3-phosphate dehydrogenase 1.2.1.12 0/1 0/4 1/2 0/1 13 2.0
Glyceraldehyde-3-phosphate dehydrogenase (NADPþ) 1.2.1.13 0/1 0/1 0
Goodwin
Glycerate dehydrogenase 1.1.1.29 1/1 0/3 0/1 20 4.0
Glycerol-3-phosphate dehydrogenase (NADþ) 1.1.1.8 0/2 0/1 0/2 0
Glyoxalase 4.4.1.5 1/1 100 2.0
Guanine deaminase 3.5.4.3 0/1 0
Hexokinase 2.7.1.1 2/5 4/8 3/4 2/3 55 1.9
Hexosaminidase 3.2.1.30 1/1 100 1.0
3-Hydroxybutyrate dehydrogenase 1.1.1.30 0/1 0/1 0/1 0
L -Iditol (sorbitol) dehydrogenase 1.1.1.14 0/3 0/1 0/1 0
Isocitrate dehydrogenase (NADP1) 1.1.1.42 5/5 0/5 5/6 2/3 63 1.8
Laccase 1.10.3.2 0/1 0
Isozyme Analysis of Fungi

L -Lactate dehydrogenase 1.1.1.27 2/5 0/1 0/3 3/3 17 2.0

Leucine dehydrogenase 1.4.1.9 0/1 0/1 0
Lysine dehydrogenase 1.4.1.15 0/1 0
Malate dehydrogenase 1.1.1.37 8/9 7/10 5/6 4/4 83 1.9
Malate dehydrogenase (NADPþ) (malic enzyme) 1.1.1.40 2/4 3/7 1/4 2/2 47 1.4
Mannitol dehydrogenase 1.1.1.67 3/3 0/2 0/1 50 1.7
Mannose-6-phosphate isomerase 5.3.1.8 1/3 3/5 2/3 3/3 64 1.6
Menadione reductase 1.6.99.2 1/1 3/6 2/3 1/2 58 1.1
NAD dehydrogenase 1.6.99.3 0/1 0
NADPH dehydrogenase 1.6.99.1 0/1 0
Nucleoside diphosphate kinase 2.7.4.6 1/1 100 2.0
Nucleoside phosphorylase 2.6.1.1 1/2 50 2.0
Octanol dehydrogenase 1.1.1.73 0/1 0
Peroxidase 1.11.1.7 1/2 0/1 33 2.0
Phosphoenolpyruvate carboxylase 4.1.1.31 0/2 0
6-Phosphofructokinase 2.7.1.11 0/1 0
Phosphoglucomutase 5.4.2.2 9/9 7/10 5/5 1/2 85 2.3
Phosphogluconate dehydrogenase 1.1.1.44 6/6 4/6 2/3 4/4 84 1.5
Phosphoglycerate kinase 2.7.2.3 0/3 1/1 25 3.0
Polyphenol oxidase 1.14.18.1 0/3 0
Pyruvate kinase 2.7.1.40 0/1 0
Shikimate dehydrogenase 1.1.1.25 1/4 0/4 0/2 0/1 9 1.0
Succinate dehydrogenase 1.3.99.1 0/3 0/1 0
Superoxide dismutase 1.15.1.1 1/5 0/2 1/3 2/3 31 2.0
Trehalase 3.2.1.28 0/2 0/1 0
Triose-phosphate isomerase 5.3.1.1 2/2 2/2 3/4 1/1 89 2.4
Uridine diphosphoglucose pyrophosphorylase 2.7.7.9 1/1 100 1.0
Uridine monophosphate kinase 2.7.4.4 0/1 0
Xanthine dehydrogenase 1.1.1.204 2/5 0/3 0/2 1/2 25 1.7
X-Pro dipeptidase 3.4.13.9 1/1 100 4.0
a
Based on an analysis of 27 published enzyme surveys plus three surveys of septoria pathogens (G. Zhang and S. B. Goodwin, unpublished). The published surveys were those of: Altomare et al. (1997);
Andrews et al. (1988); Burdon and Roelfs (1985a); Damaj et al. (1993); Gaur et al. (1991); Goodwin et al. (1993); Huss (1996); Leuchtmann and Clay (1989); (1990); Leung and Williams (1986); Linde
et al. (1990); Nyassé et al. (1999); Old et al. (1984); Otrosina and Cobb (1987); Otrosina et al. (1992); Oudemans and Coffey (1991a); Riley et al. (1998); Royse and May (1982a); Six and Paine (1999);
Surve-Iyer et al. (1995); Tooley and Fry (1985); Tuskan and Walla (1989); Vogler et al. (1991); Welz et al. (1994); Yoon et al. (1990); Zambino and Harrington (1989); Zhu et al. (1988).
b
Enzyme Commission number. International Union of Biochemistry and Molecular Biology (1992). Enzyme Nomenclature. Academic Press, Inc.
c
Ascomycates.
d
Basidiomycetes.
e
Deuteromycetes.
43
44 Goodwin

segregation have been noted (Spielman et al. 1990). It is Phytophthora infestans (Spielman et al. 1991; Tooley et al.
important to remember that no isozyme should be referred to 1985). Populations of fungi infecting conifer hosts in the
as an allozyme until a proper genetic analysis has been western United States also appeared to be highly clonal and
completed. specific clones are characterized by fixed heterozygosity
(Otrosina et al. 1992; Vogler et al. 1991).
Differences in levels of genetic variation among locations
5.3 Population Biology can help identify the center of origin of a species. The most
diverse populations genetically should occur at or near the
Gene flow is the movement of individuals among populations center of origin, as these populations will have had the longest
and can be estimated indirectly through isozyme analysis as time in which mutations can accumulate; derived populations
the number of migrants per generation (Slatkin and Barton usually will contain only a subset of the total species
1989) or by analysis of “private” alleles—those limited to a diversity. This idea has been combined with isozyme analysis
single species or population. Different species usually have to indicate that the edible mushroom Agaricus bisporus
highly differentiated allele frequencies and a higher number may be indigenous to North America (Kerrigan and Ross
of private alleles compared to individuals from a single 1989) and that Phytophthora infestans, P. palmivora, and
panmictic population. The computer program POPGENE , P. megakarya probably originated in central Mexico
among others, can perform this type of analysis. (Tooley et al. 1985), southeast Asia (Mchau and Coffey
Applications of isozyme analysis to epidemiology have 1994), and central Africa (Nyassé et al. 1999), respectively.
included monitoring the spread of specific genotypes of Very limited genetic diversity indicated that the plant
Phytophthora infestans (Goodwin et al. 1995; Legard and Fry pathogens Puccinia graminis f. sp. tritici, Phytophthora
1996), determining the influence of various evolutionary cinnamomea, and Dilophospora alopecuri had only very
forces on the composition of inoculated populations of the limited introductions into Australia (Burdon et al. 1982; Old
barley scald pathogen Rhynchosporium secalis (Goodwin et al. 1984; Riley et al. 1998), although the original source
et al. 1994), and estimating the size of and boundaries populations for the introductions could not be identified for
between individuals of the puffball Lycoperdon pyriforme on certain.
decaying logs (Huss 1993).
The first real attempt to use isozymes to analyze the
genetic structure of fungal populations was for Neurospora 6 CONCLUSIONS
intermedia by Spieth (1975). That analysis revealed a high
level of genetic variation within but low differentiation Isozyme analysis is an extremely powerful and versatile
among populations. Spieth’s pioneering work was followed technique that can answer many questions about fungal
by additional studies within a decade and this has accelerated genetics, taxonomy, and population biology. Compared to
during the 1990s. Many of these studies have had similar alternative technologies, isozymes are cheap, fast, and easy to
results and demonstrated high gene diversity within but low interpret. They only assay expressed genes and, by analysis of
differentiation among populations of a wide diversity of dimeric enzymes, can distinguish heterozygous individuals
fungi (Andrews et al. 1988; Goodwin et al. 1993; Huss from physical mixtures of cultures—an advantage that is not
1996; Tuskan et al. 1990). Other authors have found shared by DNA-based markers. With newer types of
varying degrees of subdivision among populations, usually electrophoresis systems, isozymes are suitable for analysis
among host-associated forms (Harvey et al. 2001; of small tissue samples and could be portable to remote
Leuchtmann and Clay 1989; 1990) and, more rarely, locations. The main disadvantage of isozyme analysis is a
among physiological races (Welz et al. 1994). lower level of polymorphism compared to DNA-based
Gene flow analysis of isozyme markers also can indicate markers, but this usually can be overcome by increasing the
when taxa have become sufficiently isolated reproductively to number of enzyme systems assayed. The many advantages of
be considered separate species. Existence of separate species isozyme analysis should ensure its place in the molecular
was shown conclusively for several closely related groups in toolbox for many years to come.
the genus Phytophthora (Goodwin et al. 1999; Man in ‘t Veld
et al. 2002; Nygaard et al. 1989) which could not have been
ACKNOWLEDGEMENTS
discovered without the use of molecular markers. A similar
approach identified distinct biological species within the
mushroom Pleurotus eryngii (Urbanelli et al. 2002). Thanks to Kristi Brikmanis and Bryan Wallace for retrieving
Isozyme analyses of some populations of fungi and references and helping with much of the typing.
oomycetes have revealed extremely low levels of genetic
variation reflective of a highly clonal population structure.
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4
Molecular Methods for Identification of Plant Pathogenic Fungi

Maren A. Klich / Edward J. Mullaney U.S. Department of Agriculture – Agricultural Research Service,
New Orleans, Louisiana, USA

1 INTRODUCTION molecular sites concur with one another and with “trees”
based on morphological and physiological data, one has fairly
1.1 Need for Molecular Methods in Fungal strong evidence that the phylogeny is accurate. Some fungi do
Identification not grow or do not sporulate in culture. Molecular
methodologies allow identification of these isolates by
For centuries, fungal identification has been based on comparison of DNA sequence data from the unknown isolate
morphological, physiological, and chemical characteristics with sequences from known species. Molecular methods have
of specimens. For the most part, these systems still work also been used to distinguish between closely related species
extremely well. They provide accurate species identification with few morphological differences and to distinguish strains
inexpensively, are not labor-intensive, and require little (or even specific isolates) within a species. In studies of fungal
equipment beyond a microscope and chemical reagents. Since metabolites, especially mycotoxins, there has been no way of
phenotype is the result of the expression of hundreds of genes, knowing whether a nonproducing strain is truly (genetically)
the higher level classifications based on morphology/ incapable of producing the metabolite or if it could possibly
physiology are generally sound. A major drawback of the produce it under different environmental conditions. Once the
traditional identification methods is that they require some genes of a metabolic pathway have been cloned, they can be
technical training in order to acquire the skills necessary to used to determine whether or not a strain possesses the genes
identify fungi or characterize strains. This training has become for production of the metabolite, providing a better indication
increasingly difficult to acquire, whereas molecular biology of the potential of a given strain to produce a metabolite.
techniques are now taught widely in secondary schools and can
be applied to a multitude of fields. Another drawback of the
traditional methods is that they can take a week or more for 1.2 Assumptions and Limitations
fungal colonies to grow and develop the characters necessary
for identification. In some cases, the necessary characters The assumptions made in making fungal identifications
never develop. Several of the molecular methods provide using molecular methods are the same as those made using
identifications more quickly and do not rely on the presence of traditional methods. The concept of a species (or any other
reproductive structures. taxonomic level) is ultimately what those working in the field
Molecular biology has brought many powerful new tools agree it to be. These decisions are based on available data, but
to fungal taxonomists including the potential for rapid there is always some degree of subjectivity in the process,
identification of isolates, methods for rapid determination of especially when considering closely related species.
virulence or toxicity of strains, and the means to elucidate the None of the current molecular methods used in fungal
relationships among fungal species. DNA sequence data identification can screen more than a minute portion of the
provide large numbers of data points that can be compared genome. There is no single method that can be used to
among fungi and analyzed to determine sequence relatedness, distinguish all species or all genera from one another.
which can be assumed to reflect phylogenetic relatedness Generally, if the data provided by a molecular method support
among species. If “phylogenetic trees” from different current taxonomic thought on a taxon, they are used.

49
50 Klich and Mullaney

A molecular method appropriate for identification of one and can be subjected to hybridization analysis. This enables
species or genus (or part thereof) may not be useful for species identification of bands with sequence similarity to a labeled
in other genera or even for all portions of one genus. probe. Several printed reference laboratory manuals [e.g.
Molecular methods provide powerful new tools to aid in Molecular cloning: A Laboratory Manual, 3rd Edition; Short
fungal identification, but they have not provided (and Protocols in Molecular Biology, 4th Edition] and Internet web
probably will never provide) a universal solution to problems sites such as www.protocol-online.net and www.nwfsc.noaa.-
associated with fungal identification. They are tools, not gov/protocols detail these techniques. In dot blots, the target
panaceas, for taxonomists. Currently molecular methods are nucleic acid sample is affixed directly to the membrane
more labor-intensive and more expensive than many of the without electrophoresis followed by hybridization with the
traditional methods of fungal identification, but the field is labeled probe. Detailed information on these protocols can
evolving and more rapid and affordable methods will also be obtained by utilizing a search engine such as Googlee
probably become available. to search the internet.
DNA fingerprinting is an umbrella term that describes
molecular identification techniques. As the term implies, the
1.3 Scope basic genetic material in the organism is utilized to determine
its identity and relationship with other isolates. One of the
It would be beyond the scope of this paper to review all of the earliest hybridization probes utilized was variable number
studies using molecular methods for identification of plant tandem repeats (VNTRs) or minisatellite DNA (Jeffreys et al.
pathogenic fungi. Instead, we will provide an overview of the 1985). The term “satellite” originated from buoyant density
methods available for identification and then give one or two centrifugation studies in which the bulk of the sample DNA
recent examples from the literature. These citations will give sediments as one main band, but smaller satellite bands were
interested readers a starting point for finding related citations also observed. Satellites are composed of tandem repeats of
in the literature. DNA arranged in consecutive repeats. They sediment apart
from the main band containing the bulk of the DNA due to
their significantly different base composition. Minisatellites
2 METHODOLOGIES range in size from 1 kb to 20 kb. The VNTR are a type of
minisatellite located in the noncoding regions of the genome.
The majority of molecularly-based identification methods The size of the repeat units varies from 9 to 80 base pairs (bp).
rely on the interaction of unique enzymes and nucleic acids. Smaller repeats consisting of only 1 –6 bp are designated
Two extensively utilized classes of enzymes are restriction microsatellites, or short tandem repeats (STR). The STR have
endonucleases and DNA polymerases. a repetitive region of less than 150 bp. Sequences of both
Restriction endonucleases cleave DNA molecules at mini- and microsatellites have been utilized in DNA
specific nucleotide sequences. Different endonucleases fingerprinting. In Meyer et al. (1991), the DNA of various
recognize and cut at different restriction recognition filamentous fungi were digested separately with different
sequences. An example of two endonucleases and the specific restriction endonucleases and the resulting fragments
sequence they recognize are Eco RI (GAATTC) and Bam HI separated by electrophoresis. Selected VNTR oligonucleo-
(GGATCC). Eco RI cleaves between the guanine and the tides were labeled with 32P and hybridized to the dried gel.
adenine, G^AATTC and Bam HI between the guanines Results confirmed that the method based on these
G^GATCC. Restriction fragment length polymorphism minisatellite DNA probes could differentiate between species
(RFLP) is one molecular technique that requires restriction and strains of fungi. Microsatellites have been used to
endonucleases to produce the unique DNA fragments which discriminate strains of the aflatoxigenic species Aspergillus
are the basis for this technique. flavus and A. parasiticus (Tran-Dinh and Carter 2000).
Polymerases are enzymes that catalyze the formation of The PCR technology provides researchers with a means to
DNA or RNA from nucleotide precursors. All DNA rapidly amplify small amounts of DNA, and frees them from
polymerases also require a preexisting template. For example, the time-consuming task of having to isolate sufficient DNA
various DNA polymerases are utilized for different for RFLP analysis. In PCR a thermostable polymerase is
applications [DNA polymerase I (Kornberg polymerase), is employed to enzymatically amplify a specific region of DNA
used to label DNA; Taq DNA polymerase is used to amplify sequence, defined by a set of two oligonucleotide primers.
DNA fragments in the Polymerase Chain Reaction (PCR); The target DNA is denatured and the primers are then
etc.]; and some have been engineered for enhanced features, annealed to the single-stranded DNA. The DNA polymerase
e.g., Sequenasee which is used in DNA sequencing. then synthesizes the complementary DNA strand across the
In RFLP the assorted DNA fragments resulting from the target region. This process is then repeated and the targeted
restriction endonuclease digestion are resolved by gel DNA may be amplified a million-fold or more.
electrophoresis. Ethidium bromide staining is then used to Randomly Amplified Polymorphic DNA (RAPD) is a PCR
reveal the fragments under UV (260 nm) light. Southern technique that yields genetic markers without the need to
blotting of the gel can transfer the DNA fragments to a obtain prior nucleotide sequence data (Williams et al. 1990).
support membrane. The DNA is then fixed to the membrane Random nucleotide sequences are annealed to the template
Molecular Methods for Identification of Plant Pathogenic Fungi 51

DNA under low stringency. This is followed by PCR four bases, of the restriction endonuclease increased the
amplification and electrophoresis to produce a DNA probability that a single base pair polymorphism could be
fingerprint. The RAPD technique is relatively easy, fast, detected.
and requires only a minimum amount of starting material. Two other methods of high-stringency PCR are micro-
However, the low stringency of the annealing process can satellite based PCR-amplification and the use of the small
produce PCR artifacts. subunit ribosomal DNA (SSU rDNA) based primers for DNA
Several methods have been developed to reduce the fingerprinting (Scott and Straus 2000). For example, Gargas
number of artifacts produced by PCR amplification. One and DePriest (1996) describe a list of PCR primers used to
method is by using two pairs of PCR primers, or nested amplify and sequence the small subunit of fungal nuclear
primers (Plikaytis et al. 1990). In this technique, the first pair rDNA. This information identifies primers for special
produces a PCR fragment. Then this fragment and the second applications (intron-spanning, intron specific, etc) and
pair of primers (nested primers) binding to DNA sequence a represents a valuable resource for further research. Groppe
few bases internal to the first pair of primers, are used in a et al. (1995) synthesized oligonucleotides corresponding to
further amplification. If the first pair of primers amplified the regions of the sequence of a microsatellite of the endophytic
correct locus, then the second pair of primers will produce a ascomycete Epichloë typhina. These were used for PCR
slightly smaller PCR fragment. This method requires a amplification of DNA from different Epichloë isolates. The
knowledge of the sequence immediately adjacent to the first DNA from most isolates produced a single PCR product. This
pair of PCR primers in order to synthesize the other “nested” study pointed to a potentially useful role for microsatellite-
pair of primers. containing loci as a molecular marker for population studies
If the host DNA sequence is not known, Amplified of Epichloë and other unrelated fungi.
Fragment Length Polymorphism (AFLP) will also reduce the DNA sequencing, while not yet extensively utilized
number of PCR artifacts (Vos et al. 1995). In this technique, because of the time and resources required, has been
the target DNA is digested with restriction endonucleases to employed to a limited degree in species identification. Wang
yield an assortment of different sized DNA fragments. et al. (2001) recently reported on the use of mitochondrial
Specific double-stranded adapter oligonucleotides are then cytochrome b gene to identify species of Aspergillus section
ligated to these fragments. PCR primers specific to the adapter Flavi. Mycelia of the Aspergillus isolates were harvested and
sequences with various selective 30 nucleotides are then their hyphae ruptured with glass beads and zymolyase. Their
utilized under high stringency PCR amplification and mitochondria were then collected by centrifugation and their
electrophoresis to produce a unique fragment profile. This mtDNA extracted. The sequence of the cytochrome b gene
technique is time-efficient and amplification is not completely has also been used to distinguish species of Aspergillus
random as in RAPD. section Fumigati (Wang et al. 2000) and for investigating the
When the sequence of the target DNA is known, several phylogentic relationships of other species of Aspergillus
other PCR procedures for identification are available, such as (Wang et al. 1998).
PCR amplification of internal transcribed spacer (ITS) of Single-Strand Conformation Polymorphism (SSCP) is
ribosomal DNA (rDNA). An example of the application of another PCR-based system that requires knowledge of the
this PCR method is Beck and Ligon (1995) who designed target DNA sequence to generate specific oligonucleotide
PCR primers to detect Stagonospora nodorum and Septoria primers. In this technique, the target DNA is concurrently
triticic in wheat. These primers were derived from species labeled and amplified by PCR using a labeled substrate. The
specific DNA sequences of the ITS of the pathogen’s PCR product is then denatured and resolved by electrophoresis.
ribosomal DNA. The PCR amplification of ITS rDNA has also Any changes (mutations, etc.) in the target DNA are detected as
been employed to identify a wider variety of fungi that were altered mobility of separated single strands in autoradiograms.
potential pathogens and allergens (Makimura et al. 2001). Precise information about the exact change can then be
Knowledge of the specific sequence of polymorphic loci obtained by eluting the targeted DNA from the gel and
permits high-stringency PCR and thus circumvents the amplifying it again for sequence determination (Hayashi 1991).
problem of artifacts and low reproducibility associated with Heteroduplex analysis, like SSCP, is a recently developed
random-primer methods (Scott and Straus 2000). One method technique that can detect a single base difference in target
of site-specific polymorphisms they reviewed was based on DNA (Keen et al. 1991). PCR amplification products from the
the sequence variability found in the introns of single copy isolates are combined after heat denaturation and then
metabolic and structural genes. Glass and Donaldson (1995) allowed to reanneal to form heteroduplexes. Any mismatched
tested several such oligonucleotide primers for their ability to nucleotides, caused by substitutions, insertions, deletions, etc,
amplify segments of DNA that span introns in a selection of will affect the DNA structure of the heteroduplex and lower
these genes. They identified primer sets that provided a useful its electrophoretic mobility. The heteroduplex is compared to
tool for phylogenic studies of filamentous ascomycetes and duplexes with complete base complementarity by electro-
related fungi. These oligonucleotide primers were utilized to phoresis. Kumeda and Asao (2001) employed this technique
differentiate Fusarium species (Donaldson et al. 1995). The for the detection of intraspecific variation in isolates of
PCR fragments generated were digested with several 4 bp Aspergillus Section Flavi. In their heteroduplex panel
recognition restriction enzymes. The short recognition site, analysis (HAP), fragments of the internal spacer (ITS)
52 Klich and Mullaney

regions of the rDNA gene of the different isolates were first culture is summarized in Table 1. Many of these studies were
amplified. Heteroduplexes were then generated with the designed to develop methods to distinguish between closely
standard ITS reference fragment and then subjected to related taxa within a single genus or species for which
electrophoresis. The results of this HAP study corresponded morphological or physiological characters overlap or take too
well with the established taxonomy of the Section Flavi. long to develop.
There are several advantages in isolating the fungus of
interest in culture before conducting molecular identifi-
3 RECENT APPLICATIONS IN PLANT cations. First, one knows immediately whether or not the
PATHOLOGY pathogen is viable in the plant. Second, working with pure
cultures lessens the possibility of errors such as accidentally
Most recent studies in molecular identification have used PCR creating a PCR product from the wrong fungus. There are also
in some form. This is not surprising given the power of this disadvantages to this approach. First, it takes longer time
tool for analyzing DNA. As the use of PCR methodologies in because the pathogen must be isolated before it is analyzed.
plant disease diagnosis was reviewed by Henson and French Second, some true (obligate) pathogens cannot be cultured in
(1993), Mills (1996), our emphasis will be on studies the laboratory. Finally, metabolites such as mycotoxins may
conducted since these reviews. The number of studies on remain in the crop even after the fungus dies and these may be
various plant pathogenic genera generally reflects the relative missed if no viable fungus is present.
importance of these genera in plant pathology. One of the
genera receiving the most attention from molecular biologists,
for both understanding phylogeny and pathogenicity, has
been Fusarium. Recent work on this genus has been reviewed 3.2 Identification of Pathogens Directly from
elsewhere (Nicholson 2001). Plant Parts
Applications of molecular techniques in plant pathology
have provided methods for identification of isolates of plant Examples of studies using molecular methods to identify
pathogens, and identification of pathogens directly from plant pathogens directly from plant parts are given in Table 2. Most
materials such as leaves, seeds, or roots. These procedures may of these involve identification of fungal species using RAPDs
be applied at almost any taxonomic level, but usually address or PCR of the ITS-rDNA.
taxa at species level and below [e.g. races of a given pathogen]. Developing methods for direct isolation of specific fungal
Molecular methods have also proved useful for distinguishing DNA from plant tissues is more difficult than isolating DNA
nontaxonomic categories such as virulence or toxicity. from a pure fungal strain, but the potential impact of the
former methods is tremendous. These assays have demon-
strated the presence of the pathogens in asymptomatic plants
3.1 Identification of Pathogens In Vitro (Doohan et al. 1998). Some of these procedures take only
seven to 24 h to perform (Lee et al. 2001; Lovic et al. 1995),
Information from some recently published studies using compared with several days to a week for traditional methods
molecular methods to identify plant pathogenic fungi from or methods requiring that the fungus be isolated prior to DNA

Table 1 Examples of studies on molecular identification of fungal plant pathogens in vitro

Fungal genus Host Level of discrimination Method Citation

Alternaria Umbellifers Species RAPD Pryor and Gilbertson 2002
Botrytis Onion Species subgroup PCR-ITS/rDNA Nielsen et al. 2001
Claviceps Sorghum Species/populations AFLP and RAM Tooley et al. 2000
Colletotrichum Alfalfa Species AFLP O’Neill et al. 1997
Elsinoe Citrus Species RAPD Hyun et al. 2001
Fusarium Tomato Virulence within race RAPD Mes et al. 1999
Fusarium Cucumber Forma specialis RAPD Vakalounakis and Fragkiadakis 1999
Gaeumannomyces Turf-grass Variety PCR-ITS/rDNA Goodwin et al. 1995
Gibberella (Fusarium) Banana/corn Species/toxicity/host RAPD Jimenez et al. 2000
Gibberella (Fusarium) Banana/corn Species PCR-ITS/rDNA Jimenez et al. 2000
Macrophomina Bean, cornþ Population AFLP Mayek-Perez et al. 2001
Rhizoctonia Various Anastomosis grp subsets PCR-ITS/rDNA Carling et al. 2002
Rhynchosporium Barley Species PCR-ITS/rDNA Lee et al. 2001
Tilletia Wheat Species TaqManPCR-MtDNA Frederick et al. 2000
Venturia Pear Species PCR-ITS/rDNA Le Cam et al. 2002
Molecular Methods for Identification of Plant Pathogenic Fungi 53

Table 2 Examples of studies on molecular identification of fungal plant pathogens in vivo

Fungal genus Host Method Level of discrimination Citation

Alternaria Carrot PCR-ITS/rDNA Species Konstantinova et al. 2002
Fusarium Wheat RAPD Species Parry and Nicholson 1996
Fusarium Wheat RAPD Species/variety Doohan et al. 1998
Leptosphaeria Crucifers PCR/GenBank M77515a Virulence Taylor 1993
Melampsora Willow RAPD Stem/leaf variants Pei et al. 1997
Monosporascus Cucurbits PCR-ITS/rDNA Species Lovic et al. 1995
Monilinia Stone fruits dot blots and PCR Species Boehm et al. 2001
Mycosphaerella Banana/plantain PCR-ITS/rDNA Species Johanson and Jeger (1993)
Peronosclerospora Sorghum dot blots-genomic DNA Species Yao et al. 1990
Phakopsora Soybean TaqMan-PCR-ITS/rDNA Species Frederick et al. 2002
Pythium 3 speciesb PCR-ITS/rDNA Species Kageyama et al. 1997
Rhynchosporium Barley PCR-ITS/rDNA Species Lee et al. 2001
a
Seed cultured in liquid medium.
b
Cucumber, sugar beet, and Chinese cabbage.

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5
The Application of Molecular Markers in the Epidemiology
of Plant Pathogenic Fungi

Paul D. Bridge* Birkbeck College, University of London, London, and Royal Botanic Gardens Kew, Surrey,
United Kingdom

Tanuja Singh / Dilip K. Arora† Banaras Hindu University, Varanasi, India

1 INTRODUCTION plant involves relatively long periods of intimate interaction

without apparent damage to the host, and where the pathogen
Fungi are present in a variety of forms, in almost every can persist in asymptomatic hosts for many years (Stanosz
habitat, where they are often specific in their occurrence on et al. 1997). The effects of fungi on living plants vary
particular types of host (or substrate) and ecological niche. considerably. At one extreme, damage may be limited to
Fungi may also become partners with higher plants and enter small lesions on leaves or stems [e.g., caused by some
complex biological relationships with the host (Clay and Alternaria species, see Ellis (1968)], while at the other
Kover 1996; Thrall and Burdon 1997). The term pathogen is extreme the plant may be rapidly killed [e.g., by some
defined as “a parasite able to cause disease in a host or range Verticillium species, see Pegg (1984)].
of hosts” (Kirk et al. 2001), and pathogenic fungi can occur on Much work has been done to control fungal disease
all plants. In this chapter this definition of plant pathogenic through selection and breeding programs, the genetic
fungi will be limited to those that cause diseases of living modification of both host and pathogen, and the introduction
plants, and therefore does not include the fungi involved in of resistant varieties [see Stukely and Crane (1994)]. The
the spoilage of stored plant materials that are often referred to success of these efforts depends largely on the understanding
as causing post harvest “diseases.” The plant pathogenic fungi of genetic variability in the fungal population and monitoring
consist of a large group of genera and species from diverse this variation in nature. Many aspects of the biology of the
areas of the fungal kingdom. Recent developments in the fungi have important consequences at the population level.
understanding of the evolution of eukaryotic organisms have This particularly applies to the mode of reproduction (i.e., the
meant that a number of important plant pathogenic organisms relative contributions of sexual and asexual, outcrossing and
have been reclassified, and are no longer considered as selfing mechanisms), and to hyphal anastomosis between
fungi sensu stricto. These include the economically important genetically different individuals (Brasier 1991; Glass and
genera of Phytophthora, Pythium, and other Oomycetes, Kuldau 1992; Milgroom et al. 1996). A further consideration
that are now placed in the Straminipila (Dick 2001). Plant is that some fungi are predominantly haploid in their
pathogenic fungi have a significant influence on crop vegetative phase, some are diploid, and some are dikaryotic.
productivity. Devastating fungal diseases such as corn smut, In the case of pathogenic fungi, genetic variability can be
potato blight, and black stem rust of wheat can destroy many introduced through a variety of mechanisms, either during
economically important crops. This situation becomes more sexual reproduction or independently of it (Kistler and Miao
critical when the interaction between pathogen and the host 1992). Such variability is significant as it can influence

Present affiliations:
*British Antarctic Survey, Cambridge, United Kingdom.
†
National Bureau of Agriculturally Important Microorganisms (NBAIM), New Delhi, India.

57
58 Bridge et al.

the host-pathogen interaction as genetic flexibility allows the In addition to knowledge of the mode of transmission of a
fungi to readily adapt to changing environmental conditions, plant pathogen, it is also important to be able to determine
including the introduction of new host genotypes. exactly what is being transmitted in terms of fungal
Understanding the epidemiology of plant pathogenic fungi populations. In some cases a single fungal species may
depends upon on the ability to unambiguously identify consist of a number of different host specific populations. An
sexually produced individuals and asexually produced clones. example of this is the vascular wilt pathogen F. oxysporum,
Classical identification traits, such as morphological, where around 170 different forms (referred to as special
physiological, and disease characters, lack the required forms) have been identified. Each of these special forms
sensitivity and accuracy needed for identifying individuals shows preferential or specific pathogenicity to different hosts,
within a population, and this has prevented detailed and so F. oxysporum special form cubense will cause vascular
population studies for many years. Recent developments in wilt of banana, but would not be expected to cause significant
molecular techniques now allow population studies on plant disease on oil palm. Therefore, in order to monitor what fungi
pathogenic fungi, and these can be performed with great are present, and may pose a risk to a crop, it is necessary to
sensitivity and accuracy. An almost unlimited number of know specific details of their pathogenicity. This situation
polymorphic loci can be used to detect individual genotypes becomes further complicated if there are subpopulations
for the direct assessment of genetic variation in a given fungal within the pathogen that show differential pathogenicity,
population. Application of molecular markers has also either in terms of the degree of damage or the particular
allowed the investigation of evolutionary processes in a cultivars attacked. Such populations are generally described
large number of agriculturally important fungi (Mitchell and as races, and the ability to differentiate these can make a
Brasier 1994; Milgroom et al. 1996; Valent and Chumley significant difference to their control. A further factor in
1991; Vilgalys and Cubeta 1994), and the number and scope considering populations in fungal plant pathogens is whether
of these studies is rapidly expanding. the population is comprised of meiotic or mitotic forms. Some
pathogenic fungi, such as Fusarium species, occur almost
exclusively in a mitotic (or imperfect) form. In this state, the
variability within the population can be assumed to be
2 REASONS FOR DETERMINING relatively low, particularly if the disease is the result of a
EPIDEMIOLOGY single introduction. Some fungal pathogens, such as
Phytophthora and the rusts and smuts, are however present
As is the case for human and animal diseases, a knowledge of on the plant in a meiotic (or perfect) form, and this allows for
the epidemiology of plant disease can provide important variability to be introduced into the population at each
information for its treatment and control, and can lead to the generation [for example see Duncan et al. (1998)].
development of forecasting systems [see Shaw 2001; Zadoks
and Schein 1979]. In the case of fungal plant diseases,
particularly those affecting agricultural crops, the two main
areas that need to be determined are the mechanism of spread 3 TYPES OF MOLECULAR MARKERS
of the disease, and the specificity and host range of the
infecting organism. Fungi are transmitted to, and between, Detection of fungi on the basis of visual examination of
plants by a number of different mechanisms. Many fungi are morphology is highly selective and species-specific identifi-
spread through the soil, some growing from previously cation of fungi and spores is therefore difficult. Molecular
infected debris in or above the soil. Others can be transmitted techniques present several advantages over the traditional
as spores and other propagules through water droplets, or ones and, most importantly, nucleic acid sequences unique
directly as airborne particles. Some plant pathogens may exist to particular organisms can generally be found. As these
on secondary hosts, such as Fusarium oxysporum (Armstrong techniques do not rely on phenotypic examination, gene
and Armstrong 1958) or on weeds [see Terry and Parker expression is not required and identification times can be
(2001)]. If these hosts are present in or near a crop, they may reduced significantly. In the fungi, molecular markers can be
then act as a reservoir that allows a crop disease to be carried derived from both variable and conserved regions of the
over successive plantings. A few plant pathogenic fungi such nuclear and mitochondrial genome, and different markers
as Ophiostoma species [see Brasier (1991)] can be spread by have been used to define populations at all levels from an
insects and other vectors, and some such as Sclerospora individual isolate upwards. Some methods have the potential
graminicola (Shetty et al. 1980) remain in the seeds of for the detection of specific genomic DNA sequences directly
infected plants, and cause disease in subsequent generations. from initial plant samples, thereby eliminating the require-
If the mechanism of transmission is known, this knowledge ment to isolate and culture the fungus. Specific molecular
may be important in the development of control or treatment markers, probes, and primers have commonly been developed
strategies. Such knowledge is particularly important in the from a variety of DNA sequences including randomly cloned
selection of planting material, the preparation and main- genomic DNA fragments and specific regions such as genes
tenance of planting areas, and the establishment of crop coding for ribosomal RNA (rRNA), virulence factors, and
successions and rotation [see Maude (1996)]. insertion sequences.
Epidemiology of Plant Pathogens 59

3.1 Ribosomal DNA studies of populations and species of plant pathogens were
carried out in this way [e.g., Jabaji-Hare et al. (1990),
The nuclear genomes of fungi have a number of particular Manicom et al. (1990)]. More recently these studies have
features. They are relatively small (approximately 13 –93 largely been replaced by polymerase chain reaction (PCR)
million nucleotide base pairs), and in comparison to higher based studies, particularly as the rRNA cluster can often be
plants and animals they have a much lower percentage of detected in old or contaminated environmental samples [see
redundant DNA (about 10 –20%) (Lu 1996). Around 30% of Bruns et al. (1990)]. The varying levels of specificity of the
the entire fungal genome consists of duplicated regions and different DNA regions in the gene cluster also mean that it is
genes (Mewes 1997). These repetitive sequences provide possible to amplify fungal DNA directly from samples of
potential targets for molecular markers due to their high copy infected plant material (Bridge et al. 2000; Gardes and Bruns
number. 1993).
Ribosomal DNA (rDNA), specifically the regions coding Both the ITS and the IGS regions have been used to
for the rRNA subunits and their associated spacers, is one of develop species-specific primers for plant pathogen detection
the most commonly used DNA regions for fungal molecular in plant material [e.g., Brown et al. (1993); Moukhamedov
markers [see Bridge and Arora 1998; Bruns et al. 1991; et al. (1993)]. It is becoming increasingly common in rRNA
www.mendel.berkeley.edu/boletus/boletus.html; www. cluster studies to obtain sequences of all the regions of
biology.duke.edu/fungi/]. The nuclear-encoded rRNA genes interest. Although knowledge of the complete sequences
(rDNA) and spacers occur as a gene cluster (typically of provides a large amount of information, useful information
8–12 kb) that is multiply repeated (see Figure 1). The basic may be obtained from simple restriction digestions of rRNA
unit consists of the genes for the small ribosomal subunit, the amplification products. This approach generally produces
5.8S subunit, and the large subunit. The three genes are relatively simple patterns containing 1 –4 bands, and in
separated by two internally transcribed spacers (ITS), and the certain cases these patterns, or individual bands, may be
repeated gene clusters are separated by an intergenic spacer specific for particular pathogens [e.g., Chen (1992)]. It is not
(IGS) that in many, but not all fungi, also contains the gene for possible to list all the work done using ITS and IGS regions to
the 5S subunit. [see Hillis and Dixon (1991)]. Several develop molecular markers. One example of this was the
restrictions sites are conserved in fungal rDNA, and this study of Mazzola et al. (1996) who developed an
makes them convenient sequences for cloning [see other oligonucleotide primer set that consistently and selectively
reviews by Gargas and DePriest (1996); Hibbett (1992)]. The amplified a 511 bp fragment in the ITS region that could
rRNA cluster has proved to be a good region for deriving be used to differentiate between Rhizoctonia solani and
molecular markers for many fungi [see Bruns et al. (1991); R. oryzae. It should, however, be remembered that RFLP
Hibbett (1992)]. The subunit genes have both conserved and analysis is essentially a one-tailed analysis of variation; and
variable domains, and can be used for comparisons of genera, although different patterns indicate that two organisms are
the spacer regions are considerably more variable and can be different, a common pattern is based on only the position of a
used for comparisons of species or in some cases specific few restriction sites. Therefore, identical RFLP band patterns
pathogenic forms (see Table 1). As the cluster is universal and do not imply that the rest of the sequence is identical.
multiply repeatedly, it is a good target for molecular studies.
Originally these studies involved obtaining RFLPs with 3.2 Protein Coding Genes
probes hybridized to total genomic DNA digests, and many
There are numerous gene sequences that have been examined
in the systematics and phylogeny of plant pathogenic fungi.
These include genes for the production of actin, tubulin,
elongation factors, cytochromes, proteases, and many others
[e.g., Glass and Donaldson (1995); Mehmann et al. (1994);
Schoch et al. (2001)]. These genes are generally highly
conserved between distant organisms; but can contain short
introns that can be very variable in insertion position and
number [see Edelmann and Staben (1994)]. This variation in
introns can be useful as a molecular marker among closely
related organisms and this has been investigated for a number
of plant associated fungi including Fusarium, Ascochyta, and
Phoma (see later).

3.3 Fingerprinting Methods

For the purposes of this chapter DNA fingerprinting methods

Figure 1 Ribosomal rRNA gene cluster arrangement. will be limited to those that have been used with plant
60 Bridge et al.

Table 1 Features of commonly used molecular markers

Marker Taxonomic level resolved Affected by meiosisa

RAPD Individuals, subspecific groups Yes
Simple repetitive PCR sequences; micro- and Individuals, subspecific groups Yes
mini-satellite probes and primers
AFLPs Individuals, subspecific groups, some closely related Yes
species
mtDNA RFLPs Subspecific groups, closely related species No
ITS/IGS region RFLPs Closely related species, some subspecific groups Not generallyb
ITS region sequencing Some subspecific groups, closely related species Not generally
rRNA gene sequences Species, genera, families, phyla No
Major structural/functional protein genes Species, genera, families, phyla No
a
For many fungi the effects of meiosis on markers have not been specifically considered. Yes and no entries refer to general assumptions.
b
One reason for the selection of rRNA gene cluster was that it was resistant to crossover. However, there is at least one report of presumed recombination in
the rDNA of fungi (see text).

pathogenic fungi to generate largely random PCR fragments genomic DNA, and these can then be separated to provide
from the total genome. These techniques have also been simple fingerprints. Three particular unrelated families of
referred to as total genome profiling. One of the oldest and such repetitive DNA sequences, BOX (54 bp), ERIC (124 bp),
most widely used of such PCR methods is random amplified and REP, (35–40 bp) have also been used to characterize
polymorphic DNA (RAPD) analysis (Welsh and McClelland subspecific populations of different plant pathogenic fungi
1990; Williams et al. 1990). Essentially RAPD analysis relies [see Arora et al. (1996); Toda et al. (1999)].
on the reduction in specificity of the PCR process at reduced A relatively recent development in fingerprinting fungi has
temperatures. Total genome DNA extractions are used, and been the introduction of amplified fragment length poly-
these are amplified with single, short (usually decamer) morphism (AFLP) analysis [see Vos and Kuipper (1997); Vos
primers at a reduced annealing temperature. These conditions et al. (1995)]. In this technique, total DNA is digested with
result in less stringent binding of the primer to the target restriction enzymes, and then short artificial oligonucleotides
DNA, and allow the amplification of a number of generally (linkers) are ligated to the restriction enzyme sites. Specific
small regions of DNA. These are separated by electrophoresis primers are then designed that show a particular degree of
to give a profile of bands. The RAPD analysis has been specificity to the linker sequences, and large fractions of the
criticized as it is not always entirely reproducible, but it has total DNA can then be amplified as fragments. The AFLP
been used extensively for profiling populations of plant analysis generates many bands, and electrophoresis is usually
pathogens [e.g., Bentley et al. (1995); Cooke et al. (1996)]. In undertaken in large polyacrylamide gels, it is however,
many cases these studies have shown correlations between possible to undertake more restricted studies that generate
band patterns and host, disease type or geographical origin, fewer bands and that can be analyzed in smaller
and band patterns have also been used to differentiate electrophoresis systems [e.g., Mueller et al. (1996)]. At the
between forms of the same fungus causing different disease conclusion of RAPD and AFLP analyses PCR bands of
symptoms [e.g., Pei et al. (1997)]. Another common PCR interest can be extracted, purified, and sequenced to produce
fingerprinting method that has been used for plant pathogenic sequence characterized amplified regions (SCARs). The
fungi is amplification of sequences based on simple repetitive sequence information obtained from SCARs can then be used
primers. In this method, single short repetitive primers are to develop more specific PCR primers for detection methods
used at moderate annealing temperatures in order to amplify [e.g., Dobrowolski and O’Brien (1993); Leclerc-Potvin et al.
largely repetitive fragments of the genome. One target site for (1999)].
this method is the flanking region of genes that can contain
variable numbers of such repeats. Repeat motifs that have
been used for primers have varied from simple 2 –3 base 3.4 Other Total Genome Approaches
repeats such as (CA)8 and (CAG)5 (Freeman and Rodriguez
1995; Latge et al. 1998) to more complex sequences including Two other methods that have been used to characterize
the M13 bacteriophage universal sequencing primer [see populations of plant pathogenic fungi are the analysis of
Bridge et al. (1997)]. Another method of fingerprinting that overall repetitive DNA, and chromosome size and number.
has been used with some fungi is based on a group of short When total fungal DNA is digested with frequent cutting
repetitive DNA sequences that have been found dispersed restriction enzymes, a large number of fragments of many
throughout the genome of diverse bacterial species [see van different sizes are generated, and these appear in a gel as a
Belkum et al. (1998)]. Primers specific to these repetitive “smear.” Brighter staining bands can be seen in these smears
sequences produce multiple products in PCR with fungal where there are multiple copies of fragments of the same size.
Epidemiology of Plant Pathogens 61

These bands are, in part, the result of multiple copy DNA such 3.6 Inserted Elements
as the rRNA genes and mitochondrial DNA (mtDNA).
Differences in the patterns of these bands can be a quick, Both nuclear and mitochondrial genomes in fungi may
simple way for differentiating some closely related fungi, and contain a wide range of inserted elements [e.g., see Edelmann
this approach has been used for race designation in and Staben (1994); chapters 11 and 12]. At the very simplest
F. oxysporum sp. f. pisi [see Coddington et al. (1987)]. these may be very short sequences left after transposon
Unlike plants and animals, fungal chromosomes can be insertion and removal, and at the more complex they include a
very variable, and in many fungal species different isolates wide range of different transposons [see Daboussi (1997);
may show differences in the number and size of their Daboussi and Langin (1994)]. Inserted elements, particularly
chromosomes. This has been investigated in some isolates of introns, have been investigated as potential molecular
Colletotrichum and special forms of F. oxysporum. In markers at both the population level and the higher
Colletotrichum variation was found in the number and size of phylogenetic level (DePriest 1993; Neuvéglise et al. 1997).
the smaller (type B) elements, and in F. oxysporum sp. f.
cubense variation in chromosome sizes was found between
different races and vegetative compatibility groups [see 3.7 Application of Individual Markers
Masel et al. (1993); Ploetz (1990)].
The suitability and resolution of individual markers will
depend very much on the fungus being considered, and a
3.5 Mitochondrial Sequences marker that is useful for differentiating species in one group
of fungi, may be considerably more conserved or variable in
The mitochondrial (mt) genome has been used extensively in other fungal groups. One example of this is the use of
the investigation of population structures in the plant presumptive mtDNA RFLPs (AT rich DNA). The mtDNA
pathogenic fungi. In fungi the mitochondrial genome is a RFLPs have been found useful for determining species and
circular structure of between 17 and 121 Kb [see Zimmer et al. subspecific populations in some species of Aspergillus,
(1984); Curole and Kocher (1999); Grossman and Hudspeth whereas in the species Metarhizium anisopliae and Verti-
(1985); Lu (1996)]. The fungal mitochondrial genome makes cillium lecanii the same approach identifies numerous
up between 1–20% of the DNA occurring in fungal cells, and subspecific groupings (Kouvelis and Typas 1997; Typas
generally contains a high proportion of sequences that lack a et al. 1998; Varga et al. 1994). In the plant pathogen
coding function. In addition it may contain many repeat F. oxysporum sp. f. cubense mtDNA RFLPs have been used to
sequences and introns, and these features can allow for distinguish between different races and supported the theory
considerable variation in mitochondrial sequences between that the recently determined race 4 was not derived from the
closely related organisms [see Clark-Walker (1992); existing race 1 or 2 (Thomas et al. 1994). In the basidiomycete
Clark-Walker et al. (1987); see chapter numbers 11 and 12 in Ganoderma boninense mtDNA RFLPs have been found
this book]. As it is present in multiple copies, mtDNA can be a useful for defining individuals (Miller et al. 1999), and in
good target for molecular methods. In most cases mito- some Phytophthora species they have been used for
chondrial DNA is inherited unilinearly during meiosis but determining parental lines (Whittaker et al. 1994).
recombination may occur in some fungi (Wolf 1996). In Differences in the level of variability seen with the same
addition, mtDNA can be transferred independently of the marker from different taxa is not restricted to mtDNA, and
nuclear genome during unstable vegetative fusion (Collins and appears to be a feature of most markers investigated for
Saville 1990). The mtDNA can contain GC rich palindromic population and species level investigations. One example of
repeats, but overall, simple GC sequences are relatively rare in this is the degree of ITS sequence difference seen between
fungal mitochondria, and this has been used in differential isolates of a single species, or between closely related species.
DNA restriction protocols to generate presumptive mitochon- As an example there is generally up to around 5% ITS
drial RFLPs [e.g., Kouvelis and Typas (1997); Lacourt et al. sequence variation within individual species of Colleto-
(1994)]. The mitochondrial genome contains both variable and trichum, and a maximum of about 23% variation between
conserved regions and so sequence information may be used at species (Sreenivasaprasad et al. 1996). In Rhizoctonia solani,
a variety of taxonomic levels [see Zhou and Stanosz (2001)]. In up to 30% variation in the ITS sequences has been reported
some cases closely related species may have very different between isolates of the same anastomosis group (Kuninaga
mitochondrial genomes, and one example of this is in the yeast et al. 1997).
Saccharomyces, where mtDNA varies from 24 to 78 Kb
between different species [see Grossman and Hudspeth
(1985)]. Analysis of mtDNA variation has been used at a 3.8 Combining Markers
variety of different population and systematic levels, and these
have correlated with subspecies, vegetative incompatibility In general terms the use of different markers can give rise to a
groups, different populations and individuals (Gordon and hierarchic system, with particular techniques giving more, or
Okamoto 1992; Jacobson and Gordon 1990; Miller et al. 1999). less, resolution than others [see Bruns et al. (1991)], and so it
62 Bridge et al.

may be possible to select an appropriate marker for the variation in other characters. Whatever the reason, there can
situation under study. An example of the way in which a be significant differences in the degree of variation seen in the
broadly hierarchic arrangement of markers can be used for the molecular markers chosen.
study of fungal plant pathogens is detailed later with Fungi occur in asexually (imperfect, anamorphic) and
G. boninense. This approach will however not always sexually (perfect, teleomorphic) reproducing forms, and in
generate consistent results, and one example of this is the some cases both forms are present at the same time
group of fungal plant pathogens known as the “Ascochyta- (holomorphic). In the imperfect state cell division is solely
complex” that occurs on beans, peas, and other legumes. In by mitosis, whereas in the perfect state recombination and
this case there are a number of distinct species currently meiosis will occur. Recombination and meiosis can have a
assigned to either the genus Ascochyta or Phoma (see significant effect on results obtained from some molecular
Table 2). Most of these species can be defined individually markers (see Table 1). Isoenzyme markers could be expected
from their ITS sequences, and some can in turn be subdivided to be subject to allelic variation under such circumstances, as
on the basis of their mtDNA RFLPs. When a single part of the would many DNA fingerprinting markers. The degree to
mitochondrial genome is considered there is less variability, which a marker will be affected will vary considerably, and
and the species can be arranged in three groups. The one example is the comparison of sibling haploid lines
groupings obtained from RFLPs derived from the b-tubulin derived from a single dikaryotic fruit body. In these
gene are less consistent and group some species together, circumstances, the haploid progeny have arisen by meiosis
while also showing subspecific groups in others (Fatehi and may show different isoenzyme or DNA fingerprints from
2000). the parental material. This has been investigated in the oil
palm pathogen G. boninense where both RAPD (Pilotti et al.
2000) and simplified AFLP (Figure 2) fingerprints differed
3.9 Selection of Molecular Markers both between siblings and between siblings and parent. This
variation can then be further compounded through subsequent
Two of the most important factors that need to be considered mating and recombination.
in the selection of molecular markers in any study are the Some molecular markers can be expected to be consistent
taxonomic rank under consideration, and the life cycle of the despite meiosis and recombination. DNA sequences of major
fungus. As detailed above, a marker that is particularly useful structural and functional proteins will be resistant to
at a certain taxonomic rank for one species may not be useful recombination events, and the rRNA gene cluster is one
at the same rank for another species. One explanation for this region generally considered to be maintained under such
is that different systematists or plant pathologists have had conditions (Hillis and Dixon 1991). There are however some
different species concepts, and so the terms species and indications that this is not always the case, and there is at least
subspecies may not be directly comparable between different one report that in some fungi, not only can the rRNA region
fungal genera. In some genera, such as Fusarium, there has be affected by crossover, but also that this may occur at a
been a tradition of placing different pathogenic forms in the higher frequency than predicted (Selosse et al. 1996).
special form category, whereas in others, such as Colleto- Nonnuclear markers may be recombination insensitive,
trichum or Phoma, there has been a general history of and mtDNA has been used to demonstrate a single hereditary
describing new species. Such differences in species concepts line, where the mtDNA was inherited unilinearly (Whittaker
may reflect evolutionary ages, or may reflect levels of et al. 1994). It should be remembered however that this will

Table 2 Features of Ascochyta complex species on legumes

Species mt SSU rRNA size b-tubulin gene RFLPa ITS sequenceb mtDNA RFLPs
P. exigua 749 bp D 1 Multiple, distinct
749 bp A 1
A. rabiei 660 bp A 2 Multiple, distinct
A. fabae 660 bp C 3 Multiple, distinct
A. fabae f. sp. lentis 660 bp C 4 Single, distinct
A. pisi 660 bp C 5 Single, distinct
P. medicaginis var. pinodella 645 bp B 6 Multiple, distinct
A. pinodes 645 bp B 6 Multiple, distinct
P. subboltshauseri 645 bp E 7 Single, distinct
645 bp F 7
645 bp G 7
a
Letters A –G designate 7 different RFLP patterns obtained by digestion of a PCR amplified fragment of the b-tubulin gene.
b
Numbers 1–7 designate 7 different RFLP patterns obtained by digestion of the complete ITS1/5.8 s/ITS2 region.
Epidemiology of Plant Pathogens 63

It should be remembered that some of these coefficients have

been described independently on more than one occasion, and
others can be related to each other by simple transformations.
For example Nei and Li’s genetic distance is equivalent to
1-Sorensen’s coefficient, and Sorensen’s coefficient is
mathematically identical to Dice’s coefficient [see Bridge
and Saddler (1998); Sneath and Sokal (1973)]. Similarly,
association coefficients can be related to distance measures,
and taxonomic distance can be defined as the square root of 1
minus the simple matching coefficient. It is therefore
important if more than one measure is used to ensure that
those selected are independent.
Although cluster analysis methods are a common way of
showing relationships within and between fungal populations,
this methodology does however have some limitations. One
obvious limitation with any tree diagram is that all the isolates
Figure 2 Simple sequence repetitively primed molecular must be linked, as there is no provision for an isolate that is
fingerprints for 4 lines of Ganoderma derived from a single not related. A second limitation is that cluster analysis is a
bracket. Lane 1, molcular size markers; lanes 2 and 3 good technique for showing the membership of a group, it is
monokaryotic culture derived from basidiospore a; lanes 4 less precise in showing relationships between groups. This
and 5, monokaryotic culture derived from basidiospore b; lanes is a particular failing of average linkage based systems, but
6 and 7, monokaryotic culture derived from basidiospore c; is also true of most other clustering approaches [see Abbott
lanes 8 and 9, dikaryotic culture obtained from b and c. et al. (1985)]. A further limitation to cluster analysis is the
tendency of isolates that are unrelated to the main
population to form a separate cluster together, even though
not always be the case, as not all fungi have unilinear they may be only distantly related to each other. Such
mitochondrial inheritance, and in some cases mitochondrial clusters are sometimes described as sharing only the single
recombination will occur during biparental inheritance (Borst property that they are not related to the main population.
and Grivell 1978). The range and type of variation associated One way in which some of these limitations can be
with molecular markers can provide many different tools that overcome is by using an ordination-based method such as
can be used for determining the epidemiology of plant principle component analysis (PCA). In these methods
pathogenic fungi. At one level, recombination insensitive correlated variance between characters is combined to
markers may be available for the detection of a particular produce a further set of axes that are essentially made up of
taxon in the environment, such as species and pathogen additive components of correlated individual characters.
specific probes and primers. At a lower level, recombination Each axis represents a proportion of the total variance in the
sensitive markers may be used to follow individuals or lines, data, and the placement of isolates is by plotting their
or to determine if a disease is spread by spores or through positions in relation to the first 2 or 3 axes [see Alderson
vegetative growth. 1985; Dudzinski 1975). A refinement of PCA is principle
coordinate analysis (PCO). PCO has been shown to be
appropriate for binary data, such as obtained from band
4 ANALYSING DATA patterns, and unlike PCA, does not require the use of
strictly metric coefficients (Gower 1966; Sneath and Sokal
Different molecular markers will generate results in different 1973). Unlike a cluster analysis, this does not produce a
forms. Simple RFLPs and some fingerprinting methods will series of groups, but a scatter-plot where similar isolates
produce generally simple band patterns, usually of the order may be placed near each other. These ordination methods
of between one and 20 bands. These patterns can be translated can behave differently from cluster analysis, and typically
into a simple binary form where each band obtained in the are better at representing between group relationships than
analysis is considered as an independent character, and is close within group relationships. One further aspect of PCA
scored as present or absent. In most cases, these binary is that under some circumstances it may filter random
records have then been compared by the use of one or more variation from a complex data set, as any correlated
distance or association calculation, and represented as some variation will tend to be included in the first few axes [see
form of dendrogram. There is a wide range of coefficients Bridge (1998)].
available for such comparisons. These include coefficients Band analysis methods are essentially the same for simple
that do not consider matching negative characters, and others and complex patterns. It however becomes necessary to
that provide for a double weighting of common bands to consider band reading software for the very complex patterns
reflect the presence of common restriction sites or primer that may be produced by techniques such as AFLP, as the
sequences at each end of the bands [e.g., Nei and Li (1979)]. large number of bands produced cannot be easily scored by
64 Bridge et al.

eye. There are a range of band reading and matching software One series of studies that has shown the range and limitations
packages available, and most of these convert band patterns in of molecular markers in following plant disease epidemiology
a gel into densitometric traces where peak presence, height, is the investigation of basal stem rot (BSR) of oil palm by
and shape correspond to band presence, intensity, and G. boninense. BSR was first recognized in West Africa in
thickness, respectively. These packages commonly have 1915, and as oil palm was distributed through out the world, it
manual and automated routines for correcting gels for shift was recorded in many other countries. The first report in SE
and stretch events, and routines for including standard size Asia was in 1931, and since that time BSR incidence has
markers and other reference bands. Trace data can be readily increased to the point where the lack of techniques for
converted to quantitative values as “x,y co-ordinates” and this management of the disease is considered a major constraint to
is suitable for largely distance based analyses. Quantitative oil palm production in SE Asia [see Ariffin et al. (2000)].
data can also be compared for overall pattern similarity Ganoderma species attack a variety of tropical perennial
through methods such as correlation coefficients, and crops including rubber, tea, and pineapple. In these instances
concentration independent calculations such as cosine theta the Ganoderma appears to be largely transmitted through the
[see Feltham and Sneath (1979)]. soil, possibly in plant debris, and spreading infection patches
Analysis of sequence data is more complicated as the may be evident in fields. The species G. boninense occurs as a
likelihood of certain events may also be included in the saprophyte on dead palms, particularly coconuts, but appears
analysis. The first stage in comparing DNA sequences is to to be pathogenic only to oil palm. For some years
align them to each other. Alignment routines will always seek transmission of G. boninense in oil palm was believed to be
the best alignment of the sequences being studied, and so the through the soil, as for other species, and disease control was
addition or deletion of sequences to a data set will require a attempted through practices that included digging large pits
new alignment to be made. In determining an alignment, and around infected palms (Turner 1981). The first attempt to use
calculating a measure of difference between the sequences molecular methods to investigate the epidemiology of BSR in
involved it is also necessary to consider the effects of oil palm was made in the 1990s, when initial studies were
transversions, transitions, and gaps. The bases in DNA strands made with iso-enzyme profiles (Miller et al. 1995). Although
pair through purine/pyrimidine bonds, and so when aligning some enzyme systems initially appeared useful for differ-
sequences, a change from purine to purine or pyrimidine to entiating species, in G. boninense it was found that in general
pyrimidine (transition) may be considered of less importance iso-enzyme profiles were either consistent, or showed
than a change from purine to pyrimidine (transversion). The considerable variation. Ganoderma is a basidiomycete that
importance given to transitions and transversions can forms polyporpoid brackets on the outer surface of infected
therefore be varied to reflect their relative importance, and palms. The brackets are dikaryotic, and basidiospores are
this may also depend on the particular sequences being produced by meiosis. The mycelial form found in infected
considered. When aligning sequences it may be necessary to tissue is also generally dikaryotic, and some of the iso-
insert a gap in some sequences to align where insertion/ enzyme variability may therefore be due to recombination
deletion events have occurred. Again, the relative importance events from the original fusion of monokaryotic basidio-
of inserting a gap, and also of extending that gap can be spores. However, pectinase isoenzyme analysis identified a
adjusted according to the perceived significance of the event characteristic enzyme profile that was consistent for nearly all
in the sequences under consideration [see Thompson et al. Ganoderma isolates obtained from palm hosts (Miller et al.
(1994)]. It is common with sequence analysis to use 1995; 2000). Given the known involvement of pectin and
phylogenetic techniques to produce trees [see Swofford and pectin degradation in plant pathology, this finding may
Olsen (1990)]. While these approaches are suitable when indicate a common mode of action for all of the palm
considering different species and genera, they are less associated Ganoderma species.
appropriate for comparisons of closely related populations. The first DNA based method to be investigated in these
Analysis of DNA sequence data is an area that is currently studies was analysis of RFLPs derived from presumptive
receiving further attention, and some of these developments mtDNA (AT rich DNA). This relatively simple technique
are described in more detail in chapter 33. gave rather unusual results, in that different RFLPs were
obtained from different cultures, suggesting considerable
heterogeneity in the mitochondrial genome (Miller et al.
1999). The RFLP profiles proved to be consistent among
5 FOLLOWING PLANT DISEASE: A CASE single spore isolates from a single basidiome, and so were
STUDY considered to provide “parental line” fingerprints, character-
istic of the dikaryon. These RFLPs could therefore be used to
Molecular markers have been used in a wide range of studies define sibling families. This assumption was supported by
with plant pathogenic fungi (see earlier). Although these can monokaryon and dikaryon intercompatibility studies (Pilotti
be reviewed, the volume of the literature available is et al. 2000).
considerable, and so a single case study is presented here that Subsequent investigation of molecular fingerprinting
illustrates how the molecular epidemiology of a plant methods including RAPDs and AFLP supported the bulk of
pathogen can be related directly to agricultural practice. the iso-enzyme studies and gave different profiles for isolates
Epidemiology of Plant Pathogens 65

derived from single spores from the same basidiome (Bridge wide range of molecular features that can be used as markers
et al. 2000; Pilotti et al. 2000). When these molecular methods at the population level for studying plant pathogenic fungi.
were applied to isolates obtained from single plantations and The different markers will often reflect different levels of
planting blocks it was found that nearly all of the isolates variation within and between plant pathogenic taxa, and may
differed from each other, including isolates obtained from also reflect changes resulting from meiosis and recombina-
adjacent palms. This finding was again supported by tion. It is therefore possible to study the spread and dynamics
intercompatibility studies (Miller et al. 1999; 2000; Pilotti of fungal populations on crop plants, and to determine the role
et al. 2000). These results could not have come about as a of populations of the same fungus occurring on secondary
result of simple mycelial spread in the soil, as vegetative hosts, in the soil or on debris. Molecular markers can also be
spread could be expected to result in at least some palms used to determine the genetic integrity of host or variety
being infected by the same isolate. It was therefore concluded specific groups, and can provide information on differences
that infection could be due to one of two mechanisms, either between pathogenic races. This information is fundamental to
singly or in combination. The first was that there might have understanding the spread of fungal plant diseases, and is
been mycelial spread from multiple inoculum sources, with important in developing disease control strategies. The choice
virtually no cross infection. This would account for the of marker will depend on the particular fungus under study,
molecular variability recorded, but would also require each and the correct choice of markers may also provide informa-
infection to be the result of different infected debris. The tion as to the role of spores or particular mating types in
second possibility was that infections were due to new epidemiology. Variation in fungal pathogen genotype is the
dikaryons formed from fusion of monokaryons from basis for developing methods to identify these pathogens
individual spores [see Sanderson and Pilotti (1997); using PCR. Recently, strains/species specific molecular
Sanderson et al. (2000)]. markers/primers have been developed for several plant
Although the molecular markers studied showed sufficient pathogenic fungi. There is unfortunately no single marker
variability to identify individual isolates for local epidemiol- system that can be guaranteed to provide the desired level of
ogy, they did not show sufficient conservation to allow the discrimination for all fungi, and some initial screening of
wider detection of the pathogen in the environment. BSR different methodologies may be required before a full study
was considered to be due to the single species G. boninense, can be initiated.
and although there is considerable uncertainty regarding
species concepts in Ganoderma, some information is
available on sequences within the rRNA gene cluster. Initial
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6
Molecular Biology for Control of Mycotoxigenic Fungi

Robert L. Brown / Deepak Bhatnagar / Thomas E. Cleveland U.S. Department of

Agriculture – Agricultural Research Service, New Orleans, Lousiana, USA

Zhi-Yuan Chen Louisiana State University, Baton Rouge, Lousiana, USA

1 INTRODUCTION for Industry: Fumonisin Levels in Human Foods and Animal

Feeds” in the November 9, 2001, Federal Registry. More than
Mycotoxins are fungal metabolites that can contaminate 50 countries have established or proposed regulations for
foods and feeds, and exhibit toxic effects in higher organisms controlling aflatoxins in foods and feeds, and at least 15
(Sharma and Salunkhe 1991) that consume the contaminated countries have regulations for levels of other mycotoxins
commodities. The regulatory guidelines and advisory limits (Haumann 1995). The FDA has set limits of 20 ppb, total
issued by the United States Food and Drug Adminstration aflatoxins, for interstate commerce of food and feed and
(FDA) on some contaminated commodities can facilitate 0.05 ppb of aflatoxin M1 for sale of milk. Because of both
severe economic losses to the growers. Therefore, mycotoxin food and feed safety concerns and the establishment of
contamination of foods and feeds is a serious food safety regulatory limits on DON and aflatoxins, it is estimated that
problem affecting the competitiveness of U.S. agriculture, over $1.5 billion in crop losses occur annually due to
both domestically and worldwide. Mycotoxins that signifi- contamination of corn, cottonseed, peanut, and treenuts with
cantly impact agriculture include aflatoxins produced by aflatoxins and of wheat and barley with DON (Robens 2001).
Aspergillus flavus and A. parasiticus, trichothecenes (in An association between mycotoxin contamination and
particular deoxynivalenol or DON) produced by Fusarium inadequate post harvest storage conditions has long been
spp., ochratoxins produced by A. ochraceus and Penicillium recognized. However, studies have revealed that seeds are
viridicatum, and fumonisins produced by F. verticillioides contaminated with mycotoxins primarily at the preharvest
(synonym, moniliforme, as used in some literature cited in the stage [reviewed in Lisker and Lillehoj (1991)]. Therefore,
present article) (Brown et al. 1998). Cyclopiazonic acid many current research strategies focus on preharvest control
produced by A. flavus, can also be included on this list of of mycotoxins [reviewed in Brown et al. (1998)]. Maintaining
significant mycotoxins. Aflatoxins, potent liver toxins, and good cultural and management practices that promote the
carcinogens comprise the most widely studied mycotoxins general health of crops can reduce but not eliminate
(CAST 1979; Diener et al. 1987; Payne 1998), because of preharvest mycotoxin contamination. For example, insect
established results in their ability to induce animal diseases, resistant germplasm, such as corn transformed with the gene
particularly liver cancer in humans [reviewed in Eaton and encoding Bacillus thuringiensis crystal protein (Bt maize),
Groopman (1994)]. However, other mycotoxins such as reduced levels of fumonisins (Dowd 2000). Irrigation of
DON, are of particular concern for the brewing industry peanut essentially prevents aflatoxin contamination of this
which has cutoff levels as low as 0.5 ppm for DON in barley crop, probably by preventing drought stress, known to induce
used in malting (Robens 2001). In addition, recognizing the contamination in peanut (Cole et al. 1985). However,
potential for fumonisins to cause animal or human health optimization of management practices to control mycotoxins
problems (Marasas 1996), the FDA has now announced the is not always possible due to production costs, geographic
availability of a final guidance document entitled “Guidance location, or the nature of the production system for the

69
70 Brown et al.

particular crop vulnerable to mycotoxins. In addition, even enhance resistance against other groups of mycotoxigenic
the best management practices are sometimes negated by fungi.
biotic and abiotic factors that are hard to control and by
extremes in environmental conditions. The complex epide-
miology of A. flavus on corn (Wicklow 1991) can drastically
affect the outcome of measures to control aflatoxin 2.1 Development of Aflatoxin-Resistance
contamination on this crop. Therefore, there is an urgent Screening Tools
need for development and utilization of strategies involving
state-of-the-art technologies to control preharvest mycotoxin Several screening tools have been developed and used to
contamination. The current article highlights recently facilitate corn breeding for developing germplasm resistant to
published and high-impact research involving molecular- fungal growth and/or aflatoxin contamination (King and Scott
based technologies that has been accomplished and that 1982). Inoculation methods employed with corn include the
enhances a host plant resistance strategy for controlling pinbar inoculation technique (for inoculating kernels through
mycotoxin contamination. husks with A. flavus conidia), the silk inoculation technique,
and infesting corn ears with insect larvae infected with
A. flavus conidia. (King and Scott 1982; Tucker et al. 1986).
Two resistant inbreds (Mp420 and Mp313E; Scott and
2 MYCOTOXIN PREVENTION THROUGH Zummo 1988; Windham and Williams 1998) were discovered
ENHANCEMENT OF HOST RESISTANCE and tested in field trials at different locations, using the pin-
IN CROPS bar technique, and released as sources of resistant germplasm.
A rapid laboratory kernel screening assay (KSA) was
developed and used to study resistance to aflatoxin production
Preharvest host resistance is a widely explored strategy for
in mature kernels (Brown et al. 1993; 1995). The results of
combatting fungal attack. By far, most studies aimed at the
this study indicated the presence of two levels of resistance: at
incorporation of antifungal resistance against mycotoxigenic
the pericarp and at the subpericarp level. The subpericarp
fungi have been applied toward improvement of resistance
level of resistance was shown to require a viable embryo
against preharvest aflatoxin contamination in corn [reviewed
(Brown et al. 1993). KSA studies further demonstrated a role
in Brown et al. (1998)]. With corn, the strategy of enhancing
for pericarp waxes in kernel resistance (Guo et al. 1995; 1996)
host resistance to aflatoxin contamination through breeding and highlighted quantitative and qualitative differences in
has gained prominence because of: (a) the successful pericarp wax between resistant and susceptible genotypes
identification of germplasm resistant to aflatoxin contami- (Gembeh et al. 2001; Russin et al. 1997). This research was all
nation [reviewed in Brown et al. (1999)] and (b) the based on the prior identification, during field studies, of a
significant advances in the identification of natural resistance resistant corn breeding population, GT-MAS:gk (Widstrom
mechanisms and traits (Brown et al. 1998; 1999; Chen et al. et al. 1987).
2001). However, these investigations indicated that resistance The KSA also confirmed sources of resistance among 31
to aflatoxin contamination involves multiple chromosome inbreds tested in Illinois field trials (Brown et al. 1995;
regions and several genes (Davis and Williams 1999). Campbell and White 1995), thus demonstrating that the KSA
Therefore, attempts to select for resistance traits in the can be used, at least initially, to rank corn for its field
development of commercial corn varieties, while maintaining resistance to aflatoxin contamination. Subsequently, the KSA
desirable agronomic characteristics, have been slowed due to was used as a preliminary screen for resistance to aflatoxin
a failure to identify expressed genes and proteins involved in contamination in kernels of maize inbreds selected for ear rot
resistance. This is especially needed since resistance, thus far resistance in West and Central Africa (Brown et al. 2001a).
identified is in poor genetic backgrounds. Therefore, research The KSA has advantages over traditional field screening
is needed to elucidate the biochemical mechanisms that techniques (Brown et al. 1995), mainly because of the rapidity
confer resistance in corn kernels and other crops that are of the assay. However, field trials are irreplaceable for
vulnerable to aflatoxin contamination. These resistance confirmation of resistance.
mechanisms could then be used to enhance germplasm Recently, the KSA was improved by including a method to
through marker-assisted breeding and/or genetic engineering, quantify fungal biomass using the b-glucuronidase (GUS) or
two methods for employing the identified traits towards the green fluorescent protein (GFP) (Du et al. 1999; Windham
development of resistant commercially-acceptable crops and Williams 1998; Windham et al. 1999) reporter gene-
(Brown et al. 1999). Gaining an understanding of the natural containing A. flavus tester strains. A. flavus tester strains were
resistance mechanisms in corn could serve as “nature’s genetically engineered with a gene construct consisting of the
lesson” about the specific requirements for seed-based GUS reporter gene linked to an A. flavus b-tubulin gene
resistance against fungal attack. This information will help promoter for monitoring fungal growth (Brown et al. 1995;
efforts to incorporate and enhance resistance in other crops 1997) or with the reporter gene linked to an aflatoxin
vulnerable to aflatoxin contamination such as cottonseed, biosynthetic pathway gene which could also provide a quick
peanut, and tree nuts, and will perhaps even help efforts to and economical way to indirectly measure aflatoxin levels
Molecular Biology for Control of Mycotoxigenic Fungi 71

(Brown-Jenco et al. 1998; Payne 1997). Thus, it is now

possible to accurately assess fungal infection levels and to
predict the corresponding aflatoxin levels in the same kernels,
as a result of fungal infection. This technology might be
applied to screening for resistance to mycotoxin contami-
nation by other mycotoxigenic fungi. Recently, an
F. verticillioides strain, containing a GUS reporter gene was
used in the KSA to demonstrate that this fungus is inhibited in
aflatoxin-resistant genotypes (Brown et al. 2001b). This
indicates that some resistance mechanisms may be generic for
ear rotting/mycotoxigenic fungi.

2.2 Identification of Resistance-Associated

Proteins (RAPs) and Natural Compounds in
Corn That Inhibit Aspergillus flavus Growth
and Aflatoxin Contamination

Developing resistance to fungal infection in wounded as well

as intact kernels would go a long way toward solving the
aflatoxin problem (Payne 1992). Studies demonstrating
subpericarp (wounded-kernel) resistance in corn kernels
have led to research for identification of subpericarp
resistance mechanisms. Examinations of kernel proteins of
several genotypes revealed differences between genotypes
resistant and susceptible to aflatoxin contamination (Guo et al.
1998). Imbibed susceptible kernels, for example, showed
decreased aflatoxin levels and contained germination-induced Figure 1 Strategy for enhancing host plant resistance to
ribosome inactivating protein (RIP) and zeamatin (Guo et al. aflatoxin contamination. The research approach being used to
1997). Both zeamatin and RIP have been shown to inhibit identify and employ resistance factors, such as resistance-
A. flavus growth in vitro (Guo et al. 1997). In another study, associated proteins (RAPs) that are identified in aflatoxin-
two kernel proteins were identified from a resistant corn resistant corn lines. After resistant germplasm is identified,
inbred (Tex6) which may contribute to resistance to aflatoxin various tools are used to characterize the expression of resistance,
contamination (Huang et al. 1997). One protein, 28 kDa in such as KSA-based studies, GUS/GFP reporter constructs, and
size, inhibited A. flavus growth, while a second, over 100 kDa seed physiology studies. These can lead to RAP identification
in size, primarily inhibited toxin formation. When a protocols such as proteome analysis. Genes corresponding to
commercial corn hybrid was inoculated with aflatoxin and RAPs can be cloned, and clones then used for QTL studies, plant
nonaflatoxin-producing strains of A. flavus at milk stage, one transformation, or marker-assisted breeding.
induced chitinase and one b-1,3-glucanase isoform was
detected in maturing infected kernels, while another
isoform was detected in maturing uninfected kernels (Ji
et al. 2000).
In another investigation, an examination of kernel protein antifungal genes through genetic engineering into other
profiles of 13 corn genotypes revealed that a 14 kDa trypsin aflatoxin-susceptible crops (Figure 1).
inhibitor protein (TI) is present at relatively high concen- A recent investigation into corn kernel resistance (Chen
trations in seven resistant corn lines, but at low concentrations et al. 2001) determined that both constitutive and induced
or is absent in six susceptible lines (Chen et al. 1998). The proteins are required for resistance to aflatoxin production. It
mode of action of TI against fungal growth may be partially also showed that one major difference between resistant and
due to its inhibition of fungal-amylase, limiting A. flavus susceptible genotypes is that resistant lines constitutively
access to simple sugars (Chen et al. 1999b) required not only express higher levels of antifungal proteins compared to
for fungal growth, but also for toxin production (Woloshuk susceptible lines. The real function of these high levels of
et al. 1997). The TI also demonstrated antifungal activity constitutive antifungal proteins may be to delay fungal
against other mycotoxigenic species (Chen et al. 1999a). The invasion, and consequent aflatoxin formation, until other
identification of these proteins may provide markers for plant antifungal proteins can be synthesized to form an active
breeders, and may facilitate the cloning and introduction of defense system.
72 Brown et al.

2.2.1 Identification of RAPs Through Proteome Steryl esters from maize significantly increased aflatoxin
Analysis production by some A. flavus strains at 0.3 and 1.0 mg/ml
(Norton and Dowd 1996). Anthocyanins and related
To increase protein resolution and detection sensitivity by flavonoids, some of which occur naturally in maize kernels,
10–20 fold and, thus, enhance ability to identify more RAPs, inhibited aflatoxin production by more than 50% at 0.76 mM
a proteomics approach was recently employed. The increased (Norton 1999). More highly glycosylated forms of the
reproducibility, reliability, and accuracy of 2-D gel anthocyanins tended to be less effective in inhibiting aflatoxin
electrophoresis is due to advances in technology, such as production (Norton 1999). Carotenoids containing an alpha-
immobilized pH gradient (IPG) gel strips and sophisticated ionone type ring tended to be more effective inhibitors of
computerized 2-D gel analysis software (Appel et al. 1997; aflatoxin production by A. flavus, with some having an I50 of
Görg et al. 1998). Endosperm and embryo proteins from about 6 mM (Norton 1997). Although most strains of A. flavus
several resistant and susceptible genotypes have been exposed to beta-carotene at 50 mg/ml had aflatoxin production
compared using large format 2-D gel electrophoresis, and inhibited by 90% or more, some peanut derived strains were
over a dozen such protein spots, either unique or 5-fold less sensitive (Wicklow et al. 1998). In vitro studies indicated
upregulated in resistant lines, have been identified, isolated plant peroxidase could greatly enhance the ability of plant
from preparative 2-D gels and analyzed using ESI-MS/MS chemicals to inhibit spore germination and hyphal growth of
after in-gel digestion with trypsin (Chen et al. 1999a; 2000; F. graminearum (Dowd et al. 1997). A. flavus was
2002). These proteins can be grouped into three categories considerably more resistant to quinone products potentially
based on their peptide sequence homology: (a) storage produced by plant peroxidases compared to F. graminearum
proteins, such as GLB1, GLB2, and late embryogenesis and F. verticillioides (moniliforme) (Dowd et al. 1997). In
abundant proteins (LEA3, LEA14); (b) stress-responsive addition, volatile compounds from corn and cotton, which are
proteins, such as aldose reductase (ALD), a glyoxalase I products of the lipoxygenase pathway, were shown to have
protein (GLX1), and a 16.9 kDa heat shock protein, and (c) effects upon aflatoxin biosynthesis and fungal development in
antifungal proteins, which include TI. vitro [reviewed in Bhatnagar et al. (2001)].
Thus far, no investigation has been conducted to determine
the possible direct involvement of stress-related proteins in
host fungal resistance. Heretofore, most RAPs identified have
had antifungal activities. However, increased temperatures 2.3 Plant Breeding Strategies for Enhancing Host
and drought, which often occur together, are major factors Resistance to Mycotoxigenic Fungi
associated with aflatoxin contamination of maize kernels
(Payne 1998). Other studies have found that drought stress Several resistant inbreds among the 31 tested in Illinois field
imposed during grain filling reduces dry matter accumulation trials (Campbell and White 1995) and highlighted through the
in kernels. This often leads to cracks in the seed and provides KSA (Brown et al. 1995), have been incorporated into an
an easy entry site to fungi and insects. Possession of unique or aflatoxin-resistance breeding program whose major objective
of higher levels of hydrophilic storage or stress-related is to improve elite Midwestern corn lines such as B73 and
proteins, such as the aforementioned, may put resistant lines Mo17. In this program, the inheritance of resistance of inbreds
in an advantageous position over susceptible genotypes in the in crosses with B73 and/or Mo17 was determined (Hamblin
ability to synthesize proteins and defend against pathogens and White 2000; Walker and White 2001; White et al. 1995b;
under stress conditions. Therefore, the necessary require- 1998), and in the case of several highly resistant inbreds,
ments for developing commercially-useful, aflatoxin-resistant genetic dominance was indicated. Overall, results indicated
maize lines may include, aside from antifungal proteins, a that selection for resistance to Aspergillus ear rot and
high level of expression of stress-related proteins. Further aflatoxin production should be effective, and that develop-
studies including physiological and biochemical characteriz- ment of resistant inbreds for use in breeding commercial
ation, genetic mapping, plant transformation using RAP hybrids should be successful (White et al. 1995a).
genes, and marker-assisted breeding should clarify the roles Chromosome regions associated with resistance to
of stress-related RAPs in kernel resistance. A. flavus and inhibition of aflatoxin production in corn have
been identified through Restriction Fragment Length
2.2.2 Natural Compounds That Affect Mycotoxin Polymorphism (RFLP) analysis in three “resistant” lines
Biosynthesis (R001, LB31, and Tex6) in the Illinois breeding program,
after mapping populations were developed using B73 and/or
Several compounds have been identified in corn which may Mo17 elite inbreds as the “susceptible” parents (White et al.
have regulatory effects on the aflatoxin and trichothecene 1995b; 1998). In some cases, chromosomal regions were
biosynthetic process. The compound 4-acetyl-benzoxazolin- associated with resistance to Aspergillus ear rot and not
2-one (ABOA), which was isolated from maize lines tolerant aflatoxin inhibition, and vice versa, whereas other chromo-
to F. graminearum, strongly inhibited acetyl-deoxynivalinol somal regions were found to be associated with both traits.
production at 5 mM, aflatoxin production at 2 mg/ml, and This suggests that these two traits may be at least partially
feeding by maize weevils at 1000 ppm (Miller et al. 1996). under separate genetic control. Also, it was observed that
Molecular Biology for Control of Mycotoxigenic Fungi 73

variation can exist in the chromosomal regions associated known about the nature of selectable resistance markers
with Aspergillus ear rot and aflatoxin inhibition in different associated with reduced aflatoxin contamination in crops
mapping populations, suggesting the presence of different other than corn, breeding for insect resistance, or better
genes for resistance in the different identified resistance management of insects which vector aflatoxigenic fungi may
germplasm. The RFLP technology may provide the basis for be a more viable immediate approach to manage aflatoxin
employing the strategy of pyramiding different types of contamination.
resistances into commercially viable germplasm, while Recent studies indicate that naturally occurring resistance
avoiding the introduction of undesirable traits. Another may reduce invasion of crops by other economically
Quantitative Trait Loci (QTL) mapping program was important mycotoxigenic fungi. For example, resistance to
undertaken using a mapping population created from a head blight in wheat varieties was correlated with a reduction
resistant inbred Mp313E and a susceptible one, Va35 (Davis in contamination with DON (Bai et al. 2001). Further
and Williams 1999), and regions on chromosomes, associated investigations utilizing differentially resistant wheat germ-
with resistance to aflatoxin contamination, were revealed. plasm may lead to the identification of selectable resistance
Other work using this technology is attempting to pyramid markers useful in breeding for reduced DON contamination in
insect and fungal resistance genes into commercial wheat.
germplasm (Guo et al. 2000; Widstrom et al. 2003).
Breeding strategies to enhance resistance to A. flavus
infection are being carried out in other crops vulnerable to
aflatoxin contamination such as peanut and tree nuts. 2.4 Genetic Engineering Strategies to Enhance
Promising sources of resistant peanut germplasm have been Host Resistance to Mycotoxin Contamination
identified from a core collection representing the entire peanut
germplasm collection (Holbrook et al. 1995), although Plant breeding for resistance is practical when a large
resistance screening has proven to be a difficult task with germplasm pool exists with differential resistance in the crop,
this crop (Holbrook et al. 1997). Promising peanut germplasm such as exists in corn. However, genetic engineering for
has less than acceptable agronomic characteristics, and is thus resistance may be essential for crops such as cotton which
being hybridized with lines with commercially acceptable seems to have little resistance to aflatoxin contamination of its
features. Resistant lines also are being crossed to pool seed (Cotty 1989). Extensive research has focused upon
resistances to aflatoxin production. Thus, some success has identifying genes encoding antifungal proteins effective
been achieved in identifying resistant peanut germplasm, and against mycotoxigenic fungi. Bacterial chloroperoxidase
field studies are being conducted by various researchers to (CPO) (Wolffram et al. 1988) and its gene have been
verify this trait. evaluated both in vitro in laboratory assays (Jacks et al. 1999)
Among tree nuts, strategies for controlling preharvest and in vivo in enhancing fungal disease resistance in
aflatoxin formation by breeding for host resistance have been transgenic tobacco plants (Rajasekaran et al. 2000b). In
studied mainly in almonds (Gradziel et al. 1995). The in vitro bioassays using A. flavus as the test organism,
approach has been to integrate multiple genetic mechanisms CPO greatly reduced the viability of A. flavus conidia
for control of not only Aspergillus spp. but also insects. (Jacks et al. 1999) and transgenic tobacco expressing the
Resistance to fungal colonization has been shown to be CPO gene demonstrated significant resistance to attack by
present in the undamaged seed coat of several advanced Colletotrichum destructivum (Rajasekaran et al. 2000b).
breeding selections and is further being pursued through In another study, a small lytic peptide, D4E1, demon-
breeding/genetic engineering of resistance to A. flavus growth strated broad spectrum antimicrobial activity and convincing
in kernel tissues. Genotypes are also under development that inhibitory activity against A. flavus in vitro (Rajasekaran et al.
produce low amounts of aflatoxin following fungal infections 2001), thus indicating the possibility of transforming plants
(Gradziel and Dandekar 1999). with the gene encoding D4E1 to reduce infection of seed with
Naphthoquinones in walnut hulls delayed germination of toxigenic fungi. In further substantiation of this strategy, the
A. flavus conidia and were capable of inhibiting growth of D4E1 gene when transformed into tobacco was shown to
the fungus at higher concentrations (Mahoney et al. 2000; greatly enhance resistance to C. destructivum (Cary et al.
Molyneux et al. 2000). These compounds also appeared to 2000). Cotton is being transformed with CPO and D4E genes
have a regulatory effect on aflatoxin biosynthesis and may be with the hope that aflatoxin contamination of cottonseed can
involved in resistance to aflatoxin contamination of walnut. be reduced (Chlan et al. 1999; Rajasekaran et al. 1999;
Results seen here could lead to breeding applications to 2000a).
enhance resistance in walnut to aflatoxin contamination using Mechanisms of mycotoxin biosynthesis and regulation
naphthoquinone derivatives as selectable markers. have been investigated extensively (Bhatnagar et al. 2002;
Investigations have also been conducted with figs and Cleveland and Bhatnagar 1992; Desjardins and Proctor 2001).
pistachios to identify the mode of infection of the crops by The goal is to identify weak links that can be exploited to
A. flavus and develop strategies to identify germplasm with control mycotoxin contamination through genetic engineer-
agronomically desirable characteristics and resistance to ing of plants. The finding that trichothecenes contribute to the
fungal infection (Doster et al. 1995). However, until more is virulence of F. graminearum on wheat and maize has
74 Brown et al.

identified such a weak link. If production of trichothecenes and are being expressed in transgenic maize to evaluate their
increases pathogen virulence, then increased plant resistance effect on fumonisin accumulation and ear rot symptoms
to the toxin should increase plant resistance to the pathogen. (Duvick 2001).
Three genes that increase plant resistance to trichothecenes The gene encoding the antifungal protein, TI, previously
have recently been identified, and whether such genes also shown to be correlated with corn kernel resistance, was
can increase plant resistance to F. graminearum is under transformed into and expressed in both tobacco and cotton.
investigation. Two trichothecene resistance genes are fungal Fungal growth inhibition assays of transgenic tobacco
genes that encode proteins that reduce the toxicity of expressing TI protein showed efficacy against A. flavus, but
trichothecenes. TRI101 from F. sporotrichioides encodes not at the levels observed with extracts from tobacco
trichothecene 3-O-acetyltransferase which converts tricho- transformed with genes encoding CPO or D4E1 (Cary et al.
thecenes to less toxic derivatives (Kimura et al. 1998; 2000; Rajasekaran et al. 2000b). The gene encoding TI also
McCormick et al. 1999). PDR5 from yeast encodes a has been transformed into cotton, but no inhibitory activity
multidrug resistance transporter protein that transports has yet been noted in extracts from transgenic plants.
trichothecenes extracellularly and is similar to TRI12, a It is well documented that insect injury can provide a port
trichothecene biosynthetic gene (Alexander et al. 1999; Balzi of entry by mycotoxigenic fungi and that crops containing the
et al. 1994). Transgenic expression of either TRI101 or PDR5 B. thuringiensis (Bt) gene encoding an insecticidal protein,
increased resistance of tobacco to trichothecenes (Muhitch have shown reduced levels of mycotoxin contamination
et al. 2000). Wheat and barley lines expressing TRI101 and (Dowd 2000). Currently, a binary vector is being used in this
PDR5 are being tested for resistance to F. graminearum laboratory to express both the antifungal D4E1 gene and a
(Okubara et al. 2000). synthetic anti-insecticidal gene, cryIA(c), of B. thuringiensis
Trichothecenes are potent inhibitors of protein synthesis in tobacco and cotton. Successful expression of these genes
and are believed to bind to the 60S ribosomal protein L3 under independent promoters should provide both fungal and
(RPL3). A rice gene encoding RPL3 was modified to change insect resistance in cotton, thus potentially reducing the
amino acid 258 from tryptophan to cysteine, a change that amount of fungal entry through insect injury sites as well as
confers trichothecene resistance to yeast. Transgenic retarding the growth of the aflatoxin producing fungus in
expression of the modified Rpl3, increased resistance of cotton bolls and seed.
tobacco to trichothecenes (Harris and Gleddie 2001). Maize,
wheat, and barley lines expressing the modified Rpl3 gene are
being tested for resistance to F. graminearum (Harris and 3 CONCLUSIONS
Gleddie 2001).
No analogous weak link in the aflatoxin biosynthetic Since it is unlikely that preharvest mycotoxin contamination
pathway has been discovered that can be exploited in a similar of crops will be reduced significantly through careful cultural
host resistance strategy, nor has a clear role for aflatoxin in practices, control of these problems will likely be dependent
fungal virulence been demonstrated. However, the aflatoxin upon the development and introduction into the commercial
biosynthetic pathway and the gene cluster comprising genes market, of germplasm, resistant to the growth of mycotoxi-
that govern this pathway, including a key regulatory gene genic species, and/or biosynthesis of toxins by these species.
(aflR), have been characterized (Bhatnagar et al. 2002). Also, The identification of resistance traits in corn and other crops
the regulation of these genes during invasion of the host plant can, through marker-assisted breeding, facilitate a more rapid
is being investigated using a genomics approach. This development of resistant, commercially-acceptable germ-
approach is based upon the fact that certain plant-derived plasm. Genetic engineering provides a tool especially useful
natural products apparently have regulatory effects on for introducing resistance genes into crops with little natural
aflatoxin biosynthesis [as recently reviewed in Bhatnagar genetic diversity (e.g., cotton), and for testing the efficacy of
et al. (2001) and reported in recent publications cited in this putative resistance genes.
article: Miller et al. 1996; Norton 1999; Norton and Dowd Studies identifying compounds that affect mycotoxin
1996; Wicklow et al. 1998]. biosynthesis offer hope to researchers. Limiting fungal
Genetic engineering may provide innovative solutions to growth in crops is an important aspect of host resistance,
prevent the accumulation of fumonisins in Fusarium-infected however, obtaining zero growth of fungi capable of exploiting
maize. One approach currently under development is a variety of different substrates, such as the facultative
detoxification of fumonisins by enzymes introduced into pathogen A. flavus, may be difficult to achieve. Therefore, the
maize via genetic engineering. Enzymes that detoxify and identification of a natural compound that blocks mycotoxin
degrade fumonisins have been identified from Exophiala biosynthesis might be the closest we come to discovering a
spinifera, a black yeast found on moldy maize kernels. The “magic bullet.” Nevertheless, the investigations discussed in
initial steps in fumonisin detoxification are ester hydrolysis this chapter, using molecular-based technologies to identify
followed by oxidative deamination to produce derivatives that and characterize various resistance mechanisms in crops
lack the free amino function that is believed to be important susceptible to mycotoxin contamination, and against different
for toxicity (Blackwell et al. 1999). Genes encoding the mycotoxigenic fungi, are building a foundation which can
deesterification and deamination enzymes have been cloned lead to the implementation of a successful gene pyramiding
Molecular Biology for Control of Mycotoxigenic Fungi 75

strategy to produce mycotoxin-resistant, commercially- Brown RL, Chen Z-Y, Menkir A, Cleveland TE, Cardwell K, Kling J,
attractive crops. and White DG (2001a). Resistance to aflatoxin accumulation in
kernels of maize inbreds selected for ear rot resistance in West
and Central Africa. J Food Prot 64:396– 400.
Brown RL, Cleveland TE, Woloshuk CP, Payne GA, and Bhatnagar
ACKNOWLEDGEMENT D (2001b). Growth inhibition of a Fusarium verticillioides GUS
strain in corn kernels of aflatoxin-resistant genotypes. Appl
Microbiol Biotechnol 57:708– 711.
We sincerely thank the editors for inviting us to contribute to Brown-Jenco CS, Obrian GR, Sloan S, and Payne GA (1998).
this edition of Handbook of Fungal Biotechnology. Identification of the DNA binding site for the Aspergillus flavus
AFLR in the NOR-1 promoter. Proceedings of the USDA-ARS
Aflatoxin Elimination Workshop, St. Louis, MO, p.102.
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Guo BZ, Chen Z-Y, Brown RL, Lax AR, Cleveland TE, Russin JS, pp 1– 17.
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Rajasekaran K, Cary JW, Jacks TJ, Stromberg KD, and Cleveland TE Cleveland TE, and Lynch RE (2003). Control of preharvest
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7
Biotechnological Potential of Entomopathogenic Fungi

Travis R. Glare AgResearch, Lincoln, New Zealand

1 INTRODUCTION fungus and applied it in the field for sugar-beet weevil

control.
Fungi have been known to attack insects and mites for Despite the early discovery of their potential, it was only
thousands of years. Although the causative agent of fungal recently that entomopathogenic fungi have been utilized
disease of insects was not always understood, insects infected successfully in biocontrol programs. The fungi are often
with fungi were recorded by the Chinese in the seventh effective as natural control agents, but their activity is very
century (Tanada and Kaya 1993) and drawings of Cordyceps dependent upon environmental conditions. Many have
infections abound in early 18th and 19th century literature. restrictive temperature ranges for germination, infection,
The first experimental demonstration of a microbe as a and sporulation, or high humidity requirements for sporula-
disease-causing organism was by Agostino Bassi, published tion and spore germination. In some cases, the infective stage
is not robust and, as many of the most promising candidate
in 1835 –1836, in Italy, with the silkworm pathogenic fungus,
fungi for pest control have lost the ability to form persistent
Beauveria bassiana. He demonstrated that the fungus causes
stages such as resting spores, storage and application can be
insect death and could be transmitted to other silkworms. It
problematic. Variation within species or clusters of species
was not long after the first demonstration of the devastating
has not been well understood, thus strain selection has not
impact of an entomopathogenic fungus on a beneficial insect
often been attempted or not been possible. The application of
that it occurred to researchers that disease may be a useful
biotechnology to the study and development of entomopatho-
method for control of insect pests. Pasteur is credited with the
genic fungi has the potential to overcome some of these
early proposition that fungi could be used to control a pest limitations. Biotechnology has contributed to all areas in the
insect. He proposed that a fungus could be used against development of entomopathogenic fungi as biocontrol agents,
Phylloxera in grapevines, a pest eventually controlled using from identification to formulation. This chapter reviews the
copper solutions. However, it was the Nobel Prize winning contribution of biotechnology to the development of
researcher, Elie Metchnikoff, who first developed a fungus as entomopathogenic fungi.
a practical control agent for application to a pest. Working in
Russia from 1878, Metchnikoff developed the fungus
Metarhizium anisopliae for control of the cereal cockchafer,
Anisoplia austriaca, then a devastating pest. Metchnikoff 2 BIOPESTICIDE POTENTIAL OF
carried out the first successful infection experiments with ENTOMOPATHOGENIC FUNGI
larvae of A. austriaca and the sugar beet weevil, Cleonus
punctiventris, and initiated mass production of the fungus for There are a number of methods for using entomopathogenic
field experiments (Zimmermann et al. 1995). With the mass fungi against insect pests. Eilenberg et al. (2001) recognized
production of M. anisopliae, Metchnikoff applied a four main strategies: (a) classical biological control, the
biotechnological approach to entomopathogenic fungi for intentional introduction of an exotic strain for long term,
the first time, a precursor to the development of biopesticides. unmanaged control, (b) inoculative biocontrol, the intentional
The first actual field application of M. anisopliae in Russia release of endemic strains for long-term unmanaged
was left to Krassilstchik (1888) who mass produced the biocontrol of endemic pests, (c) inundative biological control,

79
Table 1 Biopesticides based on entomopathogenic fungi (Milner 2000; Reddy et al. 2001; Shah and Goettel 1999)
80
Species and product name Targets Company Main countries and reference
B. bassiana
AGO Biocontrol Coleoptera, Homoptera, Lepidoptera, Diptera Ago Biocontrol Columbia
Bassiana
Biorin Lepidopteran caterpillars Biotech International India www.biotech-int.com
BotaniGard and Homoptera/Heteroptera, thrips, Coleoptera, Emerald BioAgriculture (ex. Mycotech USA
Mycotrol Lepidoptera and Orthoptera Corporation)
Beauveria Schweizer Turf/grassland, fruit growing, viticulture and Eric Schweizer Seeds Ltd. Switzerland
horticulture
Dispel Podborers India
Ostrinil Corn earworm O. nubalis Natural plant protection France
B. brongniartii
AGO Biocontrol Coleoptera, Homoptera, Lepidoptera, Diptera Ago Biocontrol Columbia
Beauveria 50
Engerlingspilz Melolontha melolontha Andermatt Biocontrol, AG Switzerland
M. anisopliae
Ago Biocontrol Lepidoptera, Coleoptera, Homoptera, Orthoptera Ago Biocontrol Colombia
Metarhizium 50
BIO 1020 Vine weevil Bayer AG/ Germany, USA
BioGreen A. couloni (red-headed cockchafer) BioCare Technology Pty. Ltd. Australia
BioCane Greyback canegrubs BioCare Pty. Ltd. Australia
Green guard Locust and grasshopper Seed Grain and Biotechnology Australia
Australia Pty. Ltd.
Green muscle Locusts and grasshoppers Biological Control Products SA PTY South Africa
Metarhizium Turf/grassland, fruit growing, viticulture and Eric Schweizer Seeds Ltd. Switzerland
Schweizer horticulture
Taenure Root weevils, grubs, ticks, immature thrips, white Earth BioSciences Inc. http://www.taensa.com/products-taenure.
flies html
L. giganteum
Laginex All mosquito larvae AgraQuest, Inc. Colombia, USA
Nomuraea rileyi
AGO Biocontrol Lepidoptera Ago Biocontrol Colombia
Nomuraea 50
P. fumosoroseus
AGO biocontrol Coleoptera, nematodes Ago Biocontrol Colombia
Paecilomyces
PFR-97 biological Whiteflies, aphids, thrips, spidermites Thermo Trilogy Corporation European Union, USA
insecticide
V. lecanii
Ago Biocontrol Homoptera, Diptera Ago Biocontrol Colombia, USA
Verticillium 50
Mycotal Whiteflies, some activity against thrips Koppert Biological Systems B.V. Netherlands, UK, Switzerland, Finland,
Norway, Denmark
Vertalec Aphids Koppert Biological Systems B.V. Netherlands, UK, Switzerland, Finland,
Norway, Denmark
Glare
Biotechnological Potential of Entomopathogenic Fungi 81

the use of fungi to limit pests when control is achieved number of reasons (Reinecke et al. 1991) and was
exclusively by the mass release of the organism, and (d) unavailable for many years. Recently, Bio1020 has
conservation biological control, modification of the environ- reappeared in the market as Taenuree, sold by Earth
ment to enhance fungal infection. Inundative biological BioSciences (http://www.taensa.com/products-taenure.html).
control usually relies on the development of biopesticides A recent success story for biopesticides has been the
based on pathogenic microbes, which is the most obvious development of novel strains of M. anisopliae var. acridium
application of biotechnology to entomopathogenic fungi. for locust control in several countries. Initially, a strain of this
fungus was developed in Africa under a program called
LUBILOSA, which led to the biopesticide “Green Muscle”
2.1 Biopesticides Based on Fungi (Lomer et al. 2001). This program has inspired development
of indigenous strains of M. anisopliae var. acridium in other
Application of fungi in mass inoculations against insect pests countries. For example, in Australia the success of the
began with Krassilstchik in 1888. Indeed, such was the LUBILOSA program has been duplicated with the develop-
optimism at that time Krassilstchik confidently predicted “the ment of Green Guarde based on an Australian isolate of
idea of controlling insects by means of artificially induced M. anisopliae var. acridium (Milner 2000).
epidemics, an idea expressed some 20 years ago by scholars, Other biopesticides based on Metarhizium are sold around
has become a practically feasible one, which in the future will the world. In Australia, an isolate of M. anisopliae has also
be perfected and broadly utilized” (Krassilstchik 1888). been developed as a commercially available biopesticide for
Unfortunately, progress has been much slower than what was the control of sugarcane scarabs, particularly the grayback
predicted. While progress in the early 1900s was promising, canegrub, Dermolepida albohirtum. BioCanee is effective
the discovery and application of effective chemical pesticides when applied at 33 kg/ha (1 £ 1010 conidia/m) before filling-
in the 1930–1940s reduced interest in the use of insect in of the planting furrow (Samson et al. 1999), giving
pathogens. Insect pathogenic fungi were more difficult to use, 50–60% control of grayback larvae (Logan et al. 2000). In
and it was not until environmental and health problems Columbia, a product based on several entomopathogenic
associated with the use of chemical insecticides became fungi (Micobiol) has been tested against Prodiplosis longifila
apparent in the 1960s that interest in fungal biopesticides (Diptera: Cecidomyiidae) infesting tomatoes, but was not as
again increased. Currently, most biopesticides based on effective as conventional control products (Delgado et al.
entomopathogenic fungi in the market include either 1999).
B. bassiana or M. anisopliae (Table 1). These two species, Several other entomopathogenic fungi have been
the so-called muscardine fungi, have broad host ranges, developed as commercially available biopesticides (Table 1).
although individual strains may be restricted in the number of Fungi such as Paecilomyces and Verticillium are similar in
insect species that they can attack. These species are action to Metarhizium and Beauveria. However, a more
relatively easy to produce, as they produce vast amounts of unusual fungus for development as a biopesticide is the
asexual conidia in culture as well as on insects. They are aquatic active, Lagenidium giganteum. This Oomycete
generally considered to have low mammalian toxicity and few fungus is active against mosquito larvae and has been
nontarget impacts have been reported (see Section 6). developed into the biopesticide Laginexe in California and is
A number of biopesticides have been based on the white now sold by AgraQuest, Inc. In field trials against the
muscardine fungus, B. bassiana (Table 1). The better known mosquito Culex quinquefasciatus, Laginexe compared
products are those of Emerald BioAgriculture (a merger favorably with Vectobace (based on Bacillus thuringiensis
between Mycotech Corporation and Auxein Corporation) israelensis), in terms of persistence of control (Hallmon et al.
such as BotaniGardw and Mycotrolw. BotaniGard is a liquid 2000). No biopesticides are currently produced using any
emulsion formulation of B. bassiana conidia while Mycotrol species of the Entomophthorales, which is a large order
is based on powdered conidia. There are a number of other containing mainly entomopathogenic fungi. These fungi,
products based on Beauveria spp. registered around the world. which typically forcibly discharge their primary conidia,
In France, Ostrinile, based on B. bassiana has been produced often cause large-scale epizootics among insects. This
for many years for corn earworm (Ostinia nubalis) control, suggests huge potential for development of this group of
while in India the biopesticide Dispel is sold for control of fungi as mass applied biopesticides. However, problems in
podborers (Reddy et al. 2001). Similarly, a number of production and stabilization of the fragile conidia or the more
biopesticides are based on the green muscardine fungus, durable resting spores have not been overcome, and economic
Metarhizium spp. Biopesticides based on Metarhizium spp. products are not feasible at this time.
have had a long (if not always successful) history. In the Biopesticide production has increased in many Central and
1980s, Bayer Corporation produced a biopesticide, Bio1020, South American nations and some are not strictly
which was a formulation of M. anisopliae with excellent shelf commercial. For example, in Cuba where, as a result of the
life and application potential. It was primarily developed for trade embargo, it has been difficult to obtain cheap chemical
control of black vine weevil (Reinecke et al. 1990), but was pesticides, a biopesticide production industry has grown to
tested against a number of other pests [e.g., Tabata (1992)]. fill the gap. Under the Cuban Ministry of Agriculture,
However, the product was not commercially successful for a decentralized laboratories provide insects, nematodes, and
82 Glare

entomopathogens (bacteria, fungi, and viruses) throughout usually obtained for rice grains, with viabilities of higher than
Cuba’s 15 provinces (Rosset and Moore 1997). These 85% (Dorta et al. 1990).
“Centres for the Production of Entomophages and Entomo- The production of Green Musclee M. anisopliae for
pathogens” (CREEs) have facilitated the rapid adoption of locusts in Africa used a two-stage production system with
IPM systems in crops previously managed under pesticide- fermenter production of inoculum used to inoculate rice
based systems. Several fungal entomopathogens are produced (Cherry et al. 1999). The process requires relatively low
for a number of pests, including B. bassiana for control of capital investment, but has high labor costs. As with
coleopteran pests and Verticillium lecanii for whitefly, production of most fungi, high variability in yield was
Bemisia tabaci. In 1994, 781 metric tonnes of B. bassiana, reported between batches, and this variability was only partly
196 of V. lecanii and 142 of M. anisopliae were produced by accounted for by temperature and duration of incubation
the production centers. Similarly, biopesticides based (Cherry et al. 1999).
M. anisopliae are produced by some Central American A method that showed some promise in the 1980s was the
sugar plantations for the control of pests. The sugar preparation of dried mycelium. Hyphal bodies were harvested
companies have their own production facilities for Metar- by filtration, washed with water to remove culture medium
hizium and Beauveria [e.g., Badilla (2000) and Grimm residue, and then coated with a sugar solution before drying.
(2001)]. These localized production facilities produce This method was used with M. anisopliae and B. bassiana
sufficient quantities of fungal inoculum for control of pests (Pereira and Roberts 1990). They found that conidial
such as the coffee berry borer, the diamondback moth, and production was similar to other methods after storage for up
spittlebugs. to 4.5 months at 48C and could be superior to other methods
with respect to storage at room temperature, however no
products at present use this technology.
The Emerald Bio production plant (previously Mycotech)
2.2 Production, Formulation, and Application in Butte, Montana, represents the technological end of the
production of entomopathogenic fungi. Largely utilized for
Production of entomopathogenic fungi has not advanced the production of B. bassiana, it is a “state of the art”
greatly beyond the use of simple grains as substrates for the dedicated facility, with in-line sterilization and large
Deuteromycete fungi, such as Metarhizium and Beauveria. temperature controlled growth facilities. The actual pro-
For many other entomopathogenic fungi, especially among duction method is a trade secret, but is based on fermented
the Entomophthorales, growth in culture is difficult or has yet starter cultures and solid substrate growth and sporulation.
to be achieved. Both liquid and solid substrates have been This highly technical facility contrasts with the numerous low
substantially investigated (Burgess 1998). Two-stage sys- technology “factories” producing fungi for insect control in
tems, where both liquid and solid substrates are used, have China and much of Latin America.
occasionally proved successful. For example, fermentation to Compatibility between production, formulation, and
produce hyphae to use as starter cultures is now a widespread application techniques is vital for the successful use of
practice. There are number of advantages to using liquid microbial biopesticides. The LUBILOSA program for locust
cultures as starter cultures: (a) the competitive ability of the control used Metarhizium in oil formulations and ULV
fungus is enhanced, reducing the risk of contamination from spraying, which required lipophilic conidia for easy
other microbes, (b) growth is more rapid in the early stages, suspension in oils (Jenkins et al. 1998). While production of
(c) the liquid culture can be screened for contamination prior submerged conidia was seen as having many advantages, the
to use, and (d) the liquid ensures even coverage of the solid resulting conidia were hydrophilic and lost viability quickly.
substrate (Jenkins et al. 1998). Liquid starter cultures are Therefore, production on grains remains the standard with the
commonly used to begin solid substrate production. However, locust products. For many years, approaches to the use of
experience with M. anisopliae in our laboratory is probably entomopathogenic fungi involved point release (“classical
typical of many other laboratories, where inoculation of rice biological control”) or simple application of conidia,
grains with fermenter broth of M. anisopliae hyphal bodies formulated in water with wetting agents. However, appro-
gave no improvement in production over the use of conidia priate formulation can advance entomopathogenic fungi from
from plate cultures (Glare et al. unpublished data). Production curiosity to effective biocontrol agents. It has been an area
on grains is generally in the range of 108 –1010 conidia/g of that has benefited from the application of biotechnology.
dry substrate [e.g., Feng et al. (1994)], taking between 2 and 3 Formulation has been important in terms of improved survival
weeks to reach maturity at optimal temperatures. Interest- during storage, persistence in the field (such as UV and
ingly, Metarhizium and Beauveria sporulate better when the desiccation tolerance), and ease of application.
substrate is relatively poor in nutrient content. When grains The LUBILOSA program, where M. anisopliae var.
were supplemented with sugars and yeast additives, less acridium was developed into a biopesticide for locust control
conidia per gram of substrate was obtained than with grains in Africa, is an excellent example of formulation overcoming
alone (Nelson et al. 1996). Similarly, in Brazil, M. anisopliae environmental constraints. As locusts live in hot, dry climates
has been found to produce conidial yields of 5 –15 times and M. anisopliae conidia require high humidity to germinate,
higher using rice bran/rice husk substrate mixtures than yields it seems impossible that an entomopathogenic fungus could
Biotechnological Potential of Entomopathogenic Fungi 83

successfully control the pest. However, formulating Meta- approach that takes advantage of the biological nature of
rhizium conidia in nonevaporative diluents such as oils entomopathogenic fungi is the “lure and infect” approach,
allowed the conidia to attach and germinate on susceptible best demonstrated by research on Z. radicans for control of
locusts. M. anisopliae oil formulations are especially useful at diamondback moth. Furlong et al. (1995) have shown that
low relative humidities (Bateman 1997). There have been using pheromone lures to attract moths to traps containing
several interesting studies on formulating hyphal material sporulating Z. radicans can result in contamination and spread
from members of the Entomophthorales. These fungi, because of the fungus through the target population. Such an approach
of the fragile nature of the mycelium and conidia, pose a has been investigated for use with scarab beetles in the Azores
much greater formulation problem than most of the (Klein and Lacey 1999). Autodissemination of entomopatho-
Deuteromycetes, which has contributed to their lack of genic fungi for control of Popillia japonica in the Azores used
commercial success. McCabe and Soper (1985) patented a a trapping system of commercially available attractants with
process of drying the mycelium of Zoophthora radicans and M. anisopliae. The viability of conidia in traps after 6 days
coating it with sugar, as a method for long-term storage. More was found to be about 35%, but the basic process was
recently, Shah et al. (1998) demonstrated algination as a successful for introducing fungi into pest populations.
method for formulating Erynia neoaphidis mycelium. An Another approach has been bait stations, such as those used
important area of formulation and production is the drying of with termites (Rath 2000). The entomopathogenic fungus is
conidia of entomopathogenic fungi. Moore et al. (1996) have placed in a trap together with a food-based bait, and the insect
shown that survival of conidia of M. anisopliae was highest at becomes contaminated when it enters the trap. The general
low (, 5%) relative humidity, therefore, this is an important approach is similar for lure-and-infect and bait stations:
aspect of producing a stable product. attract the insect to an inoculum source, rather than broadcast
Use of appropriate application techniques that are suited application to secure contact between pest and disease.
for the application of biopesticide to the target pest is an Use of attractants is not restricted to luring to a single trap.
obvious, but often neglected aspect of biopesticide use. Smith et al. (1999) investigated the use of vegetable fat pellets
Advances in chemical pesticide applications have slowly formulated with pheromone and B. bassiana to control the
filtered through to use with biopesticides, such as ultra-low larger grain borer, Prostephanus truncates. Significantly
volume (ULV) application of M. anisopliae for locust control higher numbers of beetles were attracted to pellets containing
(Lomer et al. 2001). Nonevaporative diluents such as oil are pheromone than those without pheromone incorporated. The
required to take advantage of ULV spraying. Rotary pellets containing pheromone and fungus could be stored for
atomizers have been used for low volume oil formulations several weeks, indicating this may be a useful strategy to
and ULVs for less than 5 l/ha. Electrostatically-charged ULV increase the utility of entomopathogenic fungi.
sprayers have been investigated for better coverage on leaf Development of biopesticides for social insects has been
undersides (Sopp et al. 1989). Generally, application of problematic because the method by which social insects
fungal-based biopesticides has been with conventional defend against disease is mainly behaviorally-based rather
equipment and research has focused on spray coverage, than biologically-based. For example, hymenopteran wasps
droplet size, and placement (i.e., penetration to the underside such as Vespula spp. have well-developed hygienic behaviour
of leaves). Hydraulic spray systems have been used to apply which includes removing all suspected material from a nest
water-based formulations on crops, air-blast and air-assist before contamination of nestmates occurs. Vespula do not
technologies are primarily used for low volume applications reuse nests and, therefore, disease in one season does not
in fields and orchards. The best success has been with large result in disease in another season. Behavioral defense against
numbers of droplets with high spore content per droplet disease requires novel application and formulation methods
(Goettel et al. 2000). for any chance of success for entomopathogenic fungi.
Introducing large amounts of fungal inoculum into the soil Similarly, termites are highly susceptible to entomopatho-
and securing an even spread remains a problem. Many genic fungi, including M. anisopliae and B. bassiana but
methods have been tested for application of fungal containing many factors such as avoidance of conidia, the removal and
granules or conidia on grains to soil, including using seed burial of fungus-killed termites, together with defensive
drills for subsurface application, and hand application. The secretions and inhibitory components in termite frass (Rath
problems of spread of conidia after application to soil has lead 2000), and grooming to remove spores (Milner and Glare,
to the Melolontha and researchers are developing an area unpublished observations) reduce field efficacy. Boucias
wide approach based on augmentative applications of et al. (1996) used a low sublethal dose of a neurotoxin,
Beauveria brongniartii for long term suppression of pest imidacloropid to disrupt the grooming behaviour of
populations (Hajek et al. 2001). termites, which then became highly susceptible to the
fungus B. bassiana.
One proposal is to use more than one pathogen to increase
2.3 Novel Strategies for Biopesticide Use the utility of entomopathogenic fungi. It is often common in
the field to find more than one pathogen exerting influence on
In some cases, preexisting application technology may not be a pest, such as both a nucleopolyhedrovirus and the fungus
well suited to the requirements of a biological agent. One Entomophaga maimaiga infecting gypsy moth (Malakar et al.
84 Glare

1999). The possibility of combining multiple species or depsipeptide metabolite which has shown toxicity to a
strains in a single biopesticide to overcome limitations number of invertebrates (Roberts 1981). Not all Beauveria
inherent in the single strain approach is intriguing. For can produce beauvericin, but it has been isolated from
example, Inglis et al. (1997) have investigated the use of both Paecilomyces fumosoroseus mycelium. B. bassiana is also
M. anisopliae and B. bassiana for control of locusts and reported to produce beauverolides, isarolides, and bassiano-
grasshoppers, to overcome the temperature limitations of both lides, all cyclotetradepsipetides. Metarhizium strains are also
species. There are potentially many methods whereby the well known for producing toxic metabolites, the best
efficacy of biopesticides could be enhanced by combinations, described of these are the destruxins. These cyclodepsi-
such as those described earlier, but the economics of peptides are toxic to a number of insects, but susceptibility
producing multiple pathogens for a single product are usually varies considerably, ranging up to 30 times between silkworm
too limiting. larvae and Galleria (Roberts 1981). Hirsutellin A is produced
by Hirsutella thompsonii and is not proteolytic, but was toxic
to a range of insects (Mazet and Vey 1995). Aspergillus
species are occasionally insect pathogens and are known to
3 BIOACTIVES FROM produce many insecticidal metabolites. However, the
ENTOMOPATHOGENIC FUNGI occurrence of aflatoxin production in many Aspergillus that
infect insects has restricted interest in this group, although it is
While the focus on the practical use of entomopathogenic by no means necessary that insecticidal strains produce
fungi has been on biocontrol using whole organisms, either as aflatoxins in any appreciable amount (Roberts 1981). Not all
inoculative or inundative agents (Eilenberg et al. 2001), these entomopathogenic fungi produce toxins in the disease
fungi are known to produce a number of toxins and enzymes. process. In some cases, toxins are suspected, but not
Some of these extracellular metabolites have been studied conclusively demonstrated. Injection of culture filtrates of
with the aim of using them as bioactives against insect pests. some entomopathogenic Entomophthorales into Galleria sp.
This biotechnological approach to utilizing entomo- resulted in blackening similar to that found in fully infected
pathogenic fungi can be demonstrated by the discovery and larvae [e.g., Roberts (1981)]. Some of the lower fungi, such as
formulation of spinosyns, insecticidal toxins produced by an Coelomomyces and the Entomophthorales, may possess only
actinomycete. From the discovery of a strain of weak toxins, if any at all. It is more likely they overcome hosts
Saccaropolyspora spinosa in the Carribbean, Dow Agrow by utilizing the nutrients and invading vital tissue (Roberts
Sciences have successfully developed a number of “green 1981).
chemistry” insecticide products, such as Successe and Some entomogenous fungi produce antibiotics. As
Naturalytee. It may be possible to utilize active components entomopathogenic fungi must compete for utilization of
from entomopathogenic fungi in a similar or novel fashion. cadavers with numerous resident and environmental bacteria,
It is not surprising that entomopathogenic fungi produce it is not surprising that a number of antibiotics are produced
extracellular enzymes and toxins. These compounds are by the various strains and species. Hirsutella and the allied
required to both assist penetration of the host cuticle and genus Cordyceps also produce a number of metabolites that
overcome other host defenses, while excluding competing may be weak toxins or antibiotics. Krasnoff and Gupta (1994)
microbes. Proteases produced by entomopathogenic fungi to described an antibiotic, phomalactone, from the H. thompsonii
degrade cuticle and assist entry into the host are similar to var. synnematosa that was also toxic to apple maggots,
proteases used by insects to degrade their own cuticle during Rhagoletis pomonella (Dipt., Tephritidae). Phomalactone was
molting (Samuels and Paterson 1995). A number of enzymes inhibitory to other entomopathogenic fungi (Beauveria,
are known from entomopathogenic fungi, such as the Tolypocladium, and Metarhizium). Cordyceps-infected cater-
proteases, lipases, and chitinases that assist in cuticular pillars are a traditional medicine in parts of Asia. This may be
breakdown. These enzymes can be thought of as bioactives partly based on the production by Cordyceps of a weak
and there has been increasing interest in use of these enzymes antibiotic, cordycepin. Zabra et al. (1996) reported that
in pest control. Screen and St Leger (2000) have reported on metabolites from Z. neoaphidis had antibacterial activity. In
the occurrence of typsins and chymotrypsins in M. anisopliae. the future, bioactives from entomopathogenic fungi may have
The novel chymotrypsin (CHY1) is similar to bacterial a role in pest insect control, either formulated as pesticides, or
chymotrypsins. Because paralogous genes for the chymo- through transgenic expression. Direct toxicity may not be the
trypsins are not found in genome sequences for yeast, gram only aim, as some toxins or metabolites have antifeedant type
eubacteria, archaebacteria, and mitochondria they hypothesis activities (e.g., http://www.item.ba.cnr.it/biopesti.htm).
that chy1 arose from horizontal gene transfer.
Entomopathogenic fungi also produce insecticidal toxins.
The early literature on toxins from entomopathogenic fungi 4 MOLECULAR GENETICS OF
was reviewed by Roberts (1981) and more recently by ENTOMOPATHOGENIC FUNGI
Strasser et al. (2000). Several metabolites from entomopatho-
genic Deuteromycetes are well known and described. For The use of molecular techniques to manipulate entomopatho-
example, Beauveria spp. are known to produce beauvericin, a genic fungi to overcome some of the limitations discussed
Biotechnological Potential of Entomopathogenic Fungi 85

earlier has been proposed for many years. In comparison with conidia with a modified polyol and trehalose content resulted
advances made in manipulation of viruses and bacteria, in conidia with increased intracellular levels of glycerol and
progress with the fungi has been slow, which is not surprising erythritol that germinated more quickly than unselected
given the multigene nature of fungal insect diseases. Most conidia and at lower water activity (Hallsworth and Magan
progress has been made with the Deuteromycete muscardine 1995). Conidia with increased trehalose germinated more
fungi, B. bassiana and M. anisopliae. slowly but stored for longer than unselected conidia. Another
approach is to use genetic modification to “improve” strains
and overcome limitations. This type of approach is in its
4.1 Transformation Systems infancy for entomopathogenic fungi, but there have been
some interesting studies indicating the utility of the process.
Modification of entomopathogenic fungi has long been Two of the more promising studies on the potential of
contemplated, but rarely reported. A limited number of biotechnology to improve entomopathogenic fungi were
studies have reported successful insertion of foreign genes published by Couteaudier et al. (1996) and Vaiud et al.
into entomopathogenic fungi. A precursor to manipulation of (1998). They demonstrated that protoplast fusion between a
entomopathogenic fungi using molecular techniques has been strain of B. bassiana from Leptinotarsa decemlineata with an
the development of transformation systems. There are several insecticidal toxin-producing strain of B. sulfurescens resulted
aims of transforming entomopathogenic fungi. These in recovery of some di-auxotrophic mutants with enhanced
techniques enable gene disruption methods to be applied, activity (faster kill) against L. decemlineata and the caterpillar
which can lead to greater understanding of the genetics of Ostrinia nubilalis. The stability of the virulence following
disease processes, or the ability to introduce DNA into fungi passage through the insect –host and stability of molecular
may allow the modification of cell processes, potentially structure for two of the fusion products suggested that asexual
allowing improvements in the use of entomopathogenic fungi genetic recombination by protoplast fusion may provide an
for insect control. attractive method for the genetic improvement of biocontrol
The first transformation of an entomopathogenic fungus efficiency in entomopathogenic fungi (Vaiud et al. 1998).
was reported by Goettel et al. (1990), where M. anisopliae The most studied genes in the entomopathogenic fungi are
was transformed to be benomyl tolerant using pBENA3, a the protease genes of M. anisopliae, particularly the Pr1 gene.
plasmid containing the benA3 allele from Aspergillus This was the first protease gene from an entomopathogenic
nidulans. Since then, there have been other reports on fungi implicated in disease and was isolated by St Leger et al.
transformation of the Dueteromycete entomopathogens (1992). Pr1 has sequence similarity to proteinase K, but was
using a variety of methods. St Leger et al. (1995) used more effective than that enzyme at degrading cuticle. It is
eletroporation and biolistic delivery to transform similar to the subtilisin subclass of serine endopeptidases.
M. anisopliae with the plasmids (pNOM102 and pBENA3) Modification of pr1 gene expression in M. anisopliae resulted
containing the b-glucuronidase and benomyl resistance in melanisation and cessation of feeding 25 –30 h earlier than
genes. The cotransformants showed normal growth rates wild-type disease in caterpillars (St Leger et al. 1996).
and retained their pathogenicity to insects (Bombyx mori). V. lecanii, B. bassiana, Tolypocladium niveum, and
Polyethylene glycol (PEG)-mediated transformation of P. farinosus also produced Pr1-type enzymes during nutrient
protoplasts is another method for transformation of deprivation (St Leger et al. 1991). Southern analysis
entomopathogenic fungi, as used with the P. fumosoroseus demonstrated that genes with significant homologies to
and P. lilacinus (Inglis et al. 1992) using benomyl as the Metarhizium pr1 were present in the entomopathogens
selective agent. More recently, a heterologous transformation A. flavus and V. lecanii but not Z. radicans (St Leger et al.
system for B. bassiana and M. anisopliae was developed 1992). More recently, 11 subtilisin proteases (Pr1s) were
based on the use of the A. nidulans nitrate reductase gene identified from one strain of M. anisopliae (St Leger et al.
(niaD) (Sandhu et al. 2001). The niaD stable mutants of 2001). Recently, intended field release of a modified
B. bassiana and M. anisopliae were selected by treatment of M. anisopliae strain was reported (St Leger 2001). The strain
protoplasts with ethane methane sulfonate (EMS) and has the pr1 cuticle degrading protease gene under control of a
regenerated on chlorate medium. constitutive promoter. The gene overproduction did not alter
the host range, but resulted in a strain with a reduced median
lethal time to kill. It also reduced the ability of transformants
4.2 Strain Improvement Through Biotechnology to sporulation. The pr1 gene expression was under dual
control of a general carbon catabolite repression/depression
Improvements in strains of entomopathogenic fungi have mechanism and a carbon source induction mechanism to
been attempted through selection as well as molecular control expression. Overexpression of extracellular chitinase,
methods. Selection of fungal strains with altered acyclic sugar an enzyme important in the cuticular penetration of insects by
alcohol (polyol) and trehalose content of the conidia may entomopathogenic fungi, has also been demonstrated for
improve the endogenous reserves to enhance viability and M. anisopliae var. anisopliae (Screen et al. 2001). They
desiccation tolerance. Cultures of B. bassiana, M. anisopliae, expressed a chitinase gene from M. anisopliae var. acridium
and P. farinosus grown under different conditions to obtain under control of an Aspergillus regulatory element to express
86 Glare

in noninducible conditions. While successful expression was While this is not a problem for strain identification or
achieved, there was no altered virulence to the caterpillar, comparison, it reduces the ability to compare between studies.
Manduca sexta, compared to the wildtype fungus. Genetic Many of the molecular studies on entomogenous fungi have
manipulation of entomopathogenic fungi has a long way to go used the nuclear ribosomal DNA, but there are a number of
before transgenic pest control strains become available, if other DNA regions used, such as mitochondrial DNA
such technology is ever acceptable to regulators and the (mtDNA) restriction fragment length polymorphisms. The
community. However, strain modification continues to MtDNA has been used to estimate intraspecies variation in
provide a wealth of data on disease processes. V. lecanii and M. anisopliae isolates.
The contribution of molecular techniques to the develop-
ment of entomopathogenic fungi has been enormous. The
5 MOLECULAR IDENTIFICATION AND techniques have been used to clarify evolutionary relation-
TRACKING ships [e.g., Driver et al. (2000) and Jensen et al. (1998)].
Molecular techniques have also allowed development of
In addition to improving our understanding of the genetics of theories of evolution around these often obligate pathogens.
disease caused by entomopathogenic fungi, molecular Generally, studies on the entomogenous fungi using
techniques have also been used to aid in identification, conserved mitochondrial or nuclear regions have failed to
classification, and environmental monitoring of fungi. It is find a link between fungal species and host species. For
now almost a standard practice to perform some form of example, Bidochka et al. (2001) found that habitat rather than
genetic characterization of fungi to specifically identify host selection drives population structure of M. anisopliae.
strains used for biocontrol purposes. The utility of molecular There have been exceptions, such as B. bassiana strains from
techniques, however, has been demonstrated beyond simple Sitona weevils (Maurer et al. 1997) and some Entomoph-
strain identification. There are a number of reviews on the use thorales [e.g., Jensen and Eilenberg (2001)]. In the order
of molecular characterization for entomogenous fungi in the Entomophthorales, sequencing of the small subunit rDNA has
literature [e.g., Driver et al. (1998) and Glare (2002)]. An been used to examine phylogenetic relationships (Jensen et al.
example of the importance of molecular characterization was 1998). The molecular studies supported the use of spore
the clarification of the taxonomic position of M. anisopliae discharge characteristics as an identifying characteristic for
strain IMI 330189 used in the LUBILOSA program for locust Entomophthorales. The role of horizontal gene transfer in
control. Often described as a M. flavoviride strain because of microbial evolution has been the topic and studies by St Leger
the morphology of the conidia and some other features, the et al. (2001) have found some evidence for the involvement of
correct classification was debated. Recently, Driver et al. horizontal gene transfer in evolution of fungal parasitism,
(2000) published a revision of the subspecific relationships finding similarity between genes in M. anisopliae and
between M. anisopliae and M. flavoviride strains, based Streptomyces bacteria. Monitoring of specific strains of
largely on the sequence of the ITS-5.8s regions of rRNA. entomopathogenic fungi in the field after release is crucial for
They demonstrated that IMI 330189 and related strains advancing and understanding of biopesticide ecology. It has
formed a discrete clade on the M. anisopliae branch of the often been difficult to conduct ecological studies on fungal
Metarhizium trees, a finding that appears to have been well persistence and spread after application, because there has
received by those working on locust control. Driver et al. been a lack of simple methods for isolation and specific strain
(2000) named the subspecies M. anisopliae var. acridium, as characterization of these fungi. The molecular characteriz-
well as describing several other subspecies, some of which ation of strains of entomogenous fungi has improved the
can only presently be distinguished by ITS sequencing. This ability to track specific fungi in the field. Specific
demonstrates a problem with molecular characterization, as identification of the B. brongniartii strain used for control
fungal species cannot be solely erected on sequence data and of the scarab pest, Hoplochelus marginalis, in the ReUnion
requires supporting morphological or biological descriptions. Islands was based on introns (insertions) in the 28s gene of the
Molecular markers have been used to characterize the rDNA (Neuvéglise et al. 1997). Genetic modification is also a
genotypes of individual fungal strains by examining gene method to allow tracking following release of a strain into the
products, but new techniques allow direct examination of environment or in a host. For example, a b-glucuronidase
variability at the DNA level. Pulsed field gel electrophoresis gene has been inserted in M. anisopliae to allow detection of
has been used to study karyotype variation in other fungi, and hyphae in infected hosts (St Leger et al. 1995) and the
could be used with entomopathogens. Several studies have expression of a green fluorescent protein-encoding gene for
examined the number of chromosomes and mapped genes on tracking purposes (St Leger 2001).
those chromosomes. Viaud et al. (1996) studied the level of
chromosome length polymorphism among nine isolates of
B. bassiana to obtain a more extensive knowledge of the 6 SAFETY OF ENTOMOPATHOGENIC
genomic organization. While extensive use of molecular FUNGI
characterization has proved useful, there are currently no
standard techniques or agreement on even how many regions Biopesticides based on entomopathogenic fungi are now
of the genome should be sampled to provide taxonomic data. available, with a range of different species and strains used.
Biotechnological Potential of Entomopathogenic Fungi 87

The growing use of these insect pathogens has raised interest 7 CONCLUSIONS
in the safety of microbial pesticides, above the level
previously required for registration purposes. This increased Biotechnological approaches to the study and development of
scrutiny of environmental and mammalian safety of entomopathogenic fungi have advanced the field in recent
entomopathogenic fungi is part of a worldwide move to years. Improvements in formulations allowing new biopes-
more awareness of potential negative impacts of bio- ticides to succeed in unexpected conditions (such as locust
technology. Few of the entomopathogenic fungi are thought control in Africa), strain selection, and identification have
to pose a direct threat to human health. There are exceptions, advanced not only biopesticide formulation, but under-
such as the entomophthoralean fungus, Conidiobolus standing of disease processes and ecology. More specific
coronatus, but it is unlikely any development of the identification systems have allowed better monitoring of
potentially hazardous strains would be contemplated. Many biopesticide applications as well as development of
products have required mammalian toxicology packages to be phylogenetic classification. Despite some success, commer-
submitted during the registration process to demonstrate cial use of entomopathogenic fungi is restricted by high cost,
safety. Generally, the entomopathogenic Deuteromycetes are inadequate or inconsistent efficacy, limited mass production
considered to have low risk of mammalian toxicity [e.g., capability, and poor shelf life. However, entomopathogenic
Donovan-Peluso et al. (1980) and Shadduck et al. (1982)].
fungi have several advantages over other microbes for
Recent papers on Metarhizium and Beauveria have raised
formulation in biopesticides as many species have a robust
some issues regarding mammalian safety of immunocom-
spore stage, capable of survival in products for many months
prised individuals (Burgner et al. 1998; Henke et al. 2002).
or years. Some are easy to grow on simple media and can be
In addition to viewing bioactives from entomopathogenic
formulated using a number of simple procedures. They can
fungi as potentially useful, there has been consideration of
often kill more than one target pest, although limited in host
their effect as potential hazards in registering entomopatho-
range enough for registration purposes. With continuing
genic fungi. There are some results showing activity against
improvements in formulation and application technology, it is
human cell lines, such as tumor cell lines for P. tenuipes
likely that many more niche biopesticides will come to
cytotoxic components (Nam et al. 2001). Production of toxic
market, especially with the increased markets due to a rise in
secondary metabolites has caused problems in the registration
of some fungi in Europe. Strasser et al. (2000) summarizes organic production and the reduction in the number of
data on specific secondary metabolites (destruxins, efra- chemical pesticides available.
peptins, oosporein, beauvericin, and beauveriolides) There are a number of new techniques and applications
produced by the genera Beauveria, Metarhizium, and that will aid in the further development of entomopathogenic
Tolypocladium. They found that fungal bioactives posed no fungi. Application of molecular biological techniques to
obvious risk to humans, although the number of detailed entomopathogenic fungi also holds the promise of strain
studies is limited. Some studies have indicated low-level improvement through genetic manipulation, or assist in strain
activity against animals of selected bioactives such as improvement without genetic modification. For example,
destruxins of Metarhizium have an intraperitoneal injection through techniques such as protoplast fusion and chromosome
LD50 of 1 –16 mg/kg in mice. However, the levels of exchange, using knowledge of desired chromosomal gene
metabolites produced during insect infection were much location, may enable superior strain qualities to be combined
lower than in culture. in single isolates. Determining the underlying genetics of host
There is a growing body of research on nontarget impacts specificity, the toxins and enzymes involved in the disease
of fungal-based insecticides [e.g., Goettel et al. (2001) and process, and genetics of fungal processes such as sporulation
Hokkanen and Hajek (2002)], which have not found increased and germination are all under study around the world.
environmental risk from their use. The present evidence is Advances in these areas may allow greater use to be made of
that mycoinsecticides are very safe in production and use entomopathogenic fungi.
from both an environmental and mammalian toxicity The potential of entomopathogenic fungi lies not just in
viewpoint (Goettel et al. 2001). However, the formulations their application as biopesticides based on the live fungus, but
being developed require stringent testing to ensure their also in the isolation and development of bioactives from these
superior safety compared with comparable chemical pesti- fungi. Toxins, enzymes, and antibiotics are all produced by
cides (Moore and Prior 1993). While the fungi themselves entomopathogenic fungi and, as techniques for their isolation
have generally not been found to be a risk through testing and and expression increase, the potential for exploiting
natural exposure, the development of novel formulations and bioactives is enhanced. In some cases these bioactives are
strain combinations will require careful evaluation to ensure not toxins, but may exert other useful effects, such as
no unexpected effects occur. This could be especially true of antifeeding activity. There is also potential in novel strategies
nontarget impacts. Similarly, any development of genetically for biopesticide use such as mixtures of behaviour-modifying
modified strains will have to be carefully studied for chemicals for enhancing control of social insects with
environmental and mammalian safety. Regulations in all pathogens.
countries are becoming more stringent on these issues, While the history of biopesticide development from
especially for genetically modified organisms. entomopathogenic fungi is littered with more failures than
88 Glare

success, the future seems brighter. As the knowledge from Driver F, Milner RJ, and Trueman JWH (2000). A taxonomic
several commercially successful products and new technol- revision of Metarhizium based on a phylogenetic analysis of
ogies are applied to biopesticide development, we can expect rDNA sequence data. Mycol Res 104:134 – 150.
to see more novel biocontrol methods applied to insect pests Eilenberg J, Hajek A, and Lomer C (2001). Suggestions for
unifying the terminology in biological control. Biocontrol
in the future using fungal species.
46:387 –400.
Feng MG, Poprawski TJ, and Khachatourians GG (1994).
Production, formulation and application of the entomopatho-
genic fungus Beauveria bassiana for insect control: current
ACKNOWLEDGEMENTS status. Biocontrol Sci Technol 4:3– 34.
Furlong MJ, Pell JK, Choo OP, and Rahman SA (1995). Field and
I thank Drs Maureen O’Callaghan Mary Christey and Trevor laboratory evaluation of a sex pheromone trap for the
Jackson for comments on the manuscript, and Lois McKay for autodissemination of the fungal entomopathogen Zoophthora
assistance with compiling the references. radicans (Entomophthorales) by the diamond-back moth,
Plutella xylostella (Lep: Yponomeutidae). Bull Entomol Res
85:331 –337.
Glare TR (2003). Molecular characterisation in the entomo-
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8
Biotechnological Potential of Ergot Alkaloids

M. Flieger / P. Mehta / A. Mehta Dr. H.S. Gour University, Saugor, India, and
Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic

1 INTRODUCTION recorded from outside this genus (Flieger et al. 1997;

Kozlovsky 1999).
Ergot alkaloids belong to the group of compounds produced Ascomycetes
by fungi, which are referred to as secondary metabolites. Eurotiales: Aspergillus fumigatus, A. flavus, A. japonicus,
They are produced by a number of fungi mainly of Claviceps A. tamarii, A. versicolor, A. nidulans, A. oryzae;
spp. but they have been also found in other fungi and higher Penicillium aurantiovirens, P. camembertii,
plants. Ergot (sclerotium of the pyrenomycete Claviceps P. chermesinum, P. fumigatus, P. clavigerum,
purpurea) develops in florets of grasses and sedges. In early P. concavorugulosum, P. crustosum, P. griseofulvum,
days, the medieval midwives used to collect the fungus from P. kapuscinskii, P. palitans, P. patulum, P. roqueforti,
naturally infected plants and used it in the induction of P. rubrum, P. rugulosum, P. sizovae, P. viridicatum
childbirth and in the control of postpartum bleeding. The role Hypocreales: Balansia claviceps, B. epichloe, B. obtecta,
of ergot has undergone important changes from a dreaded B. strangulans, Epichloe typhina, Neotyphodium
toxic parasite to an important source of biologically effective coenophialum, N. lolii, Hypomyces aurantius,
substances. C. purpurea is, apart from yeast, the first fungus, Sepedonium sp.
which was biotechnologically exploited without its existence Basidiomycetes Corticium caeruleum, Lenzites trabea,
known. The beginning of modern ergot research dates back to Pellicularia filamentosa
the extraction of the first alkaloid mixture from sclerotia in Zygomycetes Cunninghamella blakesleana, Mucor
1875, isolation of ergotoxine (mixture of ergocornine, hiemalis, Rhizopus arrhizus, R. nigricans
ergocristine, and ergokryptine) in 1907, and the discovery Higher plants
of the first clinically used compound, ergotamine in 1918. At Convolvulaceae Argyreia nervosa, Ipomoea argyro-
the beginning of fifties the chemistry, biosynthesis, phylla, I. rubro-coerulea, I. piurensis, Rivea
physiology, biochemistry, genetics, biotechnology, and corymbosa, Stictocardia tiliifolia
therapeutical applications of ergot alkaloids have been
extensively studied (Berde and Sturmer 1978; Mukherjee
and Menge 2000; Rehacek and Mehta 1993; Tudzynski et al.
2001). The present review gives an overview of biotechno- 3 STRUCTURE
logical potential of ergot alkaloids with a perspective for the
future.
The ergot alkaloids constitute the largest known group of
nitrogenous fungal metabolites and over 80 alkaloids have
been isolated from diverse natural material. The common part
2 SOURCES of chemical structure of the most ergot alkaloids is a tetra-
cyclic ergoline ring system (Figure 1), which is biosynthe-
In nature, the ergot alkaloids are formed primarily by various sized from tryptophan (Taber and Vining 1959), and
species of Claviceps. However, ergot alkaloids have also been mevalonic acid (Groger et al. 1961). The ergot alkaloids

91
92 Mehta et al.

natural substances having the unique structure called cyclol

(Figure 4) that consists of three aminoacids. The ergopeptines
are further divided to (a) ergotamine, (b) ergoxine, (c)
ergotoxine, and (d) b-ergoannine group (Flieger et al. 1997).

4 BIOGENESIS

Ergot alkaloids are derived from tryptophan, mevalonic acid,

and methionine (Birch et al. 1960; Groger et al. 1960; Taylor
and Ramsted 1960). The origin of the ergoline part from
Figure 1 Structure of ergoline. tryptophan and dimethylallylpyrophosphate was established
at an enzymic level. The cell free biosynthesis of
chanoclavines-I and II, agroclavine, and elymoclavine from
can be separated into three main structural groups: (a) clavine precursors has been reported by Sajdl and Rehacek (1975) and
alkaloids, (b) simple lysergic and paspalic acid derivatives, Cavender and Anderson (1970). Tryptophan has also been
and (c) peptide alkaloids (ergopeptines). The clavine found to be a factor in the induction and depression of
alkaloids are tricyclic (secoergolines) or tetracyclic (ergo- enzymes catalyzing alkaloid formation (Krupinski et al.
lines) compounds usually substituted with methyl, hydroxyl 1976). The first enzyme of alkaloid biosynthesis is
or hydroxymethyl group in position C-8 and in many cases dimethylallyltryptophan synthase (DMATS) (Heinstein et al.
have a double bond in positions 8,9 (D8,9-ergolenes) or 9,10 1971) and the encoding gene was cloned from C. fusiformis
(D9,10-ergolenes) of the ergoline skeleton (Figure 2). The and C. purpurea (Tsai et al. 1995; Tudzynski et al. 1999).
important feature of all D9,10-ergolenes (including simple Recent data of Tudzynski et al. (2001) show that in
derivatives of lysergic acid and ergopeptines) is an easy C. purpurea, all genes involved in alkaloid biosynthesis are
isomerization on C(8) resulting in formation of two isomers. organized in a cluster and are regulated by phosphate and pH
Clavine alkaloids represent the largest group of ergot level. This finding verified the previously published data on
alkaloids due to the action of various enzymes, which direct the negative role of phosphate in the induction of enzymes
agroclavine and/or elymoclavine from the main biosynthetic catalyzing the alkaloid synthesis (Krupinski et al. 1976) and
route agroclavine – elymoclavine – paspalic acid – lysergic on inhibition of alkaloid synthesis by high concentration of
acid. Such type of shunt activity is particularly evident in phosphate in the culture medium (Mehta 1984; Pazoutova
microorganisms that lack the complete pathway and are et al. 1983). Gene for peptide synthetase homologous to that
unable to synthesize substituted lysergic acid (Vining 1980). of C. purpurea was also detected in Epichloe and
The simple derivatives of paspalic and lysergic acid Neotyphodium species (Annis and Panaccione 1998;
(Figure 3) are mostly amides, in which the amide part is either Panaccione et al. 2001).
small peptide or a simple alkylamide. Amides of lysergic and Ergot alkaloid synthesis requires changes in differen-
paspalic acids found in ergot are ergometrine, lysergic acid tiation. In C. purpurea the formation of conidia is inversely
2-hydroxyethylamide, lysergic acid amide (ergine), paspalic dependent on the synthesis of alkaloids for those saprophytic
acid, and 10-hydroxy-paspalamide (Flieger et al. 1993). strains, which partially retain parasitic development, i.e.,
Ergopeptines are derivatives of lysergic acid and are the only differentiation of the sphacelial phase to the conidial or the

Figure 2 General structure of secoergolenes (1), D8,9-ergolenes (2), and D9,10-ergolenes (3).
Biotechnological Potential of Ergot Alkaloids 93

Figure 4 General structure of ergopeptines.

Figure 3 General structure of lysergic (1) and paspalic (2) acid
derivatives.

concentrated on production of lysergic acid and other

precursors of semisynthetic ergot preparations (lysergol,
sclerotial phase or both (Mehta 1984). In strains with not elymoclavine, ergine, and other simple derivatives of lysergic
clearly separated conidial phase, vegetative growth, and the acid). In the last decade the production of ergopeptines
alkaloid phase, the conidia formation occurs simultaneously (Table 1) remained confined due to their limited therapeutic
with alkaloid synthesis. The morphological development of use while the production of semisynthetic ergot preparations
the mycelium in submerged Claviceps cultures shows is gaining importance due to the development of new drugs
significant differences between high-yielding and degener- with new and more specific therapeutical applications (Berde
ated cultures (Flieger et al. 1982). The character of fat hyphae and Sturmer 1978; Eich and Pertz 1999; Pertz and Eich 1999).
and sclerotia-like cells of submerged mycelium is reminiscent It is evident from the data (Table 1) that lysergic acid is the
of the plectenchymatic structure of parasitic cultures and have main precursor of semisynthetic ergot preparations, which
been found to influence the alkaloid production in can be obtained by chemical decomposition of ergopeptines
C. fusiformis (Dickerson et al. 1970). or simple derivatives of lysergic acid. So far, very limited
Cultivation conditions convenient for primary metabolism amount of ergot preparations have been synthesized from
are not suitable for high alkaloid production (Taber and clavine alkaloids. Field production of ergot alkaloids is still an
Vining 1963). To obtain overproduction of alkaloids, high important source of ergopeptines. In the last decades, the
citrate or Krebs cycle intermediate level in the medium is major effort was devoted to selection of strains producing
required (Pazoutova et al. 1981). The inability of submerged defined spectrum of alkaloids. Recently, the average yield of
Claviceps cultures to grow on hexoses in absence of Krebs ergot reached the level of 1000 kg/ha with content of alkaloids
cycle intermediates comes from the parasitic way of life above 1% (Cvak 1999).
where the level of citrate and malate in the host plant phloem
sap is high. The rate of oxidative metabolism of saccharides
and the activity of alkaloid synthesis were proved to be
proportional (Pazoutova et al. 1981). Another important fact 5.2 Saprophytic Cultivation of Claviceps
is that clavine alkaloids are extracellular products with high
5.2.1 History
solubility in the culture medium and feedbacks regulate their
own biosynthesis (Flieger et al. 1988). Ergopeptines are The first saprophytic cultivation of Claviceps on artificial
mostly intracellular and are accumulated in lipid droplets nutritional media dates back to 1922 (Bonns 1922). The first
(oleosomes) with no influence on the metabolism (Neumann attempt for the industrial production of ergot alkaloids was
et al. 1979). isolation of clavine alkaloids from submerged cultures of
different Claviceps spp. (Abe and Yamatodani 1954; 1955;
Abe et al. 1952; 1956). Later, conditions for saprophytic
5 INDUSTRIAL PRODUCTION production of simple derivatives of lysergic acid were
developed using different strains of C. paspali (Arcamone
5.1 Industrial Production of Ergot Alkaloids et al. 1960; 1961). It took only 5 years more when new isolate
of C. purpurea was found to produce ergotamine under
The overall word annual production of ergot alkaloids was submerged conditions (Amici et al. 1966). Since that time all
estimated at about 20,000 kg (Cvak 1999) in which the types of ergot alkaloids for direct use as therapeutic agents or
production of ergopeptines and their dihydroderivatives precursors for the preparation of semisynthetic drugs can be
forms less then one third. The rest of the production is obtained by fermentation.
94 Mehta et al.

5.2.2 Strain

Synthesized from lysergic acid amide/dihydrolysergol

Field production, submerged production, synthetic
Field production, submerged production, synthetic

Hydrogenation of field/fermented a-ergokryptine

As with other fermentation processes the key to successful

Hydrogenation of field/fermented ergot mixtures

production of ergot alkaloids is in obtaining the proper strain

Hydrogenation of field/fermented ergocristine

Hydrogenation of field/fermented ergotamine of the fungus. There are three main processes used for the

Synthesized from lysergic acid/ erginine

preparation of Claviceps strains for saprophytic culture. (a)

Synthesized from lysergic acid/lysergol

Synthesized from dihydrolysergic acid

Plating of plectenchymatic tissue from the surface sterilized
sclerotia on an agar growth medium (Mantle 1969), (b)

Synthesized from dihydrolysergol

Bromination of a-ergokryptine Plating of honeydew drops containing conidia formed at early

Synthesized from lysergic acid

Synthesized from lysergic acid
Source

stage of Claviceps infection (Janardhanan and Husain 1984;

Synthesized from lisuride

Pazoutova et al. 2002), and (c) Trapping of sexual ascospores
ejected from fruiting bodies on germinated sclerotia. By this
method monosporic culture can be obtained (Vasarhelyi et al.
1980).

5.2.3 Strain Improvement

The classical methods (selection pressure, mutagenesis, and
recombination) used for the strain improvement are, to some
extent, more complicated with Claviceps due to incomplete
Serotonine antagonist, prolactin inhibitor, antiparkinsonian
Antiparkinsonian, prolactine inhibitor, cerebral vasodilator

information on cell nucleus. Strains used for saprophytic

Dopamine agonist, antiparkinsonian, prolactine inhibitor

Dopamine agonist, prolactine inhibitor, antiparkinsonian

Dopamine agonist, prolactine inhibitor, antiparkinsonian
Dopamine agonist, prolactine inhibitor, antiparkinsonian
Serotonine antagonist, antimigraine, prolactine inhibitor

cultivation might be heterokaryotic and homokaryotic

Uterotonic, antimigraine, vasoconstrictor, hemostatic

(Didek-Brumec et al. 1991; Mantle and Nisbet 1976).

Recently it was found that the number of chromosomes in
C. purpurea is variable so that haploid as well as aneuploid
Antimigraine, symphatolitic, vasoconstrict

nuclei may be encountered (Hüsgen et al. 1999). Mutagenesis

Table 1 Annual production, therapeutic use, and source of recently used ergot preparations

followed by subsequent selection of strain is an important

Therapeutic use

Sympatholitic, peripheric vasodilator

technique in increasing the yield of alkaloids (Didek-Brumec

Serotonine antagonist, antimigraine
Cerebral and peripheral vasodilator

et al. 1987). An ergocristine producing C. purpurea strain

showed 180-fold increase in alkaloid production after eight-
step mutation-selection with different mutagens (Kobel and
Sanglier 1978). Mutagenesis of sporulating strain results in
Uterotonic, oxytoxic

monosporic isolates. To increase the mutation frequency the

Uterotonic, oxytocic
Cerebral vasodilator

protoplasts prepared from spores of selected strains were used

(Olasz et al. 1982; Zalai et al. 1990). More complicated
situation is with mutagenesis of asporogenic strains. Hyphal
fragments are rather unsuitable for mutagenesis due to the
higher number of nuclei. Even protoplast formation from
young mycelium and subsequent regeneration without any
mutagenic treatment yielded strains with different properties
Annual production (kg)

(Schumann et al. 1987). Protoplast fusion technique is

beginning to find useful applications either in producing
1000 – 1500

1500 – 2000
1000 – 1500
1000 – 1500

improved mutant strains by intraspecific crosses or in

100 – 200

formation of novel spectrum of products by interspecific

500
1000
10000
50
150
50
30
10
50
30

hybrids (Socic and Gaberc-Porekar 1992). Relevant structural

and regulatory genes of the alkaloid biosynthesis in
C. purpurea form a cluster of about 50 kbp in length
(Tudzynski et al. 2001), therefore, isolation and cloning of the
entire pathway to more rapidly growing fungus would be
Dihydro-a-ergokryptine

difficult.
Dihydroergocristine
Dihydroergotamine

Methylergometrine

5.2.4 Maintenance Improvement and Long-Term

Bromokryptine
Dihydrotoxine

Methysergide

Preservation
Ergometrine

Metergoline

Cabergoline
Nicergoline
Ergotamine
Substance

Terguride
Pergolide
Lisuride

Degeneration, loss of production capabilities, is a general

problem of high-yielding strains of Claviceps (Kobel 1969).
Conservation and systematically performed selection of the
Biotechnological Potential of Ergot Alkaloids 95

isolates is the only way to eliminate the biological effects of sufficient amount of inoculum for further cultivation step.
given by the transfer of cultures, ageing, and other influences. During the inoculum preparation, an optimal state of the
Methods applied for long-term preservation were recently culture for biosynthesis of ergot alkaloids in the production
reviewed (Hunter-Cervera and Belt 1996). For preservation of step can be established (Socic et al. 1985; 1986). Usually, the
sporulating cultures two main methods are applied: deep- whole industrial process consist of four steps i.e., (a) shaker
freezing and maintaining of cultures on rye grains or agar culture, (b) preinoculating fermentation, (c) seed fermenta-
slants placed in refrigerator. Nonsporulating strains due to tion, and (d) production fermentation followed by down-
higher sensitivity to conservation procedure are frequently stream processing (Malinka 1999). Industrial production of
preserved as cultures on the agar plates. The universal clavine alkaloids, agroclavine, and elymoclavine by sub-
technique applied for preservation is keeping of lyophilized merged fermentation of different strains of Claviceps sp. was
cultures and cultures frozen, in liquid nitrogen (Baumert et al. reported in a number of patents (Kren et al. 1988; Rehacek
1979). et al. 1986; Trinn et al. 1990), reaching the maximum
production of alkaloidal mixture about 6 g/l (Pazoutova and
Tudzynski 1999; Pazoutova et al. 1987). In contrast to high
5.3 Fermentation production and well elaborated procedure of submerged
cultivation the industrial production of clavine alkaloids does
All technologies developed for industrial scale have the same not receive much attention due to limited amount of
aims: maximal production of desired ergot alkaloids, procedures leading to preparation of desirable final products
minimum amount of accompanying contaminants (other (cf. Table 1). Production of lysergic acid and its simple
alkaloids, toxins, etc.), the shortest possible time of derivatives is of high technological and industrial interest due
fermentation, minimized cost of medium, energy, equip- to relatively simple procedure of chemical modifications to
ments, and labor. For the production of ergot alkaloids, semisynthetic ergot preparations. The main producers of these
different fermentation technologies have been employed (a) alkaloids are strains of C. paspali. The basic studies on
stationary cultivation on liquid or solid medium (Kybal and biosynthesis, physiology, and production of simple deriva-
Vlcek 1976; Trejo-Hernandez and Lonsane 1993), (b) tives of lysergic acid were done on relatively limited number
submerged cultivations (Kobel and Sanglier 1986) also of strains including C. paspali 31 (Rosazza et al. 1967),
adapted to semicontinuous or continuous processes (Kopp C. paspali ATTC 13892 (Socic et al. 1986), and C. paspali
and Rehm 1984). In some cases the immobilized microor- MG-6 (Bumbova-Linhartova et al. 1991). The concentration
ganisms were used for production of ergot alkaloids under
of produced alkaloids reached nearly 3 g/l of fermentation
condition of submerged fermentation (Komel et al. 1985;
broth (Pertot et al. 1990) with the strain C. paspali L-52. One
Kopp and Rehm 1983; Kren 1991).
of the very interesting features of C. paspali strains is their
ability to convert clavine alkaloids added to the cultivation
5.3.1 Stationary Surface Cultivation medium to the simple derivatives of lysergic acid (Mothes
In the beginning of sixties, the development of processes for et al. 1962). Flieger et al. (1989a, b) and Harazim and Malinka
production of ergot alkaloids under conditions of stationary (1989) used this capability of C. paspali CCM 8061 and
cultivation on liquid media started (Adams 1962; Kybal et al. developed technology of aggressive bioconversion of clavine
1960; Molnar et al. 1964; Rochelmeyer 1965). The stationary alkaloids to simple derivatives of lysergic acid with total
surface cultivation on agar slants was commonly used for production of nearly 6 g/l in batch cultivation and about 3 g/l
preparation of starting cultures. When transformed to in industrial fermentor.
industrial scale this technology showed some limitations Different strains of C. purpurea, as the only producers of
mostly due to difficulties with control of aseptic conditions of ergopeptines, were described for their submerged production.
large surfaces. Vlcek and Kybal (1974) developed technology Ergotamine producing strains and their cultivation are the
for stationary cultivation of C. purpurea in plastic bags best-studied processes due to direct therapeutical use of
partially filled with inoculated liquid medium. This procedure ergotamine. Industrial technologies were developed for the
was used for production of ergotoxins and later adapted to following strains: (a) F.I. 32/17 producing 2 g/l of ergotamine
production of asexual spores of C. purpurea for field and a-ergokryptine mixture (Amici et al. 1966), (b) IBP 47,
production. IMET PA135 producing 1.5 g/l [mixture of alkaloids
containing 75% of ergotamine (Baumert et al. 1979)], (c)
L-4 (ATTC 20103) producing 1.5 g/l of ergotamine (Komel
5.3.2 Submerged Cultivation
et al. 1985). Another type of ergopeptines produced by
Submerged fermentation in laboratory scale, i.e., shaker submerged cultivation belongs to group of ergotoxines, i.e.,
cultivation, is a primary step in getting knowledge of ergocristine, ergocornine, and a-ergokryptine. Between many
production microorganism physiology, biosynthesis, sporula- published procedures and industrial technologies (Malinka
tion, stability, and influence of medium composition on 1999) the process developed for strain C. purpurea L-17
production of alkaloids. In industrial scale, submerged resulted in relatively high production of ergotoxines (2.4 g/l).
fermentation in shaker culture is mostly used for preparation This strain was further studied and intermediary metabolism
96 Mehta et al.

and production of secondary metabolites were correlated modified ergopeptines was described by Bianchi et al. (1982)
(Gaberc-Porekar et al. 1992). and Crespi-Perellino et al. (1992).
The growth of the fungus and alkaloid formation in
submerged batch fermentation was described by mathemat-
5.3.3 Solid Substrate Fermentation ical model, which can be further used in automatic process
Robinson et al. (2001) has recently proposed solid substrate control and optimization. Grm et al. (1980) proposed for
fermentation (SSF) for the production of enzymes and C. purpurea growth model based on morphological features
secondary metabolites. The production of ergot alkaloids by of a cell population during the fermentation process. Votruba
C. fusiformis using SSF procedure was found to be 3.9 times and Pazoutova (1981) proposed another model accentuating
higher than that obtained by submerged liquid fermentations the antagonistic effects of phosphate on growth and alkaloid
(SLF) (Hernandez et al. 1993). One of the reasons could be production. A mathematical simulation of different techno-
the necessity of use of antifoam chemicals and the shear logical alternatives of clavine alkaloid production was done
stresses caused by stirring in SLF. Also, better air circulation on this basis (Pazoutova et al. 1981). The activation–
can be achieved in SSF thus further increasing the ergot inhibition kinetics of clavine alkaloids production was
alkaloid yields (Balakrishnan and Pandey 1996). The SSF has evaluated for two C. fusiformis strains (Flieger et al. 1988)
been shown to produce a more stable, requiring less energy in and it was found that feed-back inhibition can be eliminated
smaller fermentors with easier downstream processing by combination of fermentation and separation units in a
measures. Also, by removal, the cost and trouble associated closed loop. Increased efficiency (more than 100%) of the
with antifoaming chemicals and by maximizing yield fermentation process was also found when inducers of
production, SSF may be seen as a viable option for industrial cytochrome P-450 were used (Rylko et al. 1988).
scale production of ergot alkaloids.

6 CONCLUSIONS
5.4 Optimization, Control, and Modeling of
Ergot Alkaloids Fermentation The biotechnological relevance of ergot alkaloids is due to
their therapeutical use, unquestionable. On the other hand,
The following facts should be taken into account for they play very important role as toxins in agricultural industry
optimization and scale-up of fermentation processes for the as products of endophytic fungi of the genus Neotyphodium
production of ergot alkaloids (Kobel and Sanglier 1978), (a) and their production is coupled with serious problems of
the production strains should be continually tested to maintain livestock grazing infected grasses. These two examples show
the optimal quality of selected production strain and thus the importance of molecular genetics of alkaloid biosynthesis.
maximally eliminate the biological effects given by transfer It could help, on one side, to develop new strategies for
of cultures, ageing, and other external influences, (b) long rational designing of ergot alkaloid based drugs, and, on the
cultivation period in absence of antibiotics require very high other one, to understand and control the production of ergot
standard of sterility in operation and equipment, (c) balanced alkaloids by endophytic fungi. The following points need
aeration and stirring are required due to the sensitivity of the more attention for modern production of ergot alkaloids and
Claviceps cultures to the stress and high oxygen tension, and its semisynthetic derivatives: (a) shortening of the initial
(d) the use of antifoam agents could cause considerable loss in nonproductive phase of alkaloid fermentation, (b) immobil-
alkaloid yield. Recently the application of oxygen vectors to ization of cells, (c) construction of plasmids which can carry
C. purpurea cultivation was published (Menge et al. 2001). selectable markers, (d) the development of transformation
The classical problem of the large-scale fermentations is an systems with drug resistance markers, (e) study of membrane
optimal supply of oxygen to growing Claviceps sp. High processes and vacuoles in the productive organism, (f) use of
oxygen demand in the exponential growth phase can be met mathematical modeling for description of phenomenon
by addition of different hydrocarbons (Gilmanov et al. 1996) observed during culture growth and alkaloid production,
or perfluorocarbons (Menge et al. 2001) to shake flask and/or and (g) further development of solid state fermentation and
stirred reactors. Perfluorocarbons were successfully applied application of oxygen vectors.
also in cultivations of other microorganisms (Lowe et al.
1998). Besides these technological aspects, other techniques
to optimize the fermentation process were described. The
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9
Fungi as Plant Growth Promoter and Disease Suppressor

M. Hyakumachi / M. Kubota Gifu University, Yanagido Gifu, Japan

1 INTRODUCTION plant-pathogenic fungi, however, some fungi that are not

normally considered as pathogens can also inhibit plant
Reduction of the use of fertilizers and fungicides in growth. These fungi have been termed indefinite pathogenic
agricultural production is necessary to help maintain fungi, and in one study, isolates of Eupenicillium javanicum,
ecosystems and to develop sustainable agriculture. The use Penicillium janthinellim, P. citreonigrum, and P. citrinum
of both bio-fertilizers and biocontrol systems can have obtained from roots of zinnia plant caused a 23 –57%
minimal affect on the environment and such strategies have inhibition of the growth of the same plant (Yuen and Schroth
been widely researched. In soils, numerous microorganisms 1986). Gamliel and Katan (1991) reported that almost all the
co-exist in association with plant roots. Some micro- fungi isolated from the rhizosphere and roots of tomato
organisms live specifically in rhizosphere or on plant root inhibited the growth of the plant. In contrast other soil-borne
surfaces, and these can have many effects on performance of fungi, such as Trichoderma sp., Rhizoctonia solani, and
the plant and may also affect the structure of the plant others, can promote significant plant growth. Most of these
community. A unique microflora is particularly present PGPF have a high rhizosphere competence as a character.
around the plant root surface, where various substances are Because the genera found to be PGPF are common soil-borne
secreted. Most of the microorganisms distributed around plant fungi, there is a possibility that fungi having a similar role of
root surface have a role in the decomposition of organic PGPF exist widely in natural ecosystems. Some examples of
matter and some may suppress deleterious microorganisms, plant growth promotion by PGPF are shown in Table 1. Most
which could inhibit plant growth. Some of the root-associated of these studies were quantified from the relative dry weights
microorganisms can promote plant growth, and they have of root or above-ground part of treated plant seedlings with
been called “plant growth-promoting rhizobacteria” (PGPR; PGPF compared to nontreated ones over periods as short as 4
Kloepper et al. 1980) or “plant growth-promoting fungi” weeks. In some cases, significant growth promoting effects of
(PGPF; Hyakumachi 1994). The PGPR and PGPF are known PGPF were observed as increased yield of plants grown in
to suppress some plant diseases. Similar effects are also fields over longer periods of 14 weeks or more (Shivanna et al.
observed in plants treated with mycorrhizal fungi, which have 1994).
a symbiotic relationship with most plant species. Endophytes
can also promote plant growth and these have recently been
considered as potential biological control agents. In this 2.1.1 PGPF in Trichoderma
chapter, fungi as known as PGPF, mycorrhizal fungi, and Isolates of Trichoderma harzianum and T. koningii have been
endophytic fungi, which act as plant growth promoters and shown to enhance seedling emergence in tomato with
disease suppressors are considered. increased shoot and root dry weights when compared to
nontreated control plants (Table 1) (Windham et al. 1986).
2 FUNGI AS PLANT GROWTH PROMOTER These species also gave rise to increased shoot and root dry
weights in tobacco (Table 1) (Windham et al. 1986). Isolates
2.1 PGPF of T. viride have been reported to increase tomato plant height
(Windham et al. 1986). Chang et al. (1986) have shown that
Many fungi isolated from soil can inhibit plant growth. isolates of T. harziamum enhanced seedling emergence in
Generally, the inhibition of plant growth is mostly caused by chilli pepper and promoted growth of tomato, chilli pepper,

101
 102

Table 1 Growth promotion on plants treated with plant growth-promoting fungi (PGPF)

Fungus Crop Growth promoting effect References

T. harzianum Tomato 2.1 – 2.8 times Increased dry weight, enhanced germination Windham et al. (1986)
Tobacco 7.9 times Increased dry weight, enhanced germination Windham et al. (1986)
Red pepper Enhanced germination Chang et al. (1986)
Periwinkle Enhanced germination Chang et al. (1986)
Bentgrass 9.1 times Increased dry weight and plant height Hyakumachi (1994)
T. koningii Tomato 2.6 – 3.2 times Increased dry weight, enhanced germination Windham et al. (1986)
Tomato 5.1 times Increased dry weight Hyakumachi (1994)
Tobacco 2.7 times Increased dry weight, enhanced germination Windham et al. (1986)
Ryegrass 4.4 times Increased dry weight and plant height Hyakumachi (1994)
T. ciride Tomato Increased plant height Windham et al. (1986)
Sterile black fungus Wheat 40% Increased shoot dry weight Speakman and Kruger (1984)
Rye 40% Increased shoot dry weight Speakman and Kruger (1984)
Sterile dark fungus Wheat 30% Increased shoot dry weight Narita and Suzui (1991)
Sterile red fungus Wheat 10 – 60% Increased shoot fresh weight Dewan and Sivasithamparam (1989)
Rye 10 – 60% Increased shoot fresh weight Dewan and Sivasithamparam (1989)
Ryegrass 10 – 60% Increased shoot fresh weight Dewan and Sivasithamparam (1989)
R. solani AG4 Radish 13.4 – 19.8% Increased shoot fresh weight Sneh et al. (1986)
28.4 – 36.0% Increased shoot dry weight Sneh et al. (1986)
Carrot 80.0 – 97.7% Increased shoot fresh weight Sneh et al. (1986)
55.0 – 150.5% Increased shoot dry weight Sneh et al. (1986)
Lettuce 58.4% Increased shoot fresh weight Sneh et al. (1986)
Cotton 28.7% Increased yield Sneh et al. (1986)
R. nigricans Tomato 42% Increased shoot dry weight Lindsey and Baker (1967)
F. roseum Tomato 54% Increased shoot dry weight Lindsey and Baker (1967)
Phoma sp. Wheat 46 – 77% Number of grain Shivanna et al. (1994)
Bean 23 – 25% Increased yield (Green house) Shivanna et al. (1994)
11 – 52% Increased yield (Field) Shivanna et al. (1994)
Cucumber 2.1 times Increased fresh weight (6 weeks) Hyakumachi (unpublished)
1.8 times Increased fresh weight (10 weeks) Hyakumachi (unpublished)
Hyakumachi and Kubota
Plant Growth Promoter 103

and cucumber. Isolates of T. harziamum have also been used nutrient-deficient one. The effect of PGPF was also observed
to enhance flowering of periwinkle and to increase the in soil that had been converted to nutrient-rich by amendment
number of flowers per plant in chrysanthemum. with NPK fertilizer.
The duration of the plant-growth promoting effect of
2.1.2 PGPF in Mycelial Fungi PGPF in the treated plants is an important factor for the earlier
application. Increased growth responses were observed in
Plant growth promotion has been obtained from isolates of wheat treated with PGPF isolates during the seedling stage
mycelial fungi that do not produce any spores. These (2 weeks after sowing), vegetative stage (4 weeks), pre-
unidentified fungi have been termed sterile black fungus flowering stage (10 weeks), and seed maturation stage (14
(SBF), sterile dark fungus (SDF), and sterile red fungus weeks) (Hyakumachi 1994; Shivanna et al. 1994). All of the
(SRF), and have been isolated from corn roots, wheat roots, isolates used increased plant height, and also significantly
and wheat and rye grass roots, respectively. Unidentified increased the ear-head length, weight, seed number, and
isolates considered to be SBF (Speakman and Kruger 1984) biomass (Table 1) (Shivanna et al. 1994).
and SDF (Narita and Suzuki 1991) were shown to increase In order to develop applications, it is important to isolate
shoot dry weight in wheat and similarly isolates termed to PGPF strains that (a) have high affinity for plants and can
SRF increased shoot wet weight in wheat (Dewan and colonize their rhizosphere, (b) show high levels of plant
Sivasithamparam 1989). Growth promotion by these growth promotion, and (c) offer consistent performance in
unidentified fungi has been reported in other plants and field trials. Although an isolate with a wide host range is an
isolates of SBF have been reported to increase shoot dry ideal candidate, there is a requirement for isolates that show
weight in barley (Speakman and Kruger 1984). Fungi high specific effectiveness with an individual host plant.
considered as SRF have been reported to promote plant
growth of rye, brome grass, chick pea, lupine, medic, pea,
ryegrass, and clover, all of which are used as typical rotation 2.2 Mechanisms of Plant Growth Promotion by
crops with wheat, and resulted in an increased shoot fresh PGPF
weight (Dewan and Sivasithamparam 1989). The mycelial
isolates used in these studies have not been identified, Several hypotheses have been put forward for the mechanisms
although isolates of SRF are thought to be Basidiomycetes of plant growth promotion by PGPF, including (a) hormone
because of the presence of clamp connection. Strains of SBF production, (b) substrate degradation (mineralization), and (c)
and SDF are easily isolated from herbal plants as well as suppression of deleterious microorganisms.
woody plants and their relationships to endophytic fungi are
being currently considered. 2.2.1 Hormone Production

2.1.3 PGPF in Rhizoctonia Culture filtrates of certain fungal species promote plant
growth, due to the production of plant growth hormones by
A particular nonpathogenic strain of R. solani has shown these fungi (Ram 1959). Growth promotion has been seen in
growth promotion and significantly increased yield for some plants after treatment with mycelial exudates from
various crops in field experiments (Sneh et al. 1986). These PGPF strains of Trichoderma and SRF, and a gibberellin-like
included increased wet and dry weights of radish roots and substance was reported to be involved (Gillespie-Sasse et al.
carrot roots, and increased weights of cotton fiber and wheat 1991; Windham et al. 1986). Some strains of Phoma species
grains. In similar experiments with potato, although increases have been found to produce abscisic acid, and this compound
were observed in shoot and tuber weight until 63 –70 days is also reported to promote plant growth. However, in general
after transplanting, there was no increase in yield at the time terms, these appear to have little relationship between the
of harvest. Some isolates of binucleate Rhizoctonia have been production of plant growth hormones and the ability of PGPF
found to be PGPF (Harris et al. 1993; Villajuan-Abgona et al. to promote plant growth.
1996).
2.2.2 Mineralization
2.1.4 Other PGPF
Close relationships have been shown between the reduction of
Isolates of Rhizopus nigricans and Fusarium roseum have barley grain weight due to PGPF, the subsequent growth
been reported to increase shoot dry weight in tomato (Lindsey promotion effect of PGPF, and their cellulase and starch
and Baker 1967). Soil conditions such as pH, water, nutrient degration activity (Hyakumachi 2000). Production of NH4-N
and organic content, together with the presence of other and NO3-N in soil can also be increased by amendment with
micro-organisms are important considerations for the PGPF-infested barley grains.
introduction of beneficial micro-organisms into soil. Hyaku- The total amount of nitrogen in PGPF infected-barley
machi (1994) reported the plant growth promotion effect of grains remains the same despite which PGPF isolate is used,
PGPF occurred in sterilized or nonsterilized nutrient-deficient however, the amount of NH4-N varies depending on the
and rich soils, potting soil, and most conspicuously in the isolate, with the highest level being seen in grains infected
104 Hyakumachi and Kubota

with Phoma. The NH4-N levels later decreased in the inoculations and their growth promoting performances were
order Phoma . Fusarium . Penicillium . Trichoderma . dependent on the host plant. Large increases in the growth of
control: The amount of NH4-N was about 7.8 times higher pines have been recorded in field experiments. For instance,
in Phoma infested-barley grains than that of control 25–100% increases in growth have been reported for three
(Hyakumachi 2000). Hyakumachi (2000) also demonstrated pine species inoculated with Pisolithus tinctorius on five
correlations between reduction of barley grain weight and reforestration sites in the southern United States (Marx et al.
cellulase activity, starch degradation activity of starch, and 1977). Inoculation with Paxillus involutus has been
the dry weight of bentgrass. These results suggest that the associated with a marked increase in stem diameter and
mineralization of organic substrates by PGPF relates to the volume, especially with sessile oak at Bouxières where the
plant-growth promoting effect of those PGPF. The PGPF volume almost doubled over 7 years (Garbaye and Churin
may therefore provide the plant with necessary mineral 1997).
nutrients in an easily assimilating form. The increase in growth resulting from inoculation with
mycorrhizal fungi has been attributed to improved nutrition of
2.2.3 Suppression of Deleterious Microorganisms the host plant in most cases. Ectomycorrhizal fungi are able to
absorb and accumulate phosphorus, nitrogen, potassium, and
A remarkable plant growth promotion effect has been calcium in the fungal mantles more rapidly and for longer
reported for field-grown cucumbers, and this was attributed to periods of time than nonmycorrhizal feeder roots. Ecto-
the suppression of indigenous pathogenic Pythium spp. in the mycorrhizal fungi improve the efficiency of phosphorus
soil by PGPF (Hyakumachi 1994). The suppression of uptake principally through the development of extramatrical
deleterious microorganisms by PGPF may therefore be one of hyphae, which increase the absorptive surface and effective
the mechanisms of plant-growth promotion. rooting density of the plant. Ectomycorrhiza are likely to
enhance N uptake where the fungus and host plant differ in
their capacity to absorb and assimilate NO3-N. Mycorrhizal
2.3 Mycorrhiza fungi generally have a preference for NH4-N, although a
number of species can also utilize NO3-N (Plassard et al.
As a definition of mycorrhizae, Smith and Read (1997) 1991).
proposed “a symbiosis in which an external mycelium of a
fungus supplies soil derived nutrients to a plant root.” 2.3.2 Arbuscular Mycorrhiza
Mycorrhizae are further divided into six types based on
anatomical characteristics, which are: (a) arbuscular mycor- Arbuscular mycorrhizal (AM) associations are due to
rhizae (AM), (b) ectomycorrhizae, (c) orchid mycorrhizae, (d) Glomales, an order of Zygomycetes (Morton and Benny
ericoid mycorrhizae, (e) monotropoid mycorrhizae, and (f) 1990). The order consists of 7 genera, Glomus, Entro-
arbutoid mycorrhizae. Some plants have requirement for phospora, Acaulospora, Archaeospora, Paraglomus,
mycorrhizae in order to complete their life cycle. Mycor- Gigaspora, and Scutellospora. Arbuscular mycorrhizal
rhizae may influence host plant survival in regeneration fungi develop arbuscules or hyphal coils within host plant
niches and mycorrhizae can also increase seed production, cortical cells, and have a wide host range including many
seed quality, and host and offspring vigor. Some types of agricultural and horticultural crops worldwide. Growth
mycorrhizae enhance host plant resistance against severe promotion has been seen in many AM-associated plants
environmental conditions. The most widely studied and including maize (Baltruchat 1987), tomato (Mohandas 1987),
most-commonly encountered mycorrhizal systems are the asparagus (Pedersen et al. 1991), Boston fern (Ponton et al.
ectomycorrhizae and arbuscular mycorrhizae. 1990), and gerbera (Wang et al. 1993). Despite the
morphological differences between ecto- and arbuscular
2.3.1 Ectomycorrhizae mycorrhizae, there appear to be many common features in
their growth-promoting effects. Arbuscular mycorrhizal fungi
About 5000 fungi, Asco- and Basidiomycetes, are known to develop extraradical hyphae that grow into the surrounding
form ectomycorrhizal association with about 2000 species of soil, increasing the potential of the root system for nutrient
woody plants (Kendrick and Berch 1985). Roughly 5% of the and water absorption, and improving the soil structure for
vascular plants are known to develop ectomycorrhizae and better aeration and water penetration. One of the mechanisms
these associations are typically seen by the intercellular of growth promotion by AM fungi involves the transport of
development of Hartig nets. Ectomycorrhizal fungi are known phosphorus by AM fungi from the soil to the plant. Direct
to enhance the uptake of water and nutrients by the host plant, measurements of phosphorus transfer by AM fungal hyphae
and to promote plant growth. Growth effects have been have been made by Jakobsen (1994) and Schweiger et al.
observed in a broad range of forest trees, such as Douglas fir, (1999). Colonization of roots by AM fungi modifies the
pine, and eucalyptus, with ectomycorrhiza associations growth response of the plant and increases supplies of
forming in nurseries and in the field. Laccaria laccota, phosphorus (Abbott et al. 1995), however, some studies have
Pisolithus tinctorius, Suillus plorans, Hebeloma cylindro- shown that effectiveness, in terms of plant growth promotion,
sporum, and H. crustuliniforme have been used as soil is not related to the extent of host root colonization (Jensen
Plant Growth Promoter 105

1982; Sanders and Fitter 1992). Efficient phosphorus uptake damping-off disease caused by virulent R. solani and R. zeae
has been found to be more closely related to the quantity of by 76 –94% on cotton, radish, and wheat. The sterile fungi,
mycelium partitioned into the extraradical phase of the fungi SBF, SDF, and SRF, have been shown to decrease the
(Abbott and Robson 1985; Morin et al. 1994). Jakobsen et al. occurrence of take-all disease of wheat caused by
(2001) reported that the phosphorus transport capacity of AM Gaeumannomyces graminis var. tritici (Dewan and Siva-
fungi is related not only to colonization rate, but also to the sithamparam 1989; Narita and Suzuki 1991; Speakman and
transport character of AM fungi themselves. The AM fungi Kruger 1984). The PGPF isolated from zoysiagrass rhizo-
cause few changes to root morphology, but the physiology of sphere have been shown high suppressive ability against
the host plant may change significantly. Tissue concentrations soil-borne diseases caused by Pythium aphanidermatum,
of growth-regulating compounds and other chemical con- P. irregulare, R. solani, Sclerotium rolfsii, Fusarium
stituents change, photosynthetis rates increase, and the oxysporum f. sp. melonis, F. o. f. sp. cucumerinum,
partitioning of photosynthate to shoots and roots changes G. graminis var. tritici and Cochliobolus sativus
(Bethlenfalvay 1992). Allen et al. (1980) demonstrated (Hyakumachi 1994). When cucumber plants inoculated with
differences in cytokinin content between Bouteloua gracilis PGPF isolates from zoysiagrass rhizozphere, disease sup-
plants with and without associated Glomus fasciculatus. They pression was observed against the air-borne pathogen,
also reported quantitative and qualitative changes in GA-like Colletotrichum orbiculare (Meera et al. 1994). In this work
substances in the leaves and roots of AM-associated plants the PGPF were applied to plant roots and leaves were used for
(Allen et al. 1982). Incleases in auxin, cytokinin, GA and pathogen inoculation thereby the PGPF and pathogen were
B-vitamin production have also been reported in plants physically separated. The result therefore suggests that
associated with ectomycorrhizal fungi (Crafts and Miller induced systemic resistance is involved as one of the
1974; Slankis 1973; Strzelczyk et al. 1977). mechanisms for disease suppression by PGPF. The PGPF
isolates from zoysiagrass rhizosphere, Trichoderma,
Fusarium, Penicillium, Phoma, and sterile fungi, all provided
2.4 Endophyte significantly protection to air-borne anthracnose caused by
C. orbiculare, bacterial angular leaf spot caused by
A widely accepted definition of an endophyte is; “endophytes Pseudomonas syringae pv. lacrymaus, and soil-borne
symptomlessly colonize the living, internal tissues of their Fusarium wilt by F. oxysporum f. sp. cucumeris (Koike et al.
host, even though the endophyte may, after an incubation or 2001). In the case of Fusarium wilt, a split-root system was
latency period, cause disease” (Petrini 1991). This definition used to ensure physical separation of PGPF and pathogen, and
includes virtually any microbe that colonizes the internal to assess induced resistance.
tissues of plants. For example, some plant-pathogenic fungi,
such as the smut fungi, can be defined as endophytes unless
the plant shows symptoms after the infection (Stone et al. 3.2 Mycorrhizae
2000). Endophytes are generally known to enhance plant
tolerance to environmental stresses, damage from harmful Initial evidence for the role of ectomycorrhizal fungi in
insects, and diseases caused by pathogens and nematodes. disease suppression was provided by a number of field
There are a few studies on the plant-growth promoting effect observations that showed mycorrhizal-associated seedlings or
of endophytic fungi. Yetes et al. (1997) observed a slight but trees of both angiosperms and gymnosperms were more
significant increase in plant weight, shoot height, and shoot resistant to root pathogens than their nonmycorrhizal
diameter in Fusarium moniliforme-infected plants, 28 days counterparts (Marx 1973). Ectomycorrhizal roots of various
after planting compared to uninoculated control plants. Pinus Pinus spp. and Sitka spruce (Picea sitchensis) seedlings were
contorta inoculated with Phialocephala fortinii increased resistant to infection by a Rhizoctonia sp. that could readily
uptake of phosphorus and nitrogen, that resulted in enhanced infected nonmycorrhizal feeder roots (Levisohn 1954).
growth of inoculated plants compared with noninoculated Richard et al. (1971) suggested that the presence of
plants (Jumpponen and Trappe 1998). ectomycorrhizal fungus, Suillus granulatus in the substratum
completely prevented any negative effect of endophytic
Mycelium radicis-atrovirens on Picea mariana seedlings.
3 FUNGI AS DISEASE SUPPRESSOR Hashimoto and Hyakumachi (2001) also suggested that
the ectomycorrhizal fungi suppressed the deleterious effect
3.1 PGPF of endophytic M. radicis-atrovirens on Betula platyphylla
var. japonica seedlings.
Almost all the PGPF reported so far have shown a pronounced Arbuscular mycorrhizae associations have been shown to
suppressive effect against soil-borne diseases. One example reduce damage caused by soil-borne plant pathogens.
of this is the suppression by Trichoderma harzianum of Although few AM isolates have been fully studied, some
damping-off disease on barley, cucumber, radish, and tomato appear to be more effective than others. Furthermore, the
caused by Pythium ultimum (Ahmad and Baker 1988). degree of protection varies with the pathogen involved, and
Nonpathogenic R. solani AG4 has been reported to suppress can be modified by soil types and other environmental
106 Hyakumachi and Kubota

conditions. Trotta et al. (1996) reported that the AM fungus, 4 MECHANISMS OF DISEASE
Glomus mosseae, reduced adventitious root necrosis and SUPPRESSION BY FUNGI
necrotic root apices caused by Phytophthora nicotianae var.
parasitica by 63 –89%. The AM associations are also 4.1 Antagonism
known to limit the damage by bacterial pathogens and
pathogenic root nematodes (Garcia-Garrido and Ocampo Many reports have shown that the growth-promoting effect of
1989; Hussey and Roncadori 1982), however, the results are PGPF is due to their ability to suppress harmful micro-
not consistent. organisms in the soil. It is generally accepted that
hyperparasitism, antibiosis, and competition are all involved
in the antagonistic activities of PGPF. The mechanisms of
3.3 Endophyte disease suppression by PGPF isolated from zoysiagrass are
shown in Table 2. The isolates of PGPF did not show
The enhanced resistance for disease shown by some hyperparasitism to other fungi. In some cases involving
endophyte-infected plants is generally considered to result Trichoderma there was a relation between antibiotic activity
from the production of defense compounds by the endophyte- and disease suppression, however in most cases, disease
plant infection. An example of this is the inoculation of maize suppression was closely related with the ability to compete for
kernels by endophytic Fusarium monilimorme, which is infection courts or nutrient on the surface of the plant root.
reported to protect against infection by pathogenic The production of antagonistic substances is thought to be
F. graminearum (Van Wyck and Scholts 1988). Nonpatho- one of the mechanisms of protection provided by ecto-
genic, endophytic strains of F. oxysporum isolated from mycorrhizal fungi. As an example of this, antibacterial
suppressive soils have been used as biological control agents activities have been demonstrated for Paxillus involutus and
for manage diseases caused by pathogenic Fusarium species Hebeloma crustuliniforme in pure culture (Marx 1973) and
on watermelon, cucumber, celery, and other crops (Larkin for Cenococcum graniforme in mycorrhizal symbiosis
et al. 1996; Schneider 1984). In each case, these fungi were (Krywolap et al. 1964). The antibiotic effect of mycorrhizal
endophytes of the hosts they protected. Endophytic Hetero- fungi was attributed to the production of organic acids as
conium chaetosprira is reported to almost completely demonstrated for P. involutus (Duchesne et al. 1989). Olsson
suppress clubroot formation in Chinese cabbage caused by et al. (1996) demonstrated that presence of the ecto-
Plasmodiophora brassicae (Narisawa et al. 1998). mycorrhizal mycelium decreased bacterial activities as

Table 2 Mechanisms of disease suppression against pathogenic fungi and bacteria by plant growth-promoting fungi (PGPF) isolated
from zoysiagrass

Pathogenic fungi PGPF Hyperparasitism Antibiosis Competition Induced resistance

R. solani T. harzianum 2* ^ þ NT**
Phoma sp. 2 2 þ NT
F. equiseti 2 2 þ NT
P. irregulare T. harzianum 2 ^ þ NT
Phoma sp. 2 2 þ NT
F. equiseti 2 2 þ NT
S. rolfsii T. harzianum 2 ^ þ NT
Phoma sp. 2 2 þ NT
F. equiseti 2 2 þ NT
F. oxysporum f. sp. cucumerinum T. harzianum 2 ^ 2 þ
Phoma sp. 2 2 þ þ
F. equiseti 2 2 2 þ
C. orbiculare T. harzianum 2 ^ 2 þ
Phoma sp. 2 2 2 þ
F. equiseti 2 2 2 þ
P. simplicissimum 2 2 2 þ
P. syringae pv. lachrimans T. harzianum 2 ^ 2 þ
Phoma sp. 2 2 2 þ
F. equiseti 2 2 2 þ
P. simplicissimum 2 2 2 þ
* þ /2: Effective/not effective; ** NT: Not tested.
Plant Growth Promoter 107

measured by using the thymidine incorporation technique. under glass house conditions and for up to 6 weeks under field
Zak (1964) suggested that ectomycorrhizal fungi may: (a) conditions (Meera et al. 1995). Lignin deposition is known as
utilize surplus carbohydrates in the root thereby reducing the one of the mechanisms of induced systemic resistance
amount of nutrients stimulatory to pathogens, (b) provide a (Hammaerschimidt and Kuć 1982). Koike et al. (2001)
physical barrier, i.e., the fungal mantle, to penetration by reported that lignification of cucumber seedling hypocotyls
pathogen, (c) secrete antibiotics inhibitory to pathogens, and was induced by culture filtrates of PGPF, following challenge
(d) support along with the root, a protective microbial inoculation with C. orbiculare. The result showed enhanced
rhizosphere population. Marx (1969) suggested that inhibitors lignin deposition in cucumber after infection by C. orbiculare
produced by symbiotically infected host cortical cells may as compared to the control. The elicitor activity of culture
also have a function as inhibitors of the infection and spread filtrates of PGPF has been evaluated by chemiluminescence to
of pathogen in ectomycorrhizal roots. determine the emission of active oxygen species from tobacco
Competition for colonization sites, direct antibiosis, callus and cucumber fruit disks (Koike et al. 2001). The
nutritional aspects, and plant defense reaction have all been oxidative burst is characterized by a rapid and transient
considered as possible mechanisms in disease suppression by generation of active oxygen species immediately following
AM fungi (Azcon-Aguilar and Barea 1996). However, these fungal elicitor treatment. From these results, the
mechanisms are still poorly understood. . 12,000 MW fraction and both . 12,000 MW fraction and
Antifungal activities are thought to be involved in lipid fraction from the culture filtrate elicited the highest
mechanisms of disease suppression by endophytic fungi. superoxide generation, respectively. A high correlation
The endophytic fungi Neotyphodium coenophialum and between superoxide generation ability and lignification
N. lolii have been shown to form an inhibition zone under ability was reported.
dual culture with the pathogenic fungi, Colletotrichum Localized and induced-systemic resistance against Phyto-
graminicola, Rhizoctonia cerealis, R. zeae, etc. (Siegel and phthora parasitica caused by the AM fungi, Glomus mosseae,
Latch 1991). These results suggest that these endophytic fungi has been observed in tomato roots (Cordier et al. 1998). The
produce antifungal substances. Volatile compounds were phenomena were demonstrated by use of a split-root
collected from both endophyte-infected and endophyte-free experimental system. Decreased pathogen development in
tall fescue, and the sheath of endophyte-infected plants was
mycorrhizal and nonmycorrhizal parts of the root system was
found to produce high levels of 1-octen-3-ol, a characteristic
associated with an accumulation of phenolics and plant cell
fungal toxic volatile compound derived from lipid per-
defense responses. G. mosseae-containing cortical cells in the
oxidation in fungi, which was absent in endophyte-free plants
mycorrhizal tissues were immune to the pathogen infection
(Yue et al. 2001). Hydroxamate siderophore synthesis by
and exhibited a localized resistance response with the
P. fortinii, a typical dark septate fungal endophyte, was
formation of cell wall appositions reinforced by callose
reported by Bartholdy et al. (2001). Iron is an essential
adjacent to intercellular hyphae. The systemically induced
micronutrient for almost every organism and siderophore
synthesis by P. fortinii may be expected to play a key role in resistance in nonmycorrhizal root parts was characterized by
iron nutrition for the plant, resulting in a lack of available iron elicitation of host wall thickenings containing nonesterified
for pathogens. pectins and PR-1a protein in reaction to the intercellular
hyphae of the pathogen. Systemic resistance was also
characterized by the formation of callose-rich encasement
4.2 Induced Resistance material around P. parasitica hyphae that were penetrating
root cells and PR-1a protein was detected in the pathogen wall
Induced-systemic resistance has been observed on cucumber only in these tissues. None of these cell reactions were
plants treated with PGPF from zoysiagrass (Hyakumachi observed in nonmycorrhizal pathogen-infected root systems,
1997; Koike et al. 2001; Meera et al. 1994). Almost all of where disease development resulted in host cell death.
these PGPF could induce resistance against anthracnose in Increased chitinase activities have also been reported in AM
cucumber. In contrast, Ishiba et al. (1981) reported that only symbiosis as part of the induced defense reaction by
1.9 – 2.4% of the soil fungi isolated from cucumber these mycorrhizal fungi. Pozo et al. (1999) studied
rhizosphere, were able to induce systemic resistance against b-1,3-glucanase in tomato roots which were either
anthracnose in cucumber plants. Different types of PGPF colonized by AM fungi and/or infected by the pathogen
have been isolated from all over the world and it would be Phytophthora parasitica. b-1,3-glucanase activity was
interesting to know if any of these have as high performance higher in mycorrhizal roots compared to the non-
of induced systemic resistance as PGPF isolated from mycorrhizal roots. Nonmycorrhizal roots infected by
zoysiagrass. Recently, induced systemic resistance caused by P. parasitica showed high levels of activity but the
binucleate Rhizoctonia and Trichoderma has been reported in pathogen did not induce b-1,3-glucanases in AM colonized
plants. Some isolates within these fungi have growth- roots. There was strong evidence to suggest that these
promoting ability and others have been used as biological hydrolases are antifungal proteins. Increased chitinase
control agents. The induced resistance on cucumber caused by activities have also been reported in ectomycorrhizal
PGPF isolated from zoysiagrass can last as long as 9 weeks symbiosis (Albrecht et al. 1994; Sauter and Hager 1989).
108 Hyakumachi and Kubota

5 CONCLUSIONS Baltruchat H (1987). Evaluation of the suitability of expanded clay as

carrier mycelial for VA mycorrhizal spores in field inoculation
of maize. Angew Bot 61:163 –169.
The original purpose of isolating beneficial microorganisms
Bartholdy BA, Berreck M, and Haselwandter K (2001). Hydro-
from soil, especially from the rhizosphere of plants, was to xamate siderophore synthesis by Phialocephala fortinii, a
obtain microorganisms, which showed a growth promotion typical dark septate fungal root endophyte. BioMetals
effect in plants. In addition to this effect, subsequent studies 14:33 – 42.
have investigated direct suppressive effects on pathogens. Bethlenfalvay GJ (1992). Mycorrhizae and crop productivity. In:
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focused on PGPF, mycorrhizal fungi, and endophytic fungi, Chang YC, Baker R, Kleifeld O, and Chet I (1986). Increased growth
all of which show efforts as plant growth promoters and of plants in the presence of the biological control agent
disease suppressors, and the mechanisms of mineralization, Trichoderma harzianum. Pl. Dis. 70:145– 148.
hormone production, antagonism, and induced resistance Cordier C, Pozo MJ, Barea JM, Gianinazzi S, and Gianinazzi-
Pearson V (1998). Cell defecse responses associated with
have been considered. These mechanisms are commonly
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suppression by the ectomycorrhizal fungus Paxillus involutus:
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10
Challenges and Strategies for Development of Mycoherbicides

Susan M. Boyetchko / Gary Peng Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan,
Canada

1 INTRODUCTION detail some of the limitations associated with bioherbicides

(Auld and Morin 1995; Makowski 1997; Mortensen 1998),
The application of biological control for the management including biological, environmental, and technological con-
of weed populations has generally been viewed as an straints. Critics have attributed these constraints to the lack of
environmentally sound additional approach to chemical further development of many biocontrol agents. This review
herbicides (Boyetchko et al. 2002; Mortensen 1998; Rosskopf will provide an update on the status of mycoherbicide research,
et al. 1999). Bioherbicides are often described as the summarize some of the challenges encountered, and provide
intentional use of plant pathogens that are mass-produced, some thoughts on potential new approaches that may be used to
formulated, and applied at high inoculum rates in a similar address these challenges in order to advance the development
fashion as chemicals. Although a variety of microbial agents of promising mycoherbicide candidates.
may be used, host-specific fungal pathogens often referred to
as mycoherbicides, have been studied more extensively for
biocontrol of weeds. In comparison, classical biological 2 STATUS OF BIOHERBICIDES
control involves the importation of natural enemies and relies
on the natural survival, dissemination, and self-perpetuation of Several recent reviews have provided an overview on various
the living agent for control of weeds below ecological bioherbicide projects being conducted around the globe
thresholds. The classical approach is often considered more (Boyetchko 1999; Boyetchko et al. 2002; Charudattan 2001;
appropriate for low management systems such as pasture and Rosskopf et al. 1999). Eight bioherbicides have been
rangeland where site disturbance is minimal, while bio- registered in various countries over the last two decades
herbicides are ideal for single-season management of with several other microbial candidates in various stages of
agricultural and forest weeds where site disturbance is the evaluation and development (Table 1). Devinew and
norm. Despite many economic, social, and environmental Collegow, the first mycoherbicides registered in the United
benefits ascribed to biological control, it is reasonable to ask States are currently marketed by Encore Technologies
why more bioherbicide or mycoherbicide products are yet to (Minnetonka, MN), while Stumpoutw, a wood-decaying
become widely available in the marketplace. Many researchers fungus used to control resprouting of Acacia spp., is
would argue that there has been a great deal of progress, with commercially available in South Africa. Chondrostereum
several additional microbial agents identified as potential purpureum is a wound pathogen that reduces regrowth of
bioherbicides and innovative improvements in mass-pro- competing hardwood tree species and is marketed as
duction, formulation, and application of living organisms. BioChone by Koppert Biological Systems in the Nether-
Despite the various accomplishments by researchers world- lands. A Canadian strain of the pathogen is also currently
wide (Boyetchko et al. 2002; Charudattan 2001), the question undergoing registration approval through the Canadian Pest
remains whether we have made significant advancements in Management Regulatory Agency and U.S. EPA and will be
bioherbicide research that would facilitate increased adoption sold as Chontrolw by MycoLogic (W.E. Hintz, MycoLogic,
of this technology. Several reviews have discussed in great Inc., personal communication). Another mycoherbicide

111
112 Boyetchko and Peng

Table 1 Examples of mycoherbicide agents at various stages of development and commercialization

Status Pathogen (Trade Name w or e) Target weed Country

Commercially available C. gloeosporioides f. sp. aeschynomene (Collegow) Northern Jointvetch USA

P. palmivora (Devinew) Stranglervine USA
C. laeve (Stumpoutw) Black & golden wattle South Africa
C. purpureum (BioChone) Hardwood tree species Netherlands
Registered, not commercially available C. gloeosporioides f. sp. malvae (Mallet WP)a Round-leaved mallow Canada, USA
P. canaliculata (Dr. Biosedgew) Nutsedges USA
Precommercial development C. purpureum (Chontrolw) Hardwood tree species Canada, USA
A. destruens (Smolderw) Dodder USA
a
Originally registered in Canada as BioMalw by Philom Bios; licensed to Encore Technology for registration as Mallet WP in Canada and the United
States. Mycoherbicide not being further developed due to technical difficulties in mass production.

currently undergoing review is Alternaria destruens, under populations, similar to chemical herbicides (Auld and
the name Smolderw, for control of dodder. Other examples of Morin 1995). The challenges that have limited the
mycoherbicides have been discussed in greater detail by advancement of bioherbicides have been categorized into
Boyetchko (1999), Boyetchko et al. (2002), and Rosskopf four constraints: (a) biological, (b) environmental, (c)
et al. (1999). Charudattan (2001) has also compiled a technological, and (d) commercial. While the commercial
comprehensive list of bioherbicide projects worldwide. While consideration is important, this review will focus on
the number of commercial bioherbicides appears to be addressing the other three constraints. Researchers can
limited, Charudattan (1991) calculated a success ratio of 20:1 make pragmatic decisions on the selection of an appropriate
for bioherbicide development compared to the success rate of target weed that may have impact on the market decisions
less than 1% for chemical herbicide compounds evaluated by industry to invest in the development of bioherbicide
and developed by chemical companies. This success ratio agents, but the regulatory environment for registration of
begins to look even more encouraging for bioherbicides when such products is often affected by political will and/or
developmental costs are taken into consideration. policy of individual governments.
Charudattan (2001) estimated that the resources and capital
required for a chemical company to conduct research and
development and register chemical herbicides is approxi- 3.1 Biological Factors
mately US$ 50 million in comparison to US$2 million for
bioherbicides. Nevertheless, a very small portion (i.e., less Weeds are inherently variable by nature, with many weed
than 1%) of the commercially available weed control products species possessing several biotypes. For this reason, the
are represented by bioherbicides and investment in these genetic diversity of weed populations can present several
microbial-based products has largely been by small-to- challenges when researchers evaluate specific isolates or
medium sized enterprises. Government and university strains of fungal pathogens (Auld and Morin 1995; Boyetchko
institutions have invested infrastructure and expertise in this et al. 2002). Such is the case with yellow nutsedge that was
area and it has been through such efforts that this technology found to have extensive genetic variability within and
has been transferred to industry. between populations and, therefore, greater variability in
susceptibility to the bioherbicide agent Puccinia canaliculata,
compared to purple nutsedge populations (Horak et al. 1987;
3 CHALLENGES IN BIOLOGICAL Okoli et al. 1997). The success of Colletotrichum gloeo-
CONTROL sporioides f. sp. aeschynomene as the mycoherbicide
Collegow was attributed to the uniform susceptibility of the
Several reviews have provided a suggested list of desirable weed northern jointvetch (Templeton et al. 1984). A better
characteristics for a bioherbicide candidate in order for it to be understanding of the target weed population structure will
successful (Charudattan 1991; Makowski 1997; Mortensen contribute to the effective search for bioherbicide agents with
1998). Generally, these traits include: (a) a narrow host range, consistent performance on genetically diverse weed species
(b) ease of use, (c) genetic stability, (d) ability to mass (Boyetchko et al. 2002). Another consideration is the view
produce inoculum cost-effectively with long shelf life, and (e) that all bioherbicide candidates must be specific to one
ability to be fast-acting with predictable field performance particular weed species. The advantage of a highly host-
and provide sufficient weed control comparable to chemical specific bioherbicide candidate is the assurance that
herbicides. Many of these traits, along with the term nontarget, beneficial plant species, or crops closely related
bioherbicide (mycoherbicide), may create unrealistic expec- to the weed will not be affected (Makowski 1997; Mortensen
tations that all bioherbicides should eradicate weed 1998). However, a strict host-range requirement may not be
Mycoherbicides 113

economically feasible because the majority of agroeco- of continuous leaf wetness. The pathogen infected the weed
systems are comprised of multispecies weed communities under a broad range of temperatures (10 –308C) when free
(Frantzen et al. 2001). Plurivorous pathogens may be used water or high relative humidity was present, but long
safely under certain circumstances when they can be durations of intermittent leaf wetness was detrimental to
separated sufficiently from nontarget hosts in space and survival of germlings and, therefore, not conducive to high
time (De Jong et al. 1999). Plant architecture and morphology bioherbicidal activity. Some pathogens such as rust fungi are
have played a role in the success or failure of bioherbicide wind-dispersed and generally require less moisture than
agents. The majority of weed species selected as targets for water-disseminated pathogens such as Colletotrichum spp.
biological control have been broad-leaved weeds using foliar where spores are contained in a mucilaginous matrix (Hasan
fungal pathogens (Charudattan 1991; 2001). Grasses are and Wapshere 1973; TeBeest 1991). Makowski (1997)
considered more difficult to control because the meristem is further reiterated that evaluating the impact of environ-
covered by a leaf sheath thereby prohibiting direct attack by mental parameters under controlled conditions may provide
the pathogen (Greaves and MacQueen 1992). Grass weeds are clues about potential performance of bioherbicide candi-
also closely related to many crops (e.g., cereals) in which they dates, but further investigations under variable field
occur, making selectivity of mycoherbicide agents more conditions where these factors are difficult to control are
challenging (Wapshere 1990). A particularly important factor more complicated.
with perennial weeds is their regeneration via rhizomes and
stolons, which makes long-term weed control difficult
(Greaves and MacQueen 1992). This often results in the 3.3 Technological Factors
weed out-growing the disease caused by foliar-applied
bioherbicide candidates. However, there are many pros- The feasibility of commercializing bioherbicide agents has
pective soilborne fungal and bacterial agents that may be often been dependent on the ease and economics of mass-
used as pre-emergent bioherbicides to control Poaceae and producing and formulating large amounts of viable, stable,
perennial weed species (Boyetchko et al. 2002). Other and highly efficacious microbial propagules (Auld and Morin
physical barriers such as leaf hairs and waxy cuticle layers 1995; Mortensen 1998; Slininger et al. 1998). It is generally
may act as impediments to infection and establishment of believed that submerged liquid fermentation is the most
fungal pathogens on the phyllosphere (Auld and Morin 1995). efficient commercial mass-production method for most
Although high inoculum applications have been used to biocontrol agents (Jackson et al. 1996). Although general
overcome biological constraints resulting from low infection guidelines or common fermentation ingredients for medium
efficiency or virulence of the pathogen, these rates may not be composition are available in public domains (Stanbury et al.
technologically feasible due to plugging of spray equipment 1995), most commercial fermentation protocols are custom
or economically viable from a production standpoint. designed for a specific organism and details are generally
treated as trade secrets. However, when a company licenses a
bioherbicide technology, the ability to mass-produce the
agent economically and market it at a cost that is affordable to
farmers, represents a strong determining factor in its
3.2 Environmental Factors development and commercialization potential. Lack of
reliable field performance due to inadequate formulation
Two major limiting factors that have an impact on and application technology has often been cited as a major
mycoherbicides are temperature and moisture requirements reason for the lack of progress in bioherbicides (Greaves et al.
(Auld and Morin 1995; Makowski 1997; Mortensen 1998). 2000; Peng et al. 2001). It has been claimed that suitable
TeBeest et al. (1992) considered temperature to be less formulation technology may help address some of the
important than moisture in most cases because many fungal environmental constraints, particularly moisture requirements
pathogens will infect plants over a broad range of that often hinder the advancement of bioherbicide candidates
temperatures. However, there is often an interaction between beyond the discovery and evaluation phase. Propagules of
temperature and moisture that has a greater effect than foliar-applied fungal agents generally require free water to
temperature alone. Free moisture and leaf wetness duration germinate and penetrate weeds. This leaf wetness requirement
can significantly affect the ability of the fungal pathogen to and its interaction with the ambient temperature often
germinate, produce penetration structures, and ultimately determine the outcome of a mycoherbicide application
cause plant infection. This requirement of leaf wetness (Zhang and Watson 1997). Often, researchers evaluate
duration often increases when temperatures are in suboptimal mycoherbicide candidates by spraying till runoff, thereby
ranges. Early evaluations of potential mycoherbicide overestimating the potential of many bioherbicide candidates
candidates have often been conducted where dew periods in at the early stage of evaluation (Lawrie et al. 1999). Less
excess of 12 h are provided to ensure a high rate of infection stringent dew requirement may be an advantage for foliar
on the weed (Lawrie et al. 1999; McRae and Auld 1988; agents, especially in semiarid climates such as the Canadian
Morin et al. 1990a). Green and Bailey (2000a,b) reported that prairies, where rainfall is infrequent at critical periods (e.g., in
A. cirsinoxia for control of Canada thistle required at least 8 h the spring). Intermittent dew occurs more often than
114 Boyetchko and Peng

continuous dew, but germinated fungal spores under short these processes may impact the others. At any stage of
dew periods are more sensitive to desiccation and UV evaluation, refinements or modifications to these processes,
irradiation (Green and Bailey 2000b), therefore, survival of even minor, may result in significant improvements in
the germlings is going to be the key to successful infection mycoherbicide performance.
under intermittent dew.

4 APPROACHES FOR OVERCOMING 4.1 Selection and Improvement of Bioherbicide

CONSTRAINTS Agents

Most mycoherbicide programs are initiated through surveys On average, chemical companies screen more than 60,000
to discover fungal pathogens exhibiting bioherbicidal compounds before a new active ingredient of pesticide can be
potential, followed by a series of biological and ecological determined. The number required for screening biocontrol
assessments to determine the feasibility of mycoherbicide agents should be less due to a relatively smaller range of
candidates. However, a pragmatic approach of selecting variations amongst naturally occurring fungal populations.
appropriate mycoherbicide candidates is required and However, if we are to identify “nature’s best,” a systematic
should be considered as a continuum amongst several approach is essential during the exploration and discovery
factors that will ultimately influence the field performance phase to thoroughly evaluate the biodiversity. This diversity
of the fungal pathogen (Figure 1). While nutritional and provides excellent opportunities for finding fungal strains
physical factors are vital during fermentation, down-stream with potential suitable traits for biocontrol (Avis et al. 2001;
processing is equally important for an efficient mass- Weidemann and TeBeest 1990). Substantial variations may
production system. Selection of appropriate formulation exist amongst different strains of a fungal species in terms of
technology is influenced by fermentation processes and its virulence and responses to environmental variables (Sands
should be based on critical limitations such as shelf life and et al. 1997; Tessmann et al. 2001). To be effective, critical
environmental constraints encountered with mycoherbicide traits for selection should be clearly identified and sensitive
development. Formulation ingredients can affect delivery bioassays developed. Pathogen strains with high levels of
and application of the mycoherbicide agent. If these virulence may exist in nature at low frequencies due to higher
ingredients result in the inability to deliver the fungal extinction rates (Yang and TeBeest 1992). Results by Yang
pathogen to the target weed (e.g., high viscosity and and TeBeest (1992; 1993) indicated that pathogens showing
ultimate plugging of equipment), effective weed control will high virulence along with important epidemiological traits
not be achieved. Often, mass-production, formulation, and such as rapid infection rates and dispersal are more likely to be
application can be interrelated, therefore, changes in one of candidates of a successful mycoherbicide agent. The success

Figure 1 Strategic framework for evaluation and development of mycoherbicides.

Mycoherbicides 115

of C. gloeosporioides f. sp. aeschynomene for control of sporulation of Phomopsis convolvulus was completely
northern jointvetch was partially attributed to its ability to inhibited when the C/N ratio was reduced from 1:1 to 1:5
easily spread as an endemic pathogen. Yang and TeBeest using modified Richards medium, but the effect on spore
(1995) further demonstrated a rapid rate of mortality of the efficacy was not reported. By understanding these impacts,
weed as the number of pathogen lesions per plant increased fermentation procedures can be fine-tuned to maximize the
from a single lesion. Therefore, more aggressive and virulent production and potency of mycoherbicide agents. One of the
isolates of a pathogen with high infection efficiency, shorter drawbacks with liquid fermentation is that down-stream
latent periods, and better sporulation from diseased tissues processing can be more complicated and costly. Large
should be selected from amongst the pathogen population. centrifuges are normally required to spin off spores and often
Chemical and physical methods have been used to create a large number of spores can be imbedded in the mycelial
fungal mutants with acquired new traits such as elevated biomass (Auld 1993a). Following recovery from the
antibiotic production (Graeme-Cook and Faull 1991) or fermentor, it is usually necessary to dry the spores for long-
increased biocontrol efficacy (Palani and Lalithakumari term storage but retaining spore viability during the drying
1999). Stability or low reversion frequency was observed process may not be easy. Generally, spores should be dried
with some mutants but, in general, stability can be a concern rapidly and gently, but the drying conditions will vary with
with chemical and physical mutagenesis (Wibowo et al. each organism. Drying methods frequently used include
1999). Ziogas et al. (1995) reported UV-induced mutants of freeze, air, spray, or fluid-bed drying, or a combination of
Nectria haematococca with variable tolerance to fungicides these methods (Churchill 1982). Solid substrate fermentation
that showed the same level of fitness as wild types as is used less commonly in commercial production of microbes
expressed by the rate of growth and virulence on squash except for the mushroom spawn industry. Often defined
seedlings. Mutagenesis is apparently a quick way of creating nutrients are added with liquids or solid materials such as
new fungal strains with variable traits. However, efficient vermiculite or paper pellets (Auld 1993a). Various cereal
bioassay systems based on the understanding of critical grains have been used to produce fungal inoculum and it is
constraints are needed for an effective selection strategy. It is relatively easy to quantify and disperse the inoculum on these
not uncommon for induced mutants to have lower competi- solid substrates (Boyette et al. 1991). In some places nutritive
tiveness than the wild type due to reduced infectivity or solid substances such as nutshells or straw may be available
reproductivity (Yang and TeBeest 1995), but judicious use of locally at low cost. Higher labor costs, difficulties in
this technique may help develop new mycoherbicide strains maintaining sterility, lack of control of cultural conditions,
that overcome critical hurdles such as those demonstrated and recovery of spores from the substrate are inherent
with the plurivorous pathogen Sclerotinia sclerotiorum problems with solid substrate fermentation (Churchill 1982).
(Miller et al. 1989). Pfirter et al. (1999) evaluated a variety of solid substrates and
found that Stagonospora convolvuli, for control of field
bindweed, sporulated the best on cous-cous (cracked hard
4.2 Fermentation/Mass Production wheat) followed by maize semolina, yielding 5 £ 108 spores/g
substrate and 3 £ 108 spores/g, respectively. Morin et al.
There are potentially three fermentation systems that may be (1990b) also reported the production of 7 £ 108 conidia/g
used for mass production of mycoherbicide agents: with P. convolvulus using pot barley grain as a solid substrate.
submerged liquid culture, solid substrate fermentation, and They also compared liquid and solid fermentation methods
two-phase system (Auld 1993a). At the industrial level, liquid and found that conidia produced using both systems were
fermentation is the most common method for economical morphologically similar and there were no differences in
production of microbial inoculum. Two commercial myco- pathogenicity. Particle size, moisture content, and tem-
herbicides, Collegow and Devinew, are manufactured this perature appear critical for successful solid substrate
way in the United States (Churchill 1982; Stowell 1991). By production. A mycoharvester developed at CABI Bioscience
understanding the importance of nutritional and environ- (www.dropdata.net/mycoharvester) appears to be a simple
mental factors on induction of fungal sporulation, spore yield, device for collecting spores of the mycoinsecticide fungus,
and bioherbicidal efficacy, rational approaches can be taken Metarhizium anisopliae, produced on rice grains. This device
to develop the most efficient liquid production procedures for has also been attempted to reduce inoculum impurity of the
mycoherbicide agents. Jackson and Bothast (1990) reported mycoherbicide Pyricularia setariae (Gary Peng, unpublished
that carbon concentrations and C/N ratios are the key to data). By reducing the proportion of large particles in the
sporulation of the mycoherbicide agent C. truncatum. Carbon inoculum, the mycoherbicide can be applied at high spore
concentrations ranging from 0.4 to 1.5% gave highest spore concentrations and low carrier volumes using common spray
yields while higher concentrations (2 –4%) inhibited sporula- equipment (Peng et al. 2001). A two-phase system produces
tion. Similarly, a C/N ratio at 15:1 was more favorable for mycelium in deep tank fermentation followed by sporulation
sporulation than 40:1 or 5:1. In a further study, it was revealed in shallow open trays. This method may be particularly useful
that nutritional aspects impacted not only spore yield, but also for fungal agents that cannot be manipulated to sporulate in
spore efficacy of C. truncatum in controlling hemp sesbania submerged culture, but this system is labor-intensive and
(Jackson et al. 1996). Morin et al. (1990b) also observed that expensive, and additional handling of the material may lead to
116 Boyetchko and Peng

contamination of the final product (Rosskopf et al. 1999). and/or enhance the rate of infection of the mycoherbicide
Liquid fermentation produces a large amount of biomass agent should be explored to address dew limitations. In the
efficiently and sporulation in a “dry phase” may circumna- literature, emulsions and hydrophilic polymers are reported
vigate costly down-stream processing issues. Walker (1980) most frequently to improve the performance of foliar-applied
used A. macrospora as a model system to first produce fungal mycoherbicide agents. Formulation research has focused
mycelium in a liquid, then homogenize and mix it with particularly on desiccation and dew requirements of fungal
vermiculite, followed by thinly spreading the homogenate on agents during the infection process and incremental to drastic
a solid surface. Using a similar system, Walker and Riley improvements have been seen in different studies (Auld
(1982) successfully produced spores of A. cassiae, a 1993b; Connick et al. 1990; Lawrie et al. 2000; Shabana
mycoherbicide agent for control of sicklepod. One area of 1997). There is a growing belief that innovations in
fermentation that can often be overlooked is the feasibility of formulation will be a vital component to the success of the
scale-up from shake flask laboratory volumes to production- next generation of bioherbicides, especially for foliar-applied
plant level. Optimization of fermentation conditions and products (Greaves et al. 1998). For best results, formulations
media components can be readily achieved in the laboratory should predispose weeds to infection by pathogens and buffer
while pilot-plant scale fermentation can help to effectively pathogen propagules against environmental extremes while
verify these selected parameters (Kwanmin 1989). Some promoting disease development. Nutrient supplements,
sophisticated pilot-scale devices with consistent designs to including simple sugars, amino acids, pectins, salts, and
large-scale facilities are now available. These units are plant extracts have been added to formulations to stimulate
particularly useful for scale-up studies and provide a wide the infection process and protect germinating propagules, but
range of pH, agitation speeds, impeller designs, aeration rates, these nutritional effects are often agent specific (Bothast et al.
choices of regulated incoming gases, variations in baffling 1993; Schisler et al. 1995; Womack and Burge 1993).
and background pressures, and temperatures (Churchill Exogenous nutrients may stimulate germination and growth
1982). However, conditions required to reach optimal yield, of many fungi, but frequently appressorial initiation is even
costs, and efficiencies in production scale fermentation can be more important to plant penetration and infection. Oversupply
more difficult to achieve. Some biological factors that need to of nutrients can lead to excessive growth of germlings,
be considered are culture stability, number of generations, and delaying, or even reducing appressorial formation and
mutation rate while chemical factors include pH, water penetration (Takano et al. 1997). Tremendous efforts have
quality, and fermentation medium quality. Physical factors been made on developing various emulsions to alleviate
that should be evaluated include aeration, agitation, pressure, moisture constraints, thereby enhancing field performance.
temperature, and medium sterilization. Kwanmin (1989) Invert emulsions showed the most impressive results by
indicated that factors that may not have been important at a reducing or even eliminating the need for dew with several
smaller scale could have significant impact on the operation fungal agents (Connick et al. 1991b; Yang et al. 1993).
and design of fermentation processes at the production plant Bioherbicidal control of hemp sesbania using C. truncatum in
level. an invert emulsion was significantly enhanced under field
conditions (Boyette et al. 1993). Invert emulsions consist of
water droplets suspended in oil and evaporation of the trapped
4.3 Formulation water is dramatically reduced and microbial propagules held
in the water are, therefore, protected (Daigle et al. 1990;
It is believed that many of the environmental challenges, Womack and Burge 1993). Despite the apparent improve-
particularly long dew period or leaf wetness requirements, can ment, invert emulsions can be very complex, difficult to apply
be tackled to a large extent with formulation technologies using existing spray equipment due to extreme viscosity and
(Boyetchko et al. 1999; Greaves et al. 2000; Green et al. may exhibit phytotoxicity in many plant species (Boyette
1998). Formulation is essentially the blending of microbial 1994; Womack et al. 1996). Although these invert emulsions
propagules with a range of carriers or adjuvants to produce a have been used to expand the host range of mycoherbicides
form that can be effectively delivered to target weeds. For (Yang and Jong 1995), this change may also affect nontarget
microbial agents, formulation may enhance pathogen survival crops. High oil content also makes invert emulsions more
and infection as well as extend propagule stability and costly, especially when high spray volumes are required.
product shelf life. Depending on the type of organism, mode Being nonevaporative, oils were considered a compatible
of action, and available spray equipment, formulation carrier with ultra-low volume application techniques for the
ingredients vary substantially. For instance, foliar-applied mycoinsecticide M. anisopliae under extremely dry con-
agents may be exposed to rain-wash, UV irradiation, and ditions (Bateman and Alves 2000). In contrast to invert
desiccation prior to germination and penetration (Rhodes emulsions, oil suspension emulsions are considered to be
1993). Therefore, various adjuvants with adhesive, sun- more practical because they have significantly lower oil
blocking, or humectant properties have been suggested to content and can be applied with most existing spray
alleviate the negative impact by these factors (Schisler et al. equipment (Green et al. 1998). Fungal propagules can be
1995; Womack and Burge 1993). Formulations that increase first suspended in oils, then mixed with a much larger volume
moisture-retaining properties, reduce the rate of evaporation of water containing an emulsifier to make stable emulsions
Mycoherbicides 117

(Auld 1993b). Klein et al. (1995) used suspension emulsions of biocontrol agents (Greaves et al. 2000). Disregard for
of C. orbiculare made from two vegetable and mineral oils appropriate methods of application can contribute to poor or
that were mixed and applied with water ranging in inconsistent field performance (Smith and Bouse 1981). In
concentrations from 0.5 to 10% for control of Xanthium most field crops, application volumes over 600 l/ha are
spinosum under field conditions. These formulations considered high (Matthews 1992), and the trend is generally
enhanced weed control in several field trials compared to toward lower volumes.
water as a carrier sprayed at similar application volumes. Oils Use of high spore concentrations can potentially reduce
may have variable effects on propagule germination and application volumes without compromising the efficacy of
performance of mycoherbicide agents. Paraffin oils were weed control (Peng et al. 2001). Increased propagule number
toxic to spores of Ascochyta pteridis, a mycoherbicide may help improve the efficiency of foliar coverage by
candidate for bracken, Pteridium aquilinum (Womack et al. reducing the proportion of “empty” droplets (Jones 1998), but
1996), while unrefined corn oil enhanced spore germination also pose high requirements on quantity and quality of the
of C. truncatum in emulsions containing 10 –50% of oil inoculum necessary to achieve the desired level of weed
(Boyette 1994; Egley and Boyette 1995). The mechanisms by control. Mycelium clumps or other impurities in extremely
which the oil suspension emulsions enhance mycoherbicidal concentrated formulations can also easily plug up the spray
activity are not well understood. Emulsions may help system. Inoculum concentration, carrier volume, and other
maintain the stability and infectivity of fungal propagules spray parameters need to be studied jointly to optimize spray
prior to onset of dew (Green et al. 1998). Spray retention is results (Jones and Burges 1998). Nordbo et al. (1993)
likely enhanced, reducing the spore dose required for suggested use of fast travel speed to improve spray retention
effective weed control. Moisture retention has also been on vertical leaf surfaces. In a recent study, Peng et al. (2001)
demonstrated with hydrophylic polymers such as Kelginw observed that a finer droplet spectrum combined with more
HV, MV, LV, Kelzanw xanthan gum, Gellan gum, N-Gele, horizontal trajectories enhanced retention efficiency on green
Metamucilw, and Evergreenw500 (Shabana et al. 1997). foxtail. Richardson (1987) and Spillman (1984) made similar
These polymers enhanced viability, germination, and efficacy suggestions on application of herbicides. These retention
of the mycoherbicide agents A. cassiae and A. eichhorniae. characteristics have also been discussed by Jones (1998) and
Humectants such as psyllium (e.g., Metamucilw) are known to Reichard (1988), and have been used to explain improved
have high moisture retention properties and reduce the rate of spray results in a number of herbicide studies (Knoche 1994).
moisture loss (Greaves et al. 2000). Coformulation of the It needs to be recognized that there are limitations with
mycoherbicide agent, A. caulina, with the skinning agent manipulation of certain spray parameters. For instance, too
polyvinyl alcohol and Metamucilw enhanced control of fine a spray may not be practical in every case depending on
Chenopodium album under reduced dew conditions. For the size of mycoherbicide propagules and due to potential
mycoherbicides applied to the soil, encapsulation of the fungi spray drift concerns (Jones 1998). Interpretation of droplet
in solid matrices is more suitable than liquid formulations. size spectra for optimal dose transfer of biopesticides is
Calcium alginate has been used to mix fungal spores with a required, and this will vary with the mycoherbicide agent,
variety of carriers such as kaolin clay, ground oatmeal, soy particularly in relation to the spore size and morphology
flour, and cornmeal (Boyette and Walker 1986; Walker and (Bateman 1999). Although fine tuning of application
Connick 1983; Weidemann and Templeton 1988). Conidial parameters can improve spray characteristics, it is more
production and field efficacy can be enhanced by amending important to determine if these improvements can be
the mixture with various nutrients (Daigle and Cotty 1992; translated into meaningful enhancements of weed control
Weidemann 1988). “Pesta” has also been used as a type of efficacy.
granular formulation where fungal propagules are entrapped
in a wheat-gluten matrix consisting of semolina flour, kaolin,
and fungal biomass (Connick et al. 1991a). Further 4.5 Herbicide Synergy
development of this process has resulted in the formation of
uniform granules using a twin-screw extruder and by As stand-alone products, mycoherbicides have achieved
controlling the moisture content through fluid-bed drying limited success in the marketplace. Technologies such as
(Connick et al. 1998). formulation and application methods can improve the
performance of biocontrol agents, but more noticeable
enhancements in weed control have been observed with the
4.4 Application Technology combined application of mycoherbicide agents and synergis-
tic herbicides (Peng et al. 2000; Sharon et al. 1992; Wymore
Delivery and retention of sufficient number of fungal et al. 1987). Limited information seems to suggest that
propagules on weeds can be very challenging. Most initial synergistic interactions are herbicide and pathogen specific.
studies on mycoherbicide efficacy spray the inoculum to the Use of glyphosate on Canada thistle assisted weed control by
weed foliage till runoff using aerosol sprayers. This spray A. cirsinoxia only marginally, especially under field
method generally applies excessive volumes (up to 3000 l/ha) conditions (Bailey et al. 2000). Peng et al. (2000) compared
that can maximize the retention and exaggerate the potential two groups of herbicides, bentazon and metribuzin, for
118 Boyetchko and Peng

interaction with a fungal pathogen on scentless chamomile, a (a) Assessment of natural strain variation and bio-
noxious weed in the Canadian prairies. Preliminary results diversity within the pathogen population based on
revealed significant fresh weight reduction by up to 150% critical epidemiological characteristics, including
with applications of herbicides plus the pathogen compared to environmental adaptation, virulence, dispersal, and
herbicides alone. Tank-mixing of C. coccodes with infection efficiency.
thidazuron (N-phenyl-N0 -1,2,3-thiadiazol-5-yl-urea) also (b) Evaluation of key areas for efficient scale-up
increased the mortality of velvetleaf when compared to the mass-production based on fundamental elements
application of the mycoherbicide alone in field trials relating to nutritional and physical requirements of
(Wymore et al. 1987). The application of a sublethal dose specific fungal agents that facilitate selection of
of glyphosate with A. cassiae resulted in an increase in economical fermentation ingredient substitutes,
susceptibility of the weed sicklepod (Sharon et al. 1992). As a along with down-stream processing procedures
result, equivalent control was achieved with five times less that are compatible with production methods.
inoculum of the mycoherbicide agent. The herbicide was (c) Selection of appropriate formulation technologies
believed to interfere with the shikimate acid pathway that is (i.e., liquid or solid-matrices) based on the mode of
involved in the elicitation of phytoalexins, low molecular attack and critical efficiencies of candidate agents.
weight antimicrobial compounds involved in a plant’s defense Over-simplification of formulation by using single
response. Subsequent interference with the plant’s defense ingredients will not likely address the complex
mechanism resulted in greater susceptibility of the target challenges that fungal organisms will encounter in
weed to the mycoherbicide (Hoagland 1996). This synergistic the environment, including moisture constraints,
interaction appears to be an attractive mechanism to enhance temperature extremes, and UV irradiation.
the effectivity and feasibility of mycoherbicide agents. (d) Critical factors in application technology related to
According to Hoagland (1996), several benefits may be placement and penetration of the crop canopy to the
captured with the application of microbe/herbicide synergy: target weed in order to maximize application
(a) when defense capabilities of weeds are lowered using efficiency. These factors include leaf-wetting
herbicides, weeds become more susceptible to pathogen properties and ability to penetrate physical barriers
attack, (b) the quantity of mycoherbicide agent or the (e.g., waxy cuticles and leaf hairs), retention and
application rate of herbicides may be reduced, and (c) host dispersal on the leaf surface, optimum dose transfer
range of a given mycoherbicide agent may be expanded with in various liquid droplets or solid-based granules,
the use of selected chemical synergists. and selection of application equipment such as
nozzle types and angle position or soil-application
placement (e.g., within furrow application, side-
banding, etc.). Application parameters should also
5 CONCLUSION be evaluated jointly with formulation ingredients.
(e) Integration of mycoherbicide agents into crop
The identification of efficacious biological control agents is production systems using several weed management
only the beginning in the development of mycoherbicide tools (e.g., synergy with chemical herbicides,
products. Continuing strain selection is essential to ensure that combinations with other biological control agents,
“nature’s best” is employed. Likely there will always be or weed control options) to optimize weed control
limitations associated with naturally occurring organisms, effectiveness. Judicious use of mycoherbicides as
therefore, enabling technologies such as fermentation, one of the components in an integrated weed
formulation, and application technology will be instrumental management system will enhance their value and
in determining whether a highly efficacious agent can be practicality for control of multispecies weed
developed into an economically feasible mycoherbicide communities in agroecosystems.
product. The ultimate goal is to incorporate mycoherbicides
into agricultural production systems. More efforts should be
directed into combining biocontrol agents with other weed
control options including chemical herbicides, cultural
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11
Biofungicides

Beom Seok Kim Institute for Structural Biology and Drug Discovery, Medical College of Virginia,
Virginia Commonwealth University, Richmond, Virginia, USA

Byung Kook Hwang College of Life and Environmental Sciences, Korea University, Seoul, South Korea

1 INTRODUCTION exposed to agricultural environment, thus leading to the low

residual level less harmful to the natural ecosystem (Tanaka
At present, approximately 200 different fungicides have been and Omura 1993).
introduced into agriculture and horticulture worldwide. Microbial metabolites can be exploited in a number of
Despite the enormous advances in chemical management of different ways for the development of new fungicides. They
fungal diseases, some of the important plant pathogens such can be directly used as fungicide products or as leads for the
as vascular wilt, anthracnoses, take-all of wheat, and other design of novel synthetic products. Alternatively, they can be
root infections remain uncontrolled by current fungicidal used to highlight novel mode of action available as a new
chemicals (Knight et al. 1997). The build-up of resistant screening target. The recent successes in fungicide develop-
strains of target pathogens and the increasing public concern ment came mainly from the discovery of potent lead
about synthetic fungicides have intensified the need for better compounds followed by chemical modifications that gave
and safer compounds in terms of novel modes of action, low additional useful features fungicides. Potent antifungal
rates of use, and low toxicological and environmental activity is not the only factor to decide whether the microbial
risk (Godfrey 1994; Tanaka and Ōmura 1993). As the metabolite can be used as a commercial fungicide. Along with
environmental and commercial requirements for new the chemical stability in the field, it should also have residual
fungicides become more demanding, it is increasingly activity enough to reduce the application time to the
difficult to discover new class of compounds to justify the economical level and low volatibility sufficient to stay on
effort and the costs of development. In order to get a chance to the surface of host plants. Overall, it is very unlikely that a
discover new fungicides that meet the mentioned character- newly discovered microbial metabolite might possess all of
istics, the exploitation of biologically active natural products the desired properties. Therefore, moving away from the
is becoming mainstream in antifungal agent research. viewpoint of antifungal metabolites as final products,
Microbial metabolites represented by antibiotics have a microbial metabolites are currently reexploited as a source
number of chemical and biological merits as fungicides. of enormously diverse chemical library that can supply lead
Microorganisms are capable of synthesizing versatile compounds for development of fungicides. As seen in the
chemical structures with diverse biological activities beyond example of the successful development of methoxyacrylates
the scope of synthetic organic chemistry (Porter 1985). An that are expected to be a major fungicide class in the future, it
unexpected and newly found chemical structure is more likely is not surprising that natural products are facing a revival as
to have new fungicidal activity and mode of action, especially lead compounds for fungicide development.
showing no cross-resistance to the commercial fungicides The advances in molecular, biological, and chemical
(Früh et al. 1996). Biodegradability is the next property of techniques made it possible to reinvestigate microbial
microbial metabolites that cannot be overlooked. They metabolites from a totally different point of view. The
degrade usually within a month or even a few days, when increasing knowledge about the complex multidisciplinary

123
124 Kim and Hwang

mystery of antifungal activity enables us to design a Alternaria kikuchiana and gray mold diseases caused by
rationalized screening system based on the mode of action. Botrytis cinerea (Isono et al. 1965).
Along with the innovative screening systems, the powerful Validamycin A produced by Streptomyces hygroscopicus
instruments available for purification and structural elucida- var. limoneus was effective in controlling rice sheath
tion of natural products have made it possible to adopt a high blight caused by Rhizoctonia solani (Iwasa et al. 1970).
throughput approach to natural product screening (Bindseil Validamycin A was found to be a pro-drug, which is
et al. 2001). converted within the fungal cell to validoxylamine A, an
In this chapter, we will review (a) microbial metabolites extremely strong inhibitor of trehalase. This mode of action
currently used as fungicides, (b) on-going efforts to discover gives validamycin A a favorable biological selectivity,
lead compounds from diverse microbial sources, and (c) because the hydrolysis of the disaccharide trehalose does not
fungal specific targets to be used for screening of potential occur in the vertebrates. The structural elucidation and total
antifungal leads. In the later part of this review, we will synthesis of validamycin A were achieved by Ogawa and
discuss (c) trends in biofungicide research and inter- coworkers (Suami et al. 1980).
disciplinary approaches to diversify their chemical library, Mildiomycin is an aminoacylated nucleoside produced by
which may yield novel antifungal compounds in the future. Streptomyces rimofaciens (Harada and Kishi 1978). It was
discovered by the method established to assay the control
efficacy of antifungal agents against powdery mildew.
Mildiomycin has been known to act as an inhibitor of the
2 MICROBIAL METABOLITES IN USE AS fungal protein biosynthesis. Its low toxicity on vertebrates
BIOFUNGICIDES allows it to be an environmentally favorable crop protection
agent.
The most important antifungal metabolites in commercial use
are listed below, which are applied to control fungal diseases
on rice, vegetable, and fruits. The relative importance of the 3 MICROBIAL METABOLITES AS
microbial compounds, when compared to synthetic fungi- ANTIFUNGAL LEADS
cides, might have been underevaluated because of several
reasons such as the limitation in their spectrum of activity and
The merits of natural products as fungicides can be a
in certain instances, the development of resistance. Never-
disadvantage in some respects. Their specific activity often
theless, the excellent activity of these biofungicides inspired
resulted in a narrow antifungal spectrum with a limited
to launch the screening programs for antifungal microbial
application and the development of resistance strains under
metabolites, which resulted in profound chemical libraries of high selection pressure. Their biodegradability can also make
natural products (Godfrey 1994; Knight et al. 1997). them fragile, which results in short residual activity under
Blasticidin S, the first microbial fungicide available for harsh field conditions. These might be the reasons why the
plant protection, has been used practically for the control of microbial metabolites used as commercial fungicide per se
rice blast disease caused by Magnaporthe grisea. Blasticidin still commands less than 1% of total fungicide market
S is a nucleoside antibiotic discovered from metabolites of (Tanaka and Ōmura 1993). Recently, a breakthrough in
Streptomyces griseochromogenes (Takeuchi et al. 1957). It biofungicide research came from the semisynthetic approach
potently inhibits the mycelial growth and conidial germina- using microbial metabolites as lead compounds. In particular,
tion of M. grisea. The successful use of the compound a far more promising and effective strategy for the
encouraged further screening of microbial fungicides that development of new biofungicides is to use knowledge of
eventually brought out kasugamycin, polyoxin, validamycin, the structure of antifungal compounds as the starting point for
and mildiomycin. the synthesis of the compounds with optimized physical,
Kasugamycin is an amino-sugar compound discovered in biological, and environmental properties. The activity of
the metabolites of Streptomyces kasugaensis and Strepto- natural products can in principle be improved by chemical
myces kasugaspinus (Umezawa et al. 1965). It has in vitro modification. However, this approach relies heavily on the
antimicrobial activity against yeast and some plant patho- ready availability of sufficient quantities of the natural
genic fungi including M. grisea. In vivo data showed that starting materials and the development of appropriate
kasugamycin efficiently suppressed the development of synthetic methodology. The biofungicides that were devel-
M. grisea mycelia on rice plants by both preventive and oped in this way are fenpiclonil and fludioxonil (Nyfeler and
curative treatments. However, it did not appear to inhibit the Ackermann 1992) and synthetic derivatives of antibiotic
spore germination. strobilurins such as b-methoxyacrylate azoxystrobin and
Polyoxins were isolated from the culture broth of kresoxim-methyl (Anke et al. 1977; Godfrey 1994). Such a
Streptomyces cacaoi var. asoensis (Suzuki et al. 1965). The derivative synthesized from microbial metabolites not only
excellent in vitro activity and in vivo efficacy led to its enhanced control efficacy but also improved properties such
commercial use for the control of fungal diseases of fruit trees as photochemical stability, low cytotoxicity, and phyto-
and vegetables such as black spot of Japanese pear caused by toxicity. These successes encouraged the fungicide
Biofungicides 125

researchers to search versatile lead compounds from diverse developed as commercial fungicides that overcame the
microbial sources with novel mode of action. problems of the lead compounds (Clough et al. 1995).
Azoxystrobin has a methyl b-methoxyacrylate toxophore,
like strobilurin A, whereas kresoxim-methyl has a methyl
3.1 Recent Success in Fungicide Development methoxyiminoacetamide structure (Figure 1). Axoxystrobin
from Antifungal Leads has a wide antifungal spectrum against all four taxonomic
groups of fungi and strong preventative activity, including
Since strobilurin A and oudemansin A were found to be inhibition of fungal germination (Heaney and Knight 1994).
fungicidal metabolites in Basidiomycete fungi Strobilurus Kresoxim-methyl is also a broad-spectrum fungicide with
tenacellus (Anke et al. 1977) and Oudemansiella mucida strong antifungal activity against powdery mildew and apple
(Musilek et al. 1969), respectively, a number of structurally scab (Brunelli et al. 1996). Considering its novel mode of
related compounds were reported to have fungicidal activity. action and amenability for synthetic approach, strobilurins are
Each member of this family incorporates a methyl expected to be a major fungicide in near future.
b-methoxyacrylate group linked at its a-position to a Pyrrolnitrin is another example of a microbial metabolite
phenylpentadienyl unit, and all the compounds except used as a lead compound. Pyrrolnitrin, a secondary metabolite
strobilurin A carry either one or two additional substituents of Pseudomonas pyrrocinia, which has a very simple
on the benzene ring that render structural complexity structure, is thought to play a significant role in biocontrol
(Figure 1). Their mode of action on mitochondrial respiration, activity of the bacterium (Arima et al. 1964). Although it
binding at a specific site on cytochrome b, is not shared by any showed excellent in vitro and in vivo activity in the
other known class of fungicides (Sauter et al. 1995). The greenhouse against B. cinerea and M. grisea, the disease-
unique mode of action may not provide a chance of cross- control efficacy in the fields was poor, because it rapidly
resistance between b-methoxylacrylates and other fungicides. decomposed when exposed to sunlight. In the extensive
Although strobilurin A has excellent in vitro activity against a synthetic programs using pyrrolnitrin as a template, feniclonil
range of fungi, it did not show any useful in vivo activity in the (Nevill et al. 1988) and fludioxonil (Gehmann et al. 1990)
greenhouse. This was due to its photochemical instability and were developed as seed-dressing agents against numerous
relatively high vapor pressure, which cause it to disappear fungal pathogens. The replacement of the chloro substituent
rapidly from a leaf surface. Through a series of synthetic in the 3-position of the pyrrole by a cyano group led to a
program to solve these problems, azoxystrobin (Godwin et al. remarkable enhancement in stability (Figure 1). Its biological
1992) and kresoxim-methyl (Ammermann et al. 1992) were activity also was optimized by appropriate substitution on the
phenyl ring. Their improved photostability over pyrrolnitrin
conferred the possibility as a foliar fungicide active against
B. cinerea, Monilinia spp. and Sclerotinia spp. (Nyfeler and
Ackermann 1992).

3.2 Screening of Potential Leads from Diverse

Microbial Sources
3.2.1 Streptomyces, the Largest Reservoir of
Diverse Chemical Structures
Actinomycetes have been a major supplier of natural products
(Huck et al. 1991; Lee and Hwang 2003). In particular,
Streptomyces is a prolific producer of versatile structures of
antibiotics. Most of antibiotics developed for agricultural uses
including pesticides were isolated from Streptomyces strains
(Tanaka and Ōmura 1993). Among antifungal antibiotics
recently discovered from Streptomyces spp., polyketide-
spiroketal spirofungins, macrolide cineromycins, and oligo-
mycin A, antimycin type kitamycins, aflatoxin inhibitor
aflastatins, aminoacetophenone family heptaene antibiotics,
and novel nikkomycin analogs were found to have potent
antifungal activity (Bormann et al. 1999; Hayashi and Nozaki
1999; Holtzel et al. 1998; Kim et al. 1999b; Ono et al. 1998;
Schiewe and Zeeck 1999; Vertesy et al. 1998).
Figure 1 Chemical structures of biofungicides in practical use Streptomyces have the ability to synthesize diverse
and their lead compounds. compounds covering the chemical structures generated by
126 Kim and Hwang

the eukaryotic organisms such as fungi, algae, and plants.

Streptomyces kurssanovii was found to have the ability to
synthesize fumaramidmycin, which is structurally very
similar to fumarimid and coniothriomycin produced by the
fungi Sordaria sp. and Conithyrium sp. (Maruyama et al.
1975). Although the frequency of rediscovery of known
compounds is relatively high, it should also be noted that
Streptomyces strains continue to provide a larger number and
wider variety of new antibiotics than any other microbial
sources (Okami and Hotta 1988). Many macrolide antibiotics,
for example, have already been introduced from a variety of
Streptomyces spp., however, new macrolide compounds are
still being discovered to be potent antifungal agents.
Faeriefungin, a polyene type macrolide, isolated from
S. griseus showed strong in vivo activity against asparagus
(Asparagus officinalis L.) pathogens Fusarium oxysporum
and Fusarium moniliforme (Smith et al. 1990). More recently,
the antifungal substances, phenylacetic acid and sodium
phenylacetate, active against Phytophthora capsici and
M. grisea were isolated from the culture filtrates of
S. humidus (Hwang et al. 2001).
Streptomyces is a sole microbial source for a certain type
of antibiotics such as members of manumycin type that
contain a multifunctional mC7N unit as a central structural
element. A manumycin type antibiotic SW-B has recently
been purified from the culture of Streptomyces flaveus strain
A11 (Hwang et al. 1996). The strain was isolated from cave
soil in Korea by an extensive screening program for the
Streptomyces strain antagonistic to P. capsici. The structure
of manumycin SW-B was determined to be 2,4,6-trimethyl
Figure 2 Potential antifungal leads from microbial sources.
deca-(2E,4E)-dienamide (molecular formular C13H23NO)
with the molecular weight of 209.178 (Figure 2). SW-B
showed a high level of inhibitory activity and broad
antifungal spectrum against several plant pathogenic Micromonospora spp. was known to be distributed widely in
oomycete and fungi such as P. capsici, M. grisea, soils of various geographical regions (Vobis 1991). Since
Colletotrichum cucumerinum, and Alternaria mali. Hyphal gentamicin, an aminoglycoside antibacterial antibiotic, was
growth of P. capsici and M. grisea was inhibited by more than isolated from M. purpurea and M. echinospora (Weinstein
50% at 10 mg ml21 and by 90% at 50 mg ml21. The simplicity et al. 1964), Micromonospora spp. has been shown to produce
of the chemical structure and its broad antifungal spectrum diverse antibiotic substances such as aminoglycosides and
provide the possibility as a lead compound for fungicide macrolides (Betina 1994). In a screening program for
development. antifungal antibiotics useful for plant disease control,
Micromonospora coerulea strain Ao58 was isolated from
3.2.2 Rare Actinomycetes, New Resource of sea-mud soils, which showed strong antifungal activity
Microbial Metabolites against P. capsici, M. grisea, C. gloeosporioides, and R. solani
(Kim et al. 1998; Kim et al. 1999a). From the culture extracts,
Since rare actinomycetes have the properties such as slow the antibiotic streptimidone (Figure 2) was purified using
growth, poor sporulation, and instability in preservation, it various chromatographic procedures. Streptimidone was
seems difficult to isolate them without applying the selective known as an inhibitor of the protein synthesis on yeast, but
isolation methods. Most of their metabolites, therefore, were little has been known about its efficacy as an antifungal agent
not subjected to the antifungal screening. However, although against filamentous fungi. In the tests for antifungal spectrum,
the antifungal agents from these non-Streptomyces groups of remarkable antifungal activities were observed against
actinomycetes have not yet been developed into commercial some plant pathogenic fungi P. capsici, M. grisea, Didymella
fungicides, they are expected to be useful microbial sources bryoniae, and B. cinerea. In vivo tests showed its potent
for diversifying chemical library of metabolites. control efficacy against phytophthora blight on pepper plants,
The genus Micromonospora, only a minor component in gray mold on cucumber leaves, and leaf blast on rice leaves.
the actinomycete population in soil, has been recognized as The compound effectively inhibited the development of these
one of the important sources for antimicrobial metabolites. plant diseases on their host plants at the concentration of
Biofungicides 127

100 mg ml21, at which the commercial fungicides showed rhamnolipid the ability to intercalate into and to disrupt
similar control efficacy against the diseases. No phytotoxicity the zoospore plasma membrane, because zoospores are
was observed on any of the host plants at the concentrations of surrounded only by plasma membrane without typical cell
500 mg ml21. wall (Stanghellini and Miller 1997). This hypothesis is
Recently, two structurally related compounds isolated well supported by the further finding that rhamnolipid B
from rare actinomycetes were found to have potent antifungal had no lytic activity on zoospore cysts surrounded with
activity against plant pathogenic fungi. Daunomycin and the cell wall (Kim et al. 2000a). In vitro growth
spartamycins were isolated from Actinomadura roseola and inhibition assay performed in the microtiter dishes showed
Micromonospora spartanea, respectively (Kim et al. 2000b; potent antifungal activities against Cercospora kikuchi,
Nair et al. 1992). Both compounds have similar anthracycline C. destructans, C. cucumerinum, Colletotrichum orbiculare,
aglycone moiety attached to one or three glycosides M. grisea, and P. capsici. In particular, rhamnolipid B had a
(Figure 2). Daunomycin noted for anticancer activity showed high level of antifungal activity (10 mg ml21 of MIC) against
substantial in vitro antimicrobial activity against P. capsici, P. capsici. In the microscopic study, most of the zoospores
R. solani, B. cinerea, Cladosporium cucumerinum, became non-motile in the presence of 25 mg ml21 of
Cylindrocarpon destructans, D. bryoniae, S. cerevisiae, and rhamnolipid B, subsequently lyzing within 1 min after
Gram positive bacteria. In particular, daunomycin showed treatment. Rhamnolipid B also was effective in inhibiting
strong inhibitory effect on the mycelial growth of P. capsici the germination of zoospore and the hyphal growth of
and Phytophthora development on pepper plants. In vivo P. capsici. The average hyphal length of germlings at the
efficiency against Phytophthora infection in pepper plants was 50 mg ml21 was reduced by 55% of that in the untreated
somewhat less effective than that of the commercial fungicide control. These results suggest that rhamnolipid B has not only
metalaxyl. the lytic effect on zoospores of P. capsici but also inhibitory
Spartamycins produced by M. spartanea were isolated effect on the growth of the oomycete. Zoospores have been
from a potted soil with A. officinalis L. plants (Nair et al. implicated in the spread of the oomycete pathogen through
1992). Between the two spartamycin analogs A and B, the irrigation water and rainwater (Hwang and Kim 1995b;
latter showed better antimicrobial activity against several Ristaino et al. 1993). The lytic effect of rhamnolipid B on
microorganisms. The minimum inhibitory concentration zoospores may provide a merit as a preventive control agent
(MIC) of spartamycin B on Aspergillus, Cladosporium, against phytophthora blight in pepper-growing fields, which
Cryptococcus, Rhodotorula, and Candida albicans ranged eliminate and/or reduce zoospore density and long-distance
from 0.2 to 1 mg ml21. However, spartamycin B was not dispersal of the pathogen. In the recirculating hydroponic
effective against the Staphylococcus aureus, Escherichia coli cultural system of crops, rhamnolipid B has been
and Citrobacter spp. In view of antifungal activity and demonstrated to be very effective in controlling the dispersal
structural similarity of the anthracycline antibiotics, their of plant diseases caused by zoosporic oomycete pathogens
analogs having different glycoside moieties may be worth- (Stanghellini et al. 1996).
while to examine their antifungal activity against various Bacillus subtilis is known to produce diverse antifungal
plant pathogenic fungi. peptides represented by inturins. A series of fungicidal
metabolites, named rhizocticines, were identified from
3.2.3 Other Microorganisms B. subtilis ATCC6633 (Figure 2) (Fredenhagen et al. 1995).
These peptides showed control efficacy against B. cinerea on
Pseudomonas aeruginosa strain B5 was isolated from pepper- apples and vines in the greenhouse. The proteolytic digestion
growing soils in Korea, which showed substantial inhibitory test of the compound revealed that L-2-amino-5-phosphono-
activity against P. capsici and other plant pathogenic fungi. 3-(Z)-pentenoic acid was the actual structure active against
From the culture broth of the antagonistic bacterial strain B5, B. cinerea. The antifungal activity was proven to be stereo
one of the antibiotic substances active against P. capsici was specific, since the corresponding 3-(E) compound did not
purified and identified as a glycolipid antibiotic rhamnolipid show any antifungal activity. The mixture of rhizocticines A,
B (Kim and Hwang 1993). Rhamnolipids containing B, and D also showed control efficacy against gray molds on
rhamnose and b-hydroxy-decanoic acid were first found in grapes in the field.
Pseudomonas pyocyanea (the old name of P. aeruginosa)
(Bergström et al. 1946). Recently, complete nuclear magnetic
resonance signal assignments of rhamnolipid B based on
intensive spectral analysis provided the evidence of 4 POTENTIAL TARGETS FOR DISCOVERY
1,2-linkage of 3-[3-[-L -rhamnopyranosyl-(1 ! 2)-a-L - OF ANTIFUNGAL LEADS
rhamnopyranosyloxy] -decanoyloxy]-decanoic acid (Moon
et al. 1996). The glycolipid antibiotic rhamnolipid B has Unlike the arena of development of antibacterial agents, in
the characteristic structure of biosurfactants, which is relative terms, where bacterial specific targets are abundant, it
comprised of a hydrophilic portion (rhamnose moiety) and seems difficult to develop antifungal agents with a specific
a hydrophobic portion (b-hydroxydecanoate moiety). The mode of action. Since, fungi as eukaryotic organism, have
biosurfactant property was supposed to render the metabolism similar to those of mammal and plant hosts, most
128 Kim and Hwang

of antifungal agents discovered to be potentially active 4.2 Sterol Biosynthesis

against plant pathogenic fungi have failed to survive during
the testing process for practical usage. The following The biosynthesis of sterols is an essential metabolism that
discussions will be of potential antifungal leads directed to produce essential constituents of cellular membranes. Most of
fungal specific targets, with the examples of antifungal agents fungi contain ergosterol as a predominant sterol (Mercer
recently developed for clinical and agricultural uses. 1991). Recent advances in our understanding of mode of
action of sterol biosynthesis inhibitors (SBI) launched a novel
approach to finding inhibitors of sterol biosynthesis, which
4.1 Cell-Wall Biosynthesis could lead to new agricultural fungicides (Barrett-Bee and
Ryder 1992). The antifungal effects of SBI have brought out a
Fungal cell wall is a crucial target for antifungal agents. great commercial success in the synthetic fungicide market.
Enzymes responsible for the biosynthesis of fungal cell wall The SBI fungicides covering about the half of the market is
include chitin and glucan synthases (Douglas et al. 1997; now practically applied to protect fruits, vegetables, and vines
Georgopapadakou 1997). Antifungal agent echinocandins from plant diseases. The major SBI are the inhibitors of
have been discovered as inhibitors of fungal cell wall 14-demethylation which correspond to many antifungal
biosynthesis (Denning 1997). They are noncompetitive compounds, referred to as azole compounds, with a wide
inhibitors of b-1,3-glucan synthase, an enzyme complex in spectrum of intrinsic activity against ascomycete, basidio-
the cell wall of many pathogenic fungi. b-1,3-Glucan mycete and deuteromycete pathogens (Aoki et al. 1993).
synthase is a fungal specific enzyme that polymerizes The discovery of restricticins and lanomycin led to the
UDP-glucose into b-1,3-glucan polymers that comprise the introduction of a new target for screening of the antifungal
major scaffolding of the fungal cell wall (Kang and natural products. The two structurally related compounds
Cabib1986). Echinocandins and its synthetic analog contain- were first isolated from the cultures of Penicillium restrictum
ing the fatty acid side chain, cilofungin, exhibited comparable (Schwartz et al. 1991) and Pycnidiophora dispersa
fungicidal activity with a narrow antifungal spectrum (O’Sullivan et al. 1992), respectively (Figure 2). Both
(Fromtling 1994). Recently, marked improvements in restricticin and lanomycin showed potent antifungal activity
antifungal activity against clinical pathogens have been through inhibition of lanosterol C14-demethylase, one of the
achieved by synthetic variations made in the lipid side chains main steps in ergosterol biosynthesis. It is interesting to
of echinocandins. LY30336 and caspofungin are the note that the structure of restricticin does not have a
examples of recently developed echinocandin analogs (phenylethyl)triazole moiety found in all azole antifungal
(Espinel-Ingroff 1998), which are generally more active agents in the market, possibly causing adverse impacts in
in vitro against a variety of yeast and filamentous fungi. They efficacy and resistance (Tuite 1996). However, restricticin
are licensed by Lilly and Merck, respectively, for clinical needed to be improved in its chemical stability, because the
usage. Such a novel mechanism of action, antifungal potency compound was found to be unstable due to the lability of the
and relatively broad-spectrum activity of echinocandins glycin ester side chain toward base-mediated hydrolysis and
provide the possibility that the inhibitors of b-1,3-glucan the tendency of the triene functionality to undergo
synthase may be available for the development of decomposition (Barrett-Bee and Ryder 1992). Along with
biofungicides effective against fungal diseases (Pfaller et al. the advances in the screening for inhibitors of other steps in
1998). sterol biosynthesis, more SBI sufficient for practical uses may
Chitin, b-1,4-N-acetylglucosamine polymer, plays a major be discovered from microbial metabolites.
structural and strengthening role in fungal cell walls. Chitin is
microfibrills consisting of hydrogen-bonded polysaccharide
chains that may be covalently cross-linked to other
polysaccharide, mainly glucan. It has been demonstrated 4.3 Acetyl-CoA Carboxylase (ACC)
that chitin synthase inhibitors and chitinase showed antifungal
activity when applied to growing cells (Lorito et al. 1993). Discovery of soraphen A from myxobacteria was an
Nikkomycins are analogs of UDP-N-acetylglucosamine important event in antifungal metabolite development,
produced by Streptomyces spp. They have potent activity because of not only enlarging microbial diversity as a source
against chitin synthase by acting as specific competitive of antifungal compounds but also introducing fungal ACC as
inhibitors (Hunter 1995). The potency of an inhibitor of a novel target for antifungal agent screening (Gerth et al.
chitinase synthase may depend on not only the isoform’s 1994). Acetyl-CoA carboxylase catalyzes carboxylation of
relative effectiveness in building a cell wall, but also its acetyl-CoA to malonyl-CoA at the expense of ATP. While the
affinity to a given enzyme. Recent research on chitin synthase functional units of ACC are usually separate proteins in
revealed that the multiple chitin synthase genes of fungi have prokaryotes, they form a multifunctional enzyme complex in
different sensitivities to the inhibitors (Munro and Gow eukaryotes. This may be the reason why soraphen A is
1995). Therefore, new antifungal compounds with higher inactive to bacteria. Soraphen A which is mainly responsible
activity and specificity to chitin synthase may be generated for antifungal activity of Sorangium cellulosum strain Soce26
from diverse chemical pool of microbial metabolites. effectively controlled powdery mildew (Erysiphe graminnis
Biofungicides 129

f. sp. hordei) in barley, snow mold (Gerlachia nivalis) in rye, biosynthesis as an antifungal target has been more
apple scab (Venturia inaequalis) on apple and gray mold challenging, because of the high degree of structural and
(B. cinerea) on grape (Reichenbach and Höfle 1995). functional identity of the components of the protein
Soraphen A has no effect on ACC of plants, thus inducing biosynthetic machinery between fungi and higher eukaryotes.
no phytotoxicity in the field (Vahlensieck et al. 1994). In As in the cases of cycloheximide, trichodermin, and
contrast, ACC from rat liver was strongly inhibited by the hygromycin B, their activities on the fungal ribosome appear
soraphen (Pridzun et al. 1995). Due to the risky side effects on to be identical to those on the mammalian ribosome (Tuite
experimental animals, soraphen A has not been practically et al. 1995). It was, therefore, of considerable interest to find
used for control of plant diseases. However, the results of out a specific antifungal agent targeting on fungal protein
soraphen research strongly suggest that fungal ACC could be biosynthesis.
a target site for antifungal agent screening. Considering the Sordarins were found to have highly specific inhibitory
numerous diversity of natural products related to the activity against the elongation factor 2 involved in the
specificity of ACC, novel biofungicides from microbial translation of several fungal species (Justice et al. 1998).
metabolites that block specifically the activity of fungal ACC Sordarins were originally isolated from the terrestrial
may be developed in the future. ascomycete Sordaria araneosa (Hauser and Sigg 1971).
The fungal specific activity of sordarins is quite interesting,
because the elongation factor 2 is a highly conserved protein.
4.4 Nucleic Acid Metabolism Recently, a mutant strain analysis revealed that sordarins had
additional interactions with the ribosome itself (Justice et al.
One of the areas that can be exploited as antifungal targets is 1999), indicating that the selectivity of these compounds was
nucleic acid metabolism. The synthesis of nucleic acids governed by multiple points of interaction between the
involves numerous biochemical reactions ranging from the compound and the ribosome. Using a high throughput
initial synthesis of purine and pyrimidine precursors to the screening (HTS) targeting on protein synthesis in Candida
final polymerization of ribonucleoside and deoxyribonucleo- spp., an analog of sordarin has recently been demonstrated to
side 50 -triphosphates into RNA and DNA. A large number of be an effective in vitro inhibitor with apparent selectivity for
compounds have been known to be inhibitors of nucleic acid fungal protein synthesis (Kinsman et al. 1998).
metabolism in fungi. However, few of these compounds have
been used as agricultural and clinical antifungal agents.
Recently, antibiotic tubercidin produced by Streptomyces
violaceoniger was discovered to have antifungal activity 5 FUTURE TRENDS IN BIOFUNGICIDE
against plant pathogenic fungi (Hwang and Kim 1995a; RESEARCH
Hwang et al. 1994). It was highly active against P. capsici,
Botryosphaeria dothidea, and R. solani. Tubercidin is an During the last two decades, there were numerous efforts
adenosine analog that interferes nucleic acid synthesis focusing on the isolation and identification of a wide range of
including de novo purine synthesis, rRNA processing, and biologically active natural products. As a result, hundreds of
tRNA methylation (Suhadolnik 1979). The potent in vivo thousands to millions of compounds became available for the
activity of tubercidin against P. capsici was compared with evaluation of their value as potential lead compounds. The
that of systemic fungicide, metalaxyl, which is one of the concept of a HTS was developed to screen a large number of
best-studied acylalanine targeting on the synthesis of chemical libraries, which overcome the limitation of
ribosomal RNA. Treatment with tubercidin on day 1 before conventional in vitro and in vivo assay. The HTS is made
inoculation of zoospores prevented phytophthora blight at possible by the advance in assay system, which was designed
500 mg ml21. Tubercidin was effective as much as metalaxyl, to target a specific biochemical event in fungal metabolism. A
irrespective of application time and concentrations, although direct measure of the activity of the compound at the target of
its antifungal activity did not persist as long as metalaxyl in interest can be done without complications arising from other
pepper plants. The potent antifungal activity of tubercidin metabolic events. These approaches can enhance the
against P. capsici suggests that possible targets for the possibility to discover new and useful biofungicides by
antifungal agent screening may be present in nucleic acid supplying unique bioassay system. This innovative procedure
metabolic pathway. was already applied in developing new fungicides such as
sordarins mentioned earlier. The target-directed screening
will be fortified by DNA sequence information that is
4.5 Protein Biosynthesis exponentially increased in recent years by a number of fungal
genome projects. The genomic information can provide a
Protein biosynthesis is available as a set of molecular targets wealth of new targets to be validated and screened for new
for antibacterial agent development. The antibiotics such as antifungal leads (DiDomenico 1999).
chloramphenicol and streptomycin have been demonstrated to Along with the innovations in screening systems, the
inactivate or alter the accuracy of the bacterial ribosome efforts to diversify the chemical library of microbial
(Cundliffe 1990). However, the use of fungal protein metabolites has been continued through combinatorial
130 Kim and Hwang

approaches. Recently, the ability to synthesize a large number actinomycetes, other eubacteria and fungi, which may be
of chemical libraries from core structure of antibiotics was available for antifungal leads. The advances in the screening
greatly enhanced by the advance of rapid combinatorial/ system directed to fungal specific targets have rendered more
parallel synthesis method (Caporale 1995). The diversity and chances to get success in biofungicide development. A
numbers of distinct compounds in combinatorial library number of useful targets have been discovered from the
enhance the possibility of finding a chemical structure fungal metabolism related to nucleic acid, protein, sterol, and
with the desired properties. Combinatorial libraries can be cell-wall biosynthesis. The recent successful example of
synthesized in many different ways as reviewed by Dolle sordarin analogs show that better understanding of biochemi-
(1999). However, most of the successes in combinatorial cal events in fungal cells would uncover more useful targets
chemistry have been accomplished by using small libraries to for the screening of antifungal leads. Combinatorial
improve the properties of a specific toxophore. The successful approaches in chemical and biochemical synthesis were
optimization of azole and oxazolidinone lead compounds suggested to diversify the chemical library of microbial
suggested a promising future of combinatorial chemistry in metabolites, which can make it easier to discover the
biofungicide research (Trias 2001). optimized antifungal compound with desired physical and
As another approach to diversify the chemical library of biological properties. These new trends in developing novel
microbial metabolites, combinatorial biosynthesis was biofungicides will be more facilitated and strengthened by
proposed to generate “unnatural” natural products, which innovative multidisciplinary approaches in the future.
use genetic information and DNA recombination techniques
to alter the biosynthetic pathway of the microorganism to
produce the designed chemical structure. This can also be
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12
Molecular Biology of Biocontrol Trichoderma

Christian P. Kubicek Institute of Chemical Engineering, Vienna, Austria

1 INTRODUCTION 2 TRICHODERMA BIOCONTROL TAXA

AND STRAINS
Plant diseases, caused primarily by fungal and bacterial
pathogens, produce severe losses to agricultural and The genus Trichoderma currently consists of more than 40
horticultural crops every year. These losses can result in known taxa, which are usually cosmopolitan, (although some
reduced food supplies, poorer quality agricultural products, species display a geographic bias: Kubicek et al. 2002;
economic hardship for growers and processors, and, Kullnig et al. 2000), and typically soilborne or wood decaying
ultimately, higher prices. For many diseases, traditional Teleomorphs of Trichoderma occur in the genera Hypocrea,
chemical control methods are not always economical nor are Podostroma, and Sarawakus of the Hypocreaceae (Gams and
they effective, and fumigation as well as other chemical control Bissett 1998; Rossman et al. 1999). The latter two genera
methods may have unwanted health, safety, and environmental thereby most likely being synonyms of Hypocrea
risks. Biological control involves the use of beneficial (GJ Samuels, personal communication). Rossman (1999)
microorganisms to attack and control plant pathogens and proposed that necrotrophy (on basidiomycetes) is the original
the diseases they cause. It offers an environmentally friendly habitat of these Hypocrea spp. and their lignicolous properties
approach to the management of plant disease and can be have developed later when the species were following their
integrated into an effective integrated disease management hosts into their habitat (wood and decaying wood in soil).
system. Thus, biological control can be an important Rossman et al. (1999) claim that the Hypocrea spp., which are
component in the development of a more sustainable found on decaying wood, actually are necrotrophic on the
agriculture. fungi in the wood. Several of the individual teleomorphic and
Trichoderma species have been investigated as bio- anamorphic partners have been detected recently, and
logical control agents for over 70 years, but it is only examples relevant to biocontrol are given in Table 1. The
relatively recently that strains have become commercially more than 100 species of Hypocrea with Trichoderma
available. The previous considerations have stimulated anamorphs which Doi and Doi (1986) described constitute
researchers to gain a better knowledge of biocontrol by this unexplored source of potential biocontrol agents.
fungus, and to understand their mechanisms of control. In Most of the isolates of the genus Trichoderma, which
view of the actuality of this research field, there are have been found to act as biocontrol agents, have been
numerous recent articles available which review the current classified as T. harzianum Rifai, leading to the fact that
state of knowledge of Trichoderma biocontrol (Chet et al. T. harzianum is generally synonymized as a “biocontrol
1998; Harman and Björkman 1998; Hjeljord and Tronsmo agent.” However, most of the Trichoderma strains used for
1998; Monte 2001 see also the atricle by A. Herrera- biocontrol were identified at the species level exclusively on
Estrella and I. Chet, this volume). In this article, the current the basis of morphological and phenotypical characters,
state of biological knowledge on Trichoderma strains showing high convergence in many cases (Kullnig-Gradinger
capable of biocontrol on a molecular level will be et al. 2003). Therefore, reports of a pronounced genetic
summarized. variability of T. harzianum isolates by analyzing carbon

135
136 Kubicek

Table 1 Teleomorphs known for Trichoderma taxa used in the soil in situ. To monitor the behavior of a given strain in the
biocontrol soil, Bae and Knudsen (2001) cotransformed T. harzianum
with genes encoding green fluorescent protein (GFP),
Anamorph Teleomorph beta-glucuronidase (GUS), and hygromycin B resistance
T. harzianum H. lixii (former H. nigricans) (hygB). One of the resulting strains was formed into calcium
T. atroviride H. atroviride alginate pellets and placed onto buried glass slides in a
T. virens H. virens nonsterile soil, and its ability to grow, sporulate, and colonize
T. asperellum Not known sclerotia of Sclerotinia sclerotiorum was compared with that
T. parceramosum Not known of the wild-type strain. The green color of cotransformant
T. longibrachiatum Not known hyphae was clearly visible with a UV epifluorescence
microscope, while indigenous fungi in the same samples were
barely visible. Green-fluorescing conidiophores and conidia
were observed within the first 3 days of incubation in soil, and
source utilization patterns (Manczinger and Polner 1985), this was followed by the formation of terminal and intercalary
secondary metabolite production (Okuda et al. 1982), chlamydospores and subsequent disintegration of older
isoenzyme polymorphism (Grondona et al. 1997; Stasz et al. hyphal segments. In addition, no significant differences
1989), RAPD profiles (Fujimori and Okuda 1994; Gomez were detected in colonization levels between wild-type and
et al. 1997; Muthumeenakshi et al. (1994); Turoczi et al. cotransformant strains; and the authors concluded that GFP
1996; Zimand et al. 1994), RFLP patterns (Bowen et al. proved a most useful tool for nondestructive monitoring of the
1996; Muthumeenakshi et al. 1994), rDNA sequence hyphal growth of the transformant in a natural soil. Also, the
(Grondona et al. 1997; Muthumeenakshi et al. 1994) and chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-
karyotype (Gomez et al. 1997) must be treated with caution. glucuronic acid (X-Gluc) could be used to monitor the
On the basis of a rigorous comparison of a pool of seventeen activity of b-glucuronidase in soil. Thus, cotransformation
bonafide “T. harzianum” biocontrol strains with the neo-ex with GFP and GUS can provide a valuable tool for the
type strain of T. harzianum, Hermosa et al. (2000) showed detection and monitoring of specific strains of T. harzianum
that they actually comprised of four different species i.e., released into the soil.
T. harzianum, T. atroviride, T. longibrachiatum and As the biological strains of Trichoderma are difficult to
T. asperellum. Consistent results were also reported by distinguish from the indigenous strains of Trichoderma found
Kullnig (2001), who by sequence analysis of the internally in the field, Hermosa et al. (2001) developed a method to
transcribed spacer regions of the rDNA (ITS1 and ITS2), the monitor these strains when applied to natural pathosystems.
small subunit of the mitochondrial DNA (mtSSUrDNA), To this end they used random amplified polymorphic
and part of the coding region of the 42-kDa endochitinase DNA (RAPD) markers to estimate genetic variation
encoding gene ech42- reassessed the species identity of among sixteen strains of the species T. asperellum,
eight T. harzianum isolates, which are being used by several T. atroviride, T. harzianum, T. inhamatum, and
laboratories for key investigations on the genetics, biochem- T. longibrachiatum. Analysis of the respective RAPD
istry, and physiology of biocontrol. Thereby the strains products generated were used to design specific primers.
T. harzianum CECT 2413, T-95, T-22, and T-11 were Diagnostic PCR performed using these primers specifically
confirmed as T. harzianum, “T. harzianum” ATCC 74058, identified one of their strains (T. atroviride 11), and clearly
IMI 206040, ATCC 36042 identified as T. atroviride, and distinguished this strain from other closely related Tricho-
“T. harzianum” T-203 assessed as T. asperellum. As outlined derma isolates, showing that SCAR (sequence-characterised
above, there may be other species capable of biocontrol amplified region) markers can be successfully used for
as well, T. virens being the most prominent example. In identification purposes.
addition, molecular proof for identity of other species as An alternative approach, suitable to monitor the presence
biocontrol agents has been presented for T. ghanense of several strains in one sample was presented by van Elsas
(previously T. parceramosum; Arisan-Atac et al. 2002) and
et al. (2000) by selecting a nested PCR approach, in which
T. stromaticum (Samuels et al. 2000).
the first PCR provided the required specificity for fungi,
whereas the second (nested) PCR served to produce
amplicons separable on denaturing gradient gels. Denatur-
3 IN SITU MOLECULAR TOOLS FOR ing gradient gel electrophoresis (DGGE) allowed the
BIOCONTROL STRAINS resolution of mixtures of PCR products of several different
fungi including Trichoderma. Although only limited
Even if the species identity is not a concern, the ability to examples have so far been published, techniques like
recognize the strain which was introduced into the field is of these and the fast current advance in PCR technology
interest. Appropriate molecular tools have thus recently been (such as real-time PCR to name only one) will stimulate
introduced for identifying Trichoderma strains in the further studies of the behavior of Trichoderma biocontrol
environment, and to follow their fate after introduction into agents in the field is now possible.
Molecular Biology of Biocontrol Trichoderma 137

4 GENOME ORGANIZATION AND interesting, as N. crassa has evolved about 200 million
REPRODUCTION years ago (Berbee and Taylor 1993), whereas H. jecorina
evolved only about 100 million years ago (Kullnig-
One of the major difficulties with Trichoderma biocontrol Gradinger et al. 2003), and thus the genomic organization
strains is their genetic instability, whose reason is only poorly of these genes has been maintained constant for about 100
understood at present. This is in part due to the fact that only million years. Hamer et al. (2001) have also recently
little is known about the genome organization and its reported that a 53-kb region of the genome of Magnaporthe
plasticity of Trichoderma. Not even the number of grisea was also syntenic to a corresponding portion of the
chromosomes is known with certainty: Fekete et al. (1996) Neurospora genome. In a comprehensive study on
separated six chromosomes in five Trichoderma biocontrol hemiascomycetous yeasts, Llorente et al. (2000) demon-
strated that even phylogenetic distant species such as
strains with sizes ranging from 3.7 to 7.7 Mb; estimated
S. cerevisiae and Yarrowia lipolytica exhibit 10.1 % of
genome sizes were between 30.5 and 35.8 Mb. When
conserved synteny. If there is indeed a high degree of
fractionated chromosomes of the five species were probed
synteny between Neurospora and Trichoderma, this may be
with a fragment of the ech42 (endochitinase-encoding) gene,
useful for studying the genomic organization of
strong hybridization signals developed, but their physical
Trichoderma biocontrol strains.
position varied among species indicating a polymorphic
Probably due to reproduction, largely via asexual
chromosomal location. Herrera-Estrella et al. (1993) com-
mechanisms, many species of Trichoderma reveal a high
pared the molecular karyotype of T. reesei with that of
level of genetic stability (cf. Kubicek et al. 2002; Kullnig
T. atroviride (named erroneously T. harzianum in their study),
et al. 2000). T. harzianum, however, is a noteworthy
and T. viride, and detected largely similar chromosomal
exception, showing a remarkable intraspecific genetic and
organization of genes in different species, although T. viride
phenotypic variation, and this may also be related to the
seemed to lack the smallest chromosome. Similarly, Hayes instability of the respective biocontrol species. The reason
et al. (1993), when karyotyping three biocontrol strains of for this has not been explained yet. As the respective
T. harzianum (one parent and two mutants derived from it), teleomorph (H. lixii) is known, the possibility of sexual
found that the smallest chromosome was not present in the recombination still needs rigorous testing. Transposons,
mutants. While all these studies revealed a low degree of which have been isolated from phylogenetically close
chromosome polymorphism at the species level, the fungal genera such as Tolypocladium or Fusarium, are
karyotypes were relatively constant. A report to the contrary another possibility. We have recently observed a very high
(Gomez et al. 1997) is probably flawed by the use noninduced mutation rate in one biocontrol strain of
“T. harzianum” strains which in fact consisted of several T. harzianum which would be compatible with the presence
different species (CP Kubicek, unpublished data). Thus, as of a mobile element (C Gallhaup, RL Mach and
expected for an asexual fungus, chromosome plasticity is CP Kubicek, unpublished).
unlikely responsible for the genetic instability of Trichoderma As far as nonchromosomal elements are concerned,
biocontrol strains. plasmids have been detected in filamentous fungi almost
Molecular genetic work with Trichoderma spp. is still exclusively in the mitochondrium (Bertrand 2002). They are
limited by the only rudimentary information about its generally stable genetic elements and vary between 1 –6 kb
genomic organization as is available for Aspergillus size. In accordance with this situation, Meyer (1991)
fumigatus (http://www.tigr.org/tdb/e2k1/afu1/) and Neuro- detected mitochondrial plasmids in strains of T. viride and
spora crassa (http://www-genome.wi.mit.edu/annotation/ the biocontrol-relevant species T. asperellum (then named
fungi/neurospora). Genetic maps could so far not be “T. viride 2”). A circular plasmid called pThr1, with a
constructed, because the teleomorphs of biocontrol species monomer size of 2.6 kb, was identified in the mitochondria
of Trichoderma (see Table 1) do not cross in axenic culture of the biocontrol isolate T. harzianum T95 (Antal et al.
(CP Kubicek, unpublished data). Also, at the time of this 2002). It revealed no DNA sequence similarity with the
writing, genome sequencing projects on selected species of mitochondrial genome of the isolate and contained a single
Trichoderma have only just been initiated at a few places, and 1818 bp open reading frame. The derived amino acid
no results from these are yet available. However, a collection sequence exhibited similarity to the reverse transcriptases of
of 1151 ESTs of T. reesei grown on glucose and the sequence the circular Mauriceville and Varkud retroplasmids of
of the complete mitochondrial genome is already available in Neurospora spp. and the linear pFOXC2 and pFOXC3
the Internet (http://trichoderma.iq.usp.br/TrEST.html), and retroplasmids of Fusarium oxysporum strains. In the
can (because of the high similarity of nucleotide sequences regions of homology all of the seven conserved amino
of protein encoding genes within the genus (unpublished acid blocks characteristic of RTs could be found. In
data) be used for picking genes from biocontrol strains as Fusarium oxysporum f. sp. conglutinans, these mito-
well). chondrial plasmids have been identified as factors
Interestingly, Seiboth and Hofmann (2002) found a similar determining the host specificity (Kistler and Leong 1986);
genomic organization of several genes of galactose unfortunately, corresponding investigations are still lacking
metabolism in T. reesei and N. crassa. This finding is highly for Trichoderma.
138 Kubicek

5 MOLECULAR GENETIC BASIS OF the cellulose-binding domain from cellobiohydrolase II of

BIOCONTROL T. reesei. The chimeric chitinases had similar activities as the
native chitinase towards soluble substrates, but higher
Arising from nectrotrophic ancestors, most of the currently hydrolytic activity on high molecular mass insoluble
known Trichoderma strains have developed highly effective substrates (chitin or fungal cell walls). Unfortunately, no
antagonistic mechanisms to survive and colonize the results from in vivo biocontrol tests were reported, and it
basidiomycete-containing competitive environment of the remains thus unclear whether the presence of such a domain
rhizosphere, soil, and decaying wood. Active parasitism on would improve the antagonistic abilities of Trichoderma
host fungi, by penetration of host hyphae is probably the biocontrol strains.
mechanism most studied (cf. Chet et al. 1998); it requires The action of chitinases and glucanases is also strongly
synergistic both with other chitinase components as well as
morphological changes of Trichoderma hyphae such as
with other components putatively involved in biocontrol, i.e.,
appressorium formation and coiling, and is further supported
antibiotics (Jach et al. 1995; Lorito et al. 1994; 1996b;
by the production of extracellular enzymes, and production of
Schirmböck et al. 1994). In the case of the peptaibols, the
antifungal antibiotics. However this mechanism has mostly
mechanism of this enzyme – antibiotic synergism has been
been observed in the laboratory, and application of
shown to be due to a synergistic effect of enzyme and the
Trichoderma in the field may involve additional mechanisms
antibiotic on the maintenance of cell wall integrity (Lorito
as well such as aggressive degradation of organic matter,
et al. 1996b). Peptaibols are linear oligopeptides of 12 –22
thereby competing for nutrients which in saprobic phases may
amino acids, which are rich in a-aminoisobutyric acid,
be a limiting factor. Also promotion of the growth and
N-acetylated at the N-terminus and containing an amino
biological activities of saprobic bacteria and mycorrhizal
alcohol (Pheol or Trpol) at the C-terminus (Rebuffat et al.
fungi, and of plant-growth and induced resistance have been
1989), and known to form voltage-gated ion channels in black
reported (for review see Herrera-Estrella and Chet, lipid membranes and modify the membrane permeability of
Chapter 57). liposomes in the absence of applied voltage (El Hadjji et al.
Among these, mycoparasitism is the only process which 1989). Hence, while the chitinases reduce the barrier effect of
has been studied on a molecular biological basis. Herrera- the cell-wall, peptaibol antibiotics inhibit the membrane
Estrella and Chet (Chapter 57) give a detailed account on this, bound chitin- and b-glucan synthases and thereby impair the
and I shall therefore treat this point here only very briefly: ability of the hyphae to repair the lytic effect of the enzymes
most attention has been paid to the enzymatic disruption on the cell walls polymers.
of the cell wall of the fungus, thereby focusing on The gene (tex1) encoding the enzyme synthesizing these
enzymes capable of hydrolysis its structural polymers peptaibols (peptaibol synthase) has recently been cloned
(chitin, b-glucan, protein and others). Genes encoding from T. virens (Wiest et al. 2002). It comprises a 62.8 kb
endochitinases, N-acetyl-b-glucosaminidases, proteases, continuous open reading frame encoding a protein structure
endo- and exo-glucan b-1,3-glucosidases, endoglucan-b- consisting of 18 peptide synthetase modules with additional
1,6-glucosidases, lipases, xylanases, amylases, phospho- modifying domains at the N- and C-terminii. Mutation of the
lipases, RNAses, and DNAses have been cloned from various gene eliminated production of all peptaibol isoforms,
biocontrol species of Trichoderma, and are listed in detail in indicating that their formation is due to a relaxed substrate
the above-mentioned chapter (also see Benitez et al. 1998; specificity of the individual synthase domains. Interestingly,
Kubicek et al. 2001; Lorito 1998). Most of these enzymes the nucleotide sequence of tex1 is 100% identical to a
showed very strong antifungal activity against a variety of 5,056-bp partial cDNA fragment of another gene ( psy1)
plant pathogenic fungi in vitro. Several of these cell wall isolated also from T. virens (Wilhite et al. 2001). These
degrading enzymes, but most notably chitinases, have thus authors observed that psy1 disruptants grew poorly under
been demonstrated to have a great potential as active low-iron conditions, and failed to produce the major T. virens
components in new fungicidal formulations or genetically siderophore, dimerum acid (a dipeptide of acylated
modified plants. N(a)-hydroxyornithine, thus suggesting that Psy1 plays a
Interestingly, the endochitinases found in Trichoderma role in siderophore production. Biocontrol activity against
belong only to one (class V) of the several classes of the damping-off diseases caused by Pythium ultimum and
chitinases known from plants (Beintema 1994). The latter Rhizoctonia solani was not reduced by the psy1disruption.
show a modular structure, and frequently contain protein The discrepancy between the results reported by Wiest et al.
domains capable of binding to chitin, which bear some (2002); Wilhite et al. (2001) need to be explained before the
resemblance to the cellulose-binding domains also found in importance of the tex1/psy1 gene in biocontrol can be
Trichoderma cellulases. In contrast, none of the chitinases estimated.
cloned from Trichoderma spp. so far has been shown to Peptaibols, however, are certainly not the only secondary
contain such a chitin-binding domain. To investigate the role metabolites with synergistic action in host cell-wall
of the latter, Limon et al. (2001) have produced hybrid degardation. Other components (e.g., gpentyl pyrone) was
chitinases with stronger chitin-binding capacity by fusing to also found to be important for antagonism in vivo (Claydon
Chit42 a ChBD from Nicotiana tabacum ChiA chitinase and et al. 1987; Howell 1998; Serrano-Carreon et al. 1993), and
Molecular Biology of Biocontrol Trichoderma 139

their mechanism of action thus awaits to be elucidated. these low-molecular weight, biocontrol-inducing molecules
6-pentylapyrone is probably the most frequently studied of released from the host cell walls, and showed that they were
these metabolites, as it also exhibits a pronounced “coconut- much more active in vitro than purified chitin or glucan
aroma” which can be used as a (for humans) nontoxic monomers.
flavoring agent. Its biosynthesis has been claimed to be The failure of T. atroviride to induce ech42 expression by
derived from linolenic acid (Serrano-Carreon et al. 1993), but chitin may be due to complex interactions within different
this conclusion was criticized by Sivasithamparam and regulatory circuits (Donzelli and Harman 2001): both ech42
Ghisalberti (1998), who consider it to be a product of and nag1 required both nitrogen starvation and the presence
polyketide biosynthesis. No other of the genes or proteins of chitin for induction, whereas gluc78 could be induced by
involved in Trichoderma secondary metabolism has as yet nitrogen starvation alone. In the presence of low levels of
been characterized. ammonium (10 mM), both chito-oligomers and chitin
triggered CHIT42 and CHIT40 (chitobiosidase) production.
CHIT73 secretion occurred in the presence of N-acetyl-
glucosamine and chito-oligomers, while chitin was less
6 BIOCONTROL-SPECIFIC GENE effective. These results indicate that the expression and
EXPRESSION IN TRICHODERMA secretion of cell wall-degrading enzymes by Trichoderma is
nitrogen repressed, and that effects of carbon and nitrogen
In the laboratory, high-level induction of extracellular cell- nutrition are interactive. The expression of ech42 from
wall lytic enzymes is usually obtained by growing T. atroviride after prolonged carbon starvation is likely not
Trichoderma on purified chitin, fungal cell walls, or mycelia due to a relieve from carbon catabolite repression, as it can
as sole carbon sources. No, or much less, induction is be observed with glucose as well as with glycerol as a carbon
normally obtained when related compounds such as chitosan, source (Mach et al. 1999). In addition, ech42 gene
cellulose, unpurified chitin, or laminarin are used. In addition, transcription was triggered by some conditions of physio-
formation of most chitinolytic enzymes does not occur or is logical stress (48C, high osmotic pressure, addition of ethanol;
even inhibited by glucose, sucrose, and chitinolytic end- Mach et al. 1999), as well as during light-induced sporulation
products (Carsolio et al. 1994; Garcia et al. 1994; Lorito et al. (Carsolio et al. 1994). Interestingly, T. harzianum chit33
1996a; Margolles-Clark et al. 1996; Peterbauer et al. 1996), expression, while being inducible by N-acetyl-b-D -gluco-
suggesting that direct induction and/or catabolic repression samine, was also triggered by carbon starvation, nitrogen
are major regulatory parameters for chitinase formation. starvation and physiological stress (de las Mercedes Dana
Some researchers also found trace quantities of some et al. 2001), suggesting that stress-mediated regulation may
chitinases (e.g. the 102-kDa N-acetyl-b-D -glucosaminidase, be a general phenomenon involved in chitinase gene
the 42-kDa endochitinase and the 33 kDa endochitinase) are expression of Trichoderma spp.
produced constitutively (Carsolio et al. 1999; Garcia et al. Some studies have so far been performed towards
1994; Haran et al. 1995; Inbar and Chet 1995; Margolles- understanding how and in which order the chitinases are
Clark et al. 1996). It should be noted that this does not rule out induced during mycoparasitic interaction. In their pioneering
regulation of the respective promoters by induction only, due studies, Inbar and Chet (1992); (1995) demonstrated that
to the fact that the binding of DNA-binding proteins to their formation of chitin-degrading enzymes in T. harzianum is
target sequences is an equilibrium, every promoter will elicited by a lectin-based physical interaction with the host,
partially be in transcriptionally active state, depending on the which was suggested to be the earliest event of interaction,
Kd and the concentrations of the respective proteins. and precede induction by possible chitooligomers (see chapter
Some of these findings have recently been supported by Herrera-Estrella and Chet). Inbar and Chet (1995) showed
the analysis of gene expression. The expression of that a 102-kDa chitinase is specifically induced by contact
T. atroviride nag1 is triggered by fungal (B. cinerea) cell with the host lectin, whereas formation of all the other
walls and the commercially available chitin monomer chitinases requires the presence of the living host. They
N-acetyl-glucosamine, and the oligomers di-N-acetyl- concluded that an N-acetyl-b-D -glucosaminidase with
chitobiose and tri-N-acetylchitotriose (Mach et al. 1999). In apparent denatured Mr of 102 kDa may be responsible for
contrast, ech42 expression in T. atroviride was also observed the first attack and induction for the other chitinases.
during growth on fungal cell walls, but could not be triggered However, Zeilinger et al. (1998), using the Aequorea victoria
by those chitin degradation products (Margolles-Clark et al. GFP as a nondisruptive reporter system, showed that ech42,
1996; Cortés et al. 1998; Mach et al. 1999), whereas in but not nag1, was formed before any detectable contact of
T. harzianum it is induced by N-acetyl-b-D -glucosamine Trichoderma with its host. Similar studies with chit33:GFP in
(Garcia et al. 1994; Schickler et al. 1998). Digestion of the T. harzianum (de las Mercedes Dana et al. 2001) showed that
host cell walls with specific combinations of purified this pre-contact gene expression did not occur with the
Trichoderma-secreted chitinases and glucanases (both endo- 33-kDa endochitinase-encoding gene chit33, and therefore
and exo-acting) released products that strongly elicited ech42 may be specific for ech42. Interestingly, ech42 gene
and nag1 gene expression and consequent mycoparasitic expression was prevented when a dialysis membrane was
activity. Lorito (2002) recently reported the purification of placed between the two fungi (Zeilinger et al. 1999), but still
140 Kubicek

occurred when a cellophane membrane was used for this microscopic examinations of the attached seed coats
purpose (Cortés et al. 1998). This led to contradicting suggested that the lack of the 42-kDa endochitinase may
conclusions regarding the nature of the molecule triggering have stimulated the colonization of the spermo- and
ech42 gene expression (Cortés et al. 1998; Zeilinger et al. rhizosphere.
1999); this issue was consequently solved by showing that the Other cell wall hydrolases, whose effect on biocontrol has
cellophane, but not the dialysis, membrane, was partially been studied, are the chitinases chit33 and nag1 and the
permeable to proteins of relatively large size (up to 100 kDa) proteinase prb1. b-Glucanases have also been tried but their
(Kullnig et al. 2000). Thus the data from both studies (Cortés overexpression appears to be counteracted by overexpression
et al. 1998; Zeilinger et al. 1999) were in perfect agreement of acid proteases (Delgado-Jarana et al. 2000). Using a
and showed that ech42 is expressed before contact of constitutively expressed pki1::chit33 fusion, Limón et al.
Trichoderma with its host, probably representing one of the (1999) obtained recombinant strains with higher antagonizing
earliest event in mycoparasitism and biocontrol. By using two activity against R. solani on agar plates. However, results
types of membranes (one permeable and one not permeable to from experiments with these mutants performed in glasshouse
proteins), which allowed the removal of either Trichoderma or soil have not been reported. T. harzianum transformants
or Rhizoctonia colony from the plate and thus the performing carrying two to ten copies of the prb1 gene significantly
of subsequent cultivations, Kullnig et al. (2000) also showed reduced the disease caused by R. solani in cotton plants under
that a chitinase activity, secreted constitutively by Tricho- greenhouse conditions (Flores et al. 1997). Interestingly,
derma, is essential for the triggering of ech42 gene culture filtrates of a T. atroviride nag1-delta strain, despite of
expression. The nature of this enzyme is still unknown; and their inability to induce chitinase gene expression (see earlier)
it could very well be either (constitutive amounts of) the exhibited a moderately reduced ability (40 –50%) to protect
42 kDa endochitinase itself or the 102 kDa protein of Inbar beans against infections by Rhizoctonia solani and
and Chet (1995), or any other constitutively formed chitinase S. sclerotiorum (Brunner et al. 2003). Therefore, while nag1
or chitinases. To this end, Brunner et al. (2002) deleted the is essential for triggering chitinase gene expression in
nag1 (73-kDa N-acetyl-b-D -glucosaminidase encoding) gene T. atroviride, the almost complete loss of chitinase activity
of T. atroviride. These strains were unable to induce ech42 only partially impairs biocontrol activity against R. solani and
gene transcription under conditions of carbon starvation or in S. sclerotiorum. It is possible that this may be compensated by
the presence of fungal cell-walls, and also lacked the an increased formation of glucanolytic enzymes in this strain
formation of other enzyme activities capable of hydrolyzing (unpublished data).
PNP-NAcGlc, PNP-NAcGlc2, and PNP-NAcGlc3. Since the A more general approach towards improvement of the
102-kDa exochitinase does not occur in T. atroviride P1 biocontrol properties of T. harzianum CECT 2413 was
(unpublished data), the 73-kDa enzyme may fulfil the role presented by Rey et al. (2001); they selected improved
of the T. harzianum 102 kDa enzyme. Unfortunately, a biocontrol mutants by testing for the ability to produce wider
characterization of the latter enzyme has not yet been haloes on pustulan, a polymer of beta-1,6-glucan, as a carbon
published. source. Interestingly, the mutants exhibited two- to four times
The obvious antifungal activity of Trichoderma chitinases more chitinase, beta-1,3- and beta-1,6-glucanase activities
has consequently lead to attempts to improve or alter than the wild type, and produced about three times more
biocontrol properties of strains by chitinase gene manipula- extracellular proteins. This mutant performed better than the
tions. Somewhat conflicting data have been reported on the wild type during in vitro experiments, overgrowing and
effect of overexpression and/or deletion of selected chitinase sporulating on R. solani earlier, killing this pathogen faster
genes of Trichoderma. Carsolio et al. (1999) found no and exerting better protection on grapes against B. cinerea.
difference between an ech42- disrupted strain and its parent
T. atroviride IMI 206040 in the biocontrol activity in
glasshouse tests against Sclerotium rolfsii and R. solani on
cotton, and therefore concluded that ech42 is not essential for 6.1 Cis and Trans-Acting Genetic Factors
biocontrol activity. In contrast, Woo et al. (1998); Baek et al. Relevant to the Expression of Biocontrol
(1999) noted pronounced effects on the biocontrol efficacy of Genes
an ech42 gene disruption mutant of T. atroviride P1 or
T. virens, respectively. The latter authors reported an Lorito et al. (1996a) first used an in vitro approach to detect
increased and decreased biocontrol activity against R. solani cis-acting motifs on the ch42 promoter being involved in
on cotton in strains of T. virens containing two ech42 copies mycoparasitism. They confronted Botrytis cinerea on agar
and a disrupted ech42 gene copy. Woo et al. (1998) also plates with T. atroviride P1, prepared crude protein extracts
observed a significant reduction in antifungal activity for the from mycelia harvested at different phases during myco-
ech42 disrupted strain and in vivo tests against B. cinerea by parasitism, and used them in electrophoretic mobility shift
leaf inoculations of bean plants revealed a significant assays. Competition experiments, using oligonucleotides
reduction of biocontrol ability of the disruptant strain. In containing functional and nonfunctional consensus sites for
contrast, a significant increase was noted for the biocontrol binding of the carbon catabolite repressor Cre1
efficacy of soils heavily infested with R. solani. Macro- and (50 -SYGGRG-30 ; Kulmburg et al. 1993) provided evidence
Molecular Biology of Biocontrol Trichoderma 141

that the complex from nonmycoparasitic mycelia involves Another cis-acting element was recently identified that
the binding of Cre1 to both fragments of the ech-42 may contribute to the regulation of ech42 gene expression: the
promoter. These findings are consistent with the presence of ech42 promoter sequence contains two short nucleotide
two and three consensus sites, respectively, for binding of sequences which resemble the consensus for binding of the
Cre1 in the two ech-42 promoter fragments used. In Aspergillus nidulans brlA (bristle) regulator (50 -MRAGGGR-
contrast, the protein-DNA complex from mycoparasitic 30 ; Chang and Timberlake 1992). The encoded BrlA protein is
mycelia does not involve Cre1, as its formation is a general regulator of conidial development, which itself
unaffected by the addition of the competing oligonucleo- responds to carbon starvation (Skromne et al. 1995). Cell-free
tides. Based on these findings, they offered a preliminary extracts of T. atroviride, prepared from mycelia subjected to
model for regulation of ech-42 expression in T. harzianum, carbon starvation, form a specific, consensus-dependent
which subsequently involves: (a) binding of Cre1 to two complex with BrlA site-containing oligonucleotide fragments
single sites in the ech-42 promoter; (b) binding of a of the ech42 promoter (K Brunner, CK Peterbauer, and CP
“mycoparasitic” protein/protein complex to the ech-42 Kubicek, unpublished data). Deletion of the promoter areas
promoter in vicinity of the Cre1 binding sites, and (c) containing the BrlA sites in vivo resulted in a derepression of
functional inactivation of Cre1 upon mycoparasitic the starvation induced expression of ech42, but had no effect
interaction to enable the formation of the “mycoparasitic” on the expression of ech42 during sporulation. This motif
protein–DNA complex (Lorito et al. 1996a). The cre1 gene therefore likely binds a new repressor of Trichoderma rather
from T. harzianum has been cloned (Ilmen et al. 1996), but than a sporulation specific regulator.
no demonstration of its effect on biocontrol in vivo was as The induction of nag1 by chitin oligomers has been
yet presented. studied in more detail, using a combination of promoter
deletion, in vivo footprinting, and EMSA experiments,
proteins binding to an AGGGG-element, to a CCAGN13-
CTGG motif and to a CCAAT-box were identified
(Peterbauer et al. 2002a,b). Disruption of either of the two
former binding sites in vivo resulted in an almost complete
reduction of induction of nag1 expression by N-acetylgluco-
samine. The nature of the proteins binding to these three
motifs is only partially understood: the spatial organization of
the CCAGN13CTGG motif would be compatible with the
binding of a Zn(II)2Cys6-type zinc finger protein (Todd and
Andrianopoulos 1997), whereas the CCAAT-box binds a
protein complex consisting of at least three proteins Hap2,
Hap3, and Hap5, which were originally described in
S. cerevisiae and more recently characterized from T. reesei
(Zeilinger et al. 2001). According to Narendja et al. (1999);
Zeilinger et al. (2003), their function is the establishment of
an open chromatin structure at the promoter.
The AGGGG-box is a motif which has been studied in
detail in Saccharomyces cerevisiae and identified as a binding
site for the Cys2His2 zinc finger proteins Msn2p and Msn4p,
which are key regulators of the transcription of a number of
genes coding for proteins with stress-protective functions
(Ruis and Schüller 1995). In Trichoderma, the occurrence of
the AGGGG-box is not restricted to the nag1 promoter but
also occurs in two other chitinase promoters, ech42 and chit33
Figure 1 Scheme illustrating the hypothesis how chitinase gene (Lorito et al. 1996a; de las Mercedes Dana et al. 2001),
expression could be triggered in T. atroviride, based on results consistent with a potential role in chitinase regulation. This
from Brunner et al. (2002); Mach et al. (1999); Kullnig et al. would be compatible with a regulation of the expression of
(2000); Peterbauer et al. (2002a,b); Zeilinger et al. (1999). chitinase genes by metabolic stress, as shown both for ech42
Circled plus and minus indicate activation and inactivation of a and chit33 (see earlier). However, nag1 is not upregulated by
process, respectively, without implying the underlying mechan- stress (CK Peterbauer, unpublished data), and the presence of
ism. Proteins A, B and C refer to the Zn(6) cluster protein this motif must therefore serve another function. In this
(Peterbauer et al. 2002a), the mycoparasitic regulator (Lorito context it is interesting to note that an AGGGG-motif was
et al. 1996a) and the BrlA-box binding starvation response also identified in the cutinase promotor of Haematonectria
repressor (see text), respectively. The black triangles indicate haematococca, where it appeared to be involved in
NAcGlc molecules, and symbolize NAcGlc, (NAcGlc)2, and maintaining the basal expression level (Kämper et al. 1994).
(NAcGlc)3, respectively. In Yarrowia lipolytica, the AGGGG motif is bound by
142 Kubicek

the Mhy1p protein, whose transcription is dramatically ACKNOWLEDGEMENTS

increased during the yeast-to-hypha transition (Hurtado and
Rachubinski 1999). Research by the author has been performed in collaboration
In order to study whether a homologue of S. cerevisiae with RL Mach, S Zeilinger, CK Peterbauer, and K Brunner.
MSN2/4 and H. haematococca AGGGG-binding protein The projects are funded to CPK by the Jubiläumsstuftung der
encoded by the open reading frame AAB04132 (which we Österreichischen Nationalbank (7817) and Austrian Science
call seb1, ¼stress element binding) has recently been Foundation (P13170-MOB).
cloned from T. atroviride (Peterbauer et al. 2002a,b). Its
zinc finger domain has high amino acid sequence identity
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Mol Gen Genomics 267:124 – 132. Trichoderma harzianum strain P1. Mol Plant-Microbe Interact
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Kubicek CP eds. Trichoderma and Gliocladium, Vol. 2. during mycoparasitic interaction of Trichoderma harzianum
Enzymes, Biological Control and Commercial Application. with its host. Fungal Genet Biol 26:131 –140.
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Stasz TE, Nixon K, Harman GE, Weeden NF, and Kuter GA (1989). 266:56– 63.
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polymorphism. Mycologia 81:391– 403. derma reesei) cellulase promoter cbh2 reveal a novel role for
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L (1996). Biological and molecular characterization of Mycol Res 98:531– 534.
13
The Biological Control Agent Trichoderma from
Fundamentals to Applications

A. Herrera-Estrella Centro de Investigación y Estudios Avanzados, Unidad Irapuato, Irapuato, México

I. Chet The Weizmann Institute of Science, Rehovot, Israel

1 INTRODUCTION Biocontrol must be effective, reliable, consistent, and

economical before it becomes an important component of
The practice of monoculture in modern agriculture enables us plant disease management. To meet these criteria, we must
to continue to provide foodstuffs for the world’s ever increase our understanding of the biology of the biocontrol
increasing population. Monoculture is, however, an eco- agent in question, which in most cases is extremely limited.
logically unnatural situation, that is inherently unstable and Furthermore, superior strains, together with delivery systems
offers considerable opportunity for the development of that enhance biocontrol activity, must be developed (Harman
diseases. Plant disease control has now therefore become et al. 1989). In this context, many biological control agents
heavily dependent on fungicides to combat the wide variety of can be modified genetically to enhance their attributes. In
fungal diseases that threaten agricultural crops. Even with addition, we can now think of microorganisms with inhibitory
intensive fungicide use, the destruction of crop plants by activity against plant pathogens as potential sources of genes
fungal pathogens is a serious problem worldwide that for disease resistance.
annually leads to losses of about 15% (Logemann and Schell
1993). The use of pesticides in general, has also resulted in
significant costs to public health and the environment. Studies 2 TRICHODERMA AS A BIOLOGICAL
aimed at replacing pesticides with environmentally safer CONTROL AGENT
methods are currently being conducted at many research
centers. In this context, control of plant pests by the The potential for the use of Trichoderma species as biocontrol
application of biological agents holds great promise as an agents was suggested 70 years ago by Weindling (1932) who
alternative to the use of chemicals. It is generally recognized was the first to demonstrate the parasitic activity of members
that biological control agents are safer and sounder of this genus towards pathogens such as Rhizoctonia solani
environmentally than is reliance on the use of high volumes (Chet 1990; Weindling 1932). Since then, several species of
of fungicides and other antimicrobial treatments. The Trichoderma have been tested as biocontrol agents; and have
heightened scientific interest in biological control of plant shown to attack a range of economically important aerial and
pathogens is a response, in part, to growing public concerns soilborne plant pathogens (Chet 1987). In many experiments,
over chemical pesticides. However, there is an equally greater showing successful biological control, the antagonistic
need for biological control of pathogens that presently go Trichoderma was found to be a necrotrophic mycoparasite
uncontrolled or only partially controlled by these “traditional” (Boosalis 1964; Chet and Elad 1982; Elad et al. 1983b).
means (Cook 1993). Thus, biological control should and can Mycoparasitism is defined as a direct attack on a fungal
be justified on its own merits, without giving it importance at thallus, followed by nutrient utilization by the parasite
the expense of chemical controls. (Chet et al. 1997). Necrotrophic mycoparasites, such as

147
148 Herrera-Estrella and Chet

Trichoderma, are those that kill the host cells before, or just D -glucose or D -mannose residues, apparently present on the
after, invasion and use the nutrients released. These cell walls of T. harzianum, inhibited the activity of a second
mycoparasites tend to be highly aggressive and destructive. lectin isolated from Sclerotium rolfsii. Inbar and Chet (1992;
They have a broad host range extending to wide taxonomic 1994) were able to mimic the fungus –fungus interaction
groups and are relatively unspecialized in their mode of in vitro using nylon fibers coated with either concanavalina A
parasitism. The antagonistic activity of necrotrophs is due to or the purified S. rolfsii lectin. During the interaction
the production of antibiotics, toxins, or hydrolytic enzymes in Trichoderma recognized and attached to the coated fibers,
such proportions as to cause death and destruction of their coiling around them and forming other mycoparasitism-
host (Manocha and Sahai 1993). In our view biocontrol by related structures, such as appresoriumlike bodies and hyphal
Trichoderma includes: (a) competition, (b) parasitism, (c) loops (Inbar and Chet 1992; 1994). Recently, using the
antibiosis, and (d) induction of defense responses in host biomimetic system we showed that different lectins induce
plants, or the combination of some of them. Parasitism is a coiling. Furthermore, coiling of Trichoderma around the
complex process including: (a) host recognition, (b) secretion fibers in the absence of lectins can be induced by applying
of hydrolytic enzymes, (c) hyphae penetration and invasion cAMP or the heterotrimeric G protein activator mastoparan
(Figure 1), and (d) lysis of the host. (Rocha-Ramı́rez et al. 2002). Transgenic lines that over-
express the Ga subunit coil at higher frequency than
untransformed controls. Furthermore, transgenic lines that
2.1 Host Recognition express an activated mutant protein with no GTPase activity
coil at an even higher frequency. In addition, lines that
In vitro, the first detectable interaction shows that the hyphae express an antisense version of the gene do not appear to coil
of the mycoparasite grow directly towards its host (Chet et al. in the biomimetic assay (Rocha-Ramı́rez et al. 2002).
1981). This phenomenon appears as a chemotropic growth of
Trichoderma in response to some stimuli produced by the host
(Chet and Elad 1983). When the mycoparasite reaches the 2.2 Host Invasion
host, its hyphae often coil around it or attach to it by forming
hooklike structures. Although not a frequent event production It has been proposed that penetration of the host mycelium
of appressoria at the tips of short branches has been observed. takes place by partial degradation of its cell wall (Elad et al.
Coiling appears to be controlled by lectins present on the host 1983b,c). Interaction sites have been stained by fluores-
hyphae. A R. solani lectin that binds to galactose and fucose ceinisothiocyanate-conjugated lectins or calcofluor. The
residues was shown to agglutinate conidia of a mycoparasitic appearance of fluorescence indicated the presence of
strain of Trichoderma harzianum but did not agglutinate two localized cell wall lysis at points of interaction between the
nonparasitic strains (Barak et al. 1985; Elad et al. 1983a). antagonist and its host (Elad et al. 1983c). Furthermore,

Figure 1 Transmission electron micrographs of Trichoderma atroviride parasiting Rhizoctonia solani. A) T. atroviride (T) penetrates
R. solani (R). B) Trichoderma (T) grows inside an R. solani (R) hyphae.
Trichoderma in Plant Protection 149

analysis by electron microscopy has shown that during the which showed that they belong to family 18, class V of the
interaction of Trichoderma spp. with either S. rolfsii or glycosyl hydrolases. Interestingly, of the eight fungal species
R. solani the parasite hyphae contacted their host and within this clade of the phylogenetic tree, all of them are
perforated their cell walls. These observations led to the either fungal or insect parasites and many of the correspond-
suggestion that Trichoderma produced and secreted mycoly- ing genes have been implicated in their parasitic activity.
tic enzymes responsible for the partial degradation of the However, the chitinolytic system of Trichoderma was
host’s cell wall. recently found to be more complex. Two genes showing
Indeed, Trichoderma produces a complex set of gluca- similarity to the one encoding the 33 kDa endochitinase
nases, chitinases, lipases, and proteases extracellularly when described by De la Cruz et al. (1992) have been cloned from
grown on cell walls of R. solani (Geremia et al. 1991; T. virens (Kim et al. 2002). These two genes are closely
Vázquez-Garcidueñas et al. 1998). Table 1 summarizes the related, according to phylogenetic analysis, and belong to
currently available information on this complex set of lytic family 18, class III of the glycosyl hydrolases (Kim et al.
enzymes produced by Trichoderma. Most attention has been 2002). Further, at least two types of N-acetyl-b- D -
paid to chitinases and several have been studied to some glucosaminidases belonging to family 20 of the glycosyl
extent in different isolates or even species of the genus. The hydrolases have been identified in T. harzianum and T. virens
purification and characterization of three endochitinases (Draborg et al. 1995; Kim et al. 2002). Chit 36 is another
secreted by T. harzianum was first reported by De la Cruz et al. antifungal chitinase recently isolated from T. harzianum TM.
(1992). They reported the isozymes to be 37, 33, and 42 kDa, This 36 kDa protein shares no significant homology to either
respectively. Only the purified 42 kDa chitinase hydrolyzed Chit33 or 42 (Viterbo et al. 2001). In addition, a 40 kDa
Botrytis cinerea purified cell walls in vitro, but this effect chitobiosidase and a 28 kDa exochitinase have been purified
was heightened in the presence of either of the other two (Deane et al. 1998; Harman et al. 1993).
isoenzymes (De la Cruz et al. 1992). The 42 kDa In 1995, Haran and co-workers identified six distinct
endochitinase has been found in most isolates. Recently, intracellular chitinases by activity on gels. This intracellular
this enzyme has been proposed to play a major role in the set of chitinases is apparently composed of two b-1,4-N-
regulatory circuits governing the expression of chitinases acetylglucosaminidases of 102 and 73 kDa, respectively, and
upon contact of Trichoderma with its host (Kubicek et al. four endochitinases of 52, 42, 33, and 31 kDa, respectively.
2001). Cloning of the genes coding for the 42 kDa secreted From this set, the 102 kDa and the 73 kDa N-acetyl
chitinases has allowed the construction of a phylogenetic tree, glucosaminidases and the 42 kDa endochitinase, were

Table 1 Trichoderma genes encoding cell-wall degrading enzymes

Gene Trichoderma spp. Strain Encoded protein References

Th-En42 T. atroviride P1 42-kDa endochitinase Hayes et al. (1994)
ech42 T. atroviride IMI206040 42-kDa endochitinase Carsolio et al. (1994)
chit42 T. harzianum CECT2413 42-kDa endochitinase Garcı́a et al. (1994)
ech1 T. virens Tv29-8 42-kDa endochitinase Kim et al. (2002)
th-ch T. harzianum Tam-61 42-kDa endochitinase Fekete et al. (1996)
ENC1 T. harzianum T25-1 42-kDa endochitinase Draborg et al. (1996)
ech2 T. virens Tv29-8 42-kDa endochitinase Kim et al. (2002)
ech3 T. virens Tv29-8 Endochitinase Kim et al. (2002)
chit33 T. harzianum CECT2413 33-kDa endochitinase Limón et al. (1995)
cht1 T. virens Tv29-8 33-kDa endochitinase Kim et al. (2002)
cht2 T. virens Tv29-8 33-kDa endochitinase Kim et al. (2002)
chit36 T. harzianum TM 36-kDa endochitinase Viterbo et al. (2001)
nag1 T. atroviride P1 73-kDa N-acetyl-b-D -glucosaminidase Peterbauer et al. (1996)
exc1 T. harzianum T25-1 73-kDa N-acetyl-b-D -glucosaminidase Draborg et al. (1995)
nag1 T. virens Tv29-8 N-acetyl-b-D -glucosaminidase Kim et al. (2002)
exc2 T. harzianum T25-1 N-acetyl-b-D -glucosaminidase ?? Draborg et al. (1995)
nag2 T. virens Tv29-8 N-acetyl-b-D -glucosaminidase Kim et al. (2002)
bgn13.1 T. harzianum CECT 2413 78 kDa b-1,3-endoglucanase De la Cruz et al. (1995)
bgn1 T. virens Tv29-8 78 kDa b-1,3-endoglucanase Kim et al. (2002)
bgn2 T. virens Tv29-8 78 kDa b-1,3-endoglucanase Kim et al. (2002)
gluc78 T. atroviride P1 78 kDa exo-b-1,3-glucosidase Donzelli et al. (2001)
bgn3 T. virens Tv29-8 b-1,6-endoglucanase Kim et al. (2002)
prb1 T. atroviride IMI206040 31-kDa subtilisinlike protease Geremia et al. (1993)
150 Herrera-Estrella and Chet

expressed differentially when Trichoderma was confronted Mach et al. 1999; Schikler et al. 1998; Ulhoa and Peberdy
with different hosts on plates (Haran et al. 1996; Inbar and 1991; Ulhoa and Peberdy 1993). Expression of ech42 in
Chet 1995). In conclusion, the complexity and diversity of T. atroviride is strongly induced during fungus –fungus
the chitinolytic system of T. harzianum involves the interaction. Its expression is repressed by glucose, may be
complementary modes of action of a diversity of enzymes, affected by other environmental factors, such as light and
all of which might be required for maximum efficiency may even be developmentally regulated (Carsolio et al.
against a broad spectrum of chitin containing plant 1994). In general, formation of most chitinolytic enzymes
pathogenic fungi. does not occur or is inhibited in the presence of glucose,
Trichoderma atroviride also secretes b-1,3-glucanases in sucrose, and chitinolytic end products (Carsolio et al. 1994;
the presence of different glucose polymers and fungal cell 1999; De la Cruz et al. 1993; Garcı́a et al. 1994; Peterbauer
walls. The level of b-1,3-glucanase activity secreted by et al. 1996; Ulhoa and Peberdy 1991). In addition, there is
T. atroviride was found to be proportional to the amount of evidence suggesting that the expression of at least ech42 of
glucan present in the inducer. The fungus produces at least T. atroviride and chit33 of T. harzianum is repressed by high
seven extracellular b-1,3-glucanases upon induction with levels of ammonium (Donzelli and Harman 2001; Mercedes
laminarin, a soluble b-1,3-glucan. The molecular weights of de las et al. 2001). In this sense, the proteinase encoding
five of these enzymes fall in the range from 60 to 80 kDa, and gene prb1 responds to carbon and nitrogen limitation. It has
their pIs are 5.0– 6.8. In addition, a 35-kDa protein with a pI also been suggested that the MRGs chit33, ech42, and prb1,
of 5.5 and a 39-kDa protein are also secreted (Vázquez- respond to other types of physiological stress (Mach et al.
Garcidueñas et al. 1998). bgn13, which encodes a 78 kDa 1999; de las Mercedes et al. 2001; Olmedo-Monfil et al.
protein from T. harzianum was the first endoglucanase gene 2002). Recently, we found that the response of ech42 and
identified (De la Cruz et al. 1995). Recently, two genes prb1 to nutrient limitation depends on the activation of
showing high homology to bgn13.1 have been identified in conserved mitogen activated protein kinase (MAPK) path-
T. virens. These endoglucanases belong to family 55 of the ways (Olmedo-Monfil et al. 2002).
glycosyl hydrolases (Kim et al. 2002). In addition, a The level of production of b-1,3-glucanases by
78 kDa exo-b-1,3-glucanase from T. harzianum has been T. atroviride is induced by the presence of cell walls of
characterized (Donzelli and Harman 2001). M. rouxii, N. crassa, S. cerevisiae, and R. solani (in ascending
From the set of glucanases produced by Trichoderma two order of efficiency) and appears to be dependent on the
b-1,6-endoglucanase genes have been identified, one in amount of b-1,3-glucan present in the cell walls of these
T. harzianum and one in T. virens (Kim et al. 2002; Lora et al. organisms (Vázquez-Garcidueñas et al. 1998). Additional
1995). These two genes encode nearly identical proteins results obtained with a filtrate of autoclaved S. cerevisiae cell
belonging to family 5 of the glycosyl hydrolases (Kim et al. walls suggest that the induction observed with cell walls may
2002). be triggered by two components, one extractable and one that
In 1993, Geremia and co-workers reported the isolation of remains cell-wall bound (Vázquez-Garcidueñas et al. 1998).
a 31 kDa basic proteinase, which is secreted by T. harzianum In general, glucanase expression is repressed by glucose and
during simulated mycoparasitism. The corresponding gene in some cases, might be repressed by primary nitrogen
( prb1) was cloned and characterized (Geremia et al. 1993) sources (Donzelli and Harman 2001).
and was the first report of the cloning of a mycoparasitism- In summary, expression of all enzymes from the cell-wall
related gene. degrading system of Trichoderma appears to be coordinated.
Suggesting a regulatory mechanism involving substrate
induction and catabolite repression. The expression of the
system is controlled at the level of transcription as indicated
by Northern analysis of the available genes (Carsolio et al.
3 EXPRESSION OF MYCOPARASITISM 1994; De la Cruz et al. 1995; Donzelli and Harman 2001;
RELATED GENES (MRGs) Flores et al. 1997; Geremia et al. 1993; Kim et al. 2002;
Limón et al. 1995; Mercedes de las et al. 2001). An exciting
Expression of extracellular chitinolytic enzymes is highly finding in terms of signaling is that the induction of at least
induced by growing Trichoderma on purified chitin, fungal two MRGs, namely prb1 and ech42, is triggered by a
cell walls, or mycelia as sole carbon source. It has been diffusible factor produced by the host (Cortés et al. 1998).
proposed that chitinolytic enzymes could be induced by Recently, it has been suggested that the activation of MRGs in
soluble chito-oligomers (Reyes et al. 1989; St. Leger et al. response to the presence of the host, through such a molecule
1986). This appears to be the case for the 73 kDa N-acetyl-b- depends on the basal expression of ech42 in T. atroviride
D -glucosaminidase of T. harzianum and T. atroviride, which (Kubicek et al. 2001). However, in T. virens, induction of four
are induced not only by chitooligomers but also by N-acetyl- MRGs in response to cell walls in ech1 (the homologue of
b-D -glucosamine. The 42 kDa endochitinase of T. harzianum ech42) knockout mutants is still observed. Whether a key
responds similarly to these compounds but ech42 expression molecule produced by the host in vivo switches on the
in T. atroviride is not induced by the products of chitin expression of MRGs remains to be proven, as well as the role
degradation (Carsolio et al. 1999; De la Cruz et al. 1993; of Ech42 in the production of such a molecule.
Trichoderma in Plant Protection 151

4 ANTIBIOSIS 1999), as well as the corresponding gene disruptants (Carsolio

et al. 1999; Woo et al. 1999). The level of extracellular
The involvement of volatile and nonvolatile antibiotics in the endochitinase activity when T. atroviride was grown under
antagonism by Trichoderma has been proposed (Dennis and inducing conditions increased up to 42 fold in multicopy
Webster 1971a,b). Indeed some isolates of Trichoderma strains as compared to the nontransformed strain. Multicopy
excrete growth inhibitory substances (Claydon et al. 1987; transformants reduced disease incidence by about 10%.
Ghisalberti and Sivasithamparam 1991; Sivan et al. 1984). Furthermore, a 30% higher degradation of the chitin content
Claydon et al. (1987) identified volatile alkyl pyrons produced in R. solani cell walls was observed during interaction with
by T. harzianum that were inhibitory to a number of fungi the overexpressing Trichoderma than with the wild type,
in vitro. When these metabolites were added to a peat-soil when quantified by transmission electron microscopy
mixture, they reduced the incidence of R. solani-induced (Carsolio et al. 1999). In the case of the gene disruptants no
damping-off on lettuce. However, there is insufficient differences in their efficiency to control R. solani or S. rolfsii
evidence to be conclusive about their contribution to pathogen were observed in greenhouse experiments, as compared to the
suppression and disease reduction in situ. Trichoderma also nontransformed control strains (Carsolio et al. 1999). In a
produces linear oligopeptides of 12–22 aminoacids (pep- second study (Woo et al. 1999), a reduction of the antifungal
taibols), which are rich in -aminoisobutyric acid, N-acetylated activity in vitro of the ech42 disrupted strains towards
at the N-terminus and containing an amino alcohol at the B. cinerea was observed. However, in vivo tests against
C-terminus (Rebuffat et al. 1989; 1991). These oligopeptides B. cinerea by leaf inoculation of bean plants revealed a
are known to form voltage-gated ion channels in black lipid significant reduction of their biocontrol capacity. Contrasting
membranes and modify the membrane permeability of with these results, ech42 gene disruptants showed increased
liposomes (El Hadjji et al. 1989). This suggested a scenario efficiency to control R. solani in soil. Similar experiments in
where cell-wall degrading enzymes weaken the cell wall and T. virens showed increased and decreased biocontrol activity
peptaibol antibiotics inhibit synthesis of cell-wall com- against R. solani on cotton using ech1 overexpressing lines
ponents, impairing the capacity of the hyphae to repair the and gene disruptants, respectively (Baek et al. 1999).
effect of cell-wall degrading enzymes (Lorito et al. 1996a,b). The role of Chit33 and Chit36 from T. harzianum in
This hypothesis is supported by the fact that the action of cell- biocontrol has also been tested by expressing the correspond-
wall degrading enzymes is synergistic with that of antibiotics ing gene at high levels using the strong constitutive pki
(Lorito et al. 1996a,b). promoter (Limón et al. 1999; Viterbo et al. 2001). Test of
R. solani control under greenhouse conditions suggested
higher efficiency of Trichoderma transformants bearing the
chit36 gene under the pki promoter, but this did not reach
5 ROLE OF MRGs IN BIOCONTROL AND statistical significance (Viterbo et al. 2001). The transgenic
STRAIN IMPROVEMENT lines generated overexpressing Chit 33 showed higher
antagonistic activity against R. solani on agar plates.
A major challenge for researchers investigating the However, in vivo experiments with these transgenic lines
mechanisms involved in the parasitic activity of Trichoderma have not been reported.
has been to establish the role of cell-wall degrading enzymes In another attempt to increase the effectiveness of
in the process. In fact, we have proposed to call all genes T. harzianum, it was transformed with a bacterial chitinase
encoding cell-wall degrading enzymes MRGs, because of gene from Serratia marcescens under the control of the
their apparent relation to the process, until their role is fully CaMV35S promoter. Two transformants showed increased
determined. Intensive efforts using genetic engineering are constitutive chitinase activity and expressed a protein of the
currently being directed at this goal. In 1997, Flores and expected size (58 kDa). When evaluated in dual cultures
coworkers generated transgenic T. atroviride lines carrying against the phytopathogenic fungus S. rolfsii both showed
multiple copies of prb1. The resulting strains produced up to higher antagonistic activity, as compared to the nontrans-
20 times more proteinase and all strains tested were more formed control (Haran et al. 1993). Unfortunately, no in vivo
effective in the control of R. solani. One strain reduced the experiments were reported using these strains.
disease incidence caused by R. solani on cotton plants to only Recently, the role of glucanases in the interaction between
6% whereas the disease incidence for the nontransformed T. harzianum and Pythium ultimum was studied (Benhamou
strain was 30% (Flores et al. 1997), demonstrating that prb1 and Chet 1997). Contact between the two fungi was
plays an important role in biocontrol and the feasibility of accompanied by the deposition of a cellulose-enriched
strain improvement through genetic engineering. material at potential penetration sites. Trichoderma was
The role of the Trichoderma 42 kDa endochitinases in able to penetrate this barrier, indicating that cellulolytic
mycoparasitism has been addressed by genetic manipulation enzymes were produced. However, cellulase production was
of the corresponding gene (ech42 and ech1) in T. atroviride not the only critical trait involved in the process. A marked
and T. virens (Baek et al. 1999; Carsolio et al. 1999; Woo et al. alteration of the b-1,3-glucan component of the Pythium cell
1999). In T. atroviride, several transgenic strains carrying wall was also observed, suggesting that b-1,3-glucanases
multiple copies of ech42 were generated (Carsolio et al. played a key role in the antagonism. In yet another study,
152 Herrera-Estrella and Chet

T. longibrachiatum transformants carrying extra copies of the in normally susceptible plants (de Wit 1985) Induced
egl1 gene (a cellulase encoding gene) were evaluated for their resistance can be localized, when it is detected only in the
biocontrol activity against P. ultimum on cucumber seedlings area immediately adjacent to the inducing factor or systemic,
(Migheli et al. 1998). The transformants showed a where resistance occurs subsequently at sites throughout the
significantly higher level of expression of the egl1 gene in plant. Both localized and systemically induced resistances are
comparison to the wild type under both inducing and nonspecific.
noninducing growth conditions. Transformants with the egl1 Recently, the potential of T. harzianum T-203 to trigger
gene under the control of a constitutive promoter had the plant defense responses was investigated by inoculating roots
highest enzymatic activity. Both the endoglucanase activity of cucumber seedlings with Trichoderma in an aseptic,
and the transforming sequences were stable under non- hydroponic system (Yedidia et al. 1999). Trichoderma-treated
selective conditions. When applied to cucumber seeds sown plants were more developed than nontreated plants through-
in P. ultimum-infested soil, T. longibrachiatum transformants out the experiment. Electron microscopy of ultrathin sections
with increased inducible or constitutive egl1 expression from Trichoderma-treated roots revealed penetration of
generally were more suppressive than the wild-type strain. T. harzianum, mainly to the epidermis and outer cortex.
Biocontrol agents tolerant to specific pesticides could be Strengthening of the epidermal and cortical cell walls was
constructed using molecular techniques. Resistance to the observed, as well as deposition of newly formed barriers.
fungicide benomyl is conferred by a single amino-acid These typical host reactions were found beyond the sites of
substitution in one of the b-tubulins of T. viride; the potential fungal penetration. Wall appositions contained large
corresponding gene has been cloned and proven to work in amounts of callose and infiltrations of cellulose. Further
other Trichoderma species (Goldman et al. 1993), thereby biochemical analyses revealed that inoculation with the
producing a biological control agent that could be applied fungus resulted in increased peroxidase and chitinase
simultaneously or in alternation with the fungicide. However, activities in roots and leaves of treated seedlings, providing
these strains have not been tested in biocontrol experiments. evidence that T. harzianum may induce systemic resistance
Molecular techniques may eventually be used to transfer mechanisms in cucumber plants (Yedidia et al. 1999).
several beneficial traits, such as the production of one or
more antibiotics and pesticide tolerance, to an aggressive
phyllosphere colonizer. 8 PLANT GROWTH PROMOTION

Microbial interactions with plant roots are known to affect

6 COMPETITION profoundly plant nutrient status and, for manganese at least, to
affect plant resistance to pathogens (Huber and McCay-Buis
Competition occurs between microorganisms when space or 1993). In addition to their biocontrol characteristics,
nutrients (i.e., carbon, nitrogen, and iron) are limiting and its Trichoderma species also exhibit plant-growth-promoting
role in the biocontrol of plant pathogens has been studied for activity (Baker 1989; Chet 1987; Harman and Bjorkman
many years, with special emphasis on bacterial biocontrol 1998; Inbar et al. 1994; Kleifeld and Chet 1992; Naseby et al.
agents (Weller 1988). Implicit in this definition is the 2000). In spite of their theoretical and practical importance,
understanding that combative interactions such as antibiotic the mechanisms responsible for the growth response due to
production, mycoparasitism, or the occurrence of induced Trichoderma have not been investigated extensively. Since
resistance in the host are excluded even though these growth enhancement has been observed in the absence of any
mechanisms may form an important part of the overall detectable disease (Chang et al. 1986; Harman and Bjorkman
processes occurring in the interaction. In the rhizosphere, 1998; Naseby et al. 2000) and in sterile soil (Windham et al.
competition for space as well as nutrients is of major 1986), it is not thought to be a side effect of suppression of
importance. Thus, an important attribute of a successful disease or minor plant pathogens. Other mechanisms,
rhizosphere biocontrol agent would be the ability to remain at including production of hormonelike metabolites and release
high population density on the root surface providing of nutrients from soil or organic matter, have been proposed
protection of the whole root for the duration of its life. (Kleifeld and Chet 1992; Windham et al. 1986).
Recently, it was found that a strain of T. harzianum (T-35) The plant-growth-promoting capacity of T. harzianum to
that controls Fusarium spp. on various crops might take solubilize in vitro some insoluble or sparingly soluble
advantage of competition for nutrients and rhizosphere minerals via three possible mechanisms: acidification of the
colonization (Sivan and Chet 1989). medium, production of chelating metabolites, and redox
activity was recently investigated (Altamore et al. 1999).
T. harzianum was able to solubilize MnO2, metallic zinc, and
7 INDUCED RESISTANCE rock phosphate (mostly calcium phosphate). Fe2O3, MnO2,
Zn, and rock phosphate were also solubilized by cell-free
Induced resistance is a plant response to challenge by culture filtrates. A size exclusion chromatographic separation
microorganisms or abiotic agents such that following the of the components of the culture filtrates indicated the
inducing challenge de novo resistance to pathogens is shown presence of a complexed form of Fe but no chelation of Mn.
Trichoderma in Plant Protection 153

In liquid culture, T. harzianum also produced diffusible increased, our understanding of the mechanisms used by
metabolites capable of reducing Fe(III) and Cu(II). Solubili- Trichoderma to antagonize phytopathogenic fungi is still very
zation of metal oxides by Trichoderma involves both limited.
chelation and reduction (Altamore et al. 1999). Little is known on the signaling pathways that determine
host recognition, although there is evidence of the involve-
ment of conserved signaling pathways such as heterotrimeric
9 TRICHODERMA AS A SOURCE OF GENES G proteins in hyphal coiling. At later stages of the interaction,
FOR CROP IMPROVEMENT MAPK pathways appear to participate in the regulation of the
expression of MRGs. However, we are still just beginning to
One of the major goals of plant genetic engineering is to untangle the networks determining host recognition.
protect plants from diseases. There are many examples of the An important number of genes encoding cell-wall
introduction of chitinase genes into plants resulting in an degrading enzymes have been cloned. In most cases, their
enhancement of resistance of the host plant to fungal expression correlates with conditions that simulate the actual
pathogens (Broglie et al. 1993; Lin et al. 1995; Vierheilig et al. interaction with the host and some of them have even been
1993). However, the desired levels of resistance for successful used to generate improved strains. However, the fact that
commercial application have not yet been reached. The use of several of the cloned MRGs respond to multiple environ-
potent chitinases with proven antifungal activity is thus an mental signals that they are subjected to catabolite repression,
attractive alternative. Because Ech42 from T. atroviride and that none of them is expressed specifically at the sites of
fulfills these criteria, the corresponding gene was introduced interaction, suggests that these genes maybe part of a
into tobacco and potato (Lorito et al. 1998). High expression specialized saprophytic response. Thus, the corresponding
levels of the fungal gene were obtained in different plant enzymes are more likely to participate in the utilization of
tissues, with no visible effect on plant growth and the host’s cellular components as a food supply at the end
development. Substantial differences in endochitinase of the parasitic process. An alternative explanation is that the
activity were detected among different transformed lines. interaction of Trichoderma with a host is interpreted by the
Transgenic lines were highly tolerant or completely resistant parasite as a stress signal and that MRGs are in fact stress
to the foliar pathogens Alternaria alternata, Alternaria solani, responsive genes. It is likely that genes coding for key
B. cinerea, and the soilborne pathogen R. solani. Interest- enzymes such as those expressed specifically at the site of
ingly, the levels of tolerance reached in these experiments interaction where penetration or cell wall perforations are
were higher than those previously achieved by expression of observed, have not been yet identified. The use of functional
bacterial or plant chitinases (Lorito et al. 1998). genomics strategies will certainly be a major step towards the
A similar strategy was used to improve scab resistance of identification of genes playing key roles in mycoparasitism by
apple (Bolar et al. 2000). The endochitinase gene (ech42), as Trichoderma. Trichoderma has already proven to be an
cDNA and genomic clones, was transferred into apple cv. important source of genes for engineering plants for pathogen
Marshall McIntosh. Eight lines propagated as grafted and resistance. Yet, there is still a complete battery of genes that
self-rooted plants were inoculated with Venturia inaequalis. should be tested for this purpose, as well as combinatory
Six transgenic lines expressing the endochitinase were more strategies using several Trichoderma genes. Induction of
resistant than controls. Disease severity in the transgenic lines defense responses in host plants and plant growth promotion
tested compared with nontransformed controls was reduced, are important attributes of Trichoderma, whose study was
as well as the number of lesions and the leaf area infected. neglected for a long time. The recent evidence on these two
However, in contrast with the results previously reported aspects makes Trichoderma an even more attractive organism
(Lorito et al. 1998), expression of the endochitinase also had for large-scale application as a biological control agent. In
negative effects on the growth of both inoculated and spite of our limited knowledge on the mechanisms underlying
uninoculated plants (Bolar et al. 2000). In a more recent the mycoparasitic activity of Trichoderma, it is clear that it is
investigation the same group introduced either an endo- an excellent model system for the study of interfungal
chitinase or an exochitinase, both from Trichoderma, into apple parasitic relationships and that it has an enormous potential
plants (Bolar et al. 2001). In agreement with their previous for a variety of biotechnological applications.
results resistance to V. inaequalis correlated with the level of
expression of either enzyme. Plants expressing both enzymes ACKNOWLEDGEMENT
simultaneously were more tolerant that plants expressing
either enzyme alone. Their results indicate that the two The authors wish to thank Dr. June Simpson for critical
enzymes acted synergistically to limit disease development. reading of the manuscript.

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77:437– 447. Microbiol 65:1858 –1863.
Inbar J and Chet I (1992). Biomimics of fungal cell – cell recognition Manocha MS and Sahai AS (1993). Mechanisms of recognition in
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174:1055 –1059. 39:269– 275.
Inbar J and Chet I (1994). A newly isolated lectin from the Mercedes de las D, Limón MC, Mejı́as R, Mach RL, Benı́tez T,
plant pathogenic fungus Sclerotium rolfsii: purification, Pintor-Toro JA, and Kubicek CP (2001). Regulation of
characterization and role in mycoparasitism. Microbiology chitinase 33 (chit33) gene expression in Trichoderma
140:651 – 657. harzianum. Curr Genet 38:335 –342.
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Migheli Q, González-Candelas L, Dealessi L, Camponogara A, and St. Leger R, Cooper RM, and Charnley AK (1986). Cuticle
Ramón-Vidal D (1998). Transformants of Trichoderma long- degrading enzymes of entomopathogenic fungi: regulation of
ibrachiatum overexpressing the beta-1,4-endoglucanase gene production of chitinolytic enzymes. J Gen Microbiol
egl1 show enhanced biocontrol of Pythium ultimum on 132:1509 – 1517.
cucumber. Phytopathology 88:673– 677. Ulhoa CJ and Peberdy JF (1991). Regulation of chitinase synthesis in
Naseby DC, Pascual JA, and Lynch JM (2000). Effect of biocontrol Trichoderma harzianum. J Gen Microbiol 137:2163 – 2169.
strains of Trichoderma on plant growth, Pythium ultimum Ulhoa CJ and Peberdy JF (1993). Effect of carbon sources on
populations, soil microbial communities and soil enzyme chitobiase production by Trichoderma harzianum. Mycol Res
activities. J Appl Microbiol 88:161– 169. 97:45 – 48.
Olmedo-Monfil V, Mendoza-Mendoza A, Gómez I, Cortés C, and Vázquez-Garcidueñas S, Leal-Morales CA, and Herrera-Estrella A
Herrera-Estrella A (2002). Multiple environmental signals (1998). Analysis of the b-1,3-glucanolytic system of the
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Gen Genom 267:703 –712. Vierheilig H, Alt M, Neuhaus J-M, Boller T, and Wiemken A (1993).
Peterbauer CK, Lorito M, Hayes CK, Harman GE, and Kubicek CP Colonization of transgenic N. sylvestris plants, expressing
(1996). Molecular cloning and expression of the nag1 gene different forms of N. tabacum chitinase, by the root pathogen,
(N-acetyl-b-D -glucosaminidase-encoding gene) from Rhizoctonia solani, and by the mycorrhizal symbiont, Glomus
Trichoderma harziaunm P1. Curr Genet 30:325– 331. mosseau. Mol Plant-Microbe Interact 6:261 –264.
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Aspergillus nidulans implicated in the autolysis of its cell wall. Windham MT, Elad Y, and Baker R (1986). A mechanism for
FEMS Microbiol Lett 60:119– 124. increased plant growth induced by Trichoderma spp.
Rocha-Ramı́rez V, Omero C, Chet I, Horwitz BA, and Herrera- Phytopathology 76:518– 521.
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Schikler H, Haran S, Oppenheim A, and Chet I (1998). Induction of pp 405 –424.
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the chitin monomer N-acetylglucosamine. Mycol Res G, and Lorito M (1999). Disruption of the ech42 (endo-
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14
Biological Control of Fungal Diseases on
Vegetable Crops with Fungi and Yeasts

Zamir K. Punja Simon Fraser University, Burnaby, British Columbia, Canada

Raj S. Utkhede Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Agassiz, British
Columbia, Canada

1 INTRODUCTION stages of seedling growth, causing seed decay and

damping-off. Examples of these fungi are Rhizoctonia
Vegetable crops may be produced as both fresh market and solani Kühn, various species of Fusarium and Pythium,
processed commodities and can be grown under field and Sclerotium rolfsii Sacc. Fungal pathogens that infect
conditions or in controlled environments, such as glasshouses the roots and crown of developing plants, causing root and
or other similar structures. There are numerous fungal crown rots and vascular wilts, have also been researched
diseases that attack a wide range of these vegetable crops for biological control strategies. These include fungi such
(Howard et al. 1994), thereby reducing crop yield and quality. as Pythium spp., Fusarium spp., and Sclerotinia sclero-
Methods for disease control have included the use of cultural tiorum (Lib.) de Bary. A third group of foliar-infecting
practices to reduce pathogen inoculum and disease incidence, fungi of vegetable crops that cause leaf spots and blights
development of resistant cultivars, as well as the application and stem infection, also have biological control strategies
of chemical fungicides to inhibit pathogen development. The developed against them. These include Botrytis cinerea
use of biological control strategies has also demonstrated the Pers. ex Fr. (gray mold), Didymella bryoniae (Auersw.)
potential of fungi and yeasts in reducing a range of fungal Rhem (gummy stem blight), S. sclerotiorum (white mold),
pathogens that cause various diseases on vegetable crops. and Sphaerotheca and Erysiphe spp. (powdery mildews).
In this chapter, some examples of recent successes in Many different fungal and yeast biological control agents
biological control of fungal diseases of vegetable crops using have been identified and evaluated for disease control potential
fungi and yeasts, and the mechanisms by which pathogen against the above-mentioned pathogens, and some have been
control was achieved will be reviewed. In addition, the formulated and brought to market to provide disease control
utilization of techniques in biotechnology to aid in the options for producers of vegetable crops. The use of biological
implementation of biological control strategies for disease control agents may be particularly attractive for vegetable
control will be reviewed. These include techniques to crops grown in glasshouses, due to the high market value of
investigate mechanism(s) of action of the biological control these crops and the possibility for control of environmental
agent, development of strains with enhanced efficacy through parameters, particularly temperature and relative humidity
genetic manipulation, monitoring the growth and spread of (Paulitz and Bélanger 2001). These are important variables
biocontrol agents using molecular techniques, and characteriz- that can significantly influence the efficacy of biological
ation of strains using genetic markers and biochemical methods. control agents under natural field conditions (Paulitz 1997).
The diseases to be considered in this chapter for which The rationale for development of biological control agents
biocontrol strategies have been described include those against fungal diseases on vegetable crops was to provide an
caused by pathogenic fungi that infect the seed and early additional/alternative approach to augment/replace the use of

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chemical fungicides, to provide a level of disease control in the 3 BIOCONTROL OF ROOT AND CROWN
absence of crop genetic resistance, and to augment cultural ROTS AND VASCULAR WILT DISEASES
control practices to further minimize the impact of these
diseases and reduce chemical residues in food. For example, Pathogenic fungi which infect the root system and crown
chemical fungicides typically have provided adequate control tissues through root hairs, natural openings, or wounds, can
of many fungal pathogens. However, fungicide resistance rapidly colonize these tissues and enter the vascular tissues,
problems, concerns regarding pesticide residues, and revoca- causing decay and death of the plants. Most of these
tion of registration of certain widely-used fungicides, have led pathogens infect during the early stages of plant development,
to increased activity in the development of biocontrol agents although disease symptoms may only be manifested later.
against foliar fungal pathogens. Fungal biological control agents have been described which
For a potential biological control agent to reach the stage when applied to the seed, planting medium, or roots of plants,
of commercial deployment, numerous criteria have to be can colonize the root system, occupy potential infection sites,
satisfied and considerable data need to be obtained to and compete with the pathogen. In addition, these agents may
demonstrate aspects of efficacy, survival, adaptability, and enhance resistance in the plants through induction of various
scale-up. These aspects are reviewed elsewhere (Avis et al. defense responses, and secrete hydrolytic enzymes and
2001a; Cook 1993; Harman 2000; Lumsden et al. 1996) and antibiotics that inhibit pathogen growth and development.
will not be discussed in this chapter. Several agricultural The most widely-researched of these biocontrol agents are
chemical companies and a number of companies with antagonistic fungi (Trichoderma and Gliocladium species)
specialized agricultural products have invested in the and nonpathogenic, closely-related fungi (Pythium,
discovery and development of biological control agents to Fusarium, and Rhizoctonia species), as well as mycoparasitic
complement synthetic pesticides for the control of diseases on distantly related fungi such as Talaromyces, Coniothyrium,
horticultural crops. These products are targeted to markets Sporidesmium, Stachybotrys, and Verticillium (Table 1).
where they have the best chance of performing and where
there is the most need, e.g., for control of seed and root-
infecting pathogens on seedlings (Whipps 2001). A range of 3.1 Trichoderma As a Biocontrol Agent of
commercially available biological control products for plant Seedling Root, Crown, and Vascular Diseases
disease control is now available (Fravel 2000) and are more
likely to be brought to market in the future. Molecular 3.1.1 Trichoderma Species Identification
methods have been described that can be adapted for use to
The genus Trichoderma contains species that occur in soils
ensure quality control and monitoring of the biocontrol agents
throughout the world. Most species are fast-growing
(Avis et al. 2001a).
saprophytes with the ability to survive under a range of
environmental conditions by utilizing different substrates for
growth (Hjeljord and Tronsmo 1998; Samuels 1996). The
2 BIOCONTROL OF SEED ROTS AND most common biological control agents in the genus
Trichoderma have been reported to be strains of Trichoderma
DAMPING-OFF DISEASES
virens, T. harzianum, and T. viride (Hermosa et al. 2000).
Characterization of 16 biocontrol strains, identified pre-
Germination of plant seeds is accompanied by the exudation viously as Trichoderma harzianum Rifai and one biocontrol
of host nutrients into the soil environment, which frequently strain recognized as T. viride, has been carried out using
attract potentially damaging fungi such as Pythium spp., several molecular techniques. A certain degree of poly-
R. solani, and Fusarium spp. These fungi utilize the seed and morphism was detected among isolates in hybridizations
root exudates as an energy source for germination and growth, using a probe of mitochondrial DNA. Sequencing of internal
and subsequently penetrate and colonize the seed and root transcribed spacers 1 and 2 (ITS1 and ITS2) of ribosomal
hairs, causing rot and damping-off of emerging seedlings. DNA revealed three different ITS lengths and four different
These fungi are favored by cool (15 –208C) and moist sequence types. Phylogenetic analysis based on ITS1
conditions. Fungal biological control agents have been sequences, including type strains of different species,
described which when applied to the surface of seed, to the clustered the 17 biocontrol strains into four groups:
planting substrate, or when applied shortly after seed T. harzianum –T. inhamatum complex, T. longibrachiatum,
germination, can utilize the host nutrient exudates and T. asperellum, and T. atroviride –T. koningii complex. ITS2
colonize the seed and developing roots to compete with and sequences were also useful for locating the biocontrol strains
exclude the pathogenic fungi. In addition, many of these in T. atroviride within the complex T. atroviride –T. koningii.
biocontrol agents secrete hydrolytic enzymes and antibiotics None of the biocontrol strains studied corresponded to
that inhibit the development of the pathogenic fungi. The biotypes Th2 or Th4 of T. harzianum that cause mushroom
most widely-researched of these biocontrol agents are species green mold. A similar study by Dodd et al. (2000) utilized
of Trichoderma and Gliocladium and to a lesser extent ITS1 and ITS2 sequence data to group 50 isolates of
Penicillium spp. (Table 1). Trichoderma species with biocontrol potential, while ITS1
Table 1 Examples of fungal biological control agents that prevent seed rots, damping-off, and root, crown, and vascular diseases caused by pathogenic fungi on vegetable
crops

Biocontrol agent Target pathogen and host References

T. harzianum F. oxysporum f. sp. radicis-lycopersici on tomato Datnoff et al. (1995), Nemec et al. (1996), and Sivan and Chet (1993)
T. harzianum, T. hamatum F. oxysporum f. sp. lycopersici on tomato Larkin and Fravel (1998)
T. harzianum P. capsici on pepper Ahmed et al. (1999)
T. harzianum P. ultimum and R. solani on bean Woo et al. (1999)
T. hamatum, T. harzianum, R. solani on eggplant Lewis et al. (1998)
T. viride, T. virens
T. harzianum S. sclerotiorum on pea Knudsen and Eschen (1991)
Biological Control of Vegetable Diseases

T. longibrachiatum P. ultimum on cucumber Migheli et al. (1998)

G. virens F. oxysporum f. sp. lycopersici on tomato Larkin and Fravel (1998)
G. virens P. ultimum on cucumber; R. solani on peas Koch (1999)
G. virens GL-3 R. solani, P. ultimum, S. rolfsii, and F. oxysporum on tomato and Mao et al. (1998)
pepper
G. virens GL-3, GL-21 S. rolfsii on carrot Ristaino et al. (1994)
G. catenulatum J1446 Pythium on cucumber Niemi and Lahdenperä (2000) and Punja and Yip (unpublished)
P. oxalicum F. oxysporum f. sp. lycopersici on tomato De Cal et al. (1999)
Nonpathogenic F. oxysporum f. sp. lycopersici on tomato Alabouvette et al. (1993), Duijff et al. (1998), and Fuchs et al. (1999)
F. oxysporum
Nonpathogenic F. oxysporum f. sp. lycopersici on tomato Larkin and Fravel (1998)
F. oxysporum,
F. solani
Nonpathogenic F. oxysporum f. sp. cucumerinum on cucumber Mandeel and Baker (1991)
F. oxysporum
Nonpathogenic R. solani R. solani and Pythium on cucumber and pepper Cubeta and Echandi (1991), Harris and Adkins (1999), and
Villajuan-Abgona et al. (1996)
Nonpathogenic Rhizocto- R. solani on bean, cabbage Jabaji-Hare et al. (1999) and Ross et al. (1998)
nia
P. oligandrum Pythium spp. on cress McQuilken et al. (1992)
P. oligandrum V. dahliae on pepper Al-Rawahi and Hancock (1998)
T. flavus S. rolfsii on bean Madi et al. (1997)
T. flavus V. dahliae on eggplant Engelkes et al. (1977) and Stosz et al. (1996)
T. flavus S. rolfsii on bean Madi et al. (1997)
C. minitans S. sclerotiorum on lettuce Budge and Whipps (2001)
S. sclerotivorum S. minor on lettuce Adams and Fravel (1990)
C. foecundissimum P. ultimum and R. solani on eggplant and pepper Lewis and Larkin (1998)
159
160 Punja and Utkhede

sequence data and RFLP analysis were used to distinguish pathogen structures, such as hyphae, causing them to be
amongst isolates of T. harzianum (Gams and Meyer 1998). degraded through the production of cell wall degrading
These studies demonstrated the utility of molecular methods enzymes, such as chitinases, glucanases, cellulases, and
to resolve the identity of strains of Trichoderma with potential proteinases (Geremia et al. 1993; Schirmbock et al. 1994;
biocontrol activity that were overlapping in morphological Thrane et al. 1997; Zeilinger et al. 1999). Trichoderma
features. This approach could also be used to develop strain- species can compete with pathogens for nutrients, rapidly
specific markers for a desired biocontrol strain. Molecular colonize a substrate and exclude pathogens from infection
markers were developed and used to detect and trace a strain sites, and colonize senescing tissues and wounds to reduce
of T. hamatum in potting mix (Abbasi et al. 1999). pathogen colonization (Hjeljord and Tronsmo 1998). Some
strains of Trichoderma are good root colonizers (rhizosphere
3.1.2 Trichoderma Biocontrol Activity competent). It has also been reported that T. harzianum (strain
T-22) has the ability to directly enhance root growth and plant
Several studies have shown that T. harzianum can control development in the absence of pathogens (Harman 2000), and
diseases caused by many root-infecting pathogens, including it has been suggested that this was due to the production of
Fusarium, Rhizoctonia, and Pythium (Table 1). T. harzianum a growth-regulating factor by the fungus (Windham et al.
strain KRL-AG2, commercially formulated as F-Stop, when 1986). Altomare et al. (1999) proposed that the ability of
added to a potting soil mix prior to seeding with tomatoes, T. harzianum to increase plant growth was partially due to the
reduced the incidence and severity of Fusarium root and organism’s ability to solubilize nutrients, thus making them
crown rot caused by Fusarium oxysporum f. sp. radicis- more available to host plants. These observations indicate the
lycopersici (Datnoff et al. 1995). T. harzianum strain T-22 versatility through which Trichoderma species can manifest
also provided control of Fusarium crown rot of tomato biological control activity. Finally, it has been reported that a
(Nemec et al. 1996), and the fungus could be recovered from strain of T. harzianum was able to trigger host defense
the roots of treated plants 26 days after application, mechanisms in cucumber plants through enhanced cell wall
suggesting it had colonized the roots. T. hamatum reduced depositions and induction of defense enzymes, suggesting an
the incidence of Fusarium wilt of tomato, caused by indirect effect in the host plant by the biocontrol agent
F. oxysporum f. sp. lycopersici, when added to potting (Yedidia et al. 1999).
medium prior to seeding (Larkin and Fravel 1998). A
commercial formulation of T. harzianum (Rootshield strain 3.1.4 Biotechnological Manipulations of
T-22) was also evaluated against this disease and was found to Trichoderma
significantly reduce it when incorporated into potting mix at
0.2% (Larkin and Fravel 1998). Strain T-22 of T. harzianum Techniques in biotechnology have been applied to elucidate
was generated by fusing a mutant strain capable of colonizing the role of hydrolytic enzymes, such as chitinases and
plant roots with a strain able to compete with bacteria under glucanases, in mycoparasitism by Trichoderma that could
iron-limiting conditions using protoplast fusion techniques lead to biological control activity. Transformants of
(Harman 2000). This new strain had the enhanced ability to T. longibrachiatum expressing extra copies of the
colonize the root system of host plants, resulting in greater b-1,4-endoglucanase gene egl1 were found to be better at
efficacy as a biological control agent for long-term root suppressing Pythium development on cucumber compared to
protection (Harman 2000; Harman and Björkman 1998; Sivan wild-type strains (Migheli et al. 1998). In addition,
and Harman 1991). transformants of T. harzianum overproducing the proteinase
A number of commercial formulations are available that gene prb1 had up to a five-fold increase in ability to protect
contain strains of T. harzianum for use against different cotton seedlings from R. solani (Flores et al. 1996).
diseases on a wide range of crops. These products are Transformants of T. harzianum overexpressing an endochi-
registered for use against a number of soilborne pathogens. tinase gene chit33 were more effective in inhibiting growth of
Among these products, RootShielde, T-22G, and T-22 the pathogen R. solani in vitro compared with wild-type
Planter Boxe contain T. harzianum strain T-22 that is able to strains (Limón et al. 1999). A mutant of T. harzianum that was
survive well in the rhizosphere of plants (Fravel 2000). selected for its enhanced ability to hydrolyze pustulan, a
polymer of b-1,6-glucan, had 2 –4 times more chitinase,
3.1.3 Trichoderma Mechanism of Action b-1,3 and b-1,6 glucanase activity compared to the wild-type,
produced three times more extracellular proteins and other
Trichoderma species can confer biological control against compounds, and showed greater inhibition of B. cinerea in
soilborne diseases through a number of mechanisms, vitro (Rey et al. 2001).These studies reaffirm the roles played
including antibiosis, parasitism, competition, and the by fungal enzymes in biocontrol of plant pathogens and also
induction of host plant resistance (Hjeljord and Tronsmo highlight the successes in manipulating biocontrol strains to
1998). Trichoderma species are known to produce a range of genetically engineer them to enhance efficacy. Specificity in
volatile and nonvolatile secondary metabolites, some of the activity of the hydrolytic enzymes was suggested in
which inhibit other microorganisms and are considered to be a study by Woo et al. (1999), in which the endochitinase
antibiotics. These fungi can also penetrate and infect ech42 gene encoding for the secreted 42 Kda endochitinase
Biological Control of Vegetable Diseases 161

(CHIT 42) was silenced by targeted disruption; it was found and Lahdenperä 2000). Primastop is currently registered in
that the endochitinase-deficient mutant had similar activity as Europe and in a number of regions of the United States, and
the wild-type strain against Pythium ultimum, but had Prestop is expected to be registered in the near future (Niemi
enhanced activity against R. solani, and reduced activity and Lahdenperä 2000). G. catenulatum strain J1446 was able
against B. cinerea. to colonize cucumber roots extensively 5 weeks following its
A genetically marked strain of T. harzianum was application, indicating that the fungus has the ability to
developed by transformation with the b-glucuronidase (uid survive and proliferate in the rhizosphere of plants. This
A) gene and the hygromycin B (hygB) gene for use in rhizosphere competence, coupled with its reported ability to
population dynamics studies (Thrane et al. 1995). Techniques act as a mycoparasite (McQuilken et al. 2001) makes
utilizing protoplast transformation as well as particle G. catenulatum a strong candidate as a biological control
bombardment of conidia have been described for Tricho- agent against a number of vegetable diseases. We have
derma (Lorito et al. 1993; Thrane et al. 1995). Population evaluated this biocontrol agent against Pythium root and
densities of the transformed strain could be monitored in a crown rot of cucumber caused by P. aphanidermatum.
potting mix (Green and Jensen 1995), and the presence of the Application at the time of seeding significantly reduced plant
biocontrol agent around wounded tissues was reported. mortality and enhanced seedling growth (Figure 1).

3.3 Nonpathogenic Fungi As Biocontrol Agents

3.2 Gliocladium As a Biocontrol Agent of of Seedling, Root, Crown, and Vascular Wilt
Seedling, Root, Crown, and Vascular Wilt Diseases
Diseases
Nonpathogenic and hypovirulent strains of fungi that are
Gliocladium virens Miller, Giddens and Foster, a biocontrol closely related taxonomically to plant pathogenic species
agent of a wide range of fungal pathogens, is now classified in have been reported to provide biological control of a number
the genus Trichoderma due to similar morphological of pathogens, including species of Fusarium, Rhizoctonia,
characteristics that are shared with members of this genus and Pythium. Competition by nonpathogenic strains for host
and DNA analysis supported the inclusion of G. virens with plant nutrients, colonization of roots and infection sites to
the Trichoderma genus (Rehner and Samuels 1994). One preclude the pathogen, parasitism of pathogen hyphae, and
characteristic of G. virens is the ability to produce the induction of host plant resistance are mechanisms through
antibiotic metabolites gliotoxin and viridin, a characteristic which these nonpathogenic strains achieved biological
not generally shared with other species of Trichoderma control (Sneh 1998).
(Papavizas 1985). G. virens (T. virens) strain GL-3 was Nonpathogenic Fusarium species (F. oxysporum and
evaluated as a seed treatment on tomato against several F. solani) provided control of Fusarium wilt of tomato
pathogens, including R. solani, P. ultimum, S. rolfsii, and
F. oxysporum f. sp. lycopersici. The treatment resulted in
significantly higher seedling establishment (Mao et al. 1998).
A commercial formulation of strain GL-21 of G. virens is
registered for use in the United States, under the trade name
SoilGarde, and can control P. ultimum and R. solani on
vegetables and ornamental seedlings (Koch 1999; Lumsden
et al. 1996). Like T. harzianum strain T-22, certain strains of
G. virens have the ability to colonize the rhizosphere of plant
roots (Harman 2000). In addition, the production of gliotoxin
occurs rapidly (within a few hours) and can persist for several
days to provide high levels of pathogen suppression
(Lumsden et al. 1992; Wilhite and Straney 1996). Gliotoxin
has also been shown to act synergistically with endochitinase
in G. virens (Di Pietro et al. 1993).
A related species, G. catenulatum Gilman and E. Abbot,
has been reported to be effective in reducing the incidence of
damping-off diseases, caused by P. ultimum and R. solani Figure 1 Effect of G. catenulatum, formulated as Prestop, on
(McQuilken et al. 2001). Incorporation of a wettable powder reducing root rot and damping-off caused by P. aphanidermatum
formulation of G. catenulatum strain JI446 into peat-based on cucumber seedlings. The plant on the left received the
growing media or application as a drench reduced damping- biocontrol agent at seeding time followed by the pathogen 10
off due to P. ultimum and R. solani. Two commercial days later; the plant in the center received the pathogen only 10
formulations of G. catenulatum strain J1446 (Prestop and days after seeding; the plant on the right is the uninoculated
Primastop) have been recently developed (Fravel 2000; Niemi control. Photograph was taken at 28 days after seeding.
162 Punja and Utkhede

(Alabouvette et al. 1993; Fuchs et al. 1999; Larkin and Fravel Verticillium biguttatum affecting R. solani (van den Boogert
1998) through systemic induction of host resistance, and an and Velvis 1994; Tweddell et al. 1995), Coniothyrium
increase in hydrolytic enzyme activity was reported in treated minitans on Sporidesmium sclerotiorum (Budge and Whipps
plants (Duijff et al. 1998; Fuchs et al. 1999). Induction of 2001), S. sclerotivorum on a number of sclerotial-forming
resistance by nonpathogenic fungi has also been reported to soilborne fungi (Mischke 1998), and Talaromyces flavus on
occur for binucleate Rhizoctonia species (Jabaji-Hare et al. S. rolfsii (Madi et al. 1997). In addition, T. flavus was reported
1999; Xue et al. 1998), P. oligandrum (Benhamou et al. to produce glucose oxidase and potentially peroxide, which
1997), and Penicillium oxalicum (DeCal et al. 1999). In plants was lethal to sclerotia of V. dahliae (Stosz et al. 1996).
treated with these biocontrol agents, induction of host defense Species of Trichoderma and Gliocladium are also known to
responses, alterations of the plant cell wall, and enhanced be mycoparasitic, as discussed in previous sections of this
expression of antifungal enzymes were reported (Benhamou chapter. All of these mycoparasitic fungi have been
et al. 1997; Jabaji-Hare et al. 1999; Xue et al. 1998). Further demonstrated to reduce diseases caused by a number of
studies on the mechanisms by which these fungi elicit the host different pathogens on a range of vegetable crop species
defense responses should provide interesting information on (Table 1).
this group of biocontrol fungi. Formulations for biocontrol
fungi such as binucleate Rhizoctonia species have been
described (Honeycutt and Benson 2001). 4 BIOCONTROL OF FOLIAR-INFECTING
FUNGI
3.3.1 Biotechnological Manipulations of
Nonpathogenic Biocontrol Fungi Pathogenic fungi which infect the leaves and stems of
developing plants may enter through senescing tissues,
Techniques in biotechnology have been applied primarily to wounded regions, or natural openings, or may penetrate host
the nonpathogenic F. oxysporum strains that provide tissues directly. These fungi can infect plants at all stages of
biocontrol of Fusarium wilt on a number of plant species. By development, and are favored by warm (20 –258C) and humid
creating strains expressing the marker gene b-glucuronidase conditions. Infection results in blighting of the foliage,
(GUS) through genetic transformation, the role of compe- premature leaf senescence, and compromised plant growth
tition for root colonization between pathogenic and and yield. Biological control agents have been described
nonpathogenic strains could be elucidated (Eparvier and which when applied to the foliage, can reduce primary
Alabouvette 1994). Nonpathogenic strains differed in their infection as well as reduce pathogen development and
ability to colonize roots and to preclude the pathogenic sporulation, and can colonize wounds and other tissues to
strains, and the use of GUS-marked strains utilizing the preclude pathogen establishment or development. Some of
glyceraldehyde-3-phosphate dehydrogenase promoter pro- the biological control agents can act as mycoparasites and
vided an estimation of fungal metabolic activity on the roots reduce pathogen growth directly, while others may secrete
(Eparvier and Alabouvette 1994). hydrolytic enzymes and antifungal compounds to reduce
A strain of nonpathogenic F. oxysporum transformed with pathogen development, or alter pathogen physiology to
the b-glucuronidase (GusA) and hygromycin B resistance reduce disease-causing potential. The most widely-researched
(Hph) genes could be detected at levels as low as 1 ng of of these biocontrol agents are fungi (Trichoderma,
mycelia and estimates of fungal biomass on tomato roots were Ulocladium, Ampelomyces, and Verticillium) and yeasts
shown to be considerably higher compared to a plating assay (Aureobasidium, Cryptococcus, Rhodosporidium, and
method (Bao et al. 2000), indicating this was a sensitive and Rhodotorula).
rapid assay for this biocontrol agent in planta. The
colonization by the transformed strain of plant roots could
be assessed in relation to the extent of colonization of a 4.1 Biocontrol of Gray Mold
pathogenic strain of F. oxysporum (Bao and Lazarovits 2001).
Botrytis cinerea Pers:Fr. is an important pathogen on many
vegetable crops grown under greenhouse conditions as well as
3.4 Mycoparasites As Biocontrol Agents of under field conditions. Under high humidity conditions or
Seedling, Root, Crown, and Vascular Wilt when free moisture is present on the plant surface, the
Diseases pathogen infects fruits, flowers, leaves, and stems causing
tissue decay. This is followed by prolific sporulation of the
Nonpathogenic fungi can act as mycoparasites, as exemplified pathogen, producing a gray mold appearance. Wounded
by P. oligandrum, P. nunn, and P. periplocum, which are tissues are especially susceptible to this pathogen. Much of
mycoparasitic on other species of Pythium and can reduce the research activity to achieve biological control of B. cinerea
pathogen infection levels and reduce disease (Berry et al. on vegetable crops has centered around the use of
1993; McQuilken et al. 1992; Paulitz and Baker 1987). Other T. harzianum, followed by Ulocladium spp. and a number
mycoparasitic fungi include Stachybotrys elegans and of yeasts, as described later.
Biological Control of Vegetable Diseases 163

4.1.1 Trichoderma As a Biocontrol Agent of against the pathogen on leaves that were spatially separated
Botrytis Cinerea from the site of application of T. harzianum. This was
attributed to induction of systemic resistance that delayed or
Isolate T-39 of T. harzianum (marketed as Trichodexe) suppressed spreading lesion formation (De Meyer et al. 1998).
provided control of gray mold as well as a number of other
fungal diseases of cucumber under commercial greenhouse
4.1.2 Saprophytic Fungi and Yeasts As Biocontrol
conditions (Elad 2000a). T. harzianum T-39 was applied as
Agents of Botrytis Cinerea
part of a gray mold management program in alternation with
chemical fungicides. The biocontrol agent was effective when The leaf surface of plants (phylloplane) is frequently
applied in formulations containing two concentrations of the colonized by a range of saprophytic fungi and yeasts, which
active ingredient (0.2 and 0.4 g/l), at around 1010 cfu/g of rely on plant nutrient exudates and a range of other carbon/
T. harzianum (Elad et al. 1993). A number of other research nitrogen sources for their survival, e.g., damaged or senescing
studies have confirmed the efficacy of T. harzianum strains in tissues, pollen grains, insect honeydew. If present in the same
reducing development of B. cinerea on crops such as niche as plant pathogenic fungi, these saprophytes may
cucumber and tomato under laboratory conditions and on compete with pathogens for nutrients, infection sites, or
greenhouse-grown plants (Dik and Elad 1999; Dik et al. 1999; reduce growth and sporulation of the pathogen on host tissues
O’Neill et al. 1996; Utkhede et al. 2000). through competition or antagonism (Fokkema 1993).
Mechanisms involved in the biological suppression of Recovery of selected fungi and yeasts and reapplication to
infection and inoculum potential of B. cinerea by Tricho- the leaf or stem surface has identified a number of potential
derma are numerous and variable and the involvement of two biological control agents that can reduce diseases caused by
or more mechanisms has been demonstrated in several B. cinerea. On onion leaf tissues, the saprophytic fungi
studies. Reported combinations include antibiosis with Alternaria alternata, Chaetomium globosum, Ulocladium
enzyme degradation of B. cinerea cell walls and parasitism atrum, and U. chartarum suppressed sporulation of the
(Bélanger et al. 1995); competition for nutrients followed by pathogen significantly when applied after pathogen inocu-
interference with pathogenicity enzymes of the pathogen or lation (Köhl et al. 1995; 1999). A monoclonal antibody-based
with induced resistance; and alteration of plant surface enzyme-linked immunosorbent assay (ELISA) has been
wettability combined with antibiosis (Elad 1996). Since, described to detect and quantify U. atrum in colonized plant
germinating B. cinerea conidia are dependent on the presence tissues (Karpovich-Tate and Dewey 2001) and could be useful
of nutrients to initiate pathogenesis, competition for nutrients in monitoring of this biocontrol agent. Application of the
is important in biocontrol. Pathogen conidial viability and saprophytic fungus Cladosporium cladosporioides to wounds
germination capacity are also potentially affected by the on tomato stems was reported to reduce infection by
presence of antibiotics produced by Trichoderma and present B. cinerea in laboratory and greenhouse experiments (Eden
in the phyllosphere. Slower in action are mechanisms et al. 1996).
involving induced resistance in the host plant and production The yeast-like fungi Aureobasidium pullulans and
of hydrolytic enzymes that degrade B. cinerea cell walls. The Cryptococcus albidis significantly reduced sporulation of
latter has been demonstrated much more convincingly in vitro B. cinerea on pruning wounds and stems of cucumber and
than in the phyllosphere. Biocontrol in established lesions and tomato under laboratory and greenhouse conditions (Dik and
reduction of sporulation of Botrytis on necrotic plant tissues is Elad 1999; Dik et al. 1999). Another yeast, Rhodosporidium
a means to minimize secondary spread of pathogen inoculum. diobovatum, when applied to tomato stems, reduced lesion
Zimand et al. (1996) also demonstrated that the presence of size due to B. cinerea and the treated plants yielded higher
T. harzianum at the site where B. cinerea infects can have an fruit when compared to the untreated controls (Utkhede et al.
adverse effect upon activity of pathogen enzymes involved in 2000). Both C. albidus and Rhodotorula glutinis reduced
pectin degradation and host cell wall destruction, e.g., sporulation of B. cinerea on bean and tomato leaves and
pectinase, cutinase, and pectate lyase. Since such enzymes are reduced disease levels (Elad et al. 1994).
intimately involved in the infection process by B. cinerea, the
effect of the biocontrol agent in reducing their activity in vitro 4.1.3 Mechanisms of Action of Yeasts Against
and on the surface of plant leaves could also limit disease Botrytis Cinerea
development by the pathogen (Kapat et al. 1998).
The inhibition of pathogen enzymes was proposed to be Yeasts can compete effectively against B. cinerea for
due to the secretion of serine proteases by T. harzianum (Elad nutrients, such as glucose and fructose (Filonow 1998;
and Kapat 1999), which could also inhibit pathogen spore Filonow et al. 1996), thereby reducing pathogen colonization
germination. The presence of protease inhibitors was found to of plant tissues and sporulation (Elad et al. 1994). Yeasts such
reduce the biocontrol activity. The potential role of induced as Aureobasidium have also been reported to produce
plant resistance by T. harzianum for control of B. cinerea was mycotoxins in culture (Schrattenholz and Flesch 1993).
demonstrated by De Meyer et al. (1998), wherein application Yeast cells may attach to pathogen hyphae, as demonstrated
of the biocontrol agent to roots or leaves of a number of for B. cinerea, and cause them to degrade (Cook et al. 1997)
different plant species was observed to provide protection through secretion of cell wall degrading enzymes
164 Punja and Utkhede

(Wisniewski et al. 1991). The production of cell wall G. catenulatum J1446 as preventative treatments to wounded
degrading enzymes, such as b-1,3-glucanases has also been stem tissues of cucumber followed by inoculation with the
documented in yeasts such as Pichia anomola that are pathogen. They demonstrated that both microbial agents
effective biocontrol agents against B. cinerea as a postharvest significantly reduced disease development when compared to
treatment (Jijakli and Lepoivre 1998). In vivo studies with plants treated with water. Anthracnose disease of cucumber,
Candida saitoana in apple demonstrated that B. cinerea caused by Colletotrichum magna, was reduced when a
hyphae had degenerated (El-Ghaouth et al. 1998), implicating nonpathogenic mutant was applied to seedlings to achieve
the possible role of toxins and/or enzymes. In addition, plant colonization and induction of defense responses that
cells in the vicinity of the yeasts appeared to have enhanced subsequently protected treated plants against the pathogen
structural defense responses, suggesting an induction of (Redman et al. 1999).
defense in the host plant may have occurred. Stimulation of
host cell defenses by the yeast C. oleophila was recently
described (Droby et al. 2002). 4.3 Biocontrol of Powdery Mildews

4.1.4 Biotechnological Techniques Applied to Powdery mildew fungi are obligate parasites of plants that
Yeasts derive nutrients and water from their host, thereby reducing
growth and yield through the acquisition of photosynthates.
Yeasts with biocontrol potential against gray mold have been The fungi penetrate into the epidermis directly and establish a
characterized using molecular techniques to provide a method parasitic relationship with the plant host through the
to distinguish between closely-related strains and to identify formation of haustoria, the nutrient-absorbing structures.
and monitor survival of strains after application (Schena et al. Mycelial growth and sporulation occur on the surface of
2000; 2002). These techniques include arbitrarily primed leaves and stems, resulting in a white fuzzy mildew
polymerase chain reaction (AP-PCR), random amplified appearance.
polymorphic DNA (RAPD-PCR) analysis, and sequence- Over the years, powdery mildew diseases have been
characterized amplification region (SCAR) analysis. In managed through the use of chemical fungicides and genetic
addition, transformation of the yeast Metschnikowia with resistance, but recent reports have highlighted the potential of
green fluorescent protein (GFP) was achieved and colonies biological control methods. Fungal and yeast biological
could be visualized under epifluorescence (Nigro et al. 1999). control agents have been described which can reduce
The transformed strains behaved similarly to the wild-type sporulation and growth of mildew pathogens, thereby
strains in biocontrol activity against B. cinerea and in growth minimizing their damaging effects to host plants. The fungal
rates. Another yeast, A. pullulans, was also transformed with biocontrol agents are mostly mycoparasites, while the yeasts
GFP and colonies were readily visible on apple leaf surfaces produce antibiotics and hydrolytic enzymes that cause the
when subjected to fluorescence and could be quantified mildew hyphae and conidia to collapse and be rendered
(Wymelenberg et al. 1997). nonviable.
Genetic transformation of the yeast Saccharomyces to
express a cecropin A-based peptide with antifungal activity 4.3.1 Fungi As Biological Control Agents of
was recently described (Jones and Prusky 2002). The Powdery Mildews
transformants inhibited the growth of Colletotrichum and
reduced fungal decay of tomato fruits when applied prior to Verticillium lecanii has been described as a mycoparasite of
pathogen inoculation. The expression of the antifungal powdery mildew fungi as well as a pathogen of insects and it
peptide in the biological control agent suggests a new has been developed as a biocontrol agent of insects on
approach for disease control. greenhouse crops. Strains of V. lecanii differed in their level
of antagonism against the powdery mildew pathogen of
cucumber, Sphaerotheca fuliginea, under laboratory con-
4.2 Biocontrol of Leaf and Stem Blights ditions (Askary et al. 1998). Application to cucumber leaves
prior to mildew infection and incubation under high (. 95%)
Didymella bryoniae (Auersw.) Rehm (anamorph Phoma relative humidity conditions reduced mildew development
cucurbitacearum Fr.:Fr.) Sacc. is an important pathogen on (Verhaar et al. 1997). The high humidity requirement for
greenhouse- and field-grown cucumbers and other cucurbits, growth of this mycoparasite was reduced by the addition of an
and causes the disease gummy stem blight. The disease is oil formulation (Verhaar et al. 1999). Infection of S. fuliginea
favored by warm, humid conditions and the pathogen infects by V. lecanii resulted in disorganized cytoplasm and
stems, fruit, leaves, and flowers of susceptible plants, plasmalemma disruption, possibly due to chitinase enzyme
especially through wounded or senescing tissues, and natural activity (Askary et al. 1997).
openings such as stomata and hydathodes. There are few Another mycoparasite, Ampelomyces quisqualis, has been
reports on the potential of using biological control agents to extensively studied as a biocontrol agent of powdery mildew
control this disease. Utkhede and Coch (unpublished) applied of cucumber. The mycoparasite infects the mildew pathogen
the yeast R. diobovatum and the biocontrol agent and forms pycnidia in association with colonized mycelium,
Biological Control of Vegetable Diseases 165

reducing growth and sporulation of the pathogen. Cells of the Molecular techniques have been used to characterize
mycoparasite grow inside the mildew hyphae, gradually strains of A. quisqualis. The RFLP analysis of the nuclear
causing them to degenerate. High levels of b-1,3-glucanase rDNA ITS region and sequence analysis among a worldwide
activity were reported in A. quisqualis (Rotem et al. 1999) and collection of isolates revealed considerable intraspecific
exposure of mildew hyphae to the enzymes caused them to variation (Kiss 1997; Kiss and Nakasone 1998). Isolates of the
degrade. same genetic background were found in widely different areas
A commercially available formulation of A. quisqualis and genetically different isolates could be found in a given
AQ10 has been extensively evaluated against powdery area.
mildew development. On cucumbers grown in the green-
house, AQ10e was very effective in reducing mildew 4.3.2 Yeasts As Biological Control Agents of
development (Elad et al. 1998). On field-grown cucurbits, Powdery Mildew
AQ10e also suppressed mildew development and increased
yield when compared to the nontreated plants (McGrath and Pseudozyma (Sporothrix) flocculosa is a yeast-like fungus
Shishkoff 1999). with demonstrated biocontrol activity against powdery

Figure 2 Effect of Tilletiopsis pallescens on development of powdery mildew (S. fuliginea) on cucumber leaves. (A) A mildew-infected
leaf showing chains of conidia and mycelium. (B) A mildew colony treated with a 3-day-old liquid culture of T. pallescens. Note
collapsed conidia and mycelium. Photograph was taken 2 days following treatment. (C) Spore masses of Tilletiopsis adjacent to mildew
conidia. Note intact mildew conidia on left and collapsed condia on the right.
166 Punja and Utkhede

mildew fungi, especially on cucumber and rose (Bélanger and electron microscopic studies have revealed that mildew
Benyagoub 1997; Belanger et al. 1994). Cytochemical hyphae and spores appeared collapsed after treatment with
investigations have shown that the yeast induces a rapid Tilletiopsis (Figure 2) (Urquhart and Punja 1997). It was
collapse of mildew spores and hyphal cells (Hajlaoui et al. postulated that extracellular antifungal compounds were
1992). Extracellular fatty acids with antifungal properties involved in biocontrol activity that included fatty acid esters
were produced by P. flocculosa and reported to be the and hydrolytic enzymes (Urquhart and Punja 2002). Various
principle mode of action in biological activity against species of Tilletiopsis have demonstrated biological control
powdery mildew (Benyagoub et al. 1996), by disrupting the activity, including T. albescens, T. minor, T. pallescens, and
cytoplasmic membrane in a range of fungi (Avis and Bélanger T. washingtonensis (Table 2). These species could be
2001). A fungicide-tolerant strain of the yeast was selected distinguished using RAPD analysis of PCR-generated DNA
which could be used in conjunction with chemical control with random primers (Urquhart et al. 1997). Intraspecific
methods to reduce powdery mildew development variation was also noted and DNA fingerprints were generated
(Benyagoub and Bélanger 1995). In a comparative study of for some isolates that could be useful for monitoring the
three biological control agents against powdery mildew of distribution and spread of certain isolates.
cucumber, i.e., V. lecanii, A. quisqualis, and P. flocculosa, it
was shown that P. flocculosa gave the best disease control
(Dik et al. 1998). 5 CONCLUSIONS
Molecular techniques have been used to characterize
strains of Pseudozyma flocculosa (Avis et al. 2001b). The numerous reports of success in the utility of fungal and
Ribosomal DNA sequences and random amplified micro- yeast biological control agents to reduce fungal diseases on
satellites were used to distinguish among different strains of vegetable corps illustrate the potential of this approach for
this species and to develop isolate-specific markers to monitor disease management. In addition, the applications of
spread and confirm genetic fidelity of the strains. A strain of techniques in biotechnology are providing numerous
P. flocculosa has been formulated and produced com- examples of how these biocontrol agents can be character-
mercially under the name Sporodexe for use in control of ized, monitored, and investigated in more depth. However,
powdery mildew on a number of crops grown under there are unique requirements in working with microbial
greenhouse conditions. biocontrol agents that must be recognized if this approach to
Species of Tilletiopsis are saprophytic yeast-like fungi that disease control is to be successful.
occur as epiphytes on the leaf surface of various plant species Environmental conditions, particularly temperature and
and which have been demonstrated to have biological control moisture, can greatly influence the degree to which fungal and
activity against powdery mildew diseases (Hijwegen 1992; yeast biological control agents can affect fungal diseases on
Knudsen and Skou 1993; Urquhart et al. 1994). Scanning vegetable crops, even in greenhouse environments. Therefore,

Table 2 Examples of fungal and yeast biological control agents that reduce foliar diseases on vegetable crops caused by pathogenic
fungi

Biocontrol agent Target pathogen and host References

T. harzianum B. cinerea on cucumber Dik and Elad (1999) and
Elad et al. (1993; 1998)
T. harzianum B. cinerea on tomato Dik and Elad (1999), Migheli
et al. (1994), and Utkhede et al. (2000)
T. harzianum C. fulvum on tomato Elad et al. (2000a)
T. harzianum S. fuliginea and S. fusca on cucumber Elad et al. (1998; 2000b)
G. catenulatum D. bryoniae on cucumber Utkhede and Coch (unpublished)
A. quisqualis AQ10 S. fusca on cucumber Elad et al. (1998)
A. pullulans B. cinerea on tomato and cucumber Dik and Elad (1999)
C. albidus B. cinerea on bean, tomato, and cucumber Dik and Elad (1999) and
Elad et al. (1994)
R. glutinis B. cinerea on bean and tomato Elad et al. (1994)
R. diobovatum B. cinerea on tomato Utkhede et al. (2000)
C. cladosporioides B. cinerea on tomato Eden et al. (1996)
S. flocculosa S. fuliginea on cucumber Dik et al. (1998)
T. albescens, T. minor, T. pallescens, S. fuliginea on cucumber Hijwegen (1992), Knudsen and Skou
and T. washingtonensis (1993), Urquhart and Punja (1997),
and Urquhart et al. (1994)
Biological Control of Vegetable Diseases 167

careful monitoring and recording of environmental variables Askary H, Benhamou N, and Brodeur J (1997). Ultrastructural and
is a requisite. The biological control agents are generally most cytochemical investigations of the antagonistic effect of
effective when applied as a preventative treatment, prior to or Verticillium lecanii on cucumber powdery mildew.
at the onset of disease, and multiple applications may be Phytopathology 87:359 –368.
Askary H, Carriere Y, Belanger RR, and Brodeur J (1998).
needed to provide longer-term disease suppression. At high
Pathogenicity of the fungus Verticillium lecanii to aphids and
levels of disease pressure, biological control agents can be powdery mildew. Biocontrol Sci Technol 8:23 – 32.
anticipated to perform less well. Some of the agents may be Avis TJ and Bélanger RR (2001). Specificity and mode of action of
used in combination with, or in alternation with, chemical the antifungal fatty acid cis-9-heptadecenoic acid produced
fungicides if it can be demonstrated that their survival is not by Pseudozyma flocculosa. Appl Environ Microbiol
adversely affected. Similarly, it may be possible that 67:956– 960.
combinations of biocontrol agents may be more effective Avis TJ, Hamelin RC, and Bélanger RR (2001a). Approaches to
than single organisms although little research has been done molecular characterization of fungal biocontrol agents: some
in this area. Biological control agents that affect more than case studies. Can J Plant Pathol 23:8 – 12.
one disease should have greater market potential than those Avis TJ, Caron SJ, Boekhout T, Hamelin RC, and Belanger RR
(2001b). Molecular and physiological analysis of the powdery
that specifically target a particular disease. It is not clear
mildew antagonist Pseudozyma flocculosa and related fungi.
whether different plant hosts may have an influence on the Phytopathology 91:249 –254.
efficacy of these biocontrol agents. Bao JR and Lazarovits G (2001). Differential colonization of tomato
Notwithstanding these conditions, the use of fungal and roots by nonpathogenic and pathogenic Fusarium oxysporum
yeast biological control agents has generated significant strains may influence Fusarium wilt control. Phytopathology
interest in both the scientific research and product develop- 91:449– 456.
ment arenas to ensure that commercially viable products will Bao JR, Velema J, Dobinson KF, and Lazarovits G (2000). Using
continue to be brought to market. GUS expression in a nonpathogenic Fusarium oxysporum strain
to measure fungal biomass. Can J Plant Pathol 22:70 – 78.
Bélanger RR and Benyagoub M (1997). Challenges and prospects for
ACKNOWLEDGEMENTS integrated control of powdery mildews in the greenhouse. Can J
Plant Pathol 19:310 –314.
Bélanger RR, Labbé C, and Jarvis WR (1994). Commercial scale
Funding for aspects of this work was provided by the British control of rose powdery mildew with a fungal antagonist. Plant
Columbia Greenhouse Growers’ Association, the National Dis 78:420 –424.
Research Council of Canada IRAP Program, the Natural Bélanger RR, Dufour N, Caron J, and Benhamou N (1995).
Sciences and Engineering Research Council of Canada Chronological events associated with the antagonistic proper-
Biocontrol Network Program, and Agriculture and Agri-Food ties of Trichoderma harzianum against Botrytis cinerea:
Canada Matching Investments Initiative Program. indirect evidence for sequential role of antibiosis and
parasitism. Biocontrol Sci Technol 5:41 –53.
Benhamou N, Rey P, Chérif M, Hockenhull J, and Tirilly Y (1997).
Treatment with the mycoparasite Pythium oligandrum triggers
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15
Control of Postharvest Diseases of Fruits Using Microbes

Wojciech J. Janisiewicz Appalachian Fruit Research Station, U.S. Department of Agriculture – Agricultural
Research Service, Kearneysville, West Virginia, USA

1 INTRODUCTION has been increasingly curtailed by the perceived hazard to

humans and the environment. This has resulted in new
Losses from postharvest diseases of fruits have been regulations restricting or eliminating their use in this country
substantial at the storage, wholesale, retail, and consumers and abroad. It has become increasing difficult to find and
levels. The total losses are very difficult to establish because register new fungicides to replace those to which postharvest
research has generally considered only one or two levels, and pathogens have developed resistance (Gullino and Kuijpers
little work has been done to determine losses at the consumer 1994; Ragsdale and Sisler 1994). Thus, there has been a need
level. Nevertheless, in the United States, losses are estimated to find effective alternatives to synthetic fungicides. None of
to range from 5% for citrus to as much as 20% for the alternative methods developed during the past two
strawberries (Cappellini and Ceponis 1984; Eckert and decades have had the broad spectrum of activity as synthetic
Ogawa 1985). Most of the fruit decay results from infection fungicides. Recently, biological control has emerged as an
through wounds made during harvest and postharvest alternative (Janisiewicz and Korsten 2002). The full potential
handling, but for some fruits, infection takes place in the of biocontrol has not yet been realized because the
mechanisms of biocontrol have not been explained. A fuller
orchard during the growing season, and remains latent. As
understanding of the antagonistic mechanisms will eventually
fruit mature in storage the pathogens become active again and
help manipulate and improve the biocontrol system. Although
invade fruit tissue. A variety of approaches have been used to
this method has some limitations, these limitations can be
reduce postharvest fruit decays, including sanitation to reduce
addressed by combining biological control with other
pathogen inoculum, gentler handling of fruit to reduce
alternative methods (Conway et al. 1999; El-Ghaouth et al.
wounding (Sommer 1982), physical treatments such as hot
2000b; Janisiewicz et al. 1998; Smilanick et al. 1999). In this
water dips and hot air treatments that kill pathogens (Falik
chapter, the key elements in the development of biological
et al. 1995; Lurie et al. 1998), storing produce at low
control of postharvest diseases (BCPD) of fruits, and the
temperatures or in modified atmosphere which stop or reduce
current status and future prospects of BCPD of fruits using
growth of the pathogens (Sommer 1982), treating fruit with examples of fungal and bacterial antagonists are discussed.
chemicals that enhance natural resistance (El-Ghaouth 1998),
with synthetic fungicides (Eckert and Ogawa 1985; Eckert
and Ogawa 1988), and, more recently, with biocontrol agents
(Droby et al. 1998; Janisiewicz and Jeffers 1997; Janisiewicz 2 PATHOGENS TARGETED FOR BCPD OF
and Korsten 2002; Korsten et al. 1995; Usall et al. 2001). FRUITS
Fungicides have been, by far, the most widely used remedy
against fruit decay because they are easy to apply and The BCPD of fruits can be approached from the perspective
generally, one fungicide is effective against most of the of the host plant (different fruits), habitat for the
pathogens on a specific crop. Storage of some fruits for microorganisms (wound, intact surface), and the pathogen’s
extended periods, e.g., citrus fruits, is totally dependent on the strategy used to infect fruit. The pathogen’s strategy has been
use of fungicides. But postharvest use of synthetic fungicides emphasized most frequently because many economically

173
174 Janisiewicz

important postharvest diseases of fruits are caused by of stone fruits caused by M. fructigena (Pusey and Wilson
necrotrophic pathogens (Dennis 1983). These pathogens 1984). Subsequent works focused on screening natural
invade mainly through wounds, and require nutrients for microflora from the aerial surfaces of apple and pear trees
spore germination and initiation of the pathogenic process, for antagonistic activity against decays caused by P. expansum
which makes them vulnerable to competition for nutrients and B. cinerea (Janisiewicz 1987). This resulted in the
from surrounding microorganisms. Other mechanisms of isolation of many bacteria and yeasts that were effective in
biocontrol described later in this chapter may also be controlling fruit decays caused by these pathogens. Isolation
involved, but the prevailing evidence suggests that their role from the fruit surfaces has become a standard practice and is
is secondary (Janisiewicz and Korsten 2002). Incipient or the most efficient source of antagonists against postharvest
latent infections that generally occur in the field are less prone fruit pathogens of temperate, subtropical, and tropical fruits
to biological control because the pathogen has already (Adikaram and Karunaratne 1998; Arras 1993; Chalutz et al.
established a parasitic relation with the host. However, these 1988; Chand-Goyal and Spotts 1996; Droby et al. 1999;
pathogens can be controlled by antagonists that prevent Guinebretiere et al. 2000; Huang et al. 1992; Kanapathipillai
infection in the field, perpetuate latency by removal of and bte Jantan 1985; Lima et al. 1998; Qing and Shiping
nutrients from areas surrounding the appressoria, or perhaps 2000; Teixidó et al. 1998a; Testoni et al. 1993; Zahavi et al.
those that can inhibit pathogen development by the 2000). A variety of enrichment procedures, employing either
production of antifungal substances or by direct parasitism fruit juice or tissue, have been used to isolate microorganisms
(Koomen and Jeffries 1993; Korsten and Jeffries 2000; best suited to colonize wounded fruit tissue (Janisiewicz
Leibinger et al. 1997). 1991; 1996; Wilson et al. 1993). The enrichment procedures
The greatest progress in BCPD of fruits has been made appear to favor isolation of the resident fruit microflora, with
against typical wound-invading necrotrophic postharvest the yeasts being isolated most frequently, followed by
pathogens such as Penicillium expansum which causes blue bacteria. Filamentous fungi have been isolated only
mold of apple, pear, and cherries, Botrytis cinerea which sporadically (Janisiewicz 1996; Wilson et al. 1993). The
causes gray mold of pome fruits (Janisiewicz and Jeffers rapid colonization of wounds by yeasts is necessary for pre-
1997), P. italicum and P. digitatum which causes blue and emptive exclusion of the wound-invading pathogens. The
green-mold of citrus fruits, respectively (Droby et al. 1998), number of species that are residents on a specific kind of fruit
and against the wound invading phase of brown rot decay of is limited, and reports from various laboratories worldwide
stone fruits caused by Monilinia fructicola (Pusey et al. 1988). increasingly describe biocontrol potential of the same
Although the likelihood of infection is dependent on the antagonist species isolated at different locations (Chand-
concentration of fungal spores, the biocontrol strategy has Goyal and Spotts 1996; 1997; Falconi and Mendgen 1994;
always focused on the preemptive colonization of the wounds Ippolito et al. 2000; Janisiewicz et al. 1994; 2001; Leibinger
by the antagonist to prevent infection, and not on reduction of et al. 1997; Lima et al. 1998; McLaughlin et al. 1992; Roberts
the pathogen inoculum. Thus, the control of these decays was 1990; Wisniewski et al. 1988). Recent studies; however,
achieved by the application of antagonists to wounds, indicate great diversity within an antagonist species, even at a
simultaneously with the pathogen or shortly after the infection single geographical location, with regard to effectiveness in
took place. Other potential candidates for this type of controlling fruit decays and other factors important in
biological control are pathogens invading through cut stem of commercializing a biocontrol agent (Janisiewicz et al. 2001;
bananas, mangos, and papayas (Eckert 1991). Since the Schena et al. 1999). Thus, investigating the same species of
development of the pathogen depends on fruit maturity and the antagonist at various locations may lead to finding an
the environment, these factors have been critical in the antagonist with superior attributes. An effective antagonist
pathogen-antagonist interaction and resulting biological may also be found by screening starter cultures used for food
control. products (Pusey 1991), various culture collections (Filonow
Significant successes were achieved with biocontrol of et al. 1996), and even by exploring an aquatic environment, as
latent infections caused by Colletotrichum spp. on mango and is the case with bacteriophages used against soft rotting
avocado (Korsten and Jeffries 2000), and to a lesser extent by bacteria (Eayre et al. 1995).
B. cinerea on strawberries (Helbig 2002; Ippolito et al. 1998; In addition to being effective in controlling fruit decays,
Peng and Sutton 1990; Takeda and Janisiewicz, unpublished antagonists should have certain attributes to make them
results). Biological control of these diseases must start in the good candidates for commercialization. These include:
field, relies on multiple application of the antagonist, and is compatibility with postharvest practices (storage tempera-
generally more difficult to achieve. tures, relative humidity-RH, storage atmosphere with
elevated CO2 and reduced O2, handling in water, heat drying
tunnels, etc.), treatments and additives (waxes, antioxidants,
3 ANTAGONIST SELECTION flotation salts), ability to grow efficiently in a commercially
used media for mass production, ease of formulation, and the
The first successful attempts in BCPD of fruits, which lack of potentially deleterious effects on human health that
stimulated research in postharvest biological control, used would disqualify them from being approved by regulatory
soil isolated bacterium, Bacillus subtilis, to control brown rot agencies. Human safety, in particular, necessitates a thorough
Biological Control of Postharvest Diseases of Fruits 175

approach in identifying an antagonist. Misidentification may enzyme also increased in apple wounds treated with the
result in abandonment of commercial development of the A. pullulans cells, but how much of this increase could be
antagonist, and, if not detected early, may be very costly. attributed to production by the antagonist, on the fruit itself,
was not resolved (Castoria et al. 2001). This antagonist can
also produce aurebasidins, antibiotics whose role has not been
4 MECHANISMS OF BIOCONTROL determined. The antagonistic yeast, Candida saitoana,
effective in reducing decays of citrus and apple, induced
Various mechanisms of biocontrol have been suggested for chitinase activity in apple (Wilson and El Ghaouth 1993).
antagonists effective in BCPD of fruits and often more than C. oleophila, used in the commercial product Aspire, induced
one mechanism was implicated for a single antagonist. In no resistance responses such as production of chitinase,
case, however, was the biocontrol mechanism fully b-1,3-endoglucanases, PAL, phytoalexins scoparone and
explained. The putative mechanisms included competition scopoletin and ethylene in flavedo tissue of grapefruit (Droby
for limiting nutrients and space, lysis, induced resistance, et al. 2002). The contribution of these induced resistance
direct parasitism, and production of inhibitory substances. responses to biocontrol was not determined. The yeast
Attachment of antagonists to a fungal hyphae was observed in C. famata, effective in reducing green mold caused by
some antagonist-pathogen interactions, but its role remains P. digitatum on oranges, increased the phytoalexins
largely speculative (Arras et al. 1998; Wisniewski et al. 1989; scopoletin and scoparone 12-fold in fruit wounds after four
1991). The main reasons for the limited knowledge in days, but the role scoparone in biocontrol is uncertain due to
mechanism of biocontrol have been a lack of appropriate its relatively slow production (Arras 1996). The antagonists
methods to study microbial interactions in wounds of fruit, Cryptococcus laurentii and Sporobolomyces roseus, effective
and the fact that progress in BCPD was driven by advances in against gray mold of apple, utilized the apple volatile, butyl
microbial ecology of the antagonists. However, recent acetate, which stimulated germination and adhesion to
advances in microbial sensing of nutrients on plants (Lindow membranes of B. cinera conidia (Filonow 1999; 2001). The
et al. 2002), molecular approaches (Bassett and Janisiewicz significance of these phenomena in the biological control was
2001; Jijakli et al. 2001; Jones and Prusky 2002; Yehuda et al. not established due to the technical difficulties in conducting
2001), and a method allowing separation of competition for this type of experiment in fruit wounds. When these
nutrient and space using natural substrates (Janisiewicz et al. antagonists were applied to harvested fruits, they colonized
2000) may lead to better explanation of the significance of fruit wounds rapidly, and competition for limiting nutrients
various biocontrol mechanisms. Progress in microbial and space was suggested as an important biocontrol
ecology of the antagonists led the commercialization of mechanism. Removal of limiting nutrients may also be
BCPD of fruits in a relatively short period of time, but further responsible for maintaining the dormancy of Colletotrichum
expansion will greatly depend on achieving the full potential spp. appresoria on fruit treated with antagonistic Bacillus spp.
of BCPD, for which knowledge of the mechanisms of (Korsten and Jeffries 2000).
biocontrol will be essential.
Bacterial antagonists such as Bacillus spp. (Gueldner
et al. 1988) or Pseudomonas spp. (Bull et al. 1998; 5 IMPROVING BCPD
Janisiewicz and Roitman 1988) produce a variety of
antifungal compounds in artificial media, which by The goal of the biocontrol improvement program is to
themselves can provide effective control of postharvest realize the full potential of biological control. This may be
decays of fruits (Bull et al. 1998; Janisiewicz et al. 1991; accomplished by more extensive strain selection of the same
Takeda and Janisiewicz 1991). But the role of these antifungal antagonist species, as indicated earlier in the case of
compounds as the biological control mechanism is uncertain, M. pulcherrima, by manipulating antagonists and/or their
because they either can not be detected in fruit wounds after environment, and by applying the antagonists before harvest
inoculation with the antagonist (Bull, personal communi- in addition to one after harvest.
cation), or pathogen mutants resistant to these inhibitory Postharvest application of the antagonists mixture of
substances are still controlled by the antagonists (Smilanick, P. syringae and S. roseus (Janisiewicz and Bors 1995), and
personal communication). C. sake and Pantoea agglomerans (Nunes et al. 2002)
Yeast antagonist such as Pichia anomala strain K improved efficacy of biocontrol of blue mold of apples
(Gravesse et al. 1998; Jijakli and Lepoivre 1998), compared to the individual antagonist. An orchard application
P. guilliermondii (Arras et al. 1998; Wisniewski et al. of a mixture of A. pullulans with Rhodotorula glutinis was
1991), or yeast like Aureobasidum pullulans (Castoria more effective than the individual antagonists, and suppressed
et al. 2001) effective in controlling gray mold of apples, apple decays caused by Penicillium spp., B. cinerea, and
produce b-1,3-glucanase, which caused lysis of the B. cinerea Pezicula malicorticis after harvest to the same level as the
hyphae. Production of this enzyme by P. anomala strain K commonly used fungicide Euparen (Leibinger et al. 1997).
was stimulated in the presence of cell wall preparations of Nutrient utilization profile of individual antagonists was
B. cinerea in apple wounds resulting in improved biocontrol successfully used to develop antagonist mixtures with a
(Gravesse et al. 1998; Jijakli and Lepoivre 1998). This minimum of nutrient overlap between the antagonists and
176 Janisiewicz

resulted in biocontrol of blue mold of apples that was superior This work demonstrated that microorganisms that colonize
to the individual antagonists (Janisiewicz 1996). fruits can be used as vehicles for providing decay control
Physiological manipulation has been focused on improv- which may include various biocontrol traits.
ing antagonist fitness by growing them under various
conditions that improved resistance to desiccation and
survival on the fruit. This is of particular importance to
antagonists that are applied in the orchard for control of 6 INTEGRATING BCPD WITH OTHER
postharvest decays. C. sake cells grown under water stress ALTERNATIVES
caused by addition of glucose or glycerol increased after
application to apple trees, while those grown on unmodified Although BCPD can provide levels of control that are
media did not (Teixidó et al. 1998b). This yeast was more commercially acceptable, the performance margin of
water-stress tolerant when grown on a molasses-based biocontrol is generally lower than for fungicides. For
medium than on a medium where water activity (aw) was example, higher concentrations of the antagonist must be
modified by the addition of NaCl (Abadias et al. 2001b). In used to achieve the same control of decay as fruit mature. To
preparing a freeze-dried formulation, viability of the C. sake increase the performance margin of biocontrol, attempts have
cells was best maintained when 10% skim milk was combined been made to integrate biocontrol with other alternatives to
with other protectants such as lactose, glucose, fructose, or synthetic fungicide methods that were developed mainly
sucrose (Abadias et al. 2001a). In general, the highest during the past two decades (Conway and Sams 1983; Falik
viability of the C. sake cells occurred when the protection and et al. 1995; Smilanick et al. 1995; 1997; 1999; Smoot and
rehydration media were the same. Melvin 1965; Spotts 1984; Spotts and Chen 1987; Tukey
Control of blue mold of apples was improved by the 1993). These methods alone did not provide commercially
addition of the amino acids L-asparagine or L-proline to the acceptable control of fruit decay, but in combination with
P. syringae antagonist treatment suspensions (Janisiewicz biocontrol increased its performance margin.
et al. 1992). These amino acids were selected after screening Infiltration of apples with calcium chloride alone reduced
various C and N sources for their effect on the antagonist and blue mold decay by approximately half (Conway and Sams
pathogen growth. Both amino acids increased population of 1983), but in combination with the antagonist, P. syringae,
the antagonist more than 10-fold in the fruit wounds. The resulted in greater reduction of fruit decay than either
addition of the glucose analog, 2-deoxy-D -glucose, to the treatment alone (Janisiewicz et al. 1998). The effects of
antagonistic yeasts S. roseus and C. saitoana significantly calcium treatment were greatest on more mature fruit,
improved decay control on apple and citrus, respectively (El- inoculated after 3 or 6 months in storage, when the
Ghaouth et al. 2000c; Janisiewicz 1994). 2-deoxy-D -glucose effectiveness of biocontrol declines (Conway et al. 1999).
can be up taken by the pathogens but it cannot be metabolized Combining biocontrol with a calcium treatment comp-
as energy source, resulting in reduced pathogen growth, lements each other to overcome the shortcomings of each,
which gives the advantage to the antagonists, and improves and may allow for reduced amounts of both products to be
biocontrol. Ammonium molybdate stimulated population of used without compromising decay control. In addition,
the antagonistic yeast C. sake (CPA-1) and improved control applying lower calcium concentrations would reduce
of blue mold of apple and pear after harvest (Nunes et al. potential calcium injury, while maintaining other benefits,
2001a). This nutrient also has fungicidal activity and inhibited including alleviating storage maladies, such as bitter pit. The
germination of P. expansum and B. cinerea spores in vitro, addition of calcium chloride to the yeast antagonist Candida
and reduced blue mold, gray mold, and Rhizopus stolonifer sp. also improved control of blue mold and gray mold on
decay of apple in pre- and postharvest applications (Nunes apples (McLaughlin et al. 1990; Wisniewski et al. 1995).
et al. 2001b). Treating apples with hot air (4 d at 388C) may virtually
Use of genetic manipulation to improve BCPD has great eliminate blue mold of apple but it has no residual effect, and
potential, but little research has been done in this area. The any inoculation with pathogens following heat treatment
appearance of decay symptoms on avocado fruit was delayed results in decay (Falik et al. 1995; Lurie et al. 1998).
when the fruit were dipped in a suspension containing a Combining antagonistic yeasts or bacteria with heat treatment
reduced-pathogenicity mutant of the avocado pathogen improved control of blue mold on apples (Conway et al.
Colletotrichum gleosporioides (Yakoby et al. 2001). This 1999). The heat treatment eradicated P. expansum infections
mutant was generated by restriction enzyme-mediated up to 12 h after inoculation, and yeast and bacterial
integration (REMI) transformation, and induced natural antagonists provided the residual effect. The heat treatment
resistance of avocado by increasing production of the complemented the lack of eradicative activity of the
antifungal diene from 700 to 1,200 mg/g fresh weight 9 days antagonists, the major shortcoming of BCPD. It may have
after inoculation. Saccharomyces cerevisiae transformed with additional benefits of eradicating pathogens from fruit bins
a cercopin A-based peptide, that inhibits germination of and storage rooms (Douglas 1998).
C. cocodes at 50 mM, was able to inhibit the growth of Substances generally regarded as safe (GRAS), such as
germinated spores, and inhibited decay development caused sodium carbonate, sodium bicarbonate, ethanol, acetic acid,
by C. cocodes on wounded tomato (Jones and Prusky 2002). hydrogen peroxide, chitosan, or some edible coatings can
Biological Control of Postharvest Diseases of Fruits 177

reduce pathogen germination and growth. They are Memorandums of Understanding, which allow private
acceptable to the consumers, and in contrast to synthetic industry the initial exploration of the commercial potential
fungicides, do not have the prospect of a lengthy and costly of an invention, or more definitive, Cooperative Research and
approval process from regulatory agencies. For example, Development Agreements (CRADA) that specify the role of
sodium carbonate has been used to treat lemons in each party in the commercial development and ownership of
commercial packinghouses (Smilanick et al. 1999). Combin- the final product. Having a private industry partner warrants
ing 3% sodium carbonate with the antagonist P. syringae closer scrutiny of the economic impact of the disease,
ESC-10 was superior to the individual treatments in importance of the disease to be controlled, and the potential
controlling green mold on citrus (Smilanick et al. 1999). for biocontrol to be competitive with the other control
This compound also improved control of blue mold and measures in terms of efficacy and cost. A CRADA was
gray mold of oranges in combination with antagonist essential in the commercialization of BioSavee by
P. agglomerans (CPA-2) (Teixido et al. 2001). Sodium EcoScience Corp., which is based on P. syringae, and
carbonate has up to 24 h of eradicative activity, but little Aspiree by Ecogen-Israel Partnership, Ltd., which is based
residual activity, which, like heat treatment, complements on C. oleophila. For example, under the CRADA, mass
biocontrol. Treatment of lemons with 10% ethanol reduced production by fermentation and biomass yield were
green mold to less than 5% (Smilanick et al. 1995). The determined for the P. syringae antagonist first, and EcoSciene
addition of 10% ethanol to suspensions of S. cerevisiae strains Corp. investigated the potential for registration and
1440 and 1749, which had little biocontrol activity, reduced formulation of the antagonist. This was followed by joint
gray mold decay of apple from more than 90% to close to 0% biocontrol feasibility and up-scale tests under simulated
(Mari and Carati 1998). Chitosan and its derivatives can commercial conditions, biocontrol tests with various formu-
reduce fungal growth and induce resistance responses in lations developed by EcoScience Corp., and the pilot test with
harvested fruits and vegetables (Allan and Hadwiger 1979; the final formulation. EcoScience Corp. developed safety data
El-Ghaouth 1998). The addition of 0.2% glycolchitosan to a and registered the product. Production, marketing, and quality
suspension of the antagonist C. saitoana increased control of control were conducted by EcoScience Corp. Also, essential
green mold of oranges and lemons, and gray and blue mold of for the success of BioSave was the well-developed
apples over that of the antagonist alone (El-Ghaouth et al. distribution of the product, skillful technical assistance, and
2000a, b). rigorous quality control. This approach could be used as a
There are many more possibilities for combining model for successful public/private sector cooperation in the
biocontrol agents with GRAS substances or other nonfungi- commercialization of other antagonists for BCPD (Stack
cidal treatments. The above examples of improving 1998).
biocontrol and integrating biocontrol with other nonfungici- The commercial development of Avogreen in South
dal treatments demonstrate that biocontrol is amenable to Africa, which contains B. subtillis and is applied in the field
manipulation and can be easily integrated with various decay for the control of postharvest diseases of avocado, followed a
control measures resulting in additive or synergistic effects. slightly different path (L. Korsten, personal communication).
Strategies must be developed for the integration of various Once registered, the approach was “to make the system work
treatments in order to maximize decay control (Conway et al. in the hands of the farmer.” First, growers tested the product
1999). Various control measures may be applied in succession on a limited scale and were encouraged to integrate the
and these applications may be customized to fit different product with existing copper sprays. By slowly phasing in
postharvest practices. biocontrol, growers gained confidence in the product. They
were provided with technical support to calculate dosages,
and develop suitable spray schedules adjusted for their spray
7 COMMERCIALIZATION OF BCPD equipment, cultivars used, age, and history of their orchards
and disease profiles. Different formulations were developed
Finding an industry partner is essential for the commerciali- to adapt biocontrol to different mixing systems, and for
zation of any antagonist for postharvest biocontrol. integration with existing chemicals and application methods.
Formulation, pilot tests, toxicology tests, and registration of Application guidelines were subsequently developed for all
the product are expensive and entail more than most research possible application systems and for different customer
programs in government laboratories or academia are needs. The wettable carrier was found to be more desirable
equipped to handle. Although there are examples of from a production point of view because it sustained excellent
commercialization of biocontrol products for plant diseases cell and biomass densities, had acceptable shelf life, and
control by individual scientists, especially from academia, was more economical. Rigorous quality control has been an
they generally involve creation of a private company that can integral part throughout the development of this product.
generate venture capital. USDA/ARS has developed a Marketing biological control products requires extensive
number of useful vehicles that allow private industry to knowledge in the fields of biological- and integrated control,
commercialize inventions created in government labora- production systems, and microbial ecosystems. The effec-
tories by working jointly with the government scientists. tiveness of implementing biocontrol alone or in integrated
These include simple Material Transfer Agreements, or systems will largely depend on knowledge of the product,
178 Janisiewicz

thorough understanding of its complexity, and transferring Candida sake grown in molasses-based media by physiological
this knowledge to the market place. These aspects are often manipulation. Can J Microbiol 47:123– 129.
neglected in the commercialization of biocontrol. Adikaram NKB and Karunaratne A (1998). Suppression of
avocado anthracnose and stem-end rot pathogens by
endogenous antifungal substances and a surface inhabiting
8 CONCLUSIONS Pestalotiopsis sp. Proceedings of an International Workshop
on Disease Resistance in Fruit, Chiang Mai, Thailand,
pp 72 –77.
Work conducted on BCPD demonstrates that in some cases Allan CR and Hadwiger LA (1979). The fungicidal effect of chitosan
biological control alone can provide adequate decay control, on fungi of varying cell wall composition. Exp Mycol
but in others it must be integrated with additional control 3:285 – 287.
measures. BCPD is compatible with many alternatives to Arras G (1993). Inhibition of postharvest fungal pathogens by
fungicide treatment, can be easily adapted to current Bacillus subtilis strains isolated from citrus fruit. Adv Hortic
postharvest practices, and be used in a cascade system, Sci 7:123– 127.
where each additional control measure further reduces fruit Arras G (1996). Mode of action of an isolate of Candida famata in
decay. Recent advances in physiological and genetic biological control of Penicillium digitatum in orange fruits.
manipulation of the yeast biocontrol agents and the Postharvest Biol Technol 8:191 – 198.
Arras G, De Cicco V, Arru S, and Lima G (1998). Biocontrol by
development of superior antagonist mixtures that led to
yeasts of blue mould of citrus fruits and the mode of action of an
significant increases in the efficacy of BCPD are only the tip isolate of Pichia guilliermondii. J Hortic Sci Biotechnol
of the iceberg in showing what can be accomplished with the 73:413 –418.
postharvest biocontrol system. They are also indicative that, Bassett CL and Janisiewicz WJ (2001). Stability of E. coli/
in the future, there will be additional situations where Pseudomonas shuttle vectors in a biocontrol strain of
biocontrol treatment alone will be adequate for the control of Pseudomonas syringae. Phytopathology 91:S7.
postharvest fruit decays. Greater effort is needed to identify Bull CT, Wadsworth ML, Sorensen KN, Tekemoto JY, Austin RK,
circumstances where currently available biocontrol can be and Smilanick JL (1998). Syringomycin E produced by
used alone. This includes not only replacing a fungicide biological control agents controls green mold on lemons. Biol
treatment but also instances where no fungicides are Control 12:89– 95.
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fruits and vegetables. In: Moline E ed. Postharvest Pathology of
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processing. There are an increasing number of situations UC, Berkeley: Agric Exp Stn. pp 24– 30.
where a fungicide is registered for postharvest use in the Castoria R, De Curtis F, Lima G, Caputo L, Pacifico S, and De Cicco
United States but not in other countries, particularly in V (2001). Aureobasidium pullulans (LS-30) an antagonist of
Europe. This may restrict export markets or increase losses if postharvest pathogens of fruits: study on its modes of action.
major decay control measures are not implemented for fruit Postharvest Biol Technol 22:7 – 17.
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control can be an acceptable treatment. Currently, the against postharvest disease of citrus fruit. Phytoparasitica
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Experience with the commercial products for BCPD such as
Biol Technol 7:51 – 64.
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Strategy for combining heat treatment, calcium infiltration, and
biological control to reduce postharvest decay of ‘Gala’ apples.
HortScience 34:700– 704.
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Janisiewicz WJ, Peterson DL, and Bors R (1994). Control of storage Conventional Methods for the Control of Postharvest Disease
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Phytopathology 92:33 –37. Pusey PL (1991). Antibiosis as mode of action in postharvest
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Korsten L, De Jager ES, De Villiers EE, Lourens A, Kotze JM, and Ragsdale NN and Sisler HD (1994). Social and political implications
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16
Arbuscular Mycorrhizal Fungi in Plant Disease Control

Lisette J.C. Xavier / Susan M. Boyetchko Saskatoon Research Centre, Agriculture and Agri-Food
Canada, Saskatoon, Saskatchewan, Canada

1 INTRODUCTION and microbiological activities occur (Lynch 1990). The

mycorrhizosphere is the region of the rhizosphere that is
Biological control of plant pathogens presents a compelling subjected to modifications following AMF colonization of the
method of increasing plant yields by suppressing or host plant (Linderman 1988). Induced biochemical changes in
destroying pathogens, enhancing the ability of plants to the plant as a result of AMF root colonization is collectively
resist pathogens, and/or protecting plants against pathogens. termed the “mycorrhizosphere effect.” The mycorrhizosphere
Micro-organisms antagonistic to plant pathogens may be effect typically results in a transient or permanent shift in the
derived from the resident microbial community or may be of resident microbial community that may favor the elimination
foreign origin. Although there are concerns towards the or proliferation of pathogens (Edwards et al. 1998; Meyer and
release of an organism of foreign extraction, in general, Linderman 1986; Nemec 1994; Paulitz and Linderman 1989).
biological control presents a myriad of benefits such as being In general, these changes are mediated by modifications in
a component of the environment, resistant to development of host root membrane permeability that subsequently leads to
chemical pesticide resistance, being relatively safe and risk modifications in root exudate composition (Graham et al.
free, and by being compatible with sustainable agriculture. 1981; Ratnayake et al. 1978). Meticulous management of the
Arbuscular mycorrhizal fungi (AMF) form one such group of mycorrhizosphere may serve as an effective, safe, and
organisms that can act as bioprotectors of plants. These environmentally friendly alternative to conventional methods
zygomycetous fungi that form specialized structures such as of plant disease control.
arbuscules and/or vesicles are obligate biotrophs and utilize
host photosynthates for their growth. They are ubiquitous
and co-exist with over 80% of terrestrial plants including 2 EXAMPLES OF AMF-MEDIATED PLANT
agricultural or horticultural crops. Their interactions with DISEASE CONTROL
rhizosphere flora and fauna influence the growth and fitness
of the associated plants (Azcon-Aguilar and Barea 1992; 2.1 Phytopathogenic Fungi
Fitter and Sanders 1992). An incompatible association
between the host plant and the indigenous AMF community Plant pathogenic fungi contribute significantly to crop
can lead to serious losses in crop yields, indicating the damage and yield loss, followed by plant pathogenic bacteria
significance of AMF in crop production. In contrast, a and viruses. The potential of AMF to control various plant
compatible association can result in enhanced plant pathogenic fungi has been clearly demonstrated (Becker et al.
productivity, through enhanced host P nutrition (Ravnskov 1999; Bodker et al. 2002; Boyetchko and Tewari 1996;
and Jakobsen 1995), prevention or control of plant diseases Duchesne et al. 1989; Kapoor et al. 1998; Kasiamdari et al.
caused by soil-borne pathogens (Caron 1989a; St-Arnaud 2002; Kegler and Gottwald 1998; Krishna and Bagyaraj
et al. 1995), and/or enhancement of plant hormonal activity 1983). In contrast, there are reports wherein AMF inoculation
(Frankenberger and Arshad 1995). did not have any effect on disease severity (Guillon et al.
The rhizosphere, a zone of soil loosely surrounding the 2002; Larsen and Bodker 2001; Wyss et al. 1991; Zambolin
roots, is a dynamic environment wherein complex chemical and Schenck 1983). In order for practical and routine use of

183
184 Xavier and Boyetchko

AMF as protectors of plants against plant pathogenic fungi, accompanied by other mechanisms of biocontrol. Using an
AMF performance must be consistent, specific, and effective. in vitro system, Filion et al. (1999) demonstrated that
Specificity of AMF for the control of crop diseases is extracts from the extraradical mycelium of G. intraradices
crucial in order to mitigate any nontarget effects to beneficial reduced the conidial germination of F. oxysporum f. sp.
micro-organisms. However, there are conflicting reports on chrysanthemi. Alternatively, alterations in the chemical
the specificity of AMF. For example, inoculation of micro- equilibrium of the mycorrhizosphere may have resulted in
propagated banana with Glomus intraradices and a Glomus pathogen control. In another study, pea mutants defective for
spp. isolate reduced rhizome necrosis and external disease mycorrhization and nodulation challenged with Aphano-
symptoms caused by Fusarium oxysporum f. sp. cubense, but myces euteiches required a fully established AMF symbiosis
differences between the two AMF isolates were not noted, for protection against the pathogen (Slezack et al. 2000).
indicating that either both AMF species were equally Several researchers have also demonstrated AMF-
effective against the pathogen or that they lacked specificity mediated reduction of root rot disease in cereal crops
(Jaizme-Vega et al. 1998). In contrast, eggplant and cucumber (Boyetchko and Tewari 1988; Grey et al. 1989; Rempel and
seedlings transplanted into soils inoculated with G. versiforme Bernier 1990; Thompson and Wildermuth 1989) and take-all
and subsequently challenged with Verticillium dahliae and disease of wheat (Graham and Menge 1982). Phytophthora
Pseudomonas lacrymans alleviated wilt symptoms caused by spp., which cause diseases in a variety of plants have been
V. dahliae, but not G. mosseae, Glomus spp.-1, or Glomus model systems for AMF-mediated plant disease control
spp.-2, indicating species-specific antagonistic symbiont – (Cordier et al. 1996; Guillemin et al. 1994; Mark and Cassells
pathogen interactions (Li et al. 1997). Pozo et al. (1999) 1999; Norman and Hooker 2000; Pozo et al. 1996; Trotta et al.
demonstrated the expression of two new basic glucanase 1996). Using the AMF species G. intraradices and pathogen
isoforms, a phytoalexin elicitor-releasing factor between F. oxysporum f. sp. lycopersici on tomato, Caron and
G. mosseae and G. intraradices used for the control of co-workers have shown that the growth medium used (Caron
Phytophothora parasitica var. nicotianae. Because of the et al. 1985), the application of P (Caron et al. 1986a), and
potential of AMF as bioprotectors against phytopathogens, pretreatment of the growth medium with AMF (Caron et al.
this is an area that needs further study. 1986c) can influence disease severity. Despite proof of AMF
In order to enhance AMF efficacy, some researchers have potential in controlling plant diseases, few published reports
used an AMF species mixture or a combination of micro- have successfully demonstrated biological control of plant
organisms including AMF that act in concert to eliminate pathogens by AMF in the field (Bodker et al. 2002; Newsham
pathogens. For example, co-inoculation of groundnut with et al. 1995; Torres-Barragan et al. 1996). Newsham et al.
G. fasciculatum, Gigaspora margarita, Acaulospora laevis, (1995) showed that pre-inoculating the annual grass Vulpia
and Sclerocystis dussii eliminated the damaging effects of ciliata var. ambigua with an indigenous Glomus sp. and
Sclerotium rolfsii (Kulkarni et al. 1997). Also, tobacco re-introducing the grass into a natural grass population
inoculated with a mixture containing G. fasciculatum and extended a favorable effect against an indigenous
Trichoderma harzianum effectively controlled damping-off F. oxysporum. Onion pretreated with Glomus sp. Zac-19
caused by Pythium aphanidermatum and black shank disease delayed the development of onion white rot caused by
caused by P. parasitica var. nicotianae (Sreeramulu et al. S. cepivorum by two weeks in the field and protected onion
1998). In some cases, microbial mixtures act synergistically plants for 11 weeks after transplanting in the field and resulted
with pesticides to result in effective control of plant diseases. in a yield increase of 22% (Torres-Barragan et al. 1996). One
A combination of wheat straw, carbendazim, G. fasciculatum, of the first reports on the effect of indigenous AMF on the
and T. viride protected safflower seedlings from the root rot development of introduced A. euteiches infection and disease
pathogen Macrophomina phaseolina, resulting in 100% development on field-grown pea (Bodker et al. 2002) showed
seedling survival (Prashanthi et al. 1997). Sharma et al. that there was no correlation between AMF root colonization
(1997) effectively managed ginger yellows disease caused by and disease incidence or severity, and emphasized the
F. oxysporum f. sp. zingiberi using a combination of importance of field evaluations for authenticating the use of
Gi. margarita, pine needles, and T. harzianum. AMF as biocontrol agents. Although the indigenous AMF
The requirement of a fully established AM symbiosis for community composition was not described, this study
elicitation of bioprotective activity by AMF has been underscores the importance of a richly diverse indigenous
disputed. The invasion of phytopathogenic fungi is said to be AMF community to defend plants from plant pathogens.
prevented by an aggressively root colonizing AMF species, Thus, there appears to be tremendous potential for AMF
indicating that AMF root colonization was satisfactory for control of plant pathogens and the need for more detailed and
control of disease. For example, Feldmann and Boyle (1998) well-planned and executed studies that will address problems
found an inverse correlation between G. etunicatum root of inconsistent and unreliable results.
colonization of begonia cultivars and susceptibility to the
foliar pathogen caused by the powdery mildew fungus 2.2 Plant Pathogenic Bacteria
Erysiphe cichoracearum. However, it was not clear whether
G. etunicatum colonization preceded infection by The AMF interact with functionally diverse bacteria such as
E. cichoracearum or whether pathogen suppression was diazotrophs, biological control agents, and other common
Plant Disease Control Using AMF 185

rhizosphere inhabitants (Nemec 1994) that often result in microscopy revealed that mycorrhizae were not viral vectors
significant alterations to plant growth, yield, and nutrition. because virus particles were absent in the AMF hyphae and
Interactions between mycorrhizal fungi and bacteria may around arbuscules, suggesting that AMF did not interact with
have detrimental (Filion et al. 1999; Shalaby and Hanna 1998; viruses (Jabaji-Hare and Stobbs 1984). Thus, potential for the
2001) or beneficial effects (Edwards et al. 1998; Gryndler and biocontrol of plant pathogenic viruses using mycorrhizae does
Hrselova 1998; Li et al. 1997; Ravnskov and Jakobsen 1999), not appear to be promising. However, it may be worthwhile to
or have no effect at all on the plant pathogenic bacterium investigate the role of viruses in the reduction of mycorrhizal
(Otto and Winkler 1995). colonization and related host plant effects.
Glomus mosseae prevented the infection of soybean plants
by P. syringae (Shalaby and Hanna 1998), by suppressing the
population density of the pathogen in soybean rhizosphere.
Li et al. (1997) also found that G. macrocarpum reduced the 3 MODES OF MYCORRHIZAE-MEDIATED
infection caused by P. lacrymans in eggplant and cucumber, DISEASE CONTROL
although no positive growth or yield effect was noted,
indicating tolerance to the pathogen as a possible mode of 3.1 Host Nutritional Effects
action. Inoculation of mulberry with G. fasciculatum or 3.1.1 Improved Plant Nutrition
G. mosseae in combination with 60–90 kg of P per hectare
per year reduced the incidence of bacterial blight caused by Mycorrhizal plants are generally able to tolerate pathogens
P. syringae pv. mori (Sharma 1995). Inoculation of grape- and compensate for root damage and photosynthate drain by
vines with AMF reduced the number of fluorescent pathogens (Azcon-Aguilar and Barea 1992; Declerck et al.
pseudomonads on the rhizoplane thereby reducing the 2002), because AMF enhance host nutrition and overall plant
incidence of grapevine replant disease (Waschkies et al. growth. For example, Declerck et al. (2002) found that
1994). Similarly, a reduction in the colonization of apple G. proliferum and a Glomus sp. isolate not only stimulated
seedling rootlets by actinomycetes causing replant disease growth and increased shoot P content of banana in the
was reported, while a proportionate increase in root presence and absence of the root rot fungus Cylindrocladium
colonization by AMF was noted (Otto and Winkler 1995). spathiphylli, but also reduced root damage by the pathogen,
indicating direct interactions between the AMF and the
pathogen. In contrast, some reports indicate that AMF are
2.3 Phytopathogenic Viruses capable of biological control activity (Boyetchko and Tewari
1988; Grey et al. 1989; Rempel and Bernier 1990). It is
Viruses remain the least studied amongst all the plant disease- believed that AMF interact equally with host plants, but in
causing target organisms listed for mycorrhizae-mediated fact AMF prefer one host or host cultivar over another, as
biocontrol. The general response of mycorrhizal plants to the shown by Grey et al. (1989) who reported that mycorrhizal
presence of viral pathogens is as follows: (a) mycorrhizal barley cultivar WI2291 not only exhibited greater control of
plants apparently enhanced the rate of multiplication of the barley common root rot pathogen Bipolaris sorokiniana
viruses in some plants (Daft and Okusanya 1973; Nemec and than a mycorrhizal cultivar Harmal, but also produced
Myhre 1984), (b) more leaf lesions were found on significantly higher yields. On the other hand, biological
mycorrhizal plants than on nonmycorrhizal plants control activity is dependent on the AMF species as
(Schönbeck 1978; Schönbeck and Dehne 1979), and (c) the demonstrated for common root rot of barley by Boyetchko
number of AMF spores in the rhizosphere was reduced and Tewari (1992). There are suggestions that root
considerably (Jayaraman et al. 1995; Nemec and Myhre colonization by natural AMF communities occurring in field
1984). Enhanced viral multiplication and activity in soils has an inverse relationship with B. sorokiniana infection,
mycorrhizal plants is speculated to be attributed to higher P indicating not only a direct interaction between the AMF and
levels compared to nonmycorrhizal plants. A similar effect the pathogen, but also an AMF-mediated improvement in host
was noted in nonmycorrhizal plants fertilized with P (Daft nutrition (Thompson and Wildermuth 1989). In contrast, there
and Okasanya 1973; Shaul et al. 1999). Some workers found are also reports suggesting a lack of interaction between AMF
that host plants were more susceptible to AMF colonization and B. sorokiniana under field conditions (Wani et al. 1991).
following infection by a virus. For example, Schönbeck and Interaction between naturally occurring AMF and pathogens
Spengler (1979) reported that following the inoculation of or the lack thereof in the field likely depends on the
mycorrhizal and nonmycorrhizal tobacco (Nicotiana distribution of the organisms particularly under the different
glutinosa L.) with tobacco mosaic virus (TMV), mycorrhizal crop rotations. Significant reductions in disease severity as a
plants exhibited higher levels of AMF colonization. In result of AMF colonization and enhanced P uptake followed
contrast, mung bean yellow mosaic bigeminivirus reduced by modifications in root exudation patterns has also been
the AMF colonization and yield of mycorrhizal plants reported for take-all disease of wheat (Graham and Menge
(Jayaraman et al. 1995), while lack of response to viral 1982). Improvement in host P nutrition is one of the earliest
infection by a mycorrhizal host was also demonstrated proposed mechanisms of AMF-mediated pathogen or disease
(Takahashi et al. 1994). Early studies using electron tolerance that is still very pertinent.
186 Xavier and Boyetchko

3.1.2 Tolerance to Pathogen collectively benefit host plants by creating favorable

conditions for the proliferation of microflora antagonistic to
Arbuscular fungi are known to enhance plant tolerance to pathogens such as Phytophthora and Pythium spp. as shown
pathogens without excessive yield losses, and in some cases, for eucalyptus seedlings by Malajczuk and McComb (1979).
enhance pathogen inoculum density. This compensation is Unfavorable conditions induced by AMF colonization
apparently related to enhanced photosynthetic capacity resulted in qualitative changes in the mycorrhizosphere that
(Abdalla and Abdel-Fattah 2000; Heike et al. 2001; Karajeh prevented P. cinnamoni sporangial induction in tomato plants
and Al-Raddad 1999) and a delay in senescence caused by the (Meyer and Linderman 1986). Proliferation of G. mosseae
pathogen, which cancels the positive relationship between inside grapevine roots was associated with a significant
disease severity and yield loss (Heike et al. 2001). For reduction in replant disease-causing fluorescent pseudomonad
example, soybean plants grown in the soil infested with inoculum in soil (Waschkies et al. 1994). Promoting AMF
M. phaseolina, Rhizoctonia solani, or F. solani exhibited diversity that will ensure that at least a component of the AMF
lower shoot and root weight and plant height compared to community may be active against pathogens can further
control plants in soil not infested with the pathogens or with enhance the benefits of this mechanism.
G. mosseae (Zambolin and Schenck 1983). The incidence of
infection by the pathogens was not affected by G. mosseae
colonization but the mycorrhizal plants were able to tolerate 3.2 Competition
infection of pathogens better than nonmycorrhizal plants. The
efficacy and efficiency of AMF in promoting plant growth The AMF spores in soil are not known to compete for
enables mycorrhizal plants to tolerate pathogens, as nutrients as spore reserves are utilized for survival until root
demonstrated by Hwang (1988) using alfalfa challenged contact is achieved. Following root entry, competition can
with P. paroecandrum and Karajeh and Al-Raddad (1999) occur for infection sites, host photosynthates, and root space
using olive seedlings. It is unclear whether mycorrhizal (Smith and Read 1997). Competition between AMF and
alfalfa tolerated P. paroecandrum or if other additional pathogens can be used for physical exclusion of pathogen
mechanisms were involved. Despite the presence of a (Davis and Menge 1980; Hussey and Roncadori 1982; Smith
pathogen benefits of AMF to susceptible hosts can occur until 1988), if the host is preinoculated with AMF. Simultaneous
a pathogen inoculum threshold level, beyond which no colonization of AMF and the pathogen may not provide a
AMF-mediated benefits can be realized (Stewart and Pfleger competitive edge for AMF for inoculum build-up (Daniels
1977). On the other hand, high tissue P levels in mycorrhizal and Menge 1980) because of its relatively slow growth rate
plants may not only improve vigor and fitness of the plant but compared with the pathogen. In contrast, some others have
also modify pathogen dynamics in the mycorrhizosphere by noted that competition may not occur between AMF and
modifying root exudation (Davis and Menge 1980; 1981; other organisms (Sempavalan et al. 1995). Competition, as a
Kaye et al. 1984). mechanism of suppressing pathogens by AMF did not receive
Tolerance of the plant to a pathogen can vary depending on much consideration, because in some cases pathogens were
the AMF species and their ability for enhancing host nutrition suppressed even in noncolonized root portions that was later
and growth, although some ineffective AMF species reduce described as induced resistance by AMF (Pozo et al. 1999). In
pathogen entry by triggering a defense reaction in plants addition, inconsistencies with regard to prerequisites and
(Davis and Menge 1981). For example, Matsubara et al. AMF effects on pathogens have contributed to a lack of
(2000) noted that there were significant differences in the interest.
ability of Gi. margarita, G. fasciculatum, G. mosseae, and
Glomus sp. R10 to not only enhance asparagus growth but
also in their ability to tolerate the severity of violet root rot 3.3 Physiological and Biochemical Alterations of
caused by Helicobasidium mompa. Asparagus seedlings the Host
inoculated with Glomus sp. R10 had the lowest incidence of
violet root rot. This important fact highlights the care that Following AMF colonization, host root tissue P levels are
needs to be exercised in the selection of AMF species for typically enhanced which modify the phospholipid compo-
biological control of diseases. sition and therefore the root membrane permeability resulting
in a reduction in the leakage of net amount of sugars,
3.1.3 Qualitative and Quantitative Alterations in carboxylic acids, and aminoacids into the rhizosphere
Pathogen Biomass (Graham and Menge 1982; Ratnayake et al. 1978; Schwab
et al. 1983). These alterations arrest the chemotactic effect of
Modifications in root exudate composition following changes pathogens to plant roots and discourage pathogen entry. Prior
in host root membrane permeability as a result of AMF inoculation of maize plants with G. mosseae decreased the
colonization (Graham et al. 1981) can enforce changes in the number of Alternaria alternata colony forming units, but
rhizosphere microbial equilibrium (Brejda et al. 1998; when both organisms were inoculated at the same time, there
Edwards et al. 1998; Kaye et al. 1984; Meyer and Linderman was no effect on pathogen inoculum density in soil
1986). Changes in the rhizosphere microfloral community can (McAllister et al. 1996). It is possible that the G. mosseae
Plant Disease Control Using AMF 187

symbiont altered membrane permeability of the host roots, their conidia. This appears to be a promising field that can be
thereby reducing the quality and quantity of substances used for the effective control of plant diseases.
exuded by the roots (Graham et al. 1981), restricting pathogen
propagule germination, indicating that the timing of 3.3.2 Phytoalexins and Phytoanticipins
inoculation can enhance biocontrol activity.
Phytoalexins are produced in response to microbial infection
3.3.1 Systemic-induced Resistance (Paxton 1981), whereas phytoanticipins are stored in plant
cells in anticipation of or prior to pathogen attack (VanEtten
Systemic-induced resistance (SIR) is typically the sustained et al. 1995). The level of phytoalexins elicited by pathogens
induction of resistance or tolerance to disease in plants by has been shown to be much higher than those elicited by
previously inoculating with a pathogen, exposing to an symbiotic organisms (Wyss et al. 1991). The function of an
environmental influence or treating with a chemical, which isoflavonoid molecule as a phytoalexin or phytoanticipin can
may or may not have antimicrobial activity (Handelsman and be predicted based on the cellular location of the molecule
Stabb 1996; Kuc 1995). Researchers have suggested that (Stafford 1992).
AMF-inoculated plants may employ SIR as a mechanism of An increase in the level of total soluble plant phenolics
biocontrol (Benhamou et al. 1994; Brendan et al. 1996; Trotta such as isoflavonoids or flavonoids, lignin, syringic, ferulic or
et al. 1996). The SIR phenomenon in mycorrhizal plants is coumaric acids, etc. have been reported as synthesis of
demonstrated as localized and systemic resistance to the phytoalexins following AMF colonization of roots (Harrison
pathogen (Cordier et al. 1998). An increase in the lignin and Dixon 1993; Morandi 1989; 1996). Some flavonoids that
deposition in plant cell walls following AMF colonization can are not true phytoalexins may also respond to AMF
restrict the spread of pathogens (Dehne and Schönbeck 1979). colonization of roots (Harrison and Dixon 1993; Morandi
Using a split root system, Cordier et al. (1998) demonstrated and Le-Quere 1991; Volpin et al. 1995).
that G. mosseae protected tomato plants against P. parasitica The production of phytoalexins as a result of pathogen
by reducing pathogen development and spread by increasing invasion in mycorrhizal plants has been explored. Tomato
cell wall appositions containing callose close to the plants inoculated with G. mosseae posed greater resistance to
intercellular hyphae and accumulation of phenolic compounds the pathogen F. oxysporum and were found to have increased
and plant cell defense responses. Root damage was observed phenylalanine and b-glucosidase activity and total phenol
in portions of mycorrhizal root systems not containing content in their roots compared to plants inoculated with
mycorrhizal structures. The SIR reaction to the pathogen in either organism alone (Dehne and Schonbeck 1979).
mycorrhizal plants was further illustrated by host wall Sundaresan et al. (1993) reported that a purified ethanol
thickenings containing nonesterified pectins and pathogenesis fraction of mycorrhizal cowpea root extract inhibited
related (PR)-1 protein in the nonmycorrhizal areas of the F. oxysporum in vitro. However, the isoflavonoid was not
roots. They also noted that the PR-1 protein was found only in identified. Production of phytoalexins in mycorrhizal plants
the pathogen-invaded tissues of pea. These responses were appears to be independent of the effect of fertilizer addition
observed in the nonmycorrhizal pathogen-infected root tissues (Caron et al. 1986b). In general, in the presence or absence of
that ultimately led to cell death. Bodker et al. (1998) reported pathogens in plant roots, phytoalexins are induced in
that the observed increased resistance to A. euteiches in mycorrhizal plants that neutralize the negative effects of
G. intraradices-inoculated pea was probably due to an pathogens.
“induced systemic factor,” induced by G. intraradices. The
AMF-mediated SIR phenomenon is speculated to play a role 3.3.3 Hydrolases
in the protection of potatoes against post-harvest suppression
of potato dry rot, wherein dry rot in G. intraradix-inoculated Differential expression of defense-related genes in
potato was reduced by up to 90% compared to uninoculated mycorrhizal plants has been the recent focus of AMF-
control (Brendan et al. 1996). This finding suggests that the mediated biocontrol (Blee and Anderson 1996; Dumas-
benefits of AMF inoculation for disease control surpasses the Gaudot et al. 1996; Lambais and Mehdy 1995; Pozo et al.
growth and reproduction phase of the host and extends to the 2002). Researchers have shown that AMF enter into host
storage phase of the product. The area of SIR response in (e.g., tomato) roots and induce a local, weak, and transient
mycorrhizal plants is still developing and several aspects activation of the host defence mechanism against pathogens
including whether all AMF species can equally elicit a SIR such as P. parasitica, which involves the induction of
response in the host are not known. hydrolytic enzymes such as chitinase, chitosanase,
Some researchers have examined the role of PR proteins in b-glucanase, and superoxide dismutase (Pozo et al. 2002).
the disease control process mediated by AMF (Liu et al. In addition, portions of the mycorrhizal root system not
1995). Enhanced levels of 10 different PR proteins were containing mycorrhizal structures appear to have alterations
detected in cotton plants inoculated with G. mosseae, in the constitutive isoforms of the enzymes indicating
G. versiforme, or Scl. sinuosa challenged with V. dahliae systemic changes following AMF colonization (Pozo et al.
compared with plants not challenged by the pathogen. The PR 2002). A high positive correlation between the level of
proteins retarded the hyphal growth of V. dahliae and killed glucanase activity in host tissues and pathogen resistance has
188 Xavier and Boyetchko

been established (Graham and Graham 1991). Further studies (Graham et al. 1981; Menge et al. 1978; Ratnayake et al.
examining the role of these glucanases will help in the 1978). Colonization of host roots by AMF is a crucial
development of strategies for control of pathogens using component in the AMF-mediated SIR response of host plants
AMF. to plant pathogens as the expression of an SIR response
requires a threshold level AMF presence within host roots
(Cordier et al. 1996; 1998). The effect of phosphorus on AMF
3.4 Antibiosis efficacy may be direct, wherein inoptimum P levels impair
AMF activity and therefore its ability to effectively control
Reports on the production of antimicrobial substances by pathogens. In contrast, soil disturbance has an indirect effect
AMF are not common. However, recently, it was shown that where AMF efficacy may be altered by a delay in mycelial
antimicrobial substances (unidentified) produced by the network initiation and diversion of carbon for the synthesis or
extraradical mycelium of the AMF species G. intraradices repair of the external mycelial network and not nutrient
reduced conidial germination of F. oxysporum f. sp. uptake. Zak et al. (1982) suggested that the ability of AMF to
chrysanthemi, which was independent of changes in pH effectively re-establish their mycorrhizal association after
(Filion et al. 1999). Budi et al. (1999) isolated a Paenibacillus disturbance might partially determine their success in a
sp. strain from the mycorrhizosphere of Sorghum bicolor disturbed site. It is not known to what extent soil disturbance
plants inoculated with G. mosseae that exhibited significant affects the biocontrol activities of AMF, but it disrupts the
antagonism against P. parasitica. Regardless of the source of external mycelial network resulting in a severe reduction in
these biocontrol activities, it is important to realize and utilize mycorrhizal efficacy (Evans and Miller 1990; Stahl et al.
the significance of AMF in plant disease control. Additional 1988).
research in this are may prove to be fruitful in the control of A richly diverse AMF community ensures qualitatively
pathogenic bacteria and fungi. and quantitatively the presence of AMF species desired for
specific activities such as biological control of plant
pathogens. However, choice of host genotype and rotation
4 CHALLENGES AND STRATEGIES TO (Bever et al. 1996; Johnson et al. 1992; Talukdar and Germida
ENHANCING ARBUSCULAR FUNGAL 1993), levels of fertilizer application (Baltruschat and Dehne
EFFICACY IN DISEASE CONTROL 1982; Jasper et al. 1979; McGonigle and Miller 1993; 1996;
Vivekanandan and Fixen 1991), tillage (Evans and Miller
4.1 Challenges 1990; McGonigle and Miller 1993; Vivekanandan and Fixen
1991), pesticide application (Manjunath and Bagyaraj 1984;
Although the efficacy of an AMF species on plant pathogens Schreiner and Bethlenfalvay 1997), and the effect of
has been assessed under controlled environments and usually associated micro-organisms (Andrade et al. 1995; Xavier
in the absence of other AMF or other organisms (Budi et al. and Germida 2002) are some critical factors that can
1999; Kasuya et al. 1996; Li et al. 1997), research indicates indirectly alter AMF diversity in soils. For example,
that the potential of AMF for control of plant pathogens is continuous cropping selectively enhances the proliferation
high. Limitations in AMF research pertaining to biological of parasitic AMF, which are relatively fast growing compared
control of plant diseases under field conditions are two fold, to beneficial AMF, leading to alterations in mycorrhizal
(a) production of large quantities of AMF inoculum is not biodiversity in the rhizosphere (Johnson et al. 1992).
feasible because of the obligate biotrophicity status of AMF, Similarly, one particular AMF host selects from an
and (b) negative interactions between the introduced AMF indigenous AMF pool resulting in the selective enrichment
and the indigenous AMF and microbial community after of certain AMF species over others (Xavier 1999).
introduction into field. Challenges posed by interactions Studies assessing the significance of AMF biodiversity in
between AMF and indigenous microbial community and soil AMF-mediated biocontrol are rare but critical. Given the
and environmental conditions often determine the success of importance of AMF to plant health and the complexity of the
AMF inoculation in disease control under field conditions. An various microbial interactions, all the relevant factors have to
appreciation of factors that influence AMF efficacy as be considered before AMF selected as biocontrol agents can
biological control agents can further enhance their survival, effectively function in the field.
competitiveness and efficacy. For example, it is known that
intraradical proliferation of AMF within roots is a host-
regulated event (Bever et al. 1996). Therefore, a highly
mycotrophic host or host cultivar may be more favorable for 4.2 Strategies
AMF proliferation and reproduction than one that is not
highly mycotrophic (Feldmann and Boyle 1998; Xavier Biological control of plant diseases by AMF under field
1999). In addition, nonconducive soil-environment combi- conditions is the effect of interactions between AMF and
nations such as high soil P levels and soil disturbance can various groups of organisms in the rhizosphere. Sikora (1997)
affect AMF colonization (Bever et al. 1996; Gazey et al. proposed “biological system management,” a holistic
1992; Stahl et al. 1988; Stutz and Morton 1996) and efficacy approach for improving plant root systems that adopts
Plant Disease Control Using AMF 189

specific cultural practices that promote plant defense (Reena and Bagyaraj 1990a,b; Talukdar and Germida 1994;
mechanisms such as tolerance and/or resistance to pathogens, Vinayak and Bagyaraj 1990). Screening procedures must
and the use of organisms that are antagonistic towards include selection pressure similar to that in which the AMF
pathogens and that target sensitive developmental stages of will be applied.
pathogens. This approach offers a viable alternative to Research shows that plant pathogens can be controlled
integrated pest management and inundative approaches such not only by the use of biocontrol agents, but also by the
as the application of high levels of microbial inocula to the induction of resistance responses in plants. Inoculating
nonrhizosphere soil for biological control purposes, and plants with AMF has been shown to induce resistance in
underscores the significance of mycorrhizae in root health plants. Such plant immunizations are a viable approach for
(Sikora 1997). transplant crops because of the ease of AMF inoculation,
while more innovative methods are required with direct-
sown crops.
4.2.1 Enhanced AMF Biodiversity
4.2.4 Superior Application Technology
A diverse AMF community contains a mycorrhizal
assemblage and species abundance that naturally aid the Research shows that “priming” plants against pathogens using
host to endure adverse conditions to ultimately enhance plant selective AMF inocula (or plant immunization) helps protect
growth. Research shows that inclusion of host crops (Bever plants by inducing a SIR response (Cordier et al. 1998). The
et al. 1996; Johnson et al. 1992) and/or cultivars that exhibit inoculum may be applied to seeds, transplanted crops, or
high mycorrhizal responsiveness can significantly improve plantlets produced through tissue culture before being
AMF functioning (Boyetchko and Tewari 1995; Xavier and transplanted into pathogen-infested fields. Application of
Germida 1998). Therefore, rotation of crops that are the agent prior to transplanting eliminates the need for
dependent on mycorrhizae will ensure early AMF root complex formulations and application techniques, guarantees
colonization and high sporulation of even the most sensitive “targeted placement,” and greater biocontrol activity, reduces
AMF species in soil. Minimal disturbance to the soil also costs associated with application and has a minimal impact on
guarantees early contact between an emerging seedling and the environment (Boyetchko 1996; Glass 1993). Inoculum
the AMF hyphal network in soil that distributes nutrients and may include one or more AMF species or other organisms
initiate early colonization of AMF propagules in soil. such as bacteria or fungi that exhibit sustained and
Excessive fertilizer and pesticide use can alter plant chemistry coordinated biocontrol activity. The application of a multiple
and cause changes in AMF assemblage and abundance, agent mixture may concurrently confer control for more than
resulting in a poor AMF community that does not benefit the one plant disease by more than one mechanism rather than
host (Gazey et al. 1992; Jasper et al. 1979; Johnson et al. single inoculants targeted for control of only one plant disease
1992). Caution in the choice of cultural practices that or pathogen. Application of two or more biocontrol agents
potentially alter AMF diversity would prove to be fruitful. targeting different life stages of a pathogen may also be more
effective than sequential application of the biocontrol agents.
In some instances, augmenting soil with organic amendments
4.2.2 Improved Understanding of Microbial (AMF) such as forest humus and charcoal compost has enabled a
Ecology and Ecosystem Functioning significant reduction in disease severity (Kobayashi 1993;
Wei et al. 1987).
It is common knowledge that AMF functioning in natural
ecosystems can be altered by various factors including
interactions with other organisms. However, specifics on the
5 CONCLUSIONS
topic are lacking. Knowledge generated from studies
addressing AMF efficacy in a typical rhizosphere community,
under moisture and salinity stress and soil disturbance, in soils Literature presents a wealth of evidence to indicate
containing extreme indigenous AMF levels, and in the potential for AMF-mediated control of plant diseases.
presence of antagonizers is required for the development of Although there are challenges in the form of noncultur-
effective AMF biocontrol agents. ability of AMF and therefore mass multiplication for
agricultural crops, there is promise for nondirect sown
crops, which is currently undervalued and underexploited.
4.2.3 Selection of Effective AMF The AMF, by increasing crop productivity using existing
resources, avoiding resistance development to chemicals,
Bagyaraj (1984) suggested that AMF species selection for a maintaining pollution and risk-free disease control, and
desired activity must be based on their ability for survival, conforming to sustainable agricultural practices, offers more
aggressive colonization of host roots, and efficacy. Use of than mere plant disease control. In the future, mycorrhizo-
AMF species originally isolated from test host roots has sphere management must become one of the viable and
proven advantageous for many plant species including ecosystem friendly solutions to managing plant diseases and
agricultural crops, forest tree species, and orchard crops reducing pathogen inoculum.
190 Xavier and Boyetchko

ACKNOWLEDGEMENTS Boyetchko SM and Tewari JP (1995). Susceptibility of barley

cultivars to vesicular-arbuscular mycorrhizal fungi. Can J Plant
Sci 75:269– 275.
The financial support provided by Western Grains Research Boyetchko SM and Tewari JP (1996). Use of VA mycorrhizal fungi
Foundation, Westco Fertilizers, and Agriculture and in soil-borne disease management. In: Utkhede RS, Gupta VK
Agri-Food Canada Matching Investment Initiative is eds. Management of Soil Borne Diseases. New Delhi: Kalyani
acknowledged. Publishers. pp 146 – 163.
Brejda JJ, Moser LE, and Vogel KP (1998). Evaluation of
switchgrass rhizosphere microflora for enhancing seedling
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17
Commercialization of Arbuscular Mycorrhizal Biofertilizer

Pragati Tiwari / Alok Adholeya The Energy and Resources Institute, New Delhi, India

Anil Prakash Barkatullah University, Bhopal, Madhya Pradesh, India

1 INTRODUCTION technological application implies a direct relevance to

commercialization for mass multiplication of the invented
Human society today demands the production of high quality bio-product so as to reach the masses. It is encouraging that
food in a most sustainable way causing least damage possible both, government and industry are becoming more responsive
to the environment. Expected benefits include increase in the to natural approaches to growing environmental problems.
efficiency of crop production, reduction in agrochemical Individuals and organizations worldwide are coming to
inputs, and an evaluation of the safety and bioethical aspects realize that excessive use of chemical fertilizers can
in relation to public acceptability. High productivity negatively impact water quality and the environment as a
agriculture exacts a high cost in terms of energy and the whole. The joint effort, if addressed properly, may lead to a
environment. Typically, fertilizer and pesticides are used at healthy environment for future generations.
high levels in the intensive production of plants. More than Biofertilizers include environment-friendly fertilizers with
150 years of over cultivation with synthetic fertilizers and organisms such as: (a) Rhizobium strains for legumes, (b)
pesticides has left our soils depleted of the natural biota Azotobacter strains for nonlegume crops, (c) VAM strains for
needed to facilitate the growth of crops. A less costly and use in agriculture, horticulture, and plantation crops, and (d)
nondestructive means of achieving high productivity rests on phosphorus solubilizing bacteria (PSB) –phosphorus dis-
a establishment of the viable low input farming system. solving bacteria strains. Most common among these are
However, to implement such a plan, we must develop plant symbiotic mycorrhiza, Rhizobium members and cyano-
systems that can efficiently scavenge and utilize soil nutrients phyceae group, which deliver plant nutrition, disease
present at low levels. The judicious use of nature’s own resistance, and tolerance to adverse soil and climatic
biofertilizers by their biotechnological applications appears to conditions. Biofertilizers also known as microbial inoculants
be a suitable answer to this problem. Role of biotechnology in may be involved in symbiotic and associative microbial
sustainable agriculture can offer a great help towards modern activities with higher plants. They are natural mini-fertilizer
agriculture improvement. In the present review, we have factories that are an economical and safer source of plant
discussed the role of biotechnology, potential biofertilizers nutrition and can increase agricultural production and
with special reference to mycorrhizal biofertilizers, their so improve soil fertility. They have great potential as a
far reported synergies, mycorrhizal potential and methodo- supplementary, renewable, and environment-friendly source
logies for its mass multiplication, different constraints in its of plant nutrients and are important components of any
commercialization and its future role in achieving sustainable integrated plant nutrient system. Research on biofertilizers
agriculture. has focussed on biological N2 fixation, plant growth
Biotechnology has been defined as the integrated use of promoting bacteria (PGPR’ B) and phosphorus solubilizing
biological, physical, and engineering sciences to achieve microbes (Hegde et al. 1999). Research and development
technological application of biological systems. The goal of activities involved in this demanding but unexplored field

195
196 Tiwari et al.

include microbiological, biochemical, serological, molecular, above and below the ground, increases. Decomposed
and ultrastructural techniques, followed by extensive field biomass, when recycled, improves the soil fertility manifold
trials for crop testing before releasing them for agricultural and this is how mycorrhiza helps in restoring ecosystems.
use. During the last decade the phenomenal increase in the Above-ground plant development is influenced by below-
production and promotion of biofertilizers in agriculture has ground microbial activity. In the presence of mycorrhizal
been the result of special attention given by the government fungi, other micro-organisms such as PSB, many free-living
and interest by entrepreneurs in setting up biofertilizer nitrogen-fixing organisms and Rhizobium work more
production facilities. Farmers have also realized the benefits efficiently, improving soil fertility and plant growth.
of biofertilizers. For ensuring the rapid growth of biofertilizer Mycorrhizas remain in the soil and form active links with
usage, constant research support is critical, as it will provide growing plants and mutually benefit each other.
the latest information on improvements of their production Papers advocating the valuable potential of mycorrhizal
technology, applications under different agro-climatic con- inoculations in plant establishments have been published
ditions and help in standardizing handling and storage norms. since the 1960s but comprehensive information on their
The role of biofertilizers like arbuscular mycorrhizal (AM) practical exploitation by multiple field trials has not been
fungi in the growth and multiplication of crop plant can prove presented so far (Findlay and Kendle 2001). Immense
to be the most effective alternative to fertilizers for enhancing potential of mycorrhiza has not been so far exploited due to its
growth and biomass production. Its application has additional uncultivable nature unlike other biofertilizers. Mycorrhizas
benefits like improved vigor and nutrient uptake, disease are conventionally propagated using pot-based methods with
resistance, and drought tolerance. The economic surplus is host trap plants. The disadvantage of this mode is the low
used to assess the impact on the overall economic growth and recovery of mycorrhizal propagules, contamination by
its contribution to economic efficiency and environmental saprobes, pathogens and other mycorrhizal fungi because of
security. improper management techniques and long gaps duration
between setup and harvest. Several alternatives to this mode
2 MYCORRHIZAL ASSOCIATIONS have been designed, but in all current methodologies of
cultivating AM fungi, host plant is indispensable. Many
variants of these methods have been proposed by various
Mycorrhizal associations include many taxas of fungi
workers to culture glomalean endomycorrhizal fungi, with a
belonging to members of Zygomycetes, Ascomycetes and
bewildering array of claims and counterclaims. These will be
Basidiomycetes and Deutromycetes. A characteristic feature
described in detail in the present review. All of these involve a
of these fungi is that they are generally widespread in soils,
plant host, either intact or as root explants. Methodological
exhibit a strong biotrophic dependence on their host plants,
differences focus mainly on differences in the cultural
and are rarely free living saprophytes. Different types of
environment, the most dramatic being the interface between
mycorrhiza are classified into seven different categories on
the basis of the extent of root penetration. Among them fungus, plant root, and external matrix. The various modes
arbuscular mycorrhizas are the most common and have include pot base techniques, hydroponic films (Elmes et al.
gained tremendous importance in present day agriculture. The 1984) or in aeroponic mist chambers (Hung and Sylvia 1988)
AM symbiosis is the association between fungi of the order and the recent in vitro root organ culture (ROC) system
Glomales (Zygomycetes) and the roots of terrestrial plants (Becard and Fortin 1988).
(Harley and Smith 1983). Conservative estimates predict that
this ancient symbiosis, dating back to the early Devonian age
(398 million years ago), occurs in approximately 90% of the
2.1 Pot-Based Techniques
Earth’s land plants (Remy et al. 1994). The AM symbiosis is
increasingly gaining recognition as an important integral
component of natural ecosystems throughout the world. The The traditional and most widely used approach has been to
AM fungus –plant association is a mutually beneficial event: grow fungus with a host plant in a solid growth medium
the plant supplies the fungus with carbon (from its fixed consisting of one or a combination of the solid growth media
photosynthates) while the fungal symbiont assists the plant in such as soil, sand, peat, vermiculite, perlite, clay, or various
phosphate uptake and also converts some unavailable mineral types of composted barks. The mycorrhizal inoculum has not
nutrients to available forms for the plant. This bidirectional been conveniently mass-produced by traditional sand-based
exchange of nutrients takes place through extensively pot culture techniques and different micro-organisms
branched haustoria, commonly called arbuscules. In addition frequently contaminate it. In addition, the volume and weight
to the nutrient uptake, mycorrhizal fungi improve the of the inoculum produced by solid growth cultures was
performance also of other beneficial microbes, help in sometimes too large and bulky to carry and utilize (Wang and
resisting root pathogens, and increase the tolerance to Tschen 1994). Alternative particle size distributions of
extremes of environmental and biological stress. With substrates vary the inoculum production, the ideal substrate
increased nutritional consumption and a higher uptake of proposed for optimum production is proposed to be low in
desirable nutrients due to mycorrhization, the biomass, both nutrients and carbon (Gaur and Adholeya 2000).
Commercialization of AM Biofertilizer 197

2.2 Aeroponic Culture Techniques Isolation and inoculum production of ectomycorrhiza

(EM) and AM fungi present very different problems. Many
It is a soil-less plant culture system in which nutrient solutions EM fungi can be cultured on artificial media. Therefore,
are intermittently or continuously misted onto plant roots. isolates of EM can be obtained by placing surface-disinfected
This system allows efficient production of AM fungi, free of a portions of sporocarps or mycorrhizal short roots on growth
physical substrate. The colonized root material can be medium. The resulting fungal biomass can be used directly as
sheared, resulting in inocula with very high propagule inoculum but, for ease of use, inoculum often consists of the
densities. Furthermore, large quantities of spores can be fungal material mixed with a carrier or bulking material such
obtained from the culture system. Aeroponic culture has as peat. They can now be produced in a fermentor and by
worked well for several species of Glomus. It typically takes entrapping the mycelium in alginate beads (Le Tacon et al.
12– 15 weeks to obtain an inoculum. There is a 3-week 1985; Mauperin et al. 1987). Obtaining isolates of AM fungi
“inoculation phase,” followed by 9 weeks of aeroponic is more difficult because they will not grow apart from their
culture for colonized roots or 12 weeks for spore formation. hosts. Spores can be sieved from soil, surface disinfected, and
This also has many disadvantages, because the system is also used to initiate “pot cultures” on a susceptible host plant in
open to other undesirable microbes, which may harbor and sterile soil or an artificial plant-growth medium. Inoculum is
propagate along side. Also, the assembly is huge and requires typically produced in scaled-up pot cultures. Alternatively,
a lot of space; and regular monitoring of the nutrient solution hydroponic or aeroponic culture systems are possible; a
and its flow is required. benefit of these systems is that plants can be grown without a
supporting substrate, allowing colonized roots to be sheared
into an inoculum of high propagule number.

2.3 Root Organ Culture Technique

The ROC system is the most attractive cultivation 3 BENEFITS OF MYCORRHIZAL

methodology for research; it uses root-inducing transfer- INOCULATION
DNA-transformed roots of a host plant to develop the
symbiosis on a specific medium in vitro (Becard and Fortin The practical application of mycorrhiza in agriculture is
1988). The pathogenic condition known as “hairy root” relatively new, though its importance has been evident for
occurs due to the transfer of root inducing Ri-plasmid from some 400 million years. The unique advantage of mycorrhizal
the bacterium Agrobacterium rhizogenes (Schenck 1992). organisms is that they not only survive in the most stressful
These techniques, though challenging have proved useful environments but also make the plant to do so. The role of
additions to our knowledge for various aspects of the AM mycorrhiza in land reclamation is most recognized these days.
fungal-host symbiosis (Douds 1997). The problem of Application of mycorrhizal biofertilizer provides a most
producing inoculum in bulk is addressed by the ROC of desirable solution to many such environmental problems.
AM isolates, which provides pure, viable, contamination-free These phosphate-solubilizing biofertilizers are suggested as
inoculum using less space and thus has an edge over the an alternative or supplements to chemical fertilizers. Some of
conventional mode of pot-culture multiplication. Pioneering the benefits offered by mycorrhizal fungi to plants and general
work on in vitro cultures was initiated in the early 1960s soil health improvement are listed below. However, these are
(Chabot et al. 1992). The use of Ri T-DNA transformed roots not discussed in detail in the present review: (a) Alleviation of
of Daucus carrota as host by A. rhizogenes has permitted an nutrient stress. Under deficiency conditions, mycorrhizal
increase in spore production of Glomus mosseae (Mugnier fungi can increase nutrient uptake. They facilitate the uptake
and Mosse 1987). Only few fungal species have being of nutrients such as phosphorus. Difference among arbuscular
successfully grown using ROC mode such as Gigaspora mycorrhizal fungi (AMF) for plant P acquisition has also been
margarita, Glomus fasciculatum, and G. macrocarpum, associated with differences in development and function of
G. intraradices, Gigaspora gigantia, G. versiforme, hyphae like intraradical and extraradical mycelia (Boddington
G. caledonium, G. clarum, G. fistulosum, and G. etunicatum and Dodd 1998; 1999). Many immobile trace elements such
(Chabot et al. 1992; Declerck et al. 1996a,b; Declerck et al. as N, S, Ca, Mg, K, Zn, Cu, etc, are also known thus providing
1998; Diop et al. 1992; Douds and Becard 1993; Gryndler better nutrition to the host plant (Clark 1997; Persad-Chinnery
et al. 1998; Karandashov et al. 2000; Pawlowska et al. 1999; and Chinnery 1996). Several reviews are available on the
Souza and Barbara 1999). Though process of bringing them enhanced acquisition of mineral nutrients in plants with
is very difficult, but the list of the new species under in vitro mycorrhization (Clark 1997; George et al. 1994; Smith and
is increasing every day. Mass production of spores is Read 1997). Turnau et al. (1993) proposed that polyphos-
prerequisite and mathematical models may be useful as phates in the fungal hyphae could sequester metals and
descriptive and predictive tools of sporulation dynamics minimize transfer to roots of the mycorrhizal plants in
(Declerck et al. 2001). This mode of culturing AM fungi also stressed conditions, (b) Enhancement of rooting and
provides an opportunity for biochemical and molecular reduction of transplant shock. Mycorrhizal fungi stimulate
investigations of AM symbiosis, which are otherwise unclear. root production and dramatically increase the volume of soil
198 Tiwari et al.

the plant can explore. This is especially important on most commonly. Also this offers wide applicability with a
disturbed sites, where nutrients and water availability might wide range of plants having little selectivity, which is
otherwise inhibit plant growth, (c) Alleviation of drought commonly reported in other biofertilizers. Though some
stress. The increased root volume allows more water to be exceptions exist with certain nonmycorrhizal families like
taken up. Mycorrhizal fungi also enhance the host’s osmotic chenopodiaceae, brassicaceae, and few nonhost plants of
adjusting capabilities, allowing some plants to continue nyctaginaceae etc. The storage conditions also are very
extracting water from soils as they become drier (Ellis et al. simple with no extra infrastructural requirements like low
1985; Morte et al. 2000), (d) Stabilization and aggregation of temperature and moisture content. Shelf-life is comparatively
soil. Mycorrhizal fungi encircle soil particles and glue larger long. Bacterial systems have short life and cause cell death
soil particles together into aggregates. This increases the easily.
surface absorbing area of roots 10 to 100 times. They release The hyphae of fungal system can extend much beyond (a
powerful chemicals in the form of exudates into the soil that few meters away) the depletion zone and thus can acquire
dissolve the hard to capture tightly bound soil nutrients. This nutrients from a much wider area. The fungal system also
improves soil structure, producing humic compounds and produces vegetative structures like chlamydospores and
organic “glues” that bind soils into aggregates and improve zygospores, which become dormant during periods of
soil porosity, increasing air and water movement though the environmental stress and germinate with the return of
soil while reducing erodibility. Reports are also available favorable conditions. Thus, they are better equipped for
suggesting the presence of a protein called Glomalin, which combating unfavorable conditions and have longer shelf-
seems to be involved in a very important hypha-mediated lives compared to bacterial systems. These biofertilizer
mechanism of soil aggregate stabilization (Borie et al. 2000; organisms are broad-spectrum and nonspecific. A single
Degens et al. 1996; Rillig et al. 2002), (e) Suppression of species is known to colonize approximately 90% of land
disease. Mycorrhizae directly and/or indirectly antagonize plants. These biofertilizers have broad ecological adapta-
disease organisms, increase the number of biocontrol agents bility and are known to occur in deserts as well as arctic,
around the roots, occupy potential infection sites on the root, temperate, tropical, and other habitats. They offer a
and increase host plant vigor to the extent that it can survive 25–50% reduction in phosphorus fertilizer application
disease (Datnoff et al. 1995; St Arnaud et al. 1997; Thomas depending on the plant.
et al. 1994). They act as biocontrol agent, (f) Enhancement of
nutrient transfer between plants. This is especially important
when nitrogen fixing and non-nitrogen fixing species are
planted together. Mycorrhizal roots exploit the soil profile
beyond the depletion zone surrounding the absorbing root and 4.1 Interaction of Natural Biofertilizers
its root hairs. Mycorrhizal modifications of the nutrient uptake
properties are dependent on the development of extramatrical Different biofertilizers have shown nitrogen-fixing, phos-
hyphae in soil, hyphal absorption of phosphate and other phorus-solubilizing, and phytohormone-producing abilities
micronutrients, their translocation through hyphae over and are used as for increasing agricultural productivity, for
considerable distances and subsequent transfer from fungus e.g., (Brady)rhizobium for legumes (grain, fodder), plant
to root cells, (g) Enhancement of beneficial interactions with growth promoting bacteria (PGPR) for cereals (wheat, rice,
other microbes. Mycorrhizal fungi increase the nitrogen made grasses, etc.), Azolla for the rice ecosystem, and actino-
available to the plant by both symbiotic and free-living mycetes (Frankia spp.) for forest trees. The AM biofertilizer
bacteria. They also increase phosphate uptake to the plant by is known to increase the nitrogen-fixing potential of the
PSB and support biocontrol agents that are antagonistic to legumes when given together with Rhizobium (Chaturvedi
pathogenic organisms, (h) Salt stress. The enhancement of and Kumar 1991) and Bradyrhizobium (Werner et al. 1994;
mineral acquisition especially that of P, K, Zn, Cu, and Fe due Xie et al. 1995). The mycorrhiza first stimulates the nodule
to AMF inoculation is more pronounced under salt-stressed bacteria in a sequential process by increasing the tissue
conditions. Studies indicate that AMF-inoculated plants have phosphorus content; this results in improved nodulation.
a greater tolerance to salt stress than un-inoculated plants There are also reports of positive interaction between
(Al-Karaki and Hammad 2001; Cantrell and Linderman Azotobacter/Azospirillum, and AM fungi (Alnahidh and
2001). Gomah 1991). The AM colonization favorably affects the
population of these free-living N-fixing bacteria and thus
stimulates better growth of plants. The AM colonization also
has a stimulatory effect with different nonlegume nitrogen-
fixing plant species. In Casuarina sp., the double inoculation
4 AM BIOFERTILIZERS HAVE AN EDGE of AM and Frankia improves plant growth and nodulation
OVER OTHER BIOFERTILIZERS (Sempavalan et al. 1995).
Two groups of bacteria, chemo-organotrophs like some
This group of biofertilizers is the only among others having Pseudomonas and Bacillus sp., and chemo-lithotrophs, such
fungal system involved. Other biofertilizers exploit bacteria as Thiobacillus sp., are able to solublize insoluble phosphates.
Commercialization of AM Biofertilizer 199

The AM when given in addition to these bacteria, improve the 6 MAJOR CONSTRAINTS AND SOLUTIONS
plant performance. The AM favors the early establishment IN COMMERCIALIZATION OF AM
and efficacy of these bacteria. The synergistic effect of these BIOFERTILIZER
fungi should thus be exploited on a large scale in the form of
biofertilizers to increase the nitrogen-fixing potential of
Perhaps the most important deterrent to the commercial use of
legumes and nonlegume plant species as well as with different
mycorrhizal fungi globally is the lack of large-scale multi-
phosphate solubilizers. Mycorrhizal fungi interact with a wide
location field trials in a variety of agricultural soils and an
assortment of organisms in the rhizosphere. The result can be
absence of general lack of awareness among the users. Without
positive, neutral, or negative on the mycorrhizal association
such an activity it will be difficult to establish a market for
or a particular component of the rhizosphere. For example,
mycorrhizal inoculum. Without a market there is little incentive
specific bacteria stimulate EM formation in conifer nurseries
for a commercial setup to initiate the production of inoculum on
and are called mycorrhization helper bacteria. In certain cases
a commercial scale, and only large-scale production will make
these bacteria eliminate the need for soil fumigation (Garbaye
large-scale field trials possible. Other important issues
1994).
responsible for the general lack of trust amongst the users in
its potential are: (a) The lack of cost-benefit analysis to
determine the economics of mycorrhizal applications, and (b)
5 ENVIRONMENTAL CONCERN ON The general trend towards excessive fertilization to substitute
CHEMICAL FERTILIZER USAGE for the lack of mycorrhizal fungi.
Once large-scale applications of the potential of
Fertilizer production is also an environmental concern. For mycorrhizal inocula are proven to common masses on
every ton of phosphoric acid produced, five tons of multi-location fields, lightweight commercial mycorrhizal
phosphogypsum are generated. Phosphogypsum is a solid formulations will need to be developed and new application
material that results from the reaction of phosphate rock methods will be devised. Most importantly, from large-scale
with sulfuric acid. Although it is nearly identical to natural field tests, cost-benefit analysis will be done accurately to
gypsum, it may contain small amounts of sand, phosphate, determine the economic benefit derived from the use of
fluorine, radium, and other elements present in phosphate mycorrhizal fungi. In the end, this will be the determining
ore. Federal regulations restrict both use and research factor in the commercial application of mycorrhizal fungi.
involving phosphogypsum because of its radium content Biological scientists are rarely able to critically assess the
and require phosphogypsum to be stacked on the ground. A economic factors involved in the application of a new
limited amount of phosphogypsum, with a minimal radium technology. It is important to design the total economic and
content, is used as an agricultural soil amendment. During infrastructure requirement for the setting up a production
the past 50 years, more than 700 MT have accumulated in facility and strict regulatory norms for the quality assessment
Florida alone. These enormous stacks, some covering an of the finished product before the release of the product in the
area of more than 300 hectares and up to 60 m high, have market. The involvement of the scientific community is
settling ponds on top that contain highly acidic water that important to define such norms.
can overflow into waterways. New regulations have been The field of applied AM research has suffered for many
enacted to guard against potential spills (Johnson and Traub years from the “chicken-and-egg” syndrome. The inoculum
1996). was not widely used because it was not readily available, and
Mycorrhiza offers an alternative to many problems in an it was not available because it was not used. The recent boom
ecofriendly, sustainable, and economical way besides in commercial AM inoculants will help break out of the cycle.
creating employment and facilitating poverty reduction. In There have been numerous inquiries about the quality of
situations where the native mycorrhizal inoculum potential is available inoculum. Unfortunately, only little data on which
low or ineffective, providing appropriate fungi for the plant to base recommendations are available. To remedy this
production system is worth considering. With the current state situation, the initiation of a quality control assay (QCA) for
of technology, inoculation is best for transplanted crops and in commercial AM products is essential which will involve
areas where soil disturbance has reduced the native inoculum conducting a standard mycorrhizal colonization percentage
potential. The first step in any inoculation program will be to (MCP) assay on commercial AM inoculum received under
obtain an isolate that is both infective, and able to penetrate before making them available in the market. This will need
and spread in the root, and effective, or able to enhance the regular supervision and knowledge of the correct mode of
growth and stress tolerance of the host. Individual isolates production, formulation, and delivery.
of mycorrhizal fungi vary widely in these properties, so Biofertilizers represent an affordable industry for many
screening trials are important to select isolates that will developing countries. In many African countries, the use of
perform successfully. Screening under actual cropping inorganic fertilizer has increased soil acidity, reducing the
conditions is best because indigenous mycorrhizal fungi, yield per ton of fertilizer. Biofertilizers are cheap to
pathogens, and soil chemical and physical properties will manufacture, suitable for small-scale farmers if produced
influence the result. locally (eliminating distribution costs), and the investment in
200 Tiwari et al.

technology is far lower than that of inorganic fertilizers. and its long-term viability in the commercial sector
Biofertilizers have been produced, packaged, and sold are listed here.
commercially in India, while in a number of African and (b) Inoculum registration. Concerns on ecological,
Latin American countries, biofertilizers have been produced biosafety, and bio-ethics demand the requirement
at national research centers. Most importantly, the demand for for microbial inoculants to be approved and
biofertilizers has outstripped production in almost all these registered. There is need for a centralized govern-
countries. It is estimated that about $40,000 –$50,000 is ment-regulated agency to provide the guidelines for
required to build a 100 – 150 MT biofertilizer plant. AM fungi-specific standards of inoculum use.
Alternatively, $500,000 for 10 plants in different locations (c) Quality control. Specific protocols for quality
could produce up to 1000 –1500 MT to meet the demand by control of AM fungal inoculum need to be
rural farmers. With increased production capacity, bioferti- developed and standardized for application. This is
lizers have a market locally and possibly internationally. essential not only as a guarantee for producers and
Biofertilizers present developing countries with a unique users but also for the protection of ecosystems. This
opportunity to enhance their crop yields. Countries like would help in quality management and assessment
Bangladesh, Brazil, Kenya, Tanzania, Zimbabwe, and of inoculum potential with every batch of inocula
Zambia have had successful pilot plants for the production produced. Quality control of commercial AM
of biofertilizers, and demand has often exceeded production. inoculum is extremely important for developing
If any of these countries built a production plant with local faith in the user community for its effectively
and regional markets in mind, they could be exporting their potential. Unless this is achieved, the potential will
products. India has developed many biofertilizers that are remain unexplored among the other biofertilizers.
currently on the market for gardeners and farmers. If these (d) Technology transfer. The product concept for AM
products are coupled with crop rotation and irrigation, it is fungal inoculum is particularly suitable for indus-
possible to increase crop yields of legumes and cereals. tries. Scaling up of production and use of AM fungal
Biopesticides, too, could help increase crop yield, reduce inoculum is only economically feasible for them if
import bills, and increase export earnings. Taken together, structures to run concerted field experiments is
they could provide an affordable source of agricultural inputs available. This needs to be offered by researchers
that would challenge chemical use in rural areas. Chemical working in the area through case studies in the areas
fertilizer and pesticide imports and exports from developing of horticulture, fruit production, and revegetation of
countries are low, and production yields are very poor, desertified ecosystems. In India, The Energy and
especially in Africa. Biotechnology will depend on renewable Resources Institute, New Delhi has developed AM
raw materials, and agriculture should play a big role in mass production technology, which was transferred
developing countries’ exports. to two leading industries, however, this is a small
move for an agricultural country where economy is
important and based on the yield production.
6.1 Mycorrhizal Commercialization Techniques
and Their Formulations
6.2 List of Producers and Formulators of
The use of AM fungi in plant biotechnology differs from that
Commercial AM Inoculum
of other beneficial soil micro-organisms because the fungi
involved are obligate symbionts and therefore recalcitrant to
pure culture. Thus specific procedures are required to culture These bio-inoculants are now formulated in different
and handle them; specific tools have to be developed and formulations. These are designed on the basis of their
provided to biotechnological producers. application to different crops and locations. They are
available in the form of powders, tablets/pellets, gel beads,
(a) Inoculum technology. Plant inoculation with AM and balls. Intraradical forms of Glomus sp. (vesicles and
fungi results in the formation of a mycorrhizosphere mycelium fragments) were entrapped in alginate and used as
with selective consequences on other important soil inocula. Isolated intraradical material was found to regenerate
micro-organisms. Therefore the use of AM fungi in in alginate beads and the regenerated mycelium infected roots
plant production needs an appropriate inoculum under controlled conditions (Declerck et al. 1996a,b; Strullu
technology compatible with that used for other and Plenchette 1991). Glass beads have also been suggested
beneficial soil micro-organisms. Development of an inoculum type with spores and mycelia inside (Redecker
second generation inocula, derived from mixing AM et al. 1995). The application in nursery plantations is normally
fungi with other inocula, is one such major activity. done using pellets or tablets placed just below the seeds or
The use of such inocula will improve plant fitness, small plantlets initiating mycorrhization in the hardening
and soil aggregation and stability, so increasing phase. Alternative approaches for inoculum disbursement
yield by biological means. Some of the important include broadcasting in the field or mycorrhizal products
issues related to AM biofertilizer commercialization often contain other ingredients designed to increase
Commercialization of AM Biofertilizer 201

Company Country
AgBio Inc., Westminster Colorado, USA
Accelerator Horticultural Products Ohio, USA
AgBio Inc., Westminster Colorado, USA
Bio-Organics Supply, Camarillo California, USA
Becker-Underwood, Ames Iowa, USA
BioScientific, Inc., Avondale Arizona, USA
EcoLife Corporation, Moorpark California, USA
First Fruits Triadelphia, West Verginia
Horticultural Alliance, Inc. Sarasota, Florida, USA
J.H. Biotech, Inc. Ventura, California, USA
Mikro-Tek Inc., Timmins Ontario, Canada
Mycorrhizal Applications. Grants Pass Oregon, USA
BioGrow TM North America
Plant Health Care, Inc. Pennsylvania, USA
MycorTM VAM MiniPlug TM North America
Premier Horticulture, Red Hill Pennsylvania, USA
Premier Tech Québec, Canada
Reforestation Technologies, Salinas California, USA
Roots Inc., Independence Montana, USA
T & J Enterprises, Spokane Washington, USA
TIPCO, Inc., Knoxville Tennessee, USA
Tree of Life Nursery, San Juan Capistrano California, USA
Tree Pro, West Lafayette Indiana, USA
Biological Crop Protection Ltd Kent, UK
Bio-Organics Medillin, Columbia
Biorize Dijon, France
Central Glass Co., Chemicals Section Tokyo, Japan
Global Horticare, Lelystad, Netherlands
Idemitsu Kosan Co. Sodegaura, Chile
MicroBio, Ltd Royston, Herts, UK
N-Viron Sdn. Bhd Malaysia
PlantWorks Ltd., Sittingbourne UK
Triton Umweltschutz GmbH Bitterfeld, Germany
KCP Sugar and Industries Corporation Ltd Andhra Pradesh, India
Cadila Pharmaceuticals Ltd Ahmedabad, India

the effectiveness of the mycorrhizal spores. For example, like pH, media manipulations (Douds 2002) can further
organic matter is often added to encourage microbial activity, increase the recovery of propagules. Recent report on the
soil structure, and root growth. Stress vitamins improve success of co-culturing two different genera together with
nutrient uptake and build root biomass. Water absorbing gels single host under in vitro as it occurs in nature, opens a new
help “plaster” beneficial mycorrhizal spores in close scope of an in vitro consortium package as inoculum, which
proximity to feeder roots and encourage favorable soil may prove more superior in varied edapho-climatic regions
moisture conditions for mycorrhizae to form and grow. where multiple mycorrhizal isolates may function better
Organic biostimulants, in general, are effective ingredients in than single isolate inoculation for future (Tiwari and
mycorrhizal products. By promoting field competitiveness, Adholeya 2002). Industry-based research documentation’s
stress resistance, and nutrient efficiency, biostimulants reduce as such are not available to the end users but a recent
barriers to rapid mycorrhizal formation especially during the brief insight into some of the potential techniques by
critical period following root initiation or transplanting. A list Moutoglis and Beland (2001) along with other alternative
of commercially available mycorrhizal inocula is provided in production techniques such as bioreactor-based production
the table above. techniques proposed by Jolicoeur et al. (1999); Jolicoeur
Recent advances in the in vitro mode of mass and Pirrier (2001) making use of ROC proposes a bright
multiplication like optimizing various growth parameters future for AM biofertilizer.
202 Tiwari et al.

7 CONCLUSIONS Becard G and Fortin JA (1988). Early events of vesicular arbuscular

mycorrhizal formation on Ri T-DNA transformed roots. New
Phytol 108:211 – 218.
A lot of research done in the past few decades has enabled
Boddington CJ and Dodd JC (1998). A comparison of the
these fungi to emerge as a potential biofertilizer, a cheap and development and metabolic activity of mycorrhizas formed by
environment friendly alternative to expensive, harmful arbuscular mycorrhizal fungi from different genera on two
chemical fertilizers. This aspect of an alternative to tropical forage legumes. Mycorrhiza 8:149 –157.
conventional route to more food grain production in a Boddington CJ and Dodd JC (1999). Evidence in differences in
sustainable manner especially gains significance for a phosphate metabolism in mycorrhizas formed by species of
developing countries where judicious and large scale Glomus and Gigaspora might be related to their life cycle
utilization of this technology can prove very useful for strategies. New Phytol 142:531 – 538.
getting maximum and long-term gains in various wasteland Borie FR, Rubio R, Morales A, and Castillo C (2000). Relationships
reclamation, reforestation, and afforestation programs apart between arbuscular mycorrhizal hyphal density and glomalin
from giving a much desirable thrust in the production of production with physical and chemical characteristics of soils
under no-tillage. Rev Chil De Hist Nat 73(4):749 –756.
important agricultural crops. The AM biofertilizer technology
Cantrell IC and Linderman RG (2001). Pre-inoculation of lettuce and
can be called poor man’s technology. Taking into account the onion with VA mycorrhizal fungi reduces deleterious effects of
amount of nutrient supplied, biofertilizers are many times soil salinity. Plant Soil 233:269 –281.
cheaper than chemical fertilizers. Biofertilizers improve the Chabot S, Becard G, and Piche Y (1992). Life cycle of Glomus
quality of produce. They are cheap and economical, the cost intraradix in root organ culture. Mycologia 84:315 –321.
benefit ratio is more than 1: 10. It is an ecofriendly practice, Chaturvedi C and Kumar A (1991). Nodulation and nitrogenase
improves natural characters of the soil. Uses of biofertilizers activity in gram (Cicer-arietinum l) as influenced by Rhizobium
maximize ecological benefits and minimize environmental and VA mycorrhiza interaction. Natl Acad Sci Lett 14:289 –292.
hazards. The demand of biofertilizers is increasing at a Clark RB (1997). Arbuscular mycorrhizal adaptations, spore
tremendous pace, which necessitates the inculcation of the germination, root colonization, and host plant growth and
more units to be established in the field to rope of the mineral acquisition at low pH. Plant Soil 192:15 –22.
Datnoff LE, Nemec S, and Pernezny K (1995). Biological-control of
outgrowing demand potential and the challenges of fabulous
Fusarium crown and root-rot of tomato in Florida using
future scope. Despite many lacunas in its commercialization Trichoderma-harzianum and Glomus intraradices. Biol Cont
and delivery to farmers for exploitation of its potential in 5:427 – 431.
agriculture, there is little doubt that AM fungi will emerge as Declerck S, Strullu DG, Plenchette C, and Guillemette T (1996a).
a potential tool for improving crop plants as a promising Entrapment of in vitro produced spores of Glomus versiforme
biofertilizer. Future upgradations in the mode of the AM in alginate beads: In vitro and in vivo inoculum potentials.
biofertilizer technology development, redefining the rate- J Biotechnol 48:51 –57.
limiting factors and exploration of possible AM combinations Declerck S, Strullu DG, and Plenchette C (1996b). In vitro mass-
along with other potential biofertilizers together as a single production of the arbuscular mycorrhizal fungus, Glomus
package for end users might bring a major boon to agriculture versiforme, associated with Ri T-DNA transformed carrot roots.
sector using nature’s biofertilizers. Mycol Res 100:1237– 1242.
Declerck S, Strullu DG, and Plenchette C (1998). Monoaxenic
culture of the intraradical forms of Glomus sp isolated from a
tropical ecosystem: a proposed methodology for germplasm
ACKNOWLEDGEMENTS collection. Mycologia 90:579 –585.
Declerck S, D’ Or D, Cranenbrouck S, and Le Boulenge E (2001).
Modelling the sporulation dynamics of arbuscular mycorrhizal
The authors wish to thank the Director General, Tata Energy fungi in monoxenic culture. Mycorrhiza 11:225 –230.
Research Institute, New Delhi, India for infrastructural and Degens BP, Sparling GP, and Abbott LK (1996). Increasing the
the Department of Biotechnology, Ministry of Science and length of hyphae in a sandy soil increases the amount of
Technology, India for financial support for development of water-stable aggregates. Appl Soil Ecol 3:149 – 159.
TERI’ s Mycorrhizal Mass Production Technology and its Diop TA, Becard G, and Piche Y (1992). Long term in vitro culture
transfer to industry for commercialization. of an endomycorrhizal fungus Gigaspora margarita on Ri
T-DNA transformed roots of carrot. Symbiosis 112:249 – 259.
Douds DD and Becard G (1993). Competitive interactions between
Gigaspora margarita and Gigaspora giganta in vitro.
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Research, Bartow, Fla., 17 p. VAM species Dianthus caryophyllus by co-culture with
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18
Control of Nematodes by Fungi

Hans-Börje Jansson / Luis V. Lopez-Llorca Universidad de Alicante, Alicante, Spain

1 INTRODUCTION 2.1 Plant-Parasitic Nematodes

Many nematodes are parasites of plants and animals causing Nearly one hundred species of plant-parasitic nematodes have
severe damage to crops and livestock. Many antihelminthics been described. They affect most crops causing important
used to control animal-parasitic nematodes have created diseases. As plant pathogens, their effects are time-dependent
resistance in the nematode population, resulting in decreased and usually do not (unlike other pathogens) kill crops, but
control efficiency (Nansen 1993). Nematicides, the chemicals reduce crop yields to noneconomical levels in spite of
which are used to control plant-parasitic nematodes, are often “good agricultural” practices (fertilization, irrigation,
toxic compounds causing both environmental and health agrochemicals, etc.).
problems. Several of these nematicides have been banned in Plant-parasitic nematodes differ regarding their feeding
many countries and the current process of phasing out the behavior (Figure 1). Migratory ectoparasites (Figure 1A)
ozone-depleting substance, methyl bromide, which is an remain in the soil and feed on the external cell layers of the
effective nematicide and insecticide, will increase the root. These nematodes cause little damage. Sedentary
nematode problem in agriculture (Nordmeyer 1992). There- ectoparasites remain feeding for long periods at the same
fore, the possibility to use nematophagous fungi for place therefore causing severe root damage. Migratory
nonhazardous biological control of parasitic nematodes endoparasites enter the root and periodically feed as they
should be encouraged. migrate through the root. Their damage to root tissue can be
To be able to use nematophagous fungi for biocontrol on a very important and may cause other soil pathogens, such as
larger scale we need to increase our research efforts to learn fungi or bacteria, to act synergistically causing complex
more about the life of these organisms, and their interactions diseases (Agrios 1997). Sedentary endoparasites (Figure 1B)
with nematodes, plants, and other soil organisms, on have developed complex feeding strategies. Essentially, their
ecological as well as cellular and molecular levels. In the females become saccate once they start feeding and deeply
present paper we will review and discuss some of the modify the root tissue in their vicinity to obtain nutrients. The
research, which has been performed on nematophagous fungi. sedentary endoparasites consist of the most important
Based on recent discoveries we will also speculate on new plant-parasitic nematode groups: the root-knot nematodes
ways to use nematophagous fungi for nematode control. (Meloidogyne spp.) and the cyst nematodes (Heterodera and
Globodera spp.).
2 WHAT ARE NEMATODES?

Nematodes are small roundworms occurring in most 2.2 Animal-Parasitic Nematodes

environments (soil, aquatic, and marine). Most nematodes
are saprotrophs or microbivores in the soil. Other nematodes There is a large group of nematodes parasitizing both
live part or most of their life cycles as parasites of plants or invertebrates and vertebrates. A number of insect-parasitic
animals. In the current review we will concentrate on parasitic nematodes, e.g., Steinernema spp., are being developed and
nematodes, mainly those attacking plants. commercially used for biological control of plant-parasitic

205
206 Jansson and Lopez-Llorca

Figure 1 Interactions amongst plant roots, plant-parasitic nematodes, and nemaphagous fungi. (A) An ectoparasitic nematode is free
living and feeds externally on plant roots by piercing the root surface with its stylet to obtain nutrients. (B) A sedentary endoparasitic
nematode (e.g., Meloidogyne spp.) performs most of its life cycle inside infected roots. The figure shows a swollen female with an egg
sack, feeding on enlarged root cells (giant cells or syncytia). (C) Root colonization by nematophagous fungi. Fungus penetration of root
cells sometimes involves formation of appressoria and inter- and intracellular growth in epidermis and cortex.

insects. Other animal-parasitic nematodes, e.g., Cooperia spp. nematophagous fungi are facultative parasites and exist in
and Ostertagia spp., cause severe infections and weight loss both saprophytic and parasitic (with infection structures,
in animal husbandry (Larsen 2000). Several tropical diseases proper lytic enzyme systems, etc.) stages induced by external
of humans are also caused by nematodes, such as and/or internal signals.
elephantiasis and river blindness (Poinar 1983). The nematophagous fungi are divided into groups
depending on their mode of infecting nematodes: nematode-
trapping (Figure 2A), endoparasitic (Figure 2B), egg- and
3 WHAT IS A NEMATOPHAGOUS FUNGUS? female-parasitic (Figure 2C), and toxin-producing fungi
(Jansson et al. 1997). Irrespective of the mode of infection,
Nematodes constitute a food source for the nematophagous the final result is always the same—a complete digestion of
(nematode-destroying) fungi. Living stages of nematodes the host. Nematode-trapping fungi capture vermiform
(eggs, juveniles, vermiform adults, and feeding sedentary nematodes in special trapping organs formed on the hyphae
females) can be attacked, penetrated, and digested by several (Barron 1977). The traps can have either an adhesive function
types of nematophagous fungi (Jansson and Lopez-Llorca where the nematodes stick to the trap, or a mechanical
2001). Dead nematodes (juveniles, vermiform adults, cysts, function. These fungi, e.g., Arthrobotrys oligospora, are
and root-knot nematode mature females) may also be characterized by low host specificity and lower parasitic
invaded by nematophagous as well as other (saprotrophic) ability. The endoparasites, e.g., Drechmeria coniospora, use
fungi, but the latter may not be regarded as proper nematode- their spores to infect the nematode hosts. The spores of these
destroying fungi. For instance, dead vermiform nematodes fungi can either be motile, e.g., zoospores, or nonmotile, e.g.,
may be invaded by nematode-trapping fungi, but then the conidia of various fungi. The endoparasites have high host
fungi enter the natural openings (mouth, anus, etc.) of the specificity and are mostly obligate parasites. The toxin-
nematodes and never penetrate their cuticles (Nordbring- producing fungi, e.g., Pleurotus ostreatus, immobilize their
Hertz and Stålhammar-Carlemalm 1978), and thus represent victims using toxins prior to penetration. These three groups
the saprophytic growth stage of these fungi. Most of fungi all attack vermiform nematode stages. Females of
Nematophagous Fungi 207

Figure 2 Scanning electron micrographs of nematophagous fungi. (A) Adhesive network trap of Arthrobotrys oligospora.
Bar ¼ 20 mm: (Courtesy of Birgit Nordbring-Hertz). (B) Adhesive conidia of the endoparasitic fungus Drechmeria coniospora adhering
to the nematode head. Bar ¼ 5 mm: (From Jansson and Nordbring-Hertz 1983, courtesy of Society of General Microbiology). (C)
Protoappressorium formed at the end of a germ-tube of Pochonia rubescens on the surface of an egg of Heterodera schachtii.
Bar ¼ 4 mm: (From Lopez-Llorca and Claugher 1990, courtesy of Pergamon Press).

sedentary endoparasitic nematodes, as well as their eggs, can attracted to the nematode-trapping and endoparasitic
be infected by facultative egg-parasites, e.g., Pochonia fungi (Jansson and Nordbring-Hertz 1988). After contact
chlamydosporia (syn. Verticillium chlamydosporium). with the infective structure (trapping organ or spore) the
nematode becomes attached, which eventually leads to
penetration of the host (Tunlid et al. 1992). Since the
4 HABITAT OF NEMATOPHAGOUS FUNGI nematodes move vigorously and also have high internal
hydrostatic pressure the production of an efficient adhesive is
4.1 Soil vital for successful infection, both as a holdfast and for
sealing the penetration area. The nonmotile nematode eggs
The nematophagous fungi are soil inhabitants found in most may be infected by egg-parasitic fungi, the hyphae of which
soil types from tropical to arctic areas of the planet, although actively grow towards the egg. Upon contact the fungus forms
they are most frequent in organic soils. One to five species of an appressorium that adhere to the eggshell. This stage is
nematophagous fungi are usually recovered from a soil followed by penetration of the egg shell and digestion of the
sample. Their abundance varies from 1.8 –150 propagules per contents of the egg (premature or mature juveniles). Infected
gram of soil (Persmark et al. 1996). These variations are due and dead nematodes may constitute a means for the
to “environmental factors,” e.g., soil type, organic matter, nematophagous fungi to survive in soil during harsh
water content, temperature, and presence of nematode hosts. environmental conditions.
Since most nematophagous fungi are facultative parasites
they also have the capacity to live saprophytically on dead
organic matter in soil. Therefore, the soil should obviously be 4.3 Fungi
seen as the ultimate sink of nematophagous fungi. The
nematophagous fungi, in spite of their saprophytic ability, are
Several species of nematode-trapping fungi, including
not especially good competitors in soil. Therefore, the
A. oligospora, can attack other fungi, e.g., Rhizoctonia solani
capacity to invade other organisms—nematodes, other fungi,
(Persson et al. 1985). This mycoparasitic behavior takes place
plant roots—is important for their survival in the soil. The
by coiling of the hyphae of the nematode-trapping fungi
interactions with these potential hosts will be discussed in the
around the host hyphae, which results in disintegration of the
following sections.
host cell cytoplasm without penetration of the host. Although
this phenomenon has never been observed in soil it may
increase the fitness of the nematode-trapping fungi in soil by
4.2 Nematodes reducing competition and providing nutrients. Moreover it
may extend the biocontrol capability of nematophagous fungi
By infecting nematodes the nematophagous fungi have the as biocontrol agents (BCAs) to fungal parasites as well as
ability to thrive in a sheltered environment protected nematodes. Furthermore, P. chlamydosporia has been
from other soil microorganisms. There are reports that described infecting propagules of important plant pathogens,
nematophagous fungi form antibiotic compounds upon such as uredospores of rust fungi (Leinhos and Buchenauer
infection of nematodes (Barron 1977). The vermiform 1992), and oospores of Phytophthora and other oomycetes
nematodes are actively moving in soil and are chemically (Sneh et al. 1977).
208 Jansson and Lopez-Llorca

4.4 Roots 5 MODE OF ACTION OF

NEMATOPHAGOUS FUNGI
The rhizosphere is a habitat with high microbial activity due to
release of nutrients from root exudates and decaying cells. The interactions between nematophagous fungi and their
Like other micro-organisms, nematophagous fungi are more hosts involve several steps from recognition (attraction
frequent in the root zone, with up to 19 times more propagules phenomena, contact), production of adhesives and lytic
than in the surrounding nonrhizosphere soil (Persmark and enzymes, differentiation of infection structures, e.g., appres-
Jansson 1997). Numbers of nematodes are also higher in the soria and trapping organs, to host penetration and digestion of
rhizosphere than in bulk soil. Since plant-parasitic nematodes the host cell contents (Tunlid et al. 1992). These events have
attack their plant hosts in the root, understanding of the been studied using a variety of techniques (microscopic,
rhizosphere biology of nematophagous fungi is important in biochemical, and molecular). In the following section we will
order to use nematophagous fungi for biological control of discuss some of these interactions in connection with two of
such nematodes. Nematodes are generally attracted to their the hosts of nematophagous fungi: the nematode and the
host roots by specific or unspecific compounds, e.g., CO2 plant.
(Green 1971). Besides, some nematophagous fungi, e.g.,
A. oligospora, grow chemotropically towards plant roots
(Bordallo et al. 2002). Recently, egg-parasitic and 5.1 Fungus– Nematode Interactions
nematode-trapping fungi have been found to invade and 5.1.1 Nematode-Trapping Fungi
grow endophytically in epidermal and cortical cells of
plant roots (Figure 1C) (Bordallo et al. 2002; Lopez- Arthrobotrys oligospora forms so-called adhesive network
Llorca 2002b). Root colonization by nematophagous fungi traps on which vermiform nematodes are captured. The
will be further discussed in “Fungi—Root –Interactions.” formation of traps in this fungus is induced chemically by

Figure 3 Top figures show infection of nematode egg by the egg parasite Pochonia sp. Germling of the fungus forms an appressorium
after contact with the egg surface (left). An adhesive is formed and the fungus penetrates the egg shell, grows inside the egg, and digests
its contents. Bottom figures illustrates capture and infection of a nematode by the nematode-trapping fungus Arthrobotrys sp. A nematode
is captured in the three-dimensional network trap (left). The middle figure (enlargement of left figure) shows the trap, covered with
adhesive, penetration of the nematode cuticle, and formation of an infection bulb. The trap and infection bulb contain dense bodies (dark
dots). The right figure shows an enlargement of the middle figure with the multilayered nematode cuticle covered with a surface coat.
Nematophagous Fungi 209

small peptides, e.g., phenylalanyl valine (Nordbring-Hertz binding, induces changes in the structure of the adhesive
1973) or by nematodes (Nordbring-Hertz 1977). The presence leading to capture of the nematode (Veenhuis et al. 1985). The
of traps is a prerequisite for infection of living nematodes, and adhesive undergoes changes from an amorphous material to a
in fact increases the ability of the fungus to chemically attract fibrillar structure, more organized with fibrils perpendicular to
nematodes (Jansson 1982). After contact between the fungal the nematode surface. This may anchor the nematode to the
trap and the nematode cuticle (Figure 3 bottom left) a possible trap thus facilitating infection. In contrast, the adhesive of the
contact recognition step occurs involving a fungal lectin endoparasitic fungus D. coniospora does not appear to change
binding to N-acetylgalactose amine (Gal-NAc) on the and has a fibrillar structure even in the absence of nematodes
nematode surface (Nordbring-Hertz and Mattiasson 1979). (Jansson and Nordbring-Hertz 1988). After the firm
The nematode surface, the cuticle (Figure 3 bottom right), attachment to the host surface, A. oligospora penetrates the
consists of several layers containing proteins (mainly nematode cuticle and forms an infection bulb (Figure 3
collagen), lipids, and carbohydrates (Bird and Bird 1991). bottom middle), from which trophic hyphae grow out to
Externally to the cuticle a surface coat (or glycocalyx) digest the nematode contents (Veenhuis et al. 1985). In the
consisting of glycoproteins is found (Bird and Bird 1991). trap and the infection bulb organelles, so-called dense bodies,
The surface coat is probably the part of the nematode surface now appear. These organelles are not present in ordinary
most relevant to recognition and adhesion of nematophagous hyphae and have been suggested to contain hydrolytic
fungi, since proteolytic removal of this structure results in enzymes used for penetration of the cuticle and digestion of
reduced adhesion of bacteria and spores of endoparasitic host (Jansson and Nordbring-Hertz 1988; Veenhuis et al.
nematophagous fungi (Bird 1985; Jansson 1993). 1985).
The trapping organ of A. oligospora contains an adhesive As in many other instances of fungal penetration of their
material. Upon contact with the nematode surface, a hosts’ surfaces, nematophagous fungi appear to use both
recognition step possibly mediated, at least partly, by lectin enzymatic and physical means. The nematode cuticle mainly

Figure 4 Hypothesis of interference with nematode chemotaxis. Conidia of the endoparasitic nematophagous fungus Drechmeria
coniospora adhere to the sensory organs at the anterior end of a nematode and block nematode attraction (left). The top right figure
illustrates the tip of the nematode head with two amphids (chemosensory organs). The chemotactic factors (black dots) are transported to
the neuron membranes where the chemoreceptors are located. (A) shows a proteinaceous chemoreceptor with carbohydrate chains, where
the terminal sugar (purportedly mannose or sialic acid, triangle) binds to a chemotactic factor leading to normal attraction of the
nematatode. (B) Illustrates blocking of terminal carbohydrates with lectins (Con A and Limulin) thereby inhibiting nematode chemotaxis.
(C) Shows enzyme (mannosidae and sialidase) obliteration of terminal sugar moieties leading to inhibition of chemotactic behavior.
Proteolytic enzymes, hydrolyzing the chemoreceptor (membrane protein), have a similar effect.
210 Jansson and Lopez-Llorca

contains proteins (Bird and Bird 1991) and therefore the immunolocalization in appressoria of the fungus infecting
action of proteolytic enzymes may be important for Heterodera schachtii eggs (Lopez-Llorca and Robertson
penetration. A serine protease, PII, from A. oligospora, has 1992b). Although pathogenesis is a complex process
been characterized, cloned, and sequenced (Åhman et al. involving many factors, inhibition of P32 with chemicals
1996). The expression of PII is increased by the presence of and polyclonal antibodies reduced egg infection and
proteins, including nematode cuticles (Åhman et al. 1996). PII penetration (Lopez-Llorca et al. 2002b). The similar species
belongs to the subtilisin family and has a molecular mass of P. chlamydosporia also produces extracellular proteases
32 kDa (for review see Jansson et al. 1997). Serine proteases (VcP1) (Segers et al. 1994), which are immunologically
will also be discussed in “Egg Parasites.” During decompo- related to P32 and similar enzymes from entomopathogenic
sition of infected nematodes A. oligospora produces a lectin fungi (Segers et al. 1995). VcP1-treated eggs were more
(A. oligospora lectin, AOL), which functions as a storage infected than untreated eggs suggesting a role of the enzyme
protein within the nematode host, and may constitute as much in eggshell penetration by egg-parasitic fungi. Recently,
as 50% of the total fungal protein. AOL is a multispecific several chitinolytic enzymes of Pochonia spp. were detected.
lectin, which binds to sugar chains common in animal One of those accounting for most of the activity was a 43-kDa
glycoproteins, including those of nematodes (Rosén et al. endochitinase (CHI43) (Tikhonov et al. 2002). When
1997). G. pallida eggs were treated with both P32 and CHI43,
damage to eggshell was more extensive than with each
5.1.2 Egg Parasites enzyme alone, suggesting a co-operative effect of both
enzymes to degrade egg shells (Tikhonov et al. 2002).
Egg-destroying fungi act on nematode eggs at two levels:
directly as true parasites by penetrating and infecting eggs, 5.1.3 Interference with Nematode Chemoreception
and indirectly by causing distortions in the larvae or embryos
they contain (Morgan-Jones and Rodrigues-Kábana 1988). Carbohydrates present on nematodes are not only involved in
The former mode of action is well documented and is largely the recognition step of lectin binding, but also appear to be
responsible for cases of soil supressiveness to nematodes. In involved in nematode chemotaxis (Jansson 1987; Zuckerman
this chapter we will describe the direct mode of fungal and Jansson 1984). The main nematode sensory organs are the
infection of nematode eggs mainly using the egg-parasitic amphids and the inner labial papillae located around the
fungi Pochonia rubescens (syn. Verticillium suchlasporium) mouth in the head region of the nematodes (Ward et al. 1975).
and P. chlamydosporia (syn. V. chlamydosporium). Figure 4 (right) schematically depicts the structure of amphids
Upon growth of germ tubes the hyphal tips swell and in the nematode head. The chemoreceptors are thought to be
differentiate into appressoria at contact with nematode eggs located on the neuron membrane. There are 28 neurons in the
(Lopez-Llorca and Claugher 1990) (Figures 2 and 4), as well nematode Caenorhabditis elegans, each with a passage to the
as on artificial, especially hydrophobic surfaces (Lopez- environment, but the number of receptors is not known (Ward
Llorca et al. 2002b). An extracellular material (ECM) 1978).
probably functions as adhesive, but possibly also seals the A hypothesis of the involvement of carbohydrates in
hole caused by the penetration hypha (Figure 3, top). This nematode chemoreception was put forward by Zuckerman
ECM can be labeled with the lectin Concanavalin A, (1983); Zuckerman and Jansson (1984). The chemoreceptors,
indicating that the ECM contains mannose/glucose moieties purportedly glycoproteins, could be blocked (Figure 4B) by
probably on side chains of glycoproteins (Lopez-Llorca et al. lectins (Concanavalin A binding to mannose/glucose
2002b). Most ECMs of fungal hyphae consist of proteins and residues, and Limulin binding to sialic acid) resulting in
carbohydrates (Nicholson 1996). loss of chemotactic behavior of bacterial-feeding nematodes
Early evidence of eggshell penetration by to bacterial exudates (Jeyaprakash et al. 1985). Furthermore,
P. chlamydosporia came from studies of plant- and animal- treatment of the nematodes with enzymes (mannosidase and
parasitic nematode egg infection (Lysek and Krajci 1987; sialidase) thus obliterating the terminal carbohydrates (Figure
Morgan-Jones et al. 1983). In eggshells, low electron dense 4C) also resulted in decreased chemotactic behavior (Jansson
areas were found in the vicinity of penetration hyphae of et al. 1984), demonstrating the importance of these
P. rubescens on cyst nematodes (Lopez-Llorca and Robertson carbohydrate moieties in nematode chemotaxis.
1992a) suggesting involvement of eggshell degradation Interfering with nematode chemotaxis, thereby inhibiting
enzymes. Nematode eggshells mostly contain protein and their host-finding behavior, may be a possible way of
chitin (Clarke et al. 1967) organized in a microfibrillar and controlling plant-parasitic nematodes. In a pot experiment
amorphous structure (Wharton 1980). Therefore, a search for using tomato as host plant and Meloidogyne incognita as
extracellular enzymes degrading those polymers was carried parasitic nematode, addition of Concanavalin A and Limax
out. A 32-kDa serine protease (P32) was first purified and flavus agglutinin (sialic acid specific lectin) resulted in
characterized from the egg parasite P. rubescens (Lopez- decreased plant damage by the nematode compared to
Llorca 1990). Involvement of the enzyme in pathogenesis was controls (Marban-Mendoza et al. 1987). Addition of lectins
suggested by quick in vitro degradation of Globodera pallida (or enzymes) on a field is not feasible, but the possibility to
egg shell proteins (Lopez-Llorca 1990), but most of all by its use, for instance, lectin-producing leguminous plants have
Nematophagous Fungi 211

been shown to reduce galling by root-knot nematodes

(Marban-Mendoza et al. 1992).
The endoparasitic nematophagous fungus D. coniospora
infects nematodes with its conidia, which adhere to the
chemosensory organs (Figures 2 and 4) (Jansson and
Nordbring-Hertz 1983). Conidial adhesion was suggested to
involve a sialic acid-like carbohydrate since treatment of
nematodes with the lectin Limulin, and treatment of the
spores with sialic acid, decreased adhesion (Jansson and
Nordbring-Hertz 1984). Furthermore, nematodes with newly
adhered spores lost their ability to respond chemotactically to
all attraction sources tested, i.e., conidia, hyphae, and
bacteria, indicating a connection between adhesion and
chemotaxis through carbohydrates on the nematode surface
(Jansson and Nordbring-Hertz 1983). The conidia of
D. coniospora adhere to the chemosensory organs of Figure 5 Arthrobotrys oligospora colonizing barley root.
Meloidogyne spp., but do not penetrate and infect the Callose deposition in cell wall papillae (arrow) stained with
nematodes. Irrespective of the lack of infection the fungus Sirofluor. Bar ¼ 20 mm: (From Bordallo et al. 2002, courtesy of
was capable of reducing root galling in tomato in a biocontrol Blackwell Science).
experiment (Jansson et al. 1985), again indicating the
involvement of chemotactic interference.
(Montfort et al. unpublished). The significance of this fact for
biological control is under investigation in our laboratory.
5.2 Fungi –Root – Nematode Interactions The growth of the two nematophagous fungi in plant roots
appears to be like that of an endophyte. Whether this
endophytic growth induces systemic resistance to nematodes
Nematophagous fungi can infect, kill, and digest living
and/or plant pathogens in plants is yet unknown, but worth
nematodes. Most of these fungi can also live saprophytically
further investigations. Endophytic rhizobacteria reducing
and some even have mycoparasitic abilities. Since most plant-
plant-parasitic nematodes have been described (Hallmann
parasitic nematodes attack plant roots, the rhizosphere
et al. 2001), as well as the reduction of root-knot nematodes
biology of nematophagous fungi is important from a
by arbuscular mycorrhizal fungi (Waecke et al. 2001). If this
biological control point of view. We previously described
is the case also in nematophagous fungi this will open up a
that nematophagous fungi were more abundant in the
new area of biocontrol using these fungi. The internal root
rhizosphere than in bulk soil (Persmark and Jansson 1997).
colonization by egg-parasitic fungi, e.g., Pochonia spp., may
In recent investigations we studied the colonization of internal
give the fungi an opportunity to infect nematode eggs in egg
cells of plant roots (Bordallo et al. 2002; Lopez-Llorca et al.
sacks of root-knot nematodes inside the roots and reduce
2002a). In these experiments we used axenic barley and
subsequent spread and infection of roots by the second
tomato plants grown in vermiculite and inoculated with the
generation of juveniles. Structures resembling trapping
nematode-trapping fungus A. oligospora or the egg-parasite
organs were observed in epidermal cells colonized by
P. chlamydosporia. Roots were sequentially sampled, cryo-
A. oligospora, and these may serve the purpose of trapping
sectioned, and observed under light- or cryo-scanning
newly hatched juveniles escaping the roots. The ability to
electron microscopes. Both fungi grew inter- and intra-
colonize plant roots may also be a survival strategy of these
cellularly, formed appressoria when penetrating plant cell
fungi and could explain soil suppressiveness to plant-parasitic
walls of epidermis and cortex cells, but never entered vascular
nematodes in nature. The colonization of plant roots is a new
tissues (Figure 2C). In contrast to Pochonia spp., appressoria
area of research that deserves in-depth investigations, not the
had never been observed previously in A. oligospora. Using
least for biocontrol purposes. This is presently underway in
histochemical stains we could show plant defense reactions,
our laboratory.
e.g., papillae, lignitubers, and other cell wall appositions
induced by nematophagous fungi, but these never prevented
root colonization. Callose depositions in papillae are shown in
Figure 5. Nematophagous fungi grew extensively especially 6 BIOLOGICAL CONTROL OF PLANT-
in monocotyledon plants producing abundant mycelia, PARASITIC NEMATODES
conidia, and chlamydospores (P. chlamydosporia). Necrotic
areas of the roots were observed at initial stages of The use of nematophagous fungi for biological control of
colonization by A. oligospora, but were never seen at later plant-parasitic nematodes has a long history (for extensive
stages even when the fungi proliferated in epidermal and review on earlier experiments see Stirling 1991). The
cortical cells. P. chlamydosporia colonized roots displayed investigations have resulted in variable and confusing
higher proteolytic activity than uninoculated control roots results—sometimes excellent control results, at other
212 Jansson and Lopez-Llorca

occasions no control at all. The reasons for these varying suppresiveness to nematodes is a very complex phenomenon.
results may be many, but the major reason is probably lack of In a study to find causes for nematode decline, Persmark et al.
knowledge, both on physiology and ecology of the (1995) did not find differences in nematophagous fungi
nematophagous fungi. Their interaction in soil with populations between putative suppressive and conducive soils
nematodes, plants, other organisms, and the soil environment in Central America.
is little understood because of difficulties of working in the Searching for nematophagous fungi in agroecosystem soils
complex soil matrix and also lack of good methods for these have yielded fungal antagonists with potential as BCAs of
purposes. We believe it is necessary to perform both plant-parasitic nematodes. For instance, cyst nematodes were
laboratory experiments and field studies using old and new infected by the entomopathogenic species Lecanicillium
techniques to understand the complexity of nematophagous lecanii (syn. Verticillium lecanii) in the United States (Meyer
fungi interactions with their environment. In the following et al. 1990). In other instances, fungal antagonists have
section we will discuss some of the possibilities to use remained undescribed and their use halted for lack of
nematophagous fungi for biological control. sporulation, e.g., ARF-18, a sterile fungus infecting cyst
nematodes in the United States (Kim and Riggs 1991). The
search for nematophagous fungi in natural “undisturbed”
6.1 Sources of Antagonists to Nematodes ecosystems is another approach for finding antagonistic
potential, e.g., the Antarctic (Gray 1982) or the tropical rain
Just as the biodiversity of the planet is becoming more and forest. Costa Rica still offers protected areas where such
more reduced, scientists and the general public are becoming studies can be carried out. Our laboratory is presently
more aware of its importance in “practical” terms. Microbial involved in such a survey.
biodiversity is little known but even the most “humble” soil Another approach of using fungi for nematode control is to
may contain strains of microorganisms with interesting identify nematophagous metabolites. These substances can be
antagonistic properties. In the past, the use of antagonists for identified from nonpreviously described nematophagous
biocontrol purposes was restricted to the culturability of the fungi, e.g., cuticular disruption of Caenorhabditis elegans
putative organisms detected. Nowadays, in the post-genomic by Byssochlamys nivea (Park et al. 2001). A search for
era, our capabilitites of handling genetic material of nematicial molecules from Arthrobotrys and other fungi was
organisms allow the detection of microorganisms or their carried out by Anke et al. (1995). In the future, genes (e.g.,
metabolites in the environment. The classical approach for toxins, enzymes) can be a source of antagonistic potential
finding antagonists has been to search for them in places against nematodes, which can be used for improvement of
where their target did not cause damage or disease in spite of BCAs.
host presence. Clear examples of these places are suppressive
soils.
The most studied sources for fungal antagonists of
nematodes are soils naturally suppressive to plant-parasitic 6.2 Fungal Biocontrol Delivery Systems for
nematodes. The first example to be discovered was the decline Nematodes
of Heterodera avenae populations in cereal monocultures.
Using previous data from nematode population dynamics and After isolation, antagonists of nematodes have to be
with a stepwise approach, Kerry (1987) and co-workers found developed for biocontrol. The first step usually involves a
fungal parasites of females (Nematophthora gynophila) and screening program including traits relevant to the perform-
eggs (P. chlamydosporia) to be the biotic causes for nematode ance of these organisms in the field. Root colonization is
suppression. Similar situations have been found elsewhere in considered as an important ability for biocontrol of plant-
the world with soils suppressive to cyst nematodes. Root-knot parasitic nematodes (Bourne et al. 1996). Evaluation of
nematodes have a much wider host range than cyst pathogenicity, especially to nematode eggs, is difficult since
nematodes. Besides, many crop hosts of root knot nematodes these stages are nonmotile. Several techniques have been used
are grown under intensive agricultural conditions with regular for this purpose (Gunasekera et al. 2000; Irving and Kerry
use of agrochemicals. Under these conditions soil antagonists 1986; Lopez-Llorca et al. 2002b). Once fungal antagonists
are not favoured. Therefore, fewer examples of natural have been rated, promising strains should be stored in a safe
suppressive soils to root-knot nematodes have been found. way. To this respect, an assessment of the existing methods of
Among these are tree orchards in the United States, where the fungal preservation on nematophagous fungi should be
egg-parasitic nematophagous fungus Dactylella oviparasitica carried out.
was first isolated (Stirling and Mankau 1978). In a similar Stirling and colleagues developed formulations
environment, ring nematodes (Criconemella spp.) were found of nematophagous fungi for use in laboratory (Stirling et al.
to be suppressed by Hirsutella rhossiliensis, an endoparasitic 1998a,b) and field experiments (Stirling and Smith 1998).
nematophagous fungus (Jaffee and Zehr 1982). Both Control of root-knot nematodes was achieved in microcosms,
antagonists are examples of organisms with an important but not in the field. The authors stressed that formulations with
ecological role but difficult to use biotechnologically. Both greater activity should be developed for field experiments.
fungi have a limited growth rate and sporulation. Soil Other factors than formulation design could also be responsible
Nematophagous Fungi 213

for poor performance of BCAs in the field. Soil microfauna, of nematophagous fungi as BCAs. A fair deal of basic
e.g., enchytraeids (Jaffee 1999) and nematodes, have been research should be invested in these fields. These efforts will
found to consume the nematophagous fungi inoculum. Soil also find applications in other fields of root health.
receptivity to inoculum of nematophagous fungi should be
assessed before delivery of large amounts of inoculations are to
be carried out (Wakelin et al. 1999).
The effects of nontarget organisms beneficial to the crop ACKNOWLEDGEMENTS
rhizosphere, e.g., mycorrhizal fungi and rhizobacteria, on
nematophagous fungi have not yet been characterized. Such We thank Alma Gámez for the illustrations and Birgit
studies would improve the design of synergistic methods of Nordbring-Hertz for supplying scanning electron micrograph.
approaching biocontrol of root diseases caused by nematodes This work has been partially financed by the NSF 0072756
and other pathogens. project from National Science Foundation (USA), and the
AGL 2001-0734 project from the Spanish Ministry of Science
and Technology.
7 CONCLUSIONS

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19
Fungi in Food Technology: An Overview

George G. Khachatourians College of Agriculture, University of Saskatchewan, Saskatoon, Canada

1 INTRODUCTION Malthus proposed in 1798 remains as controversial as

when they first appeared. Malthus’s hypothesis remained
The concepts and practical applications of fungal bio- an important unanswered question for years after the
technology continue to make significant contributions to food bicentennial of Malthus’s paper. Brown et al. (1999) point
science. Several recent books and monograms have been out what additional issues complicate the simplistic
dedicated to this topic (Hui and Khachatourians 1995; Malthusian thought are additional constraints of public
Khachatourians and Arora 2001; Khachatourians and Arora health and global economy. One major intervening force
2002; Khachatourians et al. 2002; Pointing and Hyde 2001; has been the emergence of new and reemerging infectious
Rajak 2000; Singh and Aneja 1999). Table 1 shows a few key diseases, which in the absence of global war have had an
food products and processes that depend on applied mycology equivalent effect in terms of human suffering and death
and biotechnology. Without exception many aspects of food worldwide. Ironically, these epidemics are occurring again
ingredient and process technologies involving fungi are in spite of advanced medical technologies and accelerated
impacted by research in biotechnology, for example, methods of health care delivery and immunization. To
functional foods, nutraceuticals, value added foods, and further complicate the issue, the conventional practice of
food pathogen and safety detection systems. Inevitably, and agriculture has not doubled and cannot double the
beyond the laboratory level research, the development of food production of plant and animal-based foods, which are
biotechnology depends on research and developments in still at the mercy of nature and its disasters. Presumably,
engineering and down stream processing. It is in this context biotechnology has emerged as a solution, or an option for
that the convergence of food science and engineering create one, at least for the moment.
crosscutting opportunities for the two disciplines, biotechnol- Disciplinary crossover of biochemistry, genetics, micro-
ogy and engineering. An obvious group of foods in this biology, nutritional sciences, engineering, and emergence of
crosscut is the large-scale production of fermented foods, biotechnology set the stage for reconsidering the paradigm of
edible mushrooms, so called, single cell proteins (SCP). and agriculture from traditional breeding for food plants
fermented beverages. Finally, fungi in food technology have (Khachatourians 2002). The strongest impact on agriculture
benefited from intellectual property rights associated with in this area occurred after the discovery of in vitro genetic
both engineered fungi and process patents. Fungal biotech- engineering and the use of transgenic plants. Biotechnology
nology should enjoy being a primary driver of world food as a new era, 20 years into its development, is showing its
production technologies. positive impacts in production agriculture and new food
crops.
It could be said that we still have some distance to travel
before a larger contextual effect can be felt. In general there is
2 CURRENT FOOD SUPPLY AND DEMAND confidence that judicious and timely applications of modern
genetics to plant science will be an important driver of world
With the geometric doubling of population and marginally agriculture. Given the rising number of people it is also
arithmetic doubling of food production, the ideas of Robert understood that abundance of transgenic food plants does not

217
218 Khachatourians

Table 1 Food production through fungal biotechnology trends for building production capacity in the next 25 years
(Khachatourians 2002; Khachatourians et al. 2002).
Amino acids Food pigments It is becoming increasingly clear that the once known
Beverages Food enzymes questions and hence answers of food production are no longer
Dairy products Mushrooms that well known or understood (Khachatourians 2001). Other
Digestive aids Organic acids unknown elements of further increase in appropriate food
Dough Single-cell protein production are the understanding of the shift that has occurred
Ethnic foods (kefir, koji, miso, tempeh, in the paradigmatic aspects of food science, from the
etc.) Vitamins delicate balance of the interplay in organismic biology,
Availability of germplasms, and Applications of biotech-
nology to co-operation of environment and issues of food
necessarily translate into abundant supply for people. To feed governance, science, safety, and economics (Phillips and
the world population, we must strive to overcome global Wolfe 2001). While plant and animal biomass based food
deficiencies in food transportation, and storage in many production is advancing more rapidly than in past decades,
countries as well as its affordability (Khachatourians 2002). new understandings of the processes involved in production
Certainly, agricultural biotechnology can provide part of the and post production events, especially by biotechnological
answer, but global sociopolitical factors, including ethics of means can and add value to post production agriculture to
farming, farmers, corporate agribusiness, world trade foods and new products by microorganisms. Innovation,
organizations and states, and international treaties and invention, and investments in biotechnology will continue to
enforcement agencies, will also be influential. impact the food industry and possibly help to maintain
equilibrium of food production and consumption (Phillips and
Khachatourians 2001).
3 THE BALANCING ROLE OF FOOD
TECHNOLOGY
4 FUNGAL BIOTECHOLOGY IN FOOD
Historically applications of agronomic practices have been PRODUCTION
the most significant exploitation for food production. Many
countries rely on importation of food grains, fruits, and
No matter how anecdotal the evidence, even the ancient
vegetables. However, many nations have shortfalls of food
societies recognized the use of fungal technology, in
crops that are exasperated by unpredictable global climate
change. The world production of grains per person has relationship with their agriculture and food. Knowledge of
remained at about 300 –340 kg since 1970s. Of the three— fungal diversity and distinguishing beneficial fungi for the
wheat, rice, and corn—the world wheat carryover stock was at biotransformation of food ingredients, helped to sustain and
78 days in 1999. The food security threshold is 70 days. The extend our food source. In spite of the powerful toxic
supply of rice stocks was down to 42 days of consumption; secondary metabolites of many fungi, humanity survived
consumption had been on the rise for 26 consecutive years these fungi and through innovative use of the beneficial micro
since 1973 (Khachatourians 2002). A decade has passed since and macro fungi found particular culinary and other uses of
the United Nations’ Framework Convention on Climate the mushrooms (see: this volume, chapter by Rai).
Change, and even with three major gatherings, Rio Earth The importance of the fungi relates to their particular
Summit, Kyoto Protocol, and this year’s Johannesburg World modes of growth and proliferation, production, and secretion
Summit on Sustainable Development, we are no where near of extracellular enzymes, peptides of novel functions (taste,
adoption of a policy that would help sustain world physiological functions, and antimicrobial activity) and
development trends, whether food and agriculture, or food secondary metabolites and antibiotics consequential to
security and safety, or population increase. prevention of infectious microbial growth. The molecular
The summer of 2002 brought drought to parts of the North biologists of the day work with a few well-studied fungi,
America and flooding to parts of Europe and Asia which (Table 2) but many others from the 72,000 species wait in line
seriously affected world wheat and other grains production, to be studied (Hawksworth 1991).
subsequent grain harvesting, and overall food production Through advances in the fungal biotechnology we
markets. If we add the role of water in irrigation, understand fungal role in the new age of applied molecular
sustainability of food and agriculture trends for production, biology. As we have a growing base of some 10,000 fungal
and its scarcity, the outlook deteriorates dramatically. A total species in various collections and the public domain literature
of 1000 ton of water is required to produce 1 ton of grain. we have some of the relevant knowledge to cross connect to
Aquifer depletion or contamination is an ignored but real biotechnology for exploitation of fungi for food and
threat to plant based food production. Various contributions ingredients manufactured for human benefit. The knowledge
of biotechnology and construction of transgenic plants and base in mycology is in a good position to lead the new
applications of microbial biotechnology are one of the known biotechnology of food.
Fungi in Food Technology: An Overview 219

Table 2 Representative fungal species in food biotechnology 1995; Khachatourians and Arora 2001; 2002). Historically,
applications of mycology in agriculture and food have been
Genus species/synonym Genus species/synonym exploited by many countries and ethnic groups and are now
being enhanced by several tools and concepts of biotechnol-
Yeasts Filamentous fungi
ogy (Hui and Khachatourians 1995; Pointing and Hyde 2001;
Candida Aspergillus
Singh and Aneja 1999). In recent years much are emerging in
Maltosa nidulans
the developed countries that serves as new learning
Antarctica niger
opportunities in application of fungal biotechnology for the
Pseudotropicalis oryzae
food and environment. As it can be seen from chapters of this
Shehatae Mucor
book, there are more rapid advances and new understandings
utilis hiemalus
of the processes and new products aided or deterred by
Debaryomyces hansenii miehei
fungal biotechnology. In general, all areas of food
Kluyveromyces pusillus
technology, whether pre- or postharvest food crop manage-
lactis Penicillium
ment, transformation and value differentiation of commod-
marxianus var. marxianus album
ities, increased production efficiency, increased value-in-use
Pachysolen tannophilus camemberti
of animal and plant food and nonfood markets will be
Phaffia rhodozyme caseicolum
affected by fungal biotechnology.
Pichia stipitis roquefortii
As evident from the first section of this compendium, a
Saccharomyces Rhizopus
large proportion of the innovations in food biotechnology
bayanus arrhizus
come from the renewed assessment of fungal physiology,
carlsbergensis cohnii
biochemistry and genetics in order to determine methods and
cerevissiae delemar
options for manipulations at the molecular level. Outside the
exiguous niveus
natural sciences, agricultural biotechnology of today also
lipolytica oligosporus
requires the convergence of several disciplines from
rosei oryzae
production strategies, process engineering, commerce, and
rouxii Edible mushrooms
international law. Indeed it is the entire process of science,
sake Agaricus bisporus
investment, inventions, and innovations that are the
uvarum Agaricus campus
interdisciplinary and transdisciplinary characteristics of the
Yarrowia lipolytica Lentinus edodes
new biotechnology (Phillips and Khachatourians 2001).
Pholiota nameke
Advances in ingredient subdisciplines of mycology as in
Pleurotus ostreatus
the past will remain the drivers of applied agricultural
Volvariella volvacea
research. With new interests there will be major investment
focused on generating discoveries and their applications
towards both conventional and biotechnology oriented useful
Changes in the functional features of the starting materials products and processes or services.
leading to food products and processes are the other side to In the sections to follow we will present certain highlights
the fungal biotechnology. It is expected that efforts in the of basic and applied mycology that are exciting developments
public and private sector research establishments should and which impact the cross connection with biotechnology for
provide new inputs for food production. On the output side, it applications to agri-food (Rajak 2000). Needless to say
is expected that increased productivity, higher value added, specific developments in fields ancillary to mycology should
improved quality and shelf life, and spoilage protection will continue to be of significant impact on the new applied
impact market situation. These products and processes for mycology and biotechnology products and processes.
food crops and agricultural practice are not only challenging
for the scientific community interested in mycology but also
for public health and commerce. From various estimates, the 5.1 Food-Use Enzyme Production
values of sales of mycology-based products can run into tens
of billion of dollars, projected by this decade, certainly not an Specific enzymes create particular functional and hence value
insignificant figure. contributions in foods. The predominant source for global
market demand of enzymes comes from fungi and bacteria.
From a multitude of enzymes synthesized and many secreted
extra-cellular, a few occupy the dominant role in food
5 FOOD BIOTECHNOLOGY: PRODUCTS ingredient production (MacCabe et al. 2002). Biotechnology
AND PROCESSES continues to enhance the yield and functional attributes of
fungal enzymes (see: this volume, chapters by Saxsena and
Whether through traditional or present day biotechnological Malhotra, and Viniegra-Gonzalez). The most important
routes, fundamental applications of fungi have made a aspect of this realization has been in the application in
substantial impact on our foods (Hui and Khachatourians the context of food production/processing including
220 Khachatourians

immobilization for catalysis and secondarily as a source for the production of novel foods and drink represents an
commercial “digestives” containing enzyme(s) for con- untapped resource for fungal biotechnology.
sumption. Yoshimaru et al. (2000) describes improvement In certain food production schemes, food additives and
of the digestion in pigs by using microencapsulated ingredients obtained through fungal fermentation technology
aspartase from Aspergillus usamii and A. shiro-usamii. contribute much to aroma and flavors. Fungi grown in either
The production of these enzymes occurs by batch liquid or liquid cultures or in solid-state fermentation have the
solid/semisolid fermentations. The choice is often deter- advantages of in situ contribution to the value of foods (see
mined by considerations for maximum quantity, activity, Arora 2004, chapters by Saxsena and Malhotra; Nigam,
purity, or for cost and efficiency. Robinson, and Singh; and Vinigera-Gonzalez). Several
specific aspects of fungal biotechnology’s contribution to
functional attributes including taste and smell of foods are
5.2 Organic Ingredients Production discussed in depth in the following chapters (see: this volume,
chapters Castrillo and Ugalde; Agrawal; and Hansen and
Organic acids, vitamins, nonvolatile and volatile flavor Jakobsens; see also chapter by Avalos, Arora 2004).
organic molecules (e.g., vanillin and 2-heptanone) are
important compounds in food production and technology.
They serve as food ingredients or as precursors for food
ingredients. The major organic acids produced and used in the 5.4 Fermentation Technology and Downstream
largest volumes function primarily as food acidulants. The Processing
means for organic acid and production methods rely on
bacteria and fungi. If these ingredients are used as a food Yeasts and filamentous fungi were traditionally employed in
ingredient, they must have a GRAS (generally regarded as the production of alcoholic beverages and fermented foods
safe) status. Although the use of Penicillium was the primary over centuries (Hui and Khachatourians 1995; Rajak 2000).
source for many organic acids, in recent years Aspergillus Yeasts (mainly Saccharomyces) have been used worldwide
niger has become the preferred organism and riboflavin for brewing and baking for thousands of years. Likewise,
oversynthesizing strains of Pichia guilliermondii the filamentous fungi have been traditionally used for preparing
preferred source. mold-ripened cheeses (mainly Penicillium spp.) in Europe
and soybean-based fermented foods (mainly Aspergillus spp.)
in the Orient. On the other hand, edible mushrooms (such as
5.3 Fermented Foods and Beverages Agaricus) have been used worldwide for direct consumption
since times immemorial (Hudler 1998; Pointing and Hyde
Industrial yeasts are involved in the production of many foods 2001; Rajak 2000; Singh and Aneja 1999). With passing time,
and drinks. The edible products, cheese and bread, and the these fermentation techniques were scaled up and made more
potable alcohol products, beer, wine, and spirits, depend on efficient with respect to engineering theories and practices.
yeast-based fermentation. The techniques of biotechnology Main outcomes of the evolution of food processing and
developed in recent years enable the engineering of industrial production activities, have been the introduction of
yeasts including the multiploid yeasts, which are extremely interdisciplinary natural and engineering concepts, for
difficult to manipulate by traditional genetics. Normal yeasts example, better equipment design, heat and mass transfer
are unable to completely convert starch to alcohol because of systems, feedstock supplementation system, product
their inability to degrade the starch beyond its branch points. recovery, effluent and waste management (Thassitou and
Starch debranching-enzymes and their genes have been Arvanitoyannis 2001, for an extended coverage see: this
identified and engineered into the industrial yeast. As a result, volume, chapter by Schliephak et al.), computerization and
it is possible to completely convert starch to alcohol, resulting automation, and finally hazard analysis critical control points
in the production in a single natural step, light beer. Other (HACCP) and quality assurance. Ancillary to the above
genes could be incorporated into industrial yeasts to improve developments has been the area of food packaging storage,
the efficiencies of manufacture of cheese, bread, wine, and transportation, and distribution system design. Overall, there
spirits and to make other useful products (Hui and has been incredible integration of food science, microbiolo-
Khachatourians 1995). New technology development in gical, engineering, and industrial R&D activities.
fermentation can be powerful in contributing to agri-food Fungi, because of their unique mycelial structures have
industries. Kourkoutas et al. (2002) described a high challenged bioengineers to reinvent fermenters and down-
temperature alcoholic fermentation of whey using Kluyvero- stream processing instruments. Basic research aimed at better
myces marxianus cells immobilized on delignified cellulosic fungal uses for food production have synergistically aided
material under batch conditions. They found that volatile many other aspects of fungal biotechnology from life sciences
byproducts and higher alcohols in very low levels led to a molecules to a variety of polymer sciences. More recently
product of improved characteristic aroma. The potential use advances in natural science and engineering have led to the
of various agri-food enzymes in processing raw material for application of biosensors; computer control, logistics, and real
Fungi in Food Technology: An Overview 221

time data collection and analysis; on line analytical fungi. A new avenue for applied research is the application
instrumentation; and the use of new materials for processing. of genomics, proteomics, and bioinformatics towards
intervention with fungal developmental genes for enhanced
functionality, for controlled ingredient delivery, or spoilage
of foods. As the knowledge of the regulation of fungal gene
6 FOODBORNE FUNGAL PATHOGENS AND expression advances, it is expected that strains will be
MYCOTOXINS designed for expression of commodities of high impact in
world trade.
It is estimated that some 400 fungi and many of the Recent advances in diagnostic biotechnology have
phytopathogenic ones reduce or threaten the availability and revolutionized the procedures used in the identification of
safety of food. From the above, at least 20 are confirmed food fungi. Biochemical identification assays have been
producers of food related mycotoxins and antinutrients (see: miniaturized and through automation and uses of robotics
this volume, Chapter 6). Many vegetables, fruits, and seeds have become faster, reliable, and cost affordable. Rapid
lose their nutritive and other qualitative values due to loss of identification of fungi and yeasts from foods has become less
moisture, infection with spoilage microorganisms, and cumbersome because of ease in sequestering of target fungi
senescence. These types of wastage can occur during from the food ingredients and interfering compounds. In
transport, handling, and redistribution. Food crops and their addition, biochemical tests which traditionally have been used
products can be infected with single or multiple species of in the identification of yeasts and filamentous fungi have been
toxigenic fungi. As a result food commodities can be greatly aided by the introduction of polymerase chain reaction
contaminated inadvertently with levels of toxic metabolite(s) (PCR) technology.
often ranging in PPB to PPM levels (Jones et al. 1995; Research and development work on understanding of
Koshinsky and Khachatourians 1992; Koshinsky and fungal presence in food products and processes are advancing
Khachatourians 1994). rapidly. For the most part, the goal of research in this area is to
Exposure to these metabolites whether singly or in decrease process costs. However, fungal biotechnology will
combination results in toxopathological manifestations and enable improvement in health and safety related issues of
even death. Even at subthreshold levels, multiple mycotoxins, foods such as better shelf life, decreased threats to human
by their interactions with multiple sites and targets often health, and increased perceived healthfulness of the food.
produce devastating synergistic effects on living cells and
whole animals (Koshinsky and Khachatourians 1992;
Koshinsky and Khachatourians 1994; Koshinsky et al. 7 CONCLUSIONS
2000). The historically significant and still prevailing food
consumption related threat to public health comes from the Clearly as we move through the 21st century, the prospects for
fungal alkaloids that lead to ergotism. The fungus, Claviceps food production, storage, transportation, and processing will
purpurea is prevalent in the cool climates where rye is grown. change by the same forces that are impacting on all other
Clavicepes africana has been found to spread through facets of economy. The benefits of R&D in fungal
sorghum in North and South America, Australia, and Africa biotechnology for enhanced food production; security and
(Bandyopadhyay et al. 1998). Sorghum is the fifth most safety will benefit everyone. The support for fostering a
important cereal crop in the world. research environment conducive to the above goal must
Huge challenges are posed by mycotoxins such as therefore be responsibilities for everyone whether in the
aflatoxins, trichothecenes, and fuminosins that can be public or private sector. Fungal biotechnology can ensure
addressed by fungal biotechnology. Identification of myco- harnessing of the potential of fungi while ensuring our foods
toxin biosynthesis and their genes, mycotoxin catabolism and are free from fungal pathogens, mycotoxins, mold allergens,
biotransformation and their genes could have tremendous and other problems of quality loss during storage. Fungal
value. So far, however, breeding of corn and cereal grain biotechnology has the potential of being a primary driver of
plants through conventional genetics for resistance have been world food production and its quality.
attempted but remains unsuccessful (Medianer 1997). This is
possibly due to multiple modes of action of some of
these toxins and hence polygenic nature of resistance
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T-2 toxin and verrucarin A in combination on Kluyveromyces Medianer T (1997). Review: breeding wheat and rye for resistance to
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20
Fungi and Fermented Food

T. B. Ng The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

1 INTRODUCTION 2 ACID-FERMENTED LEAVENED BREAD

AND PANCAKES
Fermentation is a process in which complex compounds
including carbohydrates, proteins, and fats are broken down These are highly accepted as food. The acid fermentation
to simpler forms under aerobic or anaerobic conditions and makes the food resistant to spoilage and development of food
have been used by the food industry for centuries. The toxins and less likely to transfer pathogens.
products of fermentation can be used as a food source itself (a) Indian Idli: Idli is the Indian equivalent of the Western
or as an additive. Fermentation has many roles including sourdough bread. It differs from sourdough bread as first
detoxification, adding nutritional value, creating aromas or leavening is achieved by bacterial instead of yeast activity and
flavors, reducing cooking time and so fuel consumption, secondly wheat or rye is not used as source of protein to retain
and some fermented foods may even have therapeutic carbon dioxide during leavening. Idli is prepared by bacterial
value. A knowledge of microbiology, genetics, and fermentation of batters produced from washed, soaked rice,
biochemistry is important in the food fermentation industry and dehulled black gram. After fermentation overnight at
as yeasts, molds, and bacteria play an integral role in food around 308C, the batter is poured into cups of an idli steamer,
fermentation. Improvement of these microorganisms by placed in a covered pan and steamed for about 15 min until the
genetic manipulation may result in higher-quality fermented idli cakes are soft and spongy. The texture and flavor can be
food and a considerable amount of work has been done in achieved by replacing rice by other cereal grains, and a
the screening of useful microorganisms. Further develop- variety of legumes can be used instead of black gram. The
ment of fermentation equipment, toxin measurement, lactic acid bacterium Leuconostoc mesenteroides is important
prevention of food spoilage, nutritional analysis, flavor for batter leavening. Yeasts are not essential, although when
and taste assessment, and addition of colors and additives added they will contribute to leavening, flavor, and increasing
are underway to attain the maximum potential from this levels of the vitamins thiamine and riboflavin. The yeasts used
process. Many of the issues are covered in a book edited by in fermentation for production of idli include Saccharomyces
Steinkraus (1996) and in another two edited by Joshi and cerevisiae, Debaromyces hansenii, Hansenula anomala,
Pandey (1999). Trichosporon beigelii, Oidium lactis, Torulopsis holmii,
Although some fungi are pathogenic to plants and animals Torulopsis candida, and Trichosporon pullulans (Soni and
and thus bring about economic losses, many of them are Sandhu 1999); (b) Philippine Puto: Puto is similar to Indian
beneficial to mankind especially in food production. In this Idli except that there are no legumes in Puto, so the flavoring
chapter, different types of fermented food with fungal is excluded. It is produced by grinding soaked rice grains in
involvement in the fermentation process are described. It is water, adding sugar and a starter culture containing
worth noting that different countries may have some similar L. mesenteroides, Streptococcus faecalis, and S. cerevisiae,
indigenous fermented food as well as varieties that are unique so allowing the mixture to ferment. This fermentation lasts for
to that country. about 15 h, before adding sodium hydroxide (lye) and more

223
224 Ng

sugar. A second fermentation continues for about 5 h before cassava roots are washed, grated, and packed into a bag that is
steaming for 30 min. S. cerevisiae is a minor component until weighted to squeeze the juice out. Natural fermentation is
the final stage of the fermentation in which it can reach as allowed to proceed for several days resulting in a lower
much as 18% of the total population, resulting in a small cyanide content and softer mushy extreme of the cassava. The
amount of ethanol. The yeast together with L. mesenteroides yeasts including Candida sp. together with bacteria
may play an important role in leavening the batter (Rosario (L. mesenteroides) are responsible for the flavor and changes
1987); (c) Sri Lankan Hopper: Rice and wheat flour, either in acidity during fermentation (Oneyekwere et al. 1989); (vii)
alone or mixed, are added to sugar, coconut water, coconut Nigerian Kamu: Kamu is a starch-cake food produced from
toddy, and inoculum (a collection of yeast and acid-producing fermented millet. The yeasts, S. cerevisiae and Candida
bacteria) or baker’s yeast (S. cerevisiae). After the mixture krusei and the lactic acid bacteria are used in fermentation.
has formed into a stiff batter, it is covered with a piece of wet The mixture of soaked millet grains, pepper, ginger, and fruit
cloth and allowed to stand at room temperature for about 12 h is wetmilled with water and sieved. Kamu is the sediment
to allow carbon dioxide to be produced by the yeast. obtained after the filtered liquor is allowed to settle for several
Following this fermentation, coconut milk and salt are added. hours and can be used to make porridge (Oyeyiola 1991); (h)
Sodium bicarbonate may also be introduced to raise the batter Nigerian Ogi: Ogi is made by fermenting cereals including
during cooking. The mixture is then poured into an oiled hot maize, sorghum, and millet that not only give them different
pan and baked for about 4 min. A longer fermentation period flavors, but also result in different colors. Fungi and bacteria
produces a better flavor. Nutritionally, the yeast provides are included in the fermentation. Aspergillus, Cephalo-
proteins and B vitamins, and the coconut supplies some sporium, Cerecospora, Fusarium, Oospora, Penicillium,
proteins, long-chain fatty acids, and flavor (Ekmon and Rhizopus, Rhodotorula, and Saccharomyces are fungi
Nagodawthana 1977); (d) Ethiopian Enjera: Enjera, also associated with fermenting maize. The yeasts contribute to
known as injera, wanjera, manjeriya, and kissra habashiya, is the flavor of Ogi. The bacteria involved are Lactobacillus
made from teff (grain of a type of grass related to love-grass), plantarum, Lactobacillus brevis, Acetobacter sp., Coryne-
wheat, barley, sorghum, corn or a mixture of them. It is a bacterium sp., and Lactobacillus plantarium, which is the
fermented sour leavened pancake-like bread. The teff flour is predominant organism producing lactic acid (Akinrele 1970);
mixed with water and starter culture before being incubated (i) Sudanese Kisra: Kisra is bread or stiff porridge prepared
for 1–3 days to produce a paste. The fermented paste is then by fermenting sorghum flour. To prepare Kisra, sorghum
mixed with water, boiled, and cooled down before baking for grains are subjected to dry milling and sieving before
several minutes to give enjera. A number of fungi including fortification with wheat or millet grains. The mixture is then
Aspergillus, Candida, Hormodendrum, Penicillium, Pullaria, milled, and mixed with water before a starter culture is added
Rhodotorula, and Torulopsis species have been identified as to start fermentation. After half a day at around 378C, water is
being used in this process. It has been suggested that Candida then added before baking. Many microbes are involved in this
guilliermondii is the primary fermenting organism although process, Yeasts, Candida intermedia, D. hansenii, and
evidence has been presented that gram-negative rod-shaped S. cerevisiae multiply throughout the fermentation. The
bacteria may be responsible for the initial fermentation molds of the fungal genera Aspergillus, Penicillium,
(Stewart and Getachew 1962); (e) Ghanian Kenkey: Kenkey Fusarium, and Rhizopus are also found. The bacteria
appears in the form of sour maize dough balls or cylinders involved include Erwinia ananas, Klebsiella pneumonia,
enclosed by maize husks or plantain leaves. To prepare and Acetobacter sp. Dough leavening is caused by
kenkey, maize kernels are washed and soaked in water before Acetobacter converting alcohol to acetic acid and yeasts
being ground into fine particles. The powder is moistened produce CO2 (Mohammed et al. 1991).
with water, packed, and covered tightly to ensure anaerobic
conditions before leaving to ferment for 1 –3 days. A portion
of the dough is then precooked, while salt is added to the rest 3 FERMENTED MILK PRODUCTS
of the mixture. The precooked dough is mixed with the
remaining dough, made into balls and wrapped with maize The advantages include easy production, improved keeping
husks or plantain leaves prior to thorough cooking. This quality, better digestibility and nutritive value, and thera-
fermentation process is uncontrolled and initially involves peutic potential. Only the acid-tolerant bacteria can grow
Aspergillus, Penicillium, and Rhizopus species. Saccharo- during milk fermentation.
myces sp. have also been detected and as yeasts esterify (a) Egyptian Kishk is a fermented milk-wheat mixture that
organic acids and alcohols, they give a distinctive aroma to is stored as dried balls. To prepare kishk, wheat grains are
kenkey. Bacteria are also involved in this process. Gram- softened by boiling, air-dried, and then ground. Milk is added
negative, catalase-positive bacteria appear in the beginning to the ground wheat to produce a paste that is rolled into balls.
but soon disappear, to be replaced by gram-positive, catalase- Spices may be added to the balls before they are dried in the
negative bacteria. This microbial activity contributes to its sun. The numbers of molds, yeasts, and bacteria have been
nutritive value by increasing the content of thiamine, examined. Penicillium and Mucor molds are found up to
riboflavin, and protein in kenkey (Christian 1970); (f) 103 g21. Yeasts are found in 104 –106 g21 concentration but
Nigerian Gari: Gari is a fermented cassava product. Peeled bacteria make up the highest numbers, which also contribute
Fungi and Fermented Food 225

to the aroma with Lactobacilli found in 108 g21 (Mahmoud leaves. The peeled bananas are mixed with grass and the juice
1977); (b) Russian Kefir is an acidic, mildly alcoholic milk is forced out by squeezing by hand. A roasted flour mixture
made from cow, goat, or sheep milk that is able to keep made from maize, sorghum and millet is mixed with the
longer. The predominant yeasts involved in fermentation banana juice and fermented in the covered pit for half a day or
include T. holmii and S. delbrueckii although Candida kefir one (Harkishor 1977); (c) African Kaffir (Kaffircorn)
and S. cerevisiae are the yeasts that are commonly isolated (Sorghum) Beer: This has a sour, yogurt-like flavor. The
(Marshall 1984). During the preparation of kefir, Kefir grains brewing process involves lactic acid fermentation and an
are added to pasteurized milk and incubated at 18–258C for alcoholic fermentation. Lactic acid fermentation by lactic
1–2 days. The mixture contains Kefir grains, which consist of acid bacteria causes souring. The yeast S. cerevisiae is used
yeasts and lactic acid bacteria held together with the help of a for alcoholic fermentation (Hesseltine 1979); (d) Nigerian
polysaccharide gum and these are removed by sieving; (c) Pito: This is a slightly bitter, sweet-sour beverage with a
Russian Koumiss: This is produced from ass milk, camel milk, fruity flavor produced by fermentation of maize or sorghum.
or mare milk. Lactic acid bacteria and the lactose fermenting The molds Rhizopus oryzae, Aspergillus flavus, Penicillium
yeasts Torula kumiss and Saccharomyces lactis cause funiculosum, and Penicillium citrinum play an undefined role
fermentation (Kosikowski 1982). Fresh milk is first heated in Pito fermentation. The bacteria Leuconotoc sp. and
at about 908C for 5 min and cooled before inoculation of Lactobacillus sp. and the yeasts Saccharomyces and Candida
starter and incubation at 288C. The incubate is agitated every spp. are also present during fermentation. Amylases from the
24 h and then cooled to form koumiss. Koumiss has both germinated maize or sorghum grains and the molds present on
nutritive and therapeutic values and has been reported to be the grains cause hydrolysis of starch in the grains to form
effective in the treatment of pulmonary tuberculosis in maltose and glucose. The yeasts ferment the sugars to form
Russia; (d) Lebanese Labneh: This is formed from goat or ethanol. The lactic acid bacteria produce lactic acid
sheep milk. Lactose-fermenting yeasts and lactic acid bacteria (Ekundayo 1977); (e) South America fermented maize chichi:
cause fermentation; (e) Cheese: Cheese-making comprises This has a ciderlike flavor. Molds including Aspergillus and
several steps: preparation of milk; addition of starter and Penicillium spp., yeasts including S. cerevisiae, S. apiculata,
ripening (souring), renneting and coagulation, and ripening/ S. pastorianus, and Mycoderma vini, and lactic acid bacteria
curing of cheese curd into the final product. Molds such as are present. Amylase needed for hydrolysis of starch is
Penicillium roqueforti, P. glauceum, P. camenberti, Geo- produced from germinated maize or from human saliva.
trichum candidum, and Mucor racemosus are used in Chicha produced using saliva reportedly has a better flavor
ripening. Different combinations of molds and bacteria are (Gomez 1949); (f) Mexican Tesguino: This is prepared by
used for different varieties of cheese; (f) Indian Dahi: Dahi is fermentation of germinated maize or maize stalk juice. To
produced by lactic fermentation of milk as a bioproduct of make Mexican Tesguino, germinated maize kernels or maize
alcoholic fermentation by yeast. Raw milk is boiled for a cane stems are first mixed with water and boiled. Catalysts,
period of a few seconds up to 10 min, then cooled before which include yeasts, vitamins, enzymes, or growth
addition of starter and incubation at room temperature for factors, are added before fermentation. S. cerevisiae,
8–16 h (Baisya and Bose 1975). C. guilliermondii, and H. anomala are important in the
alcoholic fermentation of Tesquino (Lappe and Ulloa 1989);
(g) Philippine Basi: This is sugar cane wine. Sugar cane juice
is boiled and fermented with a mixture of molds, yeasts, and
4 ALCOHOLIC FOOD AND BEVERAGES bacteria in an earthenware jar for 6 months to 1 year (Sakai
(MOSTLY CEREAL-BASED) and Caldo 1984); (h) Philippine Tapuy: This is a sweet rice
wine. To make Philippine Tapuy, the rice is washed, cooked,
These are primitive beers and wines prepared by villages in cooled, and placed in a clay pot. Powdered bubod is then
the developing countries. They are cloudy slurries containing inoculated for fermentation which lasts for 2 –3 days. The rice
residues and microorganisms including yeasts, and hence are and the liquid collected on top can be consumed separately or
a source of B vitamins, proteins, amino acids, and calories. together. The amylase-producing yeasts Saccharomyces
(a) Ethiopian Tej: This is a home-processed honey wine uvarum and Endomycopsis (Saccharomycopsis) and the lactic
fermented by Saccharomyces species present in the acid bacteria are the dominant organisms present (Sakai and
environment that convert sugars to ethanol. To make Caldo 1983); (i) Japanese Sake: This is a rice wine made from
Ethiopian tej, honey, water, and hop stems are fermented in steamed rice overgrown with Aspergillus oryzae mycelium.
a pot for about 1 week at high ambient temperature or 2 weeks The yeast Saccharomyces sake also plays a role. The process
at low temperature with stirring daily. The mixture is then of sake brewing first involves the preparation of polished,
filtered through cloth several times and the final filtrate is steeped, and steamed rice, followed by the preparation of
collected (Vogel and Gobezie 1977); (b) Kenyan Urwaga: starter consisting of yeast and lactic acid bacteria.
This is a slightly sour drink prepared from bananas, maize, Consecutive addition of more steamed rice, rice koji
millet, or sorghum. Yeasts and lactic acid bacteria are (A. oryzae) and water are then followed by the main
involved in the process of fermentation. To make Urwaga, fermentation, which takes place in an open system without the
green bananas are ripened in a covered pit lined with banana exclusion of nonstarter microorganisms. The combination of
226 Ng

hydrolysis of starch by A. oryzae and slow fermentation by the Jambal fruits are diluted and the must ameliorated with cane
yeast Saccharomyces sake at a temperature below 108C is sugar. Diammonium hydrogen phosphate, sulfur dioxide, and
referred to as parallel fermentation, which gives rise to the pectinol enzyme are introduced, then follows fermentation
high (15–20%) ethanol content of sake (Murakami 1972); (j) with the yeast S. cerevisiae (Joshi et al. 1999); (c) Coconut
Tea Fungus/Kombucha: Several types of yeast including toddy: This is produced by naturally fermenting the
C. guilliermondiil, Pichia membranefaciens, Saccharomyces influorescence sap of coconut palm in open pots for 2 days.
sp., and Torulopsis formata are present in Japanese tea Candida spp., Kloeckera javanice, Saccharomyces chevielier,
fungus. Candida obtuse and Kloeckera apiculata are present S. exiguus, S. marxianus, Schizosaccharomyces pombe, and
in Formosan tea fungus (Kozaki et al. 1972). Reiss (1994) Pichia ohmeri are involved in the fermentation process; (d)
reported that tea fungus consists of Acetobacter xylimum and Palm wine: S. cerevisiae is the yeast usually instrumental in
the following yeasts in symbiosis: Pichia sp., Saccharomyces fermentation. Other fungi including Schizosaccharomyces
ludwigii, and Saccharomyces pombe. The yeasts produce pombe, Candida, Mycoderma, Aspergillus, Mucor, Pichia sp.,
ethanol from the sugars added and Acetobacter oxidizes and Rhizopus sp. are detected together with lactic acid
ethanol to acetic acid. It is believed that tea fungus enhances bacteria in Nigerian wine (Faparusi 1977). Atputharajah et al.
hepatic detoxification and inhibits tumorigenesis; (k) Chinese (1986) identified Candida, Pichia and Saccharomyces species
Spirits, Wines, and Beers: Some are made from barley or as the major yeasts responsible for the natural fermentation of
wheat and brans overgrown with Aspergillus while others are coconut palm sap. The wine is a milky suspension of live
made from rice or rice bean overgrown with Mucor or bacteria and yeasts. It has a sweet taste and exhibits vigorous
Rhizopus (Chen and Ho 1989); (l) Chinese Lao-Chao: This is effervescence due to fermentation. The wine is made from sap
made from fermentation of glutinous rice. The fungi and collected from a cut on middle-aged flowering palm trees. It is
yeasts grown on rice flour include R. oryzae, Rhizopus collected in earthenware pots containing the bacteria and
chinensis, and Amylomyces rauxii (Wang and Hesseltine yeasts and any left-over toddy; (e) Indian jackfruit wine: To
1970). The yeasts ferment the starch; (m) Indian Ruhi: This is make jackfruit wine, seeds are removed from peeled ripe
prepared by fermentation of boiled rice. Boiled rice is spread, jackfruits and the pulp soaked and ground in a bamboo basket.
cooled, and mixed with the inoculum, which is comprised of The extract is collected in earthenware pots. A small amount
molds belonging to genera Rhizopus and Mucor and yeasts of fermented juice is added as inoculum and the extract is
(Dahiya and Prabhu 1977). It is then poured into a basket. The allowed to ferment at 18–308C for a week. The yeast involved
rice liquefies upon fermentation and the liquid is collected in a in fermentation appears to be Endomycopsis (Dahiya and
pot beneath the basket. (n) Indian Madhu: This is made by Prabhu 1977); (f) Date wines: Different types of date wine
fermentation of boiled rice by lactic acid bacterial spp., involve different fungi that include Torulopsis, Saccharo-
Mucor and Rhizopus spp. (Dahiya and Prabhu 1977). Sugars myces, and Candida spp. and also different bacterial species
formed by the hydrolysis of starch are fermented to form including Acetobacter, Bacillus, Gluconobacter, Klebsiella,
alcohol and lactic acid; (o) Whisky: S. cerevisiae is involved. and Leuconostoc. Dates are either soaked in lukewarm water
Corn, rye, and barley are used. There are several types (a) and allowed to ferment for about 4 days, or boiled to form a
Scotch whisky produced from water and malted barley to syrup which is then allowed to ferment for about 3 days in a
which only whole grains of other cereals may be added. (b) cloth bag of either sorghum malt or a mixture of ginger and
Irish whisky made from unmalted barley. Compared with cinnamon immersed in the syrup (Ali and Dirar 1984); (g)
Scotch whisky it has higher ethanol content and a stronger Plum wine: To prepare plum wine, water and starter culture
flavor but lacks the peat characteristics. (c) Canadian whisky are added to the plums and fermented for about 10 days before
with a light flavor and made from corn, rye, and barley malt. pressing for juice. The yeast S. pombe is used for
The spirits must be aged for a minimum of three years. (d) deacidification of the acidic plum pulp. S. cerevisiae is used
American rye whisky containing at least 51% rye, American for alcohol production (Joshi et al. 1999); (h) Mead (honey
corn whisky containing at least 80% corn, American light wine): S. cerevisiae is used for alcoholic fermentation in the
whisky containing a large percentage of corn, and Bourbon production of mead and wine from apples, pears and plums
whisky containing at least 51% corn (Russell and Stewart where honey is utilized as a source of sugar (Joshi et al. 1999);
1999). (i) Kiwi fruit wine: Kiwi fruit juice is clarified with the help of
pectolytic enzyme with the resulting generation of an intense
fruity aroma. The juice is highly acidic and has low sugar
5 FRUIT-BASED ALCOHOLIC BEVERAGES content, making it necessary to ameliorate the juice.
S. cerevisiae is added to the must before fementation. The
A variety of alcoholic beverages is made from fruits. The ascorbic acid content is preserved by SO 2 at low
quality of fruit wine depends on the fruit variety, maturity of concentration (Joshi et al. 1999); (j) Apricot wine, litchi
fruit, yeast strains, other vinification practices, and the wine, sparkling plum wine: S. cerevisiae is involved. (a)
method of preservation. Apricot wine: made by diluting apricot pulp with water (1:2
(a) Mango Wine: Mangoes are first pulped, Pectinase is by volume), addition of 0.5% pectinol and 0.1% diammonium
then added followed by fermentation with S. cerevisiae var. hydrogen phosphate and fermentation with S. cerevisiae, (b)
ellipsoideus (Joshi et al. 1999); (b) Jambal wine: Crushed litchi wine: peeled litchi fruits are dipped in sugar solution for
Fungi and Fermented Food 227

4 h at 508C. The fruits are pulped and water is added. Yeast is yeasts are involved in fermentation (Poesponegoro and
then added to the litchi juice and fermentation allowed to Tanuwidjaja 1977).
proceed, and (c) sparkling plum wine: plums preserved in
sodium benzoate, a sugar concentration of 1.5%, a
diammonium hydrogen phosphate concentration of 2% and 7 FERMENTED SOYBEAN PASTES
the yeast S. cerevisiae strain UCD 595 are used to yield
optimal results (Joshi et al. 1999); (k) Brandy: This refers to The three soybean pastes described in the following are used
the distillate obtained by distillation of wine or any other in making soup or side dishes. Japanese Miso and Indonesian
fermented fruit juice or residue. Grape products are most Tauco have greater economic significance than Korean
commonly used, but apple, peach, plum, cashew apple, and Doenjang and Kochuzang.
apricot products can also be used. Various yeasts are involved (a) Japanese Miso: This is prepared by fermentation of
in fermentation including S. cerevisiae, S. capensis, soybeans, with or without addition of rice or barley, using
S. ludwigii, S. rosei, and S. uvarum. SO2 is not used in A. oryzae or A. soyae and S. rouxii (Hesseltine and Shibasaki
order to prevent formation of sulfuric acid which would 1961); (b) Korean Doenjang and Kochujang: A. oryzae,
considerably lower the pH (Joshi et al. 1999). Mucor sp., Penicillium sp., Rhizopus sp., R. flava, and
T. dattila are some of the essential microorganisms used in
fermentation (Chang et al. 1977); (c) Indonesian Tauco:
A. oryzae, Rhizopus oligosporus, and Hansenula sp. are
6 SOY SAUCES involved. Soaked soybeans are boiled, dehulled, washed,
boiled again, and covered to encourage fungal growth.
It is a dark-colored liquid that adds a meatlike and salty flavor Alternatively, they are inoculated with ragi tempe and mixed
as well as color to food. It is made by the hydrolysis of with rice flour, and incubated for several days following
soybeans with or without wheat added. the second boiling. They are then dried in the sun, put in
(a) Japanese Shoyu: The molds A. oryzae or A. soyae are salt brine, fermented for 3 – 4 weeks before the addition of
involved in fermentation. The yeasts Saccharomyces rouxii, palm sugar, cooked, then bottled or packed (Winarno et al.
Torulopsis versatile, or T. etchellsii are also involved. Salted 1977).
cooked soybeans are mixed with ground roasted wheat and
the mixture is inoculated with A. oryzae seed culture. Pure
cultures of Pediococcus soyae and S. rouxii are added at the 8 LEGUME-BASED FERMENTED FOODS
start and again one month later. Fermentation proceeds at
ambient temperature for a period of up to 3 years. Filtration, Legumes are used for food production mainly in Asia.
pasteurization, and bottling are then carried out (Yokotsuka Fermentation improves digestibility of legumes by hydro-
1977); (b) Korean soy sauce Kanjang: Molds of the genera lyzing proteins, and breaking down antinutritional molecules
Aspergillus, Mucor, Penicillium, Rhizopus, and Sco- like trypsin inhibitors.
pulariopsis are involved in brine fermentation (Lee and (a) Tempe (Tempeh) Kedele: This is a fermented soybean-
Cho 1971). The bacteria Bacillus subtilis, B. pumilis, and based food, popular with American vegetarians and also
L. mesenteroides, and the yeasts Rhodotorula flava, S. rouxii, available in Canada, the West Indies, Holland, Indonesia, and
and Torulopsis dattila are also present. Steamed soybeans are Malaysia. It is supplied in the form of a white, moldy cake.
crushed and left to mold without addition of inoculum. The beans are cleaned, soaked, dehulled, partially cooked,
Fermentation is allowed to proceed for a couple of weeks, drained, inoculated, packed in banana leaves or perforated
before filtration, pasteurization, and bottling (Lee and Cho plastic bags, and incubated for 2 days to produce tempe. A
1971); (c) Chinese jiang: Molds of the Aspergillus type variety of fungi have been isolated from Malaysian tempe
overgrow soaked, steamed soybeans coated with wheat flour. including various species of Aspergillus, Mucor, Penicillium,
Fermentation is carried out at high temperatures and in the and Rhizopus by Yeoh and Merican (1977). In tempe of other
presence of salt brine; (d) Malaysian soy sauce Kicap: origins bacteria such as Bacillus and Micrococcus sp. may
Soybeans are boiled, mixed with wheat flour, spread on also be present; (b) Tempelike foods from broad beans and
bamboo trays to allow fungal growth without addition of cowpeas: Rhizopus arrhizus is used in the production of
inoculum, transferred to earthenware jars, covered with salt tempe products from broad beans. R. oligosporus, R. oryzae,
brine and incubated in the sun. After 3 months of and R. arrhizus are used for tempe products from cowpeas.
fermentation, the sauce can be extracted. Brine can be Different Rhizopus species give products with different
added to the remaining mash for further extractions. Sugar, aromas and flavors (Djurtoft and Jensen 1977); (c) Oncom
molasses, caramel, monosodium glutamate, and benzoic (Ontjon): A mixed culture of microorganisms with Rhizopus
acid may be added to the sauce. A. oryzae, A. soyae, or Neurospora species predominating is used to produce this
A. nigar, A. flavus, Rhizopus sp., and Pencillium sp. are cakelike product formed by fermenting peanut presscake.
involved in solid-substrate fermentation in soy sauce Peanut presscake is soaked, drained, crumbled, mixed
factories (Ong 1977); (e) Indonesian soy sauce Kecap: thoroughly with solid waste from tapioca production,
The molds Aspergillus and Rhizopus spp. and various steamed, cooled and formed into flat cakes, inoculated with
228 Ng

molds, covered with banana leaves, and fermented at room with Bacillus natto, and allowing fermentation to occur for
temperature for about 2 days to form oncom (Fardiaz 1987); about a day at 40 –458C (Kiuchi et al. 1976).
(d) West African Dawadawa: This is prepared by
fermenting locust beans and consumed mainly in West
Africa. Yeasts, spore forming bacilli and lactic acid bacteria 9 CEREAL-BASED FERMENTED FOOD
are involved in the fermentation. The pulp is removed from
the seeds before they are boiled and then dehulled. The Cereals including wheat and rice form the largest class of
dehulled seeds are soaked, washed, cooked, spread on a food. Bread is the most commonly found cereal-based
tray, and covered with leaves then fermented for 2 –3 days fermented food.
(Padmaja and George 1999). (e) Chinese Chee-fan: This is (a) Chinese Minchin: This is made from wheat gluten and
in solid form, prepared from soybean whey curd in China used as a solid condiment. The fungal species involved in
and eaten like cheese. Mucor spp. and A. glauca are fermentation include Aspergillus sp., Chadosporium sp.,
involved in fermentation (Padmaja and George 1999); (f) Fusarium syncephalastum, and Paecilomyces sp. (Padmaja
Chinese Meitauza: This is Chinese soybean cake. It can be and George 1999); (b) Chinese red rice (Anka): This is
fried in oil or cooked with vegetables. Actinomucor elegans produced by fermenting rice with various strains of
is the microorganism involved in fermentation (Padmaja M. purpureus Went. It is used to color foods such as fish,
and George 1999); (g) Chinese Sufu (Tau-hu-yi): Soybean rice wine, red soybean cheese, pickled vegetables, and salted
milk is made from ground soybeans strained through meats. To make Anka, polished rice is washed, steamed,
cheesecloth and boiled. CaSO4 or MgSO4 is added to cooled, inoculated with M. purpureas, and allowed to ferment
induce curdling (protein coagulation). The cake remaining for a few weeks. Anka has been reported to be effective in
after pressing is known as tofu. Sufu is a highly flavored treating indigestion and dysentery (Su and Wang 1977); (c)
creamy bean paste prepared by growing soybean curd with Jalabies: These are syrup-filled confectionery available in
Actinomucor, Mucor, or Rhizopus species and fermenting India, Nepal, and Pakistan made from wheat flour.
Saccharomyces bayanus and bacteria are involved in
the curd in a salt brine/rice wine mixture. Red sufu is
fermentation (Padmaja and George 1999); (d) Indian Kanji:
colored with a derivative from the culture of Monascus
This is made from rice and carrots. It is a sour liquid added to
purpureus on rice while the white sufu is untreated (Wai
vegetables. H. anomala is involved in fermentation (Padmaja
1929); (h) Korean Meju: This is soybean paste used for
and George 1999); (e) Indian Torani: This is prepared from
seasoning. A. oryzae and Rhizopus sp. are involved in
rice and used as a seasoning for vegetables. H. anomala,
fermentation (Padmaja and George 1999); (i) Javan
C. tropicalis, C. guilliermondii, and G. candidum are involved
Bongkrek: This is coconut presscake popular in Central
in fermentation (Padmaja and George 1999). The prevalence
Java. R. oligosporus is involved in fermentation (Padmaja of bacteria and yeasts in Indian fermented foods during
and George 1999). (j) Indian Papadem: This is solid crisp different seasons varies. Yeasts such as Candida vortiovaarai,
condiment made from black gram (Phaseolus mungo) in C. krusei, and Kluyveromyces marxianus are frequently
India. Saccharomyces sp. is involved (Padmaja and George present in the winter. H. anomala, P. membranefaciens,
1999); (k) Indian Warries: Dehulled black gram grains are S. cerevisiae, and T. beigelii are present in both summer and
ground to a paste, spiced, and molded into small balls. winter (Soni and Sandhu 1999).
These are then fermented for 4 – 10 days at room
temperature and air-dried. The yeasts Candida spp.,
Debaryomyces hansenii, H. anomala, Rhodotorula lactosa, 10 MIXED FERMENTED FOOD
S. cerevisiae, and Wingea roberstii are involved in warri
fermentation in addition to bacteria (Soni and Sandhu They may give a higher protein content and a better balanced
1999); (l) Indian Dosa: This is a fried pancake-like staple ratio of amino acids.
food of South India prepared by fermenting a paste formed Nigerian Burukutu: This is a creamy drink made from
from rice and dehulled black gram. S. cerevisiae is the most sorghum and cassava. Candida sp. and S. cerevisiae are
predominant yeast involved in fermentation followed by involved in addition to lactic acid bacteria (Padmaja and
D. hansenii, H. anomala, Oosporidium margaritiferum, George 1999).
T. pullulans, Kluyerveromyces marxianus, Candida kefyr, and
C. krusei. Bacterial species belonging to Leuconostoc,
Bacillus, and Streptococcus genera are also involved (Soni 11 TUBER CROP-BASED FERMENTED
and Sandhu 1999); (m) Yukiwari natto: This is a kind of FOOD
fermented whole soybean product made by mixing Itohiki
natto with salt and rice koji. Rice koji, prepared by using Tuber crops include cassava, yams, taros, potatoes, and sweet
A. oryzae, is the source of enzymes to hydrolyze the potatoes. With the exception of cassava, tuber crops keep well
soybean components in fermentation, produced by and hence not many fermented products are prepared.
A. oryzae. Itohiki natto is produced by inoculating (a) African Fufu: This is made from cassava roots and
soybeans which have been soaked, steamed and cooled eaten with soup, sauce, or stew. Peeled cassava roots are
Fungi and Fermented Food 229

washed, cut up, soaked in water to release HCN into water, and 50 -mononucleotides. The protein content of jeotkal is
disintegrated, and sieved. The filtered starchy particles are higher than that in vegetable foods while the vitamin content
allowed to settle and collected, rolled into balls, cooked, and is dependent on the type of jeotkal.
formed into a paste called fufu. S. cerevisiae and various
bacteria are involved in the fermentation (Padmaja and
George 1999); (b) West African Gari: This is made from 14 FERMENTED MEAT PRODUCTS
cassava roots and eaten as a staple food. Candida sp. and (SAUSAGES)
bacteria are involved in fermentation. Roots are fermented,
broken up, sun-dried, milled into flour and made into a paste The starter culture used in meat fermentation may have
with boiling water before consumption (Padmaja and George bacteria, the yeasts Deboryomyces hansanii and Candida
1999). (c) Nigerian Lafun: This is a fine, powdery cassava famata and the fungi Penicillium chrysogenum and
product. Candida sp. and bacteria are involved in fermenta- P. nalgiovense. Yeasts encourage color development and
tion (Padmaja and George 1999); (d) Indonesian Tape: This is improve aroma in sausages. Molds contribute to the
a staple food made from cassava roots. To make Tape, characteristic aroma. During fermentation, the fall in pH
cassava roots are peeled, cut up, boiled to soften, cooled, due to glycolysis by lactic acid bacteria helps to preserve the
spread in trays, inoculated, covered with banana leaves, and fermented product and inhibit the growth of pathogenic
fermented for a couple of days. The microorganisms involved microorganisms, and the production of nitric oxide due to
in fermentation include S. cerevisiae, H. anomala, R. oryzae, activity of the nitrate and nitrite reducing bacteria results in
Mucor sp., and Endomycopsis fibuliger (Padmaja and George the production of nitrosomyoglobin that accounts for the odor
1999); (e) Hawaiian Poi: This is made from taro corms. It is a of the meat product. During ripening, proteolytic activity due
semisolid dish served with fish or meat. Lactobacilli and to enzymes in meat and bacterial starter cultures, and lipolytic
Candida vini and G. candidum are involved in the enzymes in molds and yeasts lead to products which may
fermentation process which is carried out for 1 –3 days at contribute to flavor (Hammes and Knauf 1994).
room temperature (Padmaja and George 1999).

15 CONCLUSIONS
12 FERMENTED FISH PRODUCTS
Fermented foods and drinks play a substantial dietary role in
Fermented fish products, with their characteristic flavors, people living in affluent nations as well as the people of
introduce variety to the South-East Asian diet. developing countries. These foods and drinks are found in
(a) Japanese katsuobushi: This is made from fish and used supermarkets and in wet markets. The prices of these products
for seasoning. A. glaucus is involved in the fermentation vary markedly: some wines may be expensive while some
(Graikoski 1973); (b) Cambodian Phaak or Mamchas: This is bean products are much cheaper. Man wisely exploited fungi
a fermented paste produced from eviscerated salted fish. in the production of tasty foods and drinks well before the
Glutinous rice pretreated with yeast is also added to the fish advent of modern biotechnology. Today some of these
(Padmaja and George 1999); (c) Vietnamese Nuoc-mam: This fermentation procedures have developed into lucrative
is a brown liquid produced by fermentation of small marine or enterprises. Some of the fermented products, e.g., red wine,
fresh water fish that are placed in earthenware vessels buried reportedly have health-promoting effects. Many of the
in the ground for several months. Bacteria and yeasts fermented products have been found to be aflatoxin-free.
contribute to proteolysis and flavor. Enzymes from A. oryzae Nevertheless, a few incidents of poisoning after consumption
can be used for reduced fermentation time to increase yield of of fermented food have been reported. It is essential that
nuoc-mam (Richard 1959). aflatoxin-free raw materials and nontoxic cultures be used for
food fermentation.
Recent research on fermented food has focused in several
13 SALTED SEAFOODS areas including the effect of fermented foods on health. The
anka mold, Monascus anka, contains the antioxidant
Seafoods including fish are nutritious and popular as food. dimerumic acid (Aniya et al. 2000). The hypocholesterolemic
However, they are highly perishable. Processes such as salting effect of fermented dairy products and their mechanism of
are used to preserve seafoods. action have been reported (St-Onge et al. 2000). A principal
Korean Jeotkal: Fish such as herring and sardines, flavor component of soy sauce, 4-hydroxy-2(or 5)-ethyl-5(or
shrimps, cuttle fish, oysters, and clams are salted and stored 2)-methyl-(2H) furanone is a potent anticarcinogen in mice
to allow aging. Halophilic bacteria exhibiting protease, RNA (Nagahara et al. 1992). Both beneficial and harmful effects
depolymerase, and 50 -phosphodiesterase activities play a role. due to kombucha (tea fungus) ingestion in animal
Saccharomyces and Torulopsis become dominant about 40 experiments and in humans have been described (Greenwalt
days after aging when halophytic bacteria disappear (Lee et al. et al. 2000).
1977). Differences in flavor between different types of jeotkal Another area of recent research is the investigation of the
are attributed to variations in the content of free amino acids chemical constituents and nutritive values of fermented foods.
230 Ng

The changes in the major components of kombucha during Djurtoft R and Jensen JS (1977). “Tempeh”-like foods produced
prolonged fermentation have been followed (Chen and Liu from broad beans (Vicia faba), cowpeas (Vigna sinensis), barley
2000). The nutritive value of the fermented Nigerian (Hordeum vulgare), wheat (Triticum aestivum), or from
beverage, burukutu and the chemical changes have been mixtures thereof. Symposium on Indigenous Fermented
Foods, Bangkok, Thailand.
examined (Odetokun 1997). The levels of ethyl carbamate in
Ekmon TD and Nagodawthana T (1977). Fermented foods of Sri
alcoholic beverages and fermented foods (bread and cheese) Lanka. Symposium on Indigenous Fermented Foods, Bangkok,
have been determined (Dennis et al. 1989). Microbial Thailand.
analyses have been conducted on fermented food and Ekundayo JA (1977). Nigerian pito. Symposium and Indigenous
beverages (Cosentino et al. 2001). The control of food- Fermented Foods, Bangkok, Thailand.
borne pathogens during sufu fermentation has been Faparusi SI (1977). Nigerian palm-wine-emu. Symposium on
investigated (Shi and Fung 2000). The stability of stored Indigenous Fermented Foods, Bangkok, Thailand.
fermented foods e.g. gari has been studied (Sanni 1996). Fardiaz D (1987). Oncom fermented peanut presscake, a unique
Hopefully all this research will help food fermentation Indonesian traditional fermented food. In: Yanagida F, Takai Y,
become a thriving industry which will introduce more Homma S, Kato S, Ando Y eds. Traditional Fermented Foods
and Their Processing in Asia. Tokyo, Japan: Nodai Research
varieties of food and also improve human health.
Institute.
Gomez PJ (1949). La Chicha, Su fabricaciony algunas sugarencias
tecnicas adicionables a las disposiciones legales en actual
vigenica. Notas Agronomicas, Estacion Agric Exp Palmira
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Graikoski JT (1973). Chichester CO, Graham HD eds. Micro-
The skilled secretarial assistance of Miss Fion Yung and Miss biological Safety of Fishery Products. New York: Academic
Grace Chan is greatly appreciated. Press.
Greenwalt CJ, Steinkraus KH, and Ledford RA (2000). Kombucha,
the fermented tea: microbiology, composition and claimed
health effects. J Food Prot 63:976 –986.
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21
Production of Edible Fungi

R. D. Rai National Research Centre for Mushroom, Solan, Himachal Pradesh, India

1 INTRODUCTION Commercial production of edible fungi represents unique

exploitation of the microbial technology wherein worthless
The world of mushrooms, owing to their sudden wastes (agricultural, industrial, forestry, and household) are
appearance in numbers, groups, rings, bunches, and also efficiently converted into nutritious food. Indoor cultivation
in isolation as a single attractive and imposing structure, of mushrooms utilizing the vertical space is the highest
has fascinated the man since time immemorial and protein producer per unit area and time, almost 100 times
references are available in the scriptures of many ancient more than the conventional agriculture and animal husbandry.
civilizations. Theofrastus (372 –227 BC), the great Greek It has promising scope to meet the worldwide food shortage,
philosopher, wrote about food value of mushroom when the without undue pressure on land, for the human population
latter found its way in the royal dishes for Greek and increasing at an alarming rate of almost 2 lakh people per day.
Of about 2000 edible fleshy fungi, 20 types are being
Roman emperors. There are indications that mushroom
artificially cultivated and about ten are being produced and
existed long before the Homo sapiens appeared on Earth,
marketed in sizeable quantities: the common button mush-
as evident from the fossil records of the lower Cretaceous
room (A. bisporus), oyster mushroom (Pleurotus spp.),
period, i.e., about 130 million years ago; it is assumed that
shiitake (Lentinula edodes), black ear mushroom (Auricularia
the primitive man also consumed mushrooms. The
spp.), paddy straw mushroom (Volvariella volvacea), winter
collection and consumption of mushrooms from the wild
mushroom (Flammulina velutipes), silver ear mushroom
is still a practice in many regions of the world but the
(Tremella fuciformis), nameko (Pholiota nameko), monkey
scenario changed after successful artificial cultivation of head mushroom (Hypsizygus marmoreus) and two famous
mushrooms. Though the Chinese are reported to have medicinal mushrooms namely Maitake (Grifola frondosa)
cultivated some specialty mushrooms like Auricularia, and the Reishi (Ganoderma lucidum).
Flammulina and Lentinula between 600– 1000 AD but,
undoubtedly, it was the artificial cultivation of the common
button mushroom (Agaricus bisporus) in France around the
year 1650 which transformed the world of mushroom 2 WORLD PRODUCTION OF MUSHROOMS
production and consumption.
Not all mushrooms are edible, but some are highly The rapid rate of development of mushroom production
poisonous. While edible fleshy fungi are called mushrooms, technology from a primitive cave culture in France to a high-
poisonous ones are termed ‘toadstools.’ It has been estimated tech industry during the last three centuries is a success story
that out of 10,000 species of fleshy fungi (Kendrick 1985) which has kept pace with the ever-increasing demand for this
about half of them are edible (Chang 1993) and as many as commodity and there is every reason to be optimistic about its
100 species are highly poisonous. Collection and consump- further growth in the years to come (Rai and Verma 1997).
tion of wild mushrooms requires knowledge and adequate From a meager 2 million tonnes in 1986, the world mushroom
precaution. Rather it is one of the most important reasons for production has registered a 3-fold increase in a decade and
popularization of artificial cultivation of proven edible was about six million tonnes in 1997, and five mushrooms,
mushrooms. namely A. bisporus, Pleurotus spp., V. volvacea, L. edodes

233
234 Rai

and Auricularia spp., the so-called leaders, accounted for 82 them suitable for specific groups suffering with certain
per cent of the total mushroom production (Table 1). physiological disorders or ailments like obesity, diabetes,
It is clear that the button mushroom (A. bisporus) is still atherosclerosis, hypertension, hyperacidity, constipation, etc
the leader contributing 31.8% to the total mushroom (Rai 1995; 1997).
production but its share that was 56.2% in 1986 has decreased Nutritional value of mushrooms has been reviewed by
over the years. China is the biggest producer of Lentinula, many workers (Chang and Miles 1989; Crisan and Sands
Pleurotus, Auricularia, Volvariella, Flammulina, and 1978; Rai 1995). Only salient features will be briefly but
Tremella. Indonesia and Thailand are the other major critically described here. It is a fact that there are wide
producers of Volvariella. Japan produces significant quan- variations in the nutritional values reported for the same
tities of Lentinula and Flammulina and almost the whole species by different workers (Beelman and Edwards 1989;
quantity of Hypsizygus and Grifola produced in the world. Rai et al. 1988). However, certain generalizations do emerge.
Agaricus production is concentrated in three geographical Owing to very high (90%) moisture content these are
regions—Europe, North America, and East Asia. In Europe, basically a low calorie food (25 –35 cal per 100 g fresh
Netherlands, France, and UK; in America, USA and Canada, weight) and this fits in well in this era of healthy eating by
and in East Asia, besides the giant China, Indonesia and reducing the intake of calories. However, fat content in
Taiwan are the other major producers of this mushroom. Asia mushrooms is very low, it is rich in polyunsaturated fatty acid
accounts for major share of US imports. It is quite interesting and is cholesterol-free (Rai 1995). Though carbohydrates
that six countries called group of six or G-6 countries (USA, constitute the major fraction (50% of dry matter), they are not
Germany, UK, France, Italy, and Canada) account for more nutritionally significant as the chitin (fungus cellulose), a
than 80% of world consumption; per capita consumption in polymer of N-acetyl glucosamine, is the structural component
these countries is very high (2 –3 Kg). One important of cell wall and constitutes the major fractions of the
indicator of demand for mushroom is the income level of carbohydrates and fiber. Chitin-N is also reported to give
populace in the G-6 nations. Rise in income level is a global inflated values of the protein content of mushrooms if
phenomenon and the demand for mushrooms is expected to estimated by quantifying nitrogen and multiplying by the
grow at a much faster rate. usual factor of 6.25. The fiber content is high in all the
mushrooms (10% of DW). Mushrooms, due to high quantity
and quality of protein have been recognized by the FAO as
3 NUTRITIONAL AND MEDICINAL VALUES food contributing to the protein nutrition of the countries
OF MUSHROOMS depending largely on cereals. Expectedly, in the nutritional
evaluation of mushrooms, proteins have been the focus of
It is primarily the flavor and texture for which the mushrooms attention of the researchers, but wide variations in the values
are devoured by the mankind, and scientific appreciation of for protein content of the mushrooms have been reported
their nutritional and medicinal attributes is a recent (Beelman and Edwards 1989). Rai et al. (1988) determined
phenomenon. Mushrooms have, from nutrition point of protein content in seven Pleurotus species (Table 2) by
view, a distinct place in human diet which otherwise consists various methods of protein determination and found that
of items either of plant or animal origin. Mushrooms are protein values obtained with Folin-Phenol method of Lowry
perhaps the only fungi deliberately and knowingly consumed were closest to N £ 4.38 values as suggested by Crisan and
by human beings, and they complement and supplement the Sands (1978). In terms of protein quantity, mushrooms
human diet with various ingredients not encountered or ranking below animal meats rank well above common
deficient in food substances of plant and animal origin. vegetables and fruits. The quality of mushroom protein is far
Besides the attributes understood in the terms of conventional superior to the vegetable proteins and is as good as or slightly
nutrition, unique chemical composition of mushrooms makes inferior to animal proteins. This is because all the essential

Table 1 World production of cultivated mushrooms

1986 1997

Mushroom Fresh ( £ 1000 T) (%) Fresh ( £ 1000 T) (%)

A. bisporus 1227 56.2 1956 31.8
L. edodes 341 14.4 1564 25.4
Pleurotus spp. 169 7.7 876 14.2
Auricularia spp. 119 5.5 485 7.9
V. volvacea 178 8.2 181 3.0
Others 175 8.0 1096 17.70
Total 2182 3763 6158 100.00
Source: Chang (1999).
Production of Edible Fungi 235

Table 2 Protein content in Pleurotus spp. by various analytical methods

Species Protein (N £ 6.25) Protein (N £ 4.38) Protein (Lowry) Protein (dye-binding)

P. eryngii 3.10 2.18 2.18 2.12
P. flabellatus 2.80 1.97 2.01 1.89
P. florida 2.24 1.57 1.61 1.45
P. membranaceus 3.04 2.13 2.10 1.91
P. ostreatus 2.61 1.83 1.91 1.66
P. sajor-caju 3.47 2.43 2.51 2.20
P. sapidus 3.18 2.23 2.37 2.01
Source: Rai et al. (1988).

amino acids are present in mushrooms and, interestingly, mushroom culture, (b) Seed or spawn preparation, (c)
most abundant is lysine (Table 3), in which cereals are Substrate preparation, (d) Growing or cropping, and (e)
deficient. It is therefore, suggested that mushrooms can Postharvest handling
supplement the cereal-based diet of the developing countries While step (a) and (b) are more or less common and similar
(Chang and Miles 1989; Rai 1995). Mushrooms are rich in B for most of the mushrooms, it is the substrate preparation,
complex vitamins and special mention is to be made of the crop raising, and post harvest technology which vary with the
presence of folic acid and B12. Though vitamin C is present, it type of mushroom. In this article after brief treatment of the
is vulnerable to postharvest losses due to very high phenol step (a) and (b), the cultivation technology of the so-called
oxidase activity (Rai and Saxena 1989b). Potassium, sodium, five leaders mentioned earlier will be briefly described and
phosphorus, and magnesium are the predominant minerals. reviewed. The successive steps of production of mushrooms
Iron is present in appreciable quantity in the available form are depicted in Figure 1.
but mushrooms are comparatively deficient in calcium.
Very significant pharmacological activities have been
observed in some mushrooms; a billion-dollar industry exists 4.1 Spawn Production
for the medicinal mushrooms namely Reishi (Ganoderma
lucidum), Maitake (Grifola frondosa), Shiitake (L. edodes), The term “spawn” is used for vegetative growth of mushroom
Trametes versicolor, etc. The medicinal mushrooms have mycelium on a suitable medium, to be used as inoculum or
been recently reviewed (Rai 1997; Wasser and Weis 1999) “seed” for the substrate in mushroom cultivation. Right kind
and will not be dealt with here. and quality of spawn is very important in the cultivation of
edible fungi. The technique of spawn preparation witnessed
many developments before Sinden developed the currently
4 PRODUCTION TECHNOLOGY OF EDIBLE used “grain spawn” on hard winter rye grain after addition of
FUNGI calcium salts and patented the process in 1932 and 1937.
However, wheat grain is now most commonly used as the
Production of edible fungi or mushrooms involves many basal medium for spawn production. Kumar (1995) has
steps, mainly the following: (a) Raising and maintenance of described other substrates used for spawn.

Table 3 Essential amino acids (% crude protein) in edible mushrooms

Amino acid A. bisporus P. sajor-caju L. edodes V. volvacea

Leucine 7.5 7.0 7.9 4.5
Isoleucine 4.5 4.4 4.9 3.4
Valine 2.5 5.3 3.7 5.4
Tryptophan 2.0 1.2 Nd 1.5
Lysine 9.1 5.7 3.9 7.1
Threonine 5.5 5.0 5.9 3.5
Phenyl alanine 4.2 5.0 5.9 2.6
Methionine 0.9 1.8 1.9 1.1
Histidine 2.7 2.2 1.9 3.8
Cystine 1.0 1.2 Nd 3.2
Source: Bano and Rajarathnam (1982).
236 Rai

Figure 1 Major steps in mushroom production.

Often, failures to get satisfactory harvest are traced to of microbial contamination have been developed. Some
spawn. If the spawn has not been made from a genetically manufacturers have developed liquid spawn also. However,
suitable fruiting culture, or is too old and degenerated, the spawn for production of wood-decaying fungi is mostly
yield is likely to be poor. Ideal environmental conditions prepared on wood chips and saw dust – cereal bran
and management cannot compensate for the genetically mixtures (Chang and Quimio 1982). Technique of spawn
inferior stock used to make spawn. In most parts of the production has been described in detail by various workers
world, there are specialized spawn manufacturing compa- (Chang and Miles 1989; Chang and Quimio 1982; Kumar
nies, which not only multiply the spawn but also are 1995). Generally, wheat grains soaked in cold water are
engaged in genetic improvement of mushroom germplasm; first boiled until these swell and become soft but do not
many high-yielding varieties/hybrids of mushrooms with rupture. After draining out excess water and surface
superior quality attributes have been developed by them. drying in shade, calcium carbonate and calcium sulfate are
The conventional techniques of growing mushroom added in such proportions to bring pH of the substrate in
mycelium on a sterilized substrate, often cereal grains, the range of 7–7.5. The substrate is then filled in
have been described by many authors (see Kumar 1995) glass bottles/polypropylene bags, cotton plugged, and
but a revolution is taking place in the containers; it is no autoclaved at 15 psi for 60–90 min. The substrate is
more glass bottles, but specially made polybags also inoculated with either pure culture for preparation of
called “breathing bags” which can withstand sterilization “mother spawn” or with mother spawn grains to prepare
heat and allow limited gaseous exchange without chances “planting spawn.”
Production of Edible Fungi 237

4.2 Maintenance and Preservation of Fungal role in the supply of pure and authentic cultures to the spawn
Cultures production units. American Type Culture Collection in USA;
International Mycological Institute in UK; Institute of
Pure culture of edible fungi is prepared either by multispore Microbial Technology and National Research Centre for
culture or tissue culture; the former is suitable for obtaining Mushroom in India are some of the reputed mushroom culture
fruiting cultures of A. bisporus but is not a suitable technique banks.
for heterothallic species. Tissue cultures derived from the
stipe or pileus of the mushrooms, both homothallic as well as 5 BUTTON MUSHROOM (AGARICUS
heterothallic species, can be used to raise fruiting cultures.
BISPORUS)
For multispore culture, a healthy and mature fruitbody of the
mushroom is first washed in sterile water, surface-sterilized
with alcohol, and is placed on a spiral wire loop kept in sterile Agaricus bisporus (Lange) Sing., popularly known as the
petriplate covered with a beaker. Mushroom sheds spores on white button mushroom, has the widest acceptability and still
petriplates from which a loopful of spores is transferred on accounts for more than 30% of total production of all
suitable growth medium, generally malt extract agar in case mushrooms. Limited quantities of A. bitorquis, a high
of A. bisporus. Spores after germination give rise to temperature species, are also produced in some countries. Its
multispore culture. In case of tissue culture, a piece from a cultivation technology has developed over the years from a
primitive cave culture in France in the 16th century to a high-
suitable place of fruitbody is cut and after surface-
tech industry in America and Europe now. Still in many parts
sterilization, the piece is transferred onto sterile growth
of the world, especially in developing Asian and African
medium slants. Different mushrooms may require different
countries, sizeable quantities are being produced in low-cost
growth medium and incubation temperatures; for example,
structures like huts under the seasonal conditions. In some
A. bisporus grows best on malt extract agar medium between
parts of the Europe, seasonal growing is done with
24– 288C while Volvariella spp. are best maintained on potato
arrangement for heating during the winters. Like any such
dextrose agar medium at 358C. It is desirable that the cultures
venture, the production systems differ in the infrastructure,
are not maintained on the same type of medium in each
level of technology, automation, and mechanization but the
subculturing for a long time. Degeneration of cultures or
basic principles and processes remain the same. The
spawn, which refers to the loss of desired traits, survival,
production technology of the white button mushroom
growth rate, and productivity, is not uncommon (Chang and
(A. bisporus) has been described earlier by several authors
Miles 1989; Stadelmann 1986) and has, of late, attracted the (van Griensven 1988; Vedder 1978; Vijay and Gupta 1995).
attention of the researchers to understand the reasons. Most important aspect of the button mushroom production
Authentic pure cultures of mushrooms should preferably be is the preparation of the selective growth medium, called
obtained from the reputed mushroom germplasm banks. Now compost, in which Agaricus mycelium thrives at the practical
a days, mushroom strains of commercial importance are exclusion of other competing organisms.
patented and thus free availability is restricted (Jong and
Birmingham 1991).
Though the pure cultures of mushrooms, once raised or 5.1 Substrate (Compost) Preparation
obtained as described above, are traditionally maintained by
periodic subculturing and/or cold storing between 2 –58C, Substrate preparation technique for the button mushroom has
however, better long-term preservation methods of fungal witnessed evolutionary changes over the years, from the long-
cultures are advisable and practiced now, which are required method of composting to the current environment-friendly
for maintenance of vigor and genetic characteristics indoor composting. However, the intermediate short-method
especially related with productivity and quality (Chang and of composting, is still the most popular method all over the
Miles 1989). Frequent subculturing is not only time world.
consuming but also costly and risky (Smith and Onions
1983). Other methods of preserving fungal cultures including
5.1.1 Long Method of Composting
mushrooms have been described (Jong 1989; Singh and
Upadhyay 2002; Smith and Kolkowski 1996; Smith and Long method of composting is the oldest method and now
Onions 1983), which include storage in mineral oil (paraffin exists only in few pockets of the world mainly because of poor
wax), lyophilisation, cryopreservation at low temperatures productivity, proneness to attack by the competitors, and also
(2708C), in liquid nitrogen or in mechanical freezers. The due to more time and labor consuming process (Vijay and
choice of method depends on many factors like requirement, Gupta 1995). This method is completely an outdoor process
resources, cost, etc. It is advisable that each mushroom strain and takes about 28 days, though production of long-method
should be maintained by at least two different methods; liquid compost in lesser duration has also been achieved. But the
nitrogen and mineral oil preservation have been found highly biomass loss in this process is very high (30 – 35%) and
suitable and are popular for preservation of mushroom the quality as well as productivity is poor, besides the
cultures. Mushroom culture repositories/banks play a vital environmental problems it creates.
238 Rai

5.1.2 Short Method of Composting end-product and above all, very high degree of selectivity
(Miller 1997).
Based upon the observations of Lambert that productive
compost came from the regions of the pile having temperature
between 50 –608C and adequate supply of oxygen, Sinden and 5.1.4 Growing or Cropping
Hauser (1950) developed the so-called short-method of Ready compost is seeded with spawn approximately at 0.5%
composting mainly because it took lesser time than the long on fresh weight basis after which the seeded substrate is either
method. The concept and process was indeed a revolution in filled in polybags or in shelves and the temperature and
the cultivation of button mushroom. The short method of humidity in the growing rooms are maintained at near
composting mainly consists of two phases: outdoor- 25–288C and 90–95% RH respectively for 12 –15 days for
composting for 10 –12 days (Phase-I) followed by pasteuri- mycelial colonization of the substrate called spawn-running.
zation and conditioning for 6 –7 days inside specialized Mushroom mycelium derives nutrition from the substrate by
insulated structures, called tunnels. secreting an array of extracellular degradative enzymes
Based upon the temperature conditions maintained inside capable of degrading cellulose, hemicellulose, lignin-humus
the tunnel, Phase-II can be divided into two sub-phases: complex, and several bacteriolytic and mycolytic enzymes.
pasteurization (57 – 608C for 6 – 8 h) and conditioning Once the compost is completely colonized, various types of
(45–488C for 5–6 days). Importance of conditioning has supplements, often suitably treated proteinaceous materials
been linked with the growth of desirable thermophilic like soybean meal is thoroughly mixed at 1% in the upper one
microorganisms; pasteurization and conditioning are essential third area of the substrate which is then layered with 4 –5 cm
for achieving the selectivity in the compost. Short method of of casing material. Depending upon the availability and
composting has many advantages over the long method: more suitability, various types of materials are used as casing in
compost per unit weight of the ingredients, higher different regions of the world; peat is of course the substrate
productivity of mushrooms, less chances for pests and of the choice in the developed countries while farmyard
diseases, shorter duration and less environment pollution. manure (FYM) in various combinations with soil and other
materials are used in many countries. Recently, choir peat
(specially decomposed and processed coir pith dust) has
5.1.3 Indoor Composting found acceptability as casing material in many Asian
countries where coconut plantations abound. Many authors
The problems of environmental pollution related with (Hayes 1974; Kurtzman 1997; Vijay and Gupta 1995) have
production of stinking gases associated with long as well as discussed materials, techniques, and the role of casing in
short methods of composting drew the attention of the cultivation of the button mushroom. Though there are varied
researchers to evolve an alternative “clean” process. Because observations and opinions on the importance of some
first time the work on such composting system started using properties of casing materials, e.g., pH, conductivity, bulk
completely indoor system, it was termed as indoor composting density, water holding capacity, and associated microflora,
(Laborde 1992); other terms like environmentally-controlled but that the button mushroom requiring a layering (casing)
composting, rapid indoor composting, and aerated rapid above the colonized compost for fruiting is a fact well-
composting have also been used for this process. Based on the established. After casing, growing rooms are again main-
temperature conditions maintained inside the tunnel, the tained at high temperature (,258C), humidity (, 90%), and
process could be divided into two categories, i.e., INRA CO2 (. 5000–10000 ppm) for mycelium to colonize casing
method and Anglo-Dutch method. In the INRA method, which layer called case-run. “CaCing” (mixing of small quantity of
is popular in France, Italy, and Belgium, phase-I is carried out colonized compost in casing material at the time of casing)
at constant temperature of 808C for 2–3 days followed by and ruffling of partially colonized casing material few days
phase-II at 508C for 5–7 days (Laborde 1991). As very high before readying for fruiting are some of the improvisations
temperature attained during phase-I of this method kills most practiced in high-tech mushroom production. After the
of the microbes including the desirable thermophiles, complete colonization of the casing layer when white
reinoculation with mature compost or thermophilic fungi mycelium becomes visible between the clumps all over the
becomes necessary in this process. However, in the bunker casing surface, room temperature is lowered down to
system used nowadays this can be dispensed with. In the 16 – 188C, fresh air is introduced/increased with slight
Anglo-Dutch method, a weeklong conditioning at 418C decrease in the humidity (85% RH) i.e., the conditions not
follows a short pasteurization phase of 4 – 6 h at 608C. The conducive for mycelial growth to continue. Under these
method has attained popularity in several European countries “adverse” circumstances mycelial aggregation takes place to
and Australia, and compost with high selectivity and form pinheads or primordia which differentiate and develop
substantial savings on raw materials (Miller 1997) is reported into mushrooms (Figure 2). After growth to the desired size,
to be produced. Indoor-composting has many advantages even mushrooms are handpicked or mechanically harvested. It is
over the short-method of composting: takes lesser time, gives common observation that during the commercial cropping of
higher yield, is environment-friendly, and conforms to A. bisporus and also of some other mushrooms, there is heavy
civic laws, lesser loss of raw materials and thus increased and synchronous appearance, called “flushes,” of sporophores
Production of Edible Fungi 239

at appropriate intervals with very little fruiting between the other than the button mushroom. Production and consumption
flushes called flush break. Fructification of mushrooms of the specialty mushrooms are very popular in the East Asian
represents an interesting phenomenon to study the countries namely China, Japan, Korea, Thailand, and
differentiation in multicellular eukaryotes (Rai and Saxena Indonesia and is picking up fast in many American and
1991). European countries where these are considered as novelty.
Varied levels of mechanization in composting and The scope, importance, and cultivation technology of
cropping and automation especially in the environment many specialty mushrooms has been covered briefly by
control of the growing rooms have been introduced in many various authors (Royse 1997; Sharma 1997; Stamets 2000)
developed countries in case of the button mushroom. but this review will cover the production technology of the
However, old manual system of seasonal growing in economically most significant specialty mushrooms namely,
makeshift cropping rooms is still practiced in many Pleurotus, Lentinus, Volvariella, and Auricularia which
developing countries to feed the domestic market. together accounted for 75% of the world production of
specialty mushrooms in 1997 (Table 1).

6 SPECIALTY MUSHROOMS
6.1 Pleurotus spp. (Oyster Mushrooms)
“Specialty mushrooms” is a term given to a group of
cultivated mushrooms which are less common in a particular Unlike most of the cultivated mushrooms, which represent one
area or country, but the term has been used to practically species, a group of species of the genus Pleurotus are
encompass all mushrooms other than the common button commercially cultivated and referred commonly as oyster
mushroom (A. bisporus). In the United States, the term mushrooms. Pleurotus ostreatus (Jack. ex.Fr.) Kummer, is
“specialty mushrooms” is used to cover all mushrooms other best known species among oyster mushrooms and the specific
than the button mushroom, which accounted for 90% of total epithet “oyster” obviously refers to its Oyster-shell like
production of 346188 MT there in 1993–1994 (Sharma appearance of the fruitbodies. Pleurotus spp. are most versatile
1997). In Japan, however, the situation is reverse to that in the of all the mushrooms, representing about fifteen species
United States where 90% of total production was of the capable of growing over a wide range of temperature (58C to
so-called specialty mushrooms and button mushroom 308C) and on almost all the lignocellulosic wastes; P. sajor-
contributed only 10%. Therefore, from the Japanese caju, P. florida, P. ostreatus, and P. flabellatus are most
perspective button mushroom could be termed as specialty popular commercial species. It is a primary rot fungi and can
mushroom. Be that as it may, the term specialty mushrooms is degrade moistened substrates directly and does not require
now well established by usage to represent all mushrooms precomposted substrates like secondary rot fungus, e.g.,

Figure 2 Button mushroom (A. bisporus) cultivation in polybags.

240 Rai

A. bisporus. Ease with which oyster mushrooms can be grown general parameters and have been described by Jandaik
has manifested itself in the production statistics where the (1997). There are wide variations among the growers about
production of oyster mushroom registered 442% increase the method and style of opening of the containers for fruiting:
during the period 1980–1991 (Chang 1993). complete removal of polycover, only top open like Japanese
Production of the oyster mushroom also involves the main method of bottle cultivation, and slashes or holes in
steps of mushroom growing described earlier: selection of the polycover. Average commercial yields obtained are 1 ton
substrate and its pretreatment, spawn preparation, spawning, fresh weight of mushrooms per ton of dry weight of the
incubation for spawn-run, and providing conditions for substrate. One peculiar and serious problem with the
fruiting of mushrooms, i.e. cropping. Oyster mushrooms can cultivation of the oyster mushroom is spore allergy, which
be grown on a large variety of lignocellulosic wastes sometimes develops among workers for which facemasks are
depending upon the availability and cost (Poppe 2000) but the generally used during the operations.
cereal straws (wheat and paddy) are the most common
substrates in many countries (Figure 3); cottonseed hulls are
also popular in the United States. Substrate pretreatment is 6.2 Lentinula edodes (Shiitake)
mainly aimed at moistening and so-called pasteurization/
sterilization to give advantage to the mushroom mycelium Shiitake is the second most important commercial mushroom;
and avoid contamination with moulds specially Trichoderma it contributed 25.4% of total mushroom production in 1997
spp. Most commonly used pretreatments are hot water dip, (Table 1). Of late, production of this mushroom has become
pasteurization with steam, chemical pasteurization and steam very popular due to not only its unique taste and flavor but
sterilization (Jandaik 1997). Zadrazil and Dube (1992) have also its unique medicinal properties, such as antitumor,
described a method for special substrate preparation for oyster hyocholesterolemic, and antiviral properties; Lentinan, a
mushroom. Pretreated substrate is mixed with grain spawn at polysaccharide, is now an established immunomodulator
2 per cent by wet weight of the substrate and then filled in (Mizuno 1995); Though Japan is the leading producer of
suitable containers, most commonly polybags. Bottle shiitake its cultivation first started in China near 1100 AD,
cultivation of the oyster mushroom is done in Japan. In and the technology was perhaps passed on to Japanese
some countries delayed-release nutrients, mostly formal- growers by the Chinese.
dehyde-treated or polymer-coated soybean meal, is added in Cultivation technology of the L. edodes has been described
the substrate to increase the yield but the method involves risk in detail by various authors (Harris 1986; Royse 2001).
of rise in bed temperature and contamination with moulds. Traditionally shiitake has been grown on natural logs of
Growth parameters for cultivation of Pleurotus differ from various species of trees but currently oak (Quercus) logs are
species to species, especially temperature requirement and most popular. The type, size, and quality of logs used have

Figure 3 Pleurotus florida on wheat straw blocks.

Production of Edible Fungi 241

been described by Royse (2001). Generally, logs of 7 –15 cm (gypsum), and sugar. Ingredients are mixed in a mixer and
dia are cut into 1 m lengths. Holes are then drilled; one row of moistened to a level of 60%. Moistened substrate is generally
holes is drilled for each 2.5 cm of log diameter and are evenly filled in polypropylene bags (2.5 Kg). Little holes are either
spaced length-wise every 15 cm along the row. Holes are made in the polybags or special breather patch made of micro-
plugged with wood piece spawn or sawdust spawn, and then porous plastic is preprovided. The bags are then sterilized for
finally sealed with hot wax; plug spawn is however preferred 2 h at 1218C in large autoclaves and, after cooling, seeded
for varied reasons. Spawn run may take 6 –18 months which with spawn. The bags after heat sealing are shaken to evenly
depends upon many factors. Logs, after the spawn-run, are distribute the spawn; sawdust spawn or cereal grain spawn is
transferred to a growing yard, which should be cooler and used in this system. Spawn-run at 218C with 4 h of light per
humid than the spawn-run area. One interesting treatment day takes 18 –23 days for optimum growth. Colonized blocks
given to induce fruiting in logs is “shocking treatment” where are taken out by slicing and peeling off the polycover and kept
logs are banged with a hammer or dropped on end (Chang and for 4 weeks in the environment conducive for browning of the
Miles 1989). Production is very good during the spring and exterior surface i.e., at temperature of about 198C and
fall. Some growers, however, use green houses for winter 2000–3000 ppm CO2 and are watered once daily; and
production when the prices are considerably higher. In the humidification may also be resorted to. As the browning
green house cultivation technology, logs are generally soaked process nears completion, pinheads start to form about
in water and vibrated mechanically prior to keeping in the 1–2 mm beneath the surface. Primordia development is
houses. After taking the first flush, logs are reincubated for stimulated by soaking the blocks in cool water (128C) for
about 3 months and the process is repeated up to five times. 3–4 h; soaking is required for second and third flushes also.
Yields obtained from log system may be as high as 33%; best Mushrooms are ready for harvesting approximately after
production occurs in second and third years. Shiitake 7–11 days of soaking (Figure 4). Shiitake are harvested by
production drops and is no longer possible after the bark is gentle twisting by hand and stem cut with sharp knife like that
lost. for button mushroom. After harvesting, blocks are soaked
To make the shiitake cultivation more environment- again for 12 h, which may be 18 h in third soaking; flush
friendly, synthetic log production system was developed breaks in shiitake are 16 –20 days long. The total production
where sawdust is the main ingredient; however, straw and cycle on synthetic logs is just 3 –4 months and biological
corncobs are also used. Basal ingredients are supplemented efficiency achieved is also very high (75 –125%) as against
with some starchy substance like cereal brans, maize, and cycle of 5–6 years and B.E. of 33% in natural log cultivation
some chemicals like calcium carbonate, calcium sulfate (Royse 2001).

Figure 4 Lentinula edodes on synthetic (sawdust) block (experimental crop).

242 Rai

6.3 Volvariella spp. (Paddy Straw Mushroom) while the more sophisticated indoor technique is preferred for
industrial scale production on cotton waste compost; the latter
Volvariella is a mushroom of the tropics and subtropics; it gives higher yield but is capital-intensive process. While the
grows at a relatively high temperature of around 358C. This is traditional paddy straw cultivation has been described in
a very fast growing mushroom, it takes about 10 days from detail by several authors (Chang 1982; Khanna 1997; Quimio
spawning to first harvesting, is easiest to cultivate with a et al. 1990), modern indoor cultivation technology under
cropping cycle of 3 weeks but does not give good economic controlled conditions on cotton waste compost will be dealt
yields and its shelf-life is poorest of all mushrooms. here (Chang 1982; Quimio 1993). Since 1973, straw
Understandably, its production in 1997 was only 0.18 million mushroom has been cultivated completely on cotton waste
tonnes and it contributed only 3% to the world mushroom compost in controlled conditions in Hong Kong and the
production while its share was 4.6% in 1986. Nevertheless, its technology is now being practiced in Thailand, Indonesia,
significance lies in East Asian countries where staple food is Vietnam, Singapore, and some parts of Malaysia and
rice and paddy straw can be utilized for growing this Philippines. It takes about 4 days to make compost of cotton
mushroom mostly for self-consumption or trade in the waste mixed with rice bran and water. Compost is filled
domestic market. Commonly cultivated species are (10 cm) on beds in a plastic mushroom house fitted with
V. volvacea and V. bombycina. blower and polyduct and pasteurized with live steam for 2 h at
There are two commercial substrates for growing this 60–628C, conditioned at 50–528C for 8 h and then allowed to
mushroom: traditional paddy straw bundles made into beds cool to 34 –368C suitable for spawning. The amount of spawn
and the other involves the use of cotton waste compost after a used is 1.4% of the dry weight of compost or 0.4% of wet
short period of fermentation. The latter has many advantages weight. Full growth is achieved within 3 –4 days at 32 –348C
(Quimio 1993; Quimio et al. 1990). While outdoor seasonal of room temperature. During spawn-running no water and
farming is still done on paddy straw by small growers, modern light is needed and a little ventilation is provided. Then white
indoor cultivation is done on fermented cotton waste. Grain light is provided with fluorescent lamps and fresh air is
(wheat, sorghum) or cereal straw spawn is used; however, introduced. After removal of plastic sheets and sprinkling
cotton waste þ tea leaves spawn has been found superior with water, growth of other fungi and actinomycetes retards
(Chang 1982). Several techniques have been used for the while V. volvacea continues to grow. On the fifth day after
cultivation of V. volvacea in the tropics, which thrives in the spawning primordia appear, which take about 4 days to reach
temperature range of 30 –358C and R.H. of 75 –85% (Quimio the harvesting stage. Straw mushrooms are not allowed to
et al. 1990). However, the traditional method of cultivation in grow to umbrella like structures but are picked for marketing
South-East Asia is on straw beds, both outdoor and indoor, at the stage before volva enclosing the cap breaks or just after

Figure 5 Proper stage for harvesting of V. volvacea.

Production of Edible Fungi 243

rupture; the former is “button stage” and the latter is “egg The substrate may be composted for up to 5 days or used
stage” (Figure 5). The first flush usually lasts for 4 –5 days; directly after mixing. Compost is prepared by mixing and
four days later second flush starts but yield is just 10% of the watering the ingredients (sawdust 78%, bran 20%; CaCO3,
first flush. For all purposes, only one flush is commercially 1%, sucrose 1%) in a large pile, which is turned twice at 2
taken and in this way, one mushroom house can give two days interval. Directly used substrate mainly has 93%
crops a month or at least three corps every two months. The cotton seed hulls, 5% wheat bran, and 1% each of sucrose
shelf life of Volvariella is very short; it liquefies even at 48C and CaCO3 which is moistened to a level of 60% and then
and also at high temperature. While canning is done at the filled into polypropylene bags and sterilized at 1218C for
industrial scale it is generally traded fresh or dried by small 60 min. Spawning is done in cooled substrate either with
farmers in the developing countries. grain or sawdust spawn, manually or mechanically. Bags
are incubated at 25 ^ 28C for 28–30 days for spawn-run
preferably in dark area (,500 lux). Temperature is then
6.4 Auricularia spp. (Wood Ear Mushroom) lowered to 20 ^ 28C and light intensity increased to
2000 lux to promote pinning for which 5 –8 holes of 2.5 cm
The species of Auricularia, commonly known as wood ear are cut in the bags for emergence and maturation of
mushroom, are morphologically and, above all, texturally mushrooms. Biological efficiency of 100% and above has
quite distinct from other mushrooms. With typical ear like been achieved. In India this mushroom has been
morphology with cartilaginous texture and gelatinous surface, successfully cultivated on wheat straw supplemented with
these are liked as well as disliked at the same time by different wheat bran (Figure 6) and B. E. of as high as 140% has
people. This mushroom is very popular in China and been recorded (Bhandal and Mehta 1989).
Southeast Asia but does not seem to attract western
consumers. It has been reported to possess many medicinal
attributes: treatment of piles, sore throat, anemia and
hypocholesterolemic effect (Quimio et al. 1990; Royse
1997). Out of about 10 recognized species of Auricularia two
main commercially cultivated species are A. auricula and
A. polytricha, the former is thin and light coloured while the
latter is the thicker, longer, hairy, and darker. A. fuscosuccinea
is also produced on a limited scale. Thailand and Taiwan are
the main producers of this mushroom.
Like shiitake (L. edodes), Auricularia are also produced on
natural logs as well as on synthetic logs (sawdust medium),
the latter is more popular, productive and profitable system.
The cultivation of Auricularia on natural logs is popular in the
areas where suitable trees are abundant. This mushroom,
unlike Lentinula, is not fastidious about the type of tree
species and almost any tree except pines can be used. A log
diameter of 3-6 cm and length of 1 m is recommended. The
time of season for log felling is similar to that for Lentinula
log cultivation i.e., when leaves are just starting to dry in
autumn then logs have sufficient sugar and moisture to
support mycelial growth. Holes are made, spawned, and
sealed like that for Lentinula and incubated (20 –288C)
outdoors in the “laying yard” during which logs are turned
upside down once a month. After about 2 months, logs are
transferred to the cropping area which may be an open area in
a forest or a green house or shed where logs are kept upright
and frequently watered; ideal temperature for cropping is
15– 258C which of course depends upon the strain used.
Mushrooms can be harvested about 30 days after exposure to
cropping conditions. During the winters, production ceases
but logs are kept protected for getting fruiting next spring by
resuming heavy watering. The logs should continue to
produce mushrooms year after year unless they become
heavily contaminated with some wood decaying fungi.
In the synthetic log cultivation, the substrate consists Figure 6 Auricularia polytricha on wheat straw blocks (mark
of sawdust, cottonseed hulls, bran, and other cereal grains. the slits).
244 Rai

7 POSTHARVEST TECHNOLOGY OF 8 EDIBLE FUNGI AND RECYCLING OF THE

MUSHROOMS WASTES/RESIDUES

Mushrooms are highly perishable and have short shelf life There are very few wastes of lignocellulosic nature of agro-
ranging from few hours to days depending upon the species forestry origin, which can not be used for growing one or the
and the storage environment. Weight loss, blackening, veil- other mushroom. Poppe (2000) has compiled the information
opening, and microbial spoilage are the common undesirable on various agro-wastes, which have been used for growing
postharvest changes besides many physiological and bio- mushrooms. Residues left after obtaining the main product
chemical changes (Bano et al. 1997; Rai and Saxena 1989a; (e.g., grains, cotton, sugar) pose problems of their disposal
Saxena and Rai 1989). Mushrooms require utmost postharvest and many may prove to be environmental hazards.
care like proper handling, packaging, precooling, cool-chain Cultivation of various edible fungi on these wastes represents
transport, and storage till consumed. Modified atmosphere one of the unique recycling mechanisms where hardly any
packaging (MAP), controlled atmosphere packaging (CAP), residue is left unexploited in one form or the other. The
and modified humidity packaging (MHP) of the button substrate left after growing the mushrooms is though often
mushroom have been described by Anantheshwaran and called “spent mushroom substrate” (SMS), which is a
Ghosh (1997). Individual Quick Frozen (IQF) mushrooms are misnomer and “post-mushroom substrate” (PMS) is a more
gaining popularity with other frozen vegetables in the super appropriate term because it is not “spent” and can be further
markets. decomposed by new set of organisms. Many efforts have been
Postharvest technology of mushrooms has been dealt in made towards profitable utilization of the PMS. The subject
detail by many authors (Bano et al. 1997; Lal Kaushal and has recently been reviewed (Ahlawat and Rai 2002; Levanon
Sharma 1995; Saxena and Rai 1989). Mushrooms are and Danai 1997a). PMS of one mushroom can be utilized for
delicate, contain 90% water, rich in phenolics and have growing other mushroom after suitable treatments. The PMS
very active phenol oxidase and protease activities. They of the oyster mushroom is fed to cattle as feed and the dung
lack protective covering of suberin or cuticle, unlike fresh obtained is used for biogas production; slurry of biogas plant
fruits and vegetables. Postharvest physiological and is used as casing material for the button mushroom and PMS
biochemical changes do take place at very fast rate. of button mushroom including casing fraction is decomposed
Storage in package films, sodium alginate coating, chemical in composting pits to be used as manure for raising the crop
preservation, lactic acid fermentation, irradiation, steeping which was used to grow the primary mushroom, in this case
preservations have been attempted to varied levels of the oyster mushroom (Table 4). In Israel, the above system of
success for short-term preservation of mushrooms. But complete recycling of wastes has been successfully developed
dehydration and canning are the most common forms of and adopted by the growers (Levanon and Danai 1997b).
long-term storage and trade in mushrooms; while canning is PMS, especially of the button mushroom, after suitable
the most common method for the button mushroom, drying treatment like recomposting, has proved very good manure
is resorted to for storage and trade of all the specialty for raising not only the horticultural crops but also the major
mushrooms. Button mushrooms are also freeze-dried in crops; it can also be used for biogas production. As mentioned
limited quantities. However, improvements in solar earlier, the PMS of the oyster mushroom is a very good cattle
dehydration, cabinet tray drying, and tunnel drying feed with improved protein content and digestibility. Post-
technologies of mushrooms are needed to produce better mushroom substrates have also been used for reclamation of
product. Of late, many attempts have been made to produce soil and bioremediation of soil and water (Ahlawat and Rai
value-added mushroom products like pickles, soup powder, 2002). Cultivation of edible fungi has the above mentioned
ready-to-use mushroom curry, nuggets, biscuits, etc.; in economic as well as environmental advantages; it
future we may witness more of such products. represents the production of food (mushroom) feed, fuel

Table 4 Recycling mushroom substrates and wastes

Recycling course Main product Waste/byproduct

Cultivation of cotton/wheat Grains/fibers Wheat and cotton straw
Cultivation of oyster mushroom on straw Oyster mushroom Spent mushroom substrate (SMS)
SMS as cattle feed Meat or milk Manure
Manure for biogas production Biogas (energy) Slurry “Cabutz”
Cabutz as casing soil for button mushroom Button mushroom SMS
SMS composted for organic farming Organic food crops No further waste from SMS
Source: Levanon and Danai (1997a).
Production of Edible Fungi 245

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Chiu S eds. Mushroom Biology and Mushroom Products. Hong
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Chang ST (1999). World production of cultivated edible and
9 CONCLUSIONS medicinal mushrooms with emphasis on Lentinus edodes
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Bhandal MS and Mehta KB (1989). Cultivation of Auricularia Levanon D and Danai O (1997b). Recycling agricultural residues in
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22
Mycoprotein and Related Microbial Protein Products

Juan Ignacio Castrillo University of Manchester, Manchester, United Kingdom

Unai Ugalde University of the Basque Country, San Sebastián, Spain

1 HISTORICAL BACKGROUND rapid development rendered mass microbial food production

uncompetitive against traditional agricultural food crops,
Microorganisms have long been used in the elaboration of which became readily available at comparatively lower
foods for human consumption. Practically, every civilization prices. This fact also led to a progressive decline in Single
has developed fermentation processes of one sort or another Cell Protein research studies in the literature (Ugalde and
as the basis of their culinary traditions. However, the culture Castrillo 2002).
of microorganisms as a source of nourishment, rather than as Notwithstanding this decline, biomass production tech-
food transformers, came with the advent of industrial nologies evolved away from microbial protein as bulk food,
fermentation technology. The first recognition of the value towards new specialities, leading to a new revitalization in the
of surplus brewer’s yeast as a feeding supplement for animals field (Figure 1). Examples of this are a wide range of food
by Max Delbrück (1910) was rapidly followed by the flavors and aromas produced by Burns Philp Food and
production of yeast for food during the ensuing First World Fermentation group (http://www.bpfoods.com), and yeast
War. The novel microbial foods may not have appealed to the products directed to human and animal consumption by
conservative human palate, but this drawback was vastly Lallemand Inc (http://www.lallemand.com). Most notable
outweighed by the logistic advantage of a high productivity of example of the evolution of microbial biomass into new
aerobic fermentations in relatively compact installations. This products is perhaps that led by Rank Hovis McDougall
overriding feature motivated military strategists to draw plans (RHM) in cooperation with Imperial Chemical Industries
to produce large amounts of yeast and fungal biomass during, (ICI), founding Marlow Foods (now part of the AstraZeneca
as well as between, the two World War periods. The cessation group), a company which started producing mycoprotein
of open worldwide hostilities in the second half of the 20th and related products under the commercial trademark
century could have meant an end to interest in microbial Quorne (http://www.quorn.com). This company produces
protein production for food. However, new preoccupations fungal biomass from Fusarium venenatum (formerly
regarding malnutrition in third world countries, or political F. graminearum) in continuous culture and the resulting
and economic isolation, as in the case of the Soviet Union and product is manipulated to achieve a texture and taste which
China, maintained the scope of microbial fermentations as a are reminiscent of meat products, covering a market as a meat
practical solution for the production of food, at least in alternative for vegetarian formulations. The mycoprotein
emergency situations. production process experienced an evolution of 20 years and
Spectacular developments in the field of agriculture, later an estimated Research and Development expenditure of $40
accompanied by important changes in international relations million, before unrestricted clearance by the UK Ministry of
leading to the opening of the world food markets, Agriculture, Fisheries, and Foods was granted in 1985. Quorn
overshadowed the worries of limited food supplies. This products are currently the only fungal-based products

247
248 Castrillo and Ugalde

proteins and protein compounds (Halász and Lásztity 1991;

Ugalde and Castrillo 2002), only a few of them are being
actually used in industrial processes at the production scale
(Table 1). The Quorn mycoprotein process is the main
microbial protein production project directed to human
consumption to date. Thus the Quorn project will be alluded
in this review as the general example. Other projects will be
specifically referred to.

2.1 Mycoprotein as Food

The microbial food product of the early 21st century does

not base its market success solely on account of its protein
Figure 1 Number of food patents including “mycoprotein” or content, as vegetable proteins sources are abundant in the
“yeast” in their title or abstract. Data from Esp@cenet network market at competitive prices. The resemblance in texture to
worldwide database (http://ep.espacenet.com). that of currently appreciated foods, and a bland taste and
light color, which renders it susceptible to the addition of
flavoring and coloring agents, are also a prerequisite. The
exclusively directed at human consumption in the market. In
filamentous nature of the organism, a feature, which was
this review, we intend to highlight the most important
considered as a technical difficulty for production at first,
developments that have taken place in the production and use
was foreseen as an advantage in the case of Quorn,
of mycoprotein, defined here as “microbial protein produced
rendering the final product a resemblance to animal or fish
from microscopic fungi,” with mention of other specialities.
meat. Rarer features, but by no means less important ones,
The study includes relevant examples already present in the
such as those favoring health in normal humans, or indeed
market and others, which have only been registered as
rendering beneficial effects in patients with high blood
patents. We will also conduct an exercise in determining what
cholesterol levels or diabetes are certainly important, as will
future lay ahead for mycoprotein and related products.
be discussed below.

2 MYCOPROTEIN AS FOOD FOR HUMAN 2.1.1 Composition

CONSUMPTION Although fungal biomass can be considered principally as a
source of protein, it also contains nucleic acids, carbohydrate
Although a number of species has been reported to present cell wall material, lipids, minerals, and vitamins. These
favorable characteristics for the production of microbial contributions are generally considered of little relevance, with

Table 1 Yeasts and filamentous fungi species accepted for production of protein
compounds and food ingredients for the food industry

Yeasts S. cerevisiae (baker’s yeast)

S. cerevisiae (brewer’s yeast)
C. utilis (Torula yeast)
K. marxianus (formerly K. fragilis, S. fragilis)
K. marxianus var. lactis (K. lactis, formerly S. lactis)
C. pseudotropicalis
P. pastoris
P. rhodozyma
Filamentous fungi F. venenatum (formerly F. graminearum) (Quorn products)
P. variotii (Pekilo process, discontinued)
A. niger; A. oryzae
R. niveus; R. oryzae
Mucor spp.
Streptomyces spp.
P. roquefortii
Data obtained from Halász and Lásztity (1991), Peppler (1983) and Centre for Food Safety and Applied
Nutrition (CFSAN) and US Food and Drug Administration (FDA) (http://www.cfsan.fda.gov).
Mycoprotein and Related Microbial Protein Products 249

Table 2 Daily requirements (g) of essential amino acids for the digestibility of various protein sources. Thus, empirical tests
human adult are usually required for feed evaluation, and the parameters
used are: the total quantity of microbial nitrogen ingested (I),
Essential amino acids FAO recommendation Minimum the nitrogen of faeces (F), and nitrogen in urine (U). From
these parameters, Digestibility, Biological Value (BV), and
Phenylalanine 2.2 1.1
Protein efficiency (PE) can be calculated. Thus, Digestibility
Methionine 2.2 1.1
(D) is the percentage of the total nitrogen consumed, which is
Leucine 2.2 1.1
absorbed from the digestive tract.
Valine 1.6 0.8
Lysine 1.6 0.8 D ¼ 100 £ ðI 2 FÞ=I ð1Þ
Isoleucine 1.4 0.7
Threonine 1.0 0.5 Biological Value (BV) is the percentage of the total nitrogen
Tryptophan 0.5 0.25 assimilated, which is retained by the organism, taking into
Total 12.7 6.35 account the simultaneous loss of endogenous nitrogen
through excretion in urine.
Data retrieved from FAO (http://www.fao.org).
BV ¼ 100 £ ðI 2 ½F þ U
Þ=ðI 2 FÞ ð2Þ
Protein Efficiency (PE) is the proportion of nitrogen retained
the exception of nucleic acids, which account for 10 –15% when the protein under test is fed and compared with that
(w/w) of the total nitrogen. Approximately 80% of total retained when a reference protein, such as egg albumin, is fed.
fungal nitrogen is composed of essential amino acids required Protein from yeast biomass such as Candida utilis has
for human growth and nutrition (Table 2). With reference to presented high digestibility values (81%), and BVs between
egg albumin, which is considered a perfectly balanced source 32 and 48%. Both parameters can be substantially increased
of essential amino acids for human nutrition, mycoprotein when supplementation with 0.5% methionine is implemented
presents a similar composition, although it is lower in sulfur (w/w, 90% in both cases). The BV of yeast has been estimated
containing amino acids. On the other hand, it is relatively rich to be of 0.9, and methionine supplementation increases it to a
in lysine and threonine if compared to other traditional protein value of 2.3 (Rivière 1977).
sources of agricultural origin, such as wheat. A comparison Quorn mycoprotein presents very high PE values, reaching
with a wide range of protein sources is provided in Table 3. 75% with respect to egg albumin. In experimental tests where
mycoprotein was supplemented with 0.2% methionine, this
2.1.2 Protein Value value rose to 100%. Quorn products are not supplemented
with methionine, but egg protein as explained below. Thus,
The amino acid composition is only a theoretical indicator of mycoprotein could be used as a total replacement for the
the protein value of foods, since the degree of digestion and human diet (Trinci 1992, 1994; US Patent 5935841 1999; WO
absorption of any one substance is determined by its Patent 9117669 1991).
susceptibility to be degraded and absorbed. The presence of Toxicity testing of Quorn mycoprotein has shown that the
inhibitors and multiple other factors in foods also modify their product can be consumed as the sole source of protein on a
nutritional value. On the other hand, the digestive system of continued basis, without any adverse effects. Given the
the organism in question is a key determinant in the unconventional nature of this product, the tests undertaken for

Table 3 Essential amino acid content (g per 100 g edible portion) of mycoprotein (Quorn), baker’s yeast, egg (whole raw fresh), beef
(ground, regular baked-medium), soybeans (mature seeds, raw) and wheat (Durum)

Amino acids Myco protein Baker’s yeast Egg Beef Soybeans Wheat
Histidine 0.39 0.22 0.30 0.78 0.98 0.32
Isoleucine 0.57 0.48 0.68 1.02 1.77 0.53
Leucine 0.95 0.67 1.10 1.80 2.97 0.93
Lysine 0.91 0.69 0.90 1.89 2.43 0.30
Methionine 0.23 0.17 0.39 0.58 0.49 0.22
Phenylalanine 0.54 0.41 0.66 0.89 1.90 0.68
Tryptophan 0.18 0.11 0.15 0.26 0.53 0.18
Threonine 0.61 0.44 0.60 0.99 1.58 0.37
Valine 0.60 0.51 0.76 1.11 1.82 0.59
Data from US Department of Agriculture, Agricultural Research Service. USDA Nutrient Database for Standard Reference, Release 14 July 2001. Nutrient
Data Laboratory (http://www.nal.usda.gov/fnic/foodcomp) and P. Collins, Marlow Foods, UK.
250 Castrillo and Ugalde

its approval were especially thorough, lasting ten years. 2.1.5 Additional Functionalities
Human trials involved 2500 people with no adverse effects.
The mycoprotein product is approved for consumption in the In addition to the nutritional effects, consumption of
European Union. The U.S. Food and Drug Administration is mycoprotein under both controlled and free-living studies
currently in the process of publishing an official response to has been shown to beneficially reduce total and low density
the Full Food Additive Petition submitted by the manu- lipoprotein (LDL) serum cholesterol levels. Studies by
facturers. This response will be made public in 2002 (FDA, Homma et al. (1995), Turnbull et al. (1992), Udall et al.
http://www.fda.gov, GRAS Notice No. GRN 000091). (1984) incorporating realistic amounts of the product
Products containing mycoprotein have been on sale in the concluded that mycoprotein consumption has a beneficial
United States since January 2002. effect in serum lipid variables. Post-meal glycemia has been
shown to be reduced after consumption of mycoprotein, by
13% with respect to controls. On the other hand, insulinemia
2.1.3 Nucleic Acid Content is reduced by 19% thirty minutes after ingestion (Turnbull
Nucleic acids are a necessary component of all cells, and are and Ward 1995). Finally, a mycoprotein lunch has a
found in relatively high levels in rapidly dividing cells. Thus, significant effect on the sensation of satiety, in ways that
the nucleic acid content of yeast (around 10% of dry weight) would help control the appetite of dieting patients, as
is approximately five times greater than in the average proposed by Burley et al. (1993), Turnbull et al. (1993).
mammalian organ. When nucleic acids are ingested, they are
first attacked in the duodenum by pancreatic nuclease. The
resulting nucleotides are then attacked by nucleotidases in the 2.2 Mycoprotein Production (Quorn Products)
intestine, resulting in nucleosides and phosphate. These in
turn are further degraded to purine and pyrimidine bases. The In order to ensure that Quorn is produced according to
degradation of purine bases in man results in the production of approved health regulations, the fermentation process is
uric acid. Accumulation of uric acid beyond the excretion currently supported on glucose (nearly all of which is
capacity of the kidney results in the formation of crystalline obtained from maize) and approved inorganic medium
deposits in the joints and soft tissues, leading to goutlike constituents and vitamins. However, the process may also
manifestations and calculi in the urinary tract. Pyrimidines are be applied with various sources of starch as the carbon source
degraded to orotic acid, the accumulation of which results in
liver damage. The administration of foods of microbial origin
is limited by the amount of nucleic acid contained within. The
administration of 130 g of yeast daily for one week results in
uric acid levels ranging between 4.8 and 8.3 mg/100 ml in
human volunteers. Normal plasma levels of uric acid range
between 2 and 7 mg/100 ml in males (Rivière 1977).
Quorn mycoprotein is obtained from a filamentous fungus
which proliferates at slower rate than yeast (Trinci 1994;
Ugalde and Castrillo 2002). Thus, the starting nucleic acid
content subject to removal is also slightly lower (8 –9%). The
RNA content reduction of mycoprotein is further effected by
a heat shock treatment that will be described below [see
section “Mycoprotein production (Quorn products”)]. RNA
levels are thus reduced well below the levels which limit
consumption to 100 g per day per person (2% of dry weight),
although this treatment also results in important losses in dry
weight (Trinci 1992).

2.1.4 Texture
Another favorable feature which differentiates Quorn
products is the advantage taken from the filamentous nature
of the microorganism in product design. Fusarium venenatum
A3/5 filaments are aligned in parallel by a specially designed
mechanical process which renders the product a texture very
similar to that of meat fibers once set in a light matrix of egg Figure 2 Diagramatic representation of the Quorne air-lift
white protein and heated. The final product has a bland taste, fermenter used by Marlow Foods for the production of
light color which renders it susceptible to the addition of mycoprotein in continuous flow culture, (Courtesy: P. Colllins,
flavoring and coloring agents (Anderson et al. 1975). Marlow Foods, UK).
Mycoprotein and Related Microbial Protein Products 251

(Anderson et al. 1975; Steinkraus 1986). The production of Studies on the incidence of the production phase duration
Fusarium venenatum strain A3/5 takes place in turbidostat on product cost and commercial viability had indicated that
culture using air-lift fermenters of 155 m3 in volume and 50 m periods above 200 h operation can be necessary to result in
tall, weighing over 250 tons each. The Quorn fermenters are consistent unit costs of production (Trilli 1977).
the largest operating air-lift fermentation facility in the world In principle, a continuous culture may run indefinitely, as
to date (Marlow Foods communication 2002). long as contamination is kept under check. However,
Each fermenter operates as a loop where culture medium is cultivation beyond 100 generations (about 400 h) of
circulating (Figure 2). As the liquid flows through the bottom F. venenatum may result in the appearance of highly
of the loop, air is pumped in. Circulation is induced by a rising branched colonial mutants which can alter the texture of the
column of air bubbles providing good oxygen transfer final product (Wiebe and Trinci 1991; Wiebe et al. 1992;
conditions. This circulation is maintained due to mean density Wiebe et al. 1995).
difference between riser and downcomer. The bottom of the The details behind the appearance of mutations in
downcomer hosts the glucose, biotin, and mineral salts intake. continuous culture, with specific reference to F. venenatum
The nitrogen supply is delivered separately in the form of strain A3/5 have been studied by external research
ammonia along with sterile air at the base of the riser. The independent to the production process (Trinci 1992; Trinci
supply of ammonia to the culture is regulated by a pH monitor 1994). From these studies, the authors obtained the
set to give a culture pH of approximately 6.0. The dilution rate conclusion that spontaneous mutant appearance may be
of the process ranges between 0.17 and 0.2 h21 and is managed by careful manipulation of the selective pressure
operated so that glucose is always in excess and the fungus imposed through culture conditions. The above-described
always grows at mmax at a biomass concentration of 10 to strategies however have been successfully tested in laboratory
15 g L21 (Trinci 1994). The culture is kept at a temperature of conditions. These strategies do not conform to the industrial
approximately 308C by a heat exchanger set into the riser. production process and are not necessarily used commercially
The dilution rate of the fermenter results in an output of (Marlow Foods communication 2002).
30 tons of liquid per hour. Harvesting by filtration and RNA
reduction ensues. The harvested biomass (Figure 3) contain-
ing 8-9% (w/w) RNA is heated to 688C for 25 min at a pH of 2.3 Related Products
5–6. This results in the reduction of RNA to ca. 1% (w/w), at
the expense of losing up to one third of the total mass, Although there is a high variety of microbial protein
including dissolved salts, RNA, internal water, carbohydrates, formulations which can be used as food ingredients, apart
and protein. from the major Quorn mycoprotein example a number of

Figure 3 Electron micrograph of F. venenatum A3/5 as collected from the outlet of the Quorne fermenter, illustrating the filamentous
nature of mycoprotein, (Courtesy: P. Collins, Marlow Foods, UK).
252 Castrillo and Ugalde

mycoprotein products are accepted and being used directly as are used as starters in the baking industry and in other
food for human consumption (FDA, http://www.fda.gov; traditional fermentation industries where the leavening
Halász and Lásztity 1991). Primary grown yeasts for use in activity, and ability to ferment different raw materials, is
human food are feed yeasts, inactive (nonfermentative) yeast necessary to improve storability, taste and flavor. At this
cells, classified as “Primary Dried Yeasts” (category 96.1, moment, companies such as Beldem (http://www.beldem.
Official Publication AAFCO 2002; Peppler 1983). The FDA com), subsidiary of the Puratos group, manufactures active
admits the utilization of dried yeast from S. cerevisiae, dry yeast for use in bakery and Lesaffre group (http://www.
K. marxianus, and dried torula yeast (C. utilis) for human lesaffre.com) and Lallemand Inc (http://www.lallemand.com)
consumption provided that the total folic acid content is not commercialize a wide range of sourdough starters and yeast
greater than 0.4 mg per g of yeast. Dried yeast derived from products for the baking, brewing and wine industry.
S. cerevisiae is also accepted for use in bakery products (Code Yeast culture is the only officially defined feed yeast
of Federal Regulations, CFR. http://www.access.gpo.gov/ ingredient which is not composed of isolated dried yeast cells
nara/cfr/index.html; Title 21. Sections 172.325 and 172.896; only. Yeast culture consists mainly of growth medium with a
Halász and Lásztity 1991). moderate content in crude protein. It is being used as an aid to
Lallemand Inc (http://www.lallemand.com) produces the ensiling process (lactic acid formation by naturally present
inactive dry yeast (baker’s yeast) as dough conditioner. bacteria), which increases feed value and improves the quality
Moreover, dry yeasts and filamentous fungi containing extra and palatability of the resulting silage, but the greater
contents of minerals or vitamins are being used to meet portion is used to fortify feeds and feed concentrates
specific nutritional requirements. Thus, Diamond Vw and the administered to livestock and poultry (Peppler and Stone
Chinese Academy of Agriculture report the use of edible 1976; Peppler 1983). As an example, Diamond Vw
selenium enriched yeast and fungi (http://www.diamondv. produces active yeast cultures for fermenting liquid and
com; CN Patent 1121531 1996), Lallemand Inc and Burns cereal grain raw ingredients for animal feeds including
Philp (http://www.bpfoods.com) produce mineral and vitamin dairy, beef, horse, poultry, aquaculture, and pet foods.
enriched yeasts for human nutrition, and Kohjin Co Ltd Yeast and fungi active cultures are also approved for
(http://www.kohjin.co.jp) and Universal Foods (subsidiary of production of commercial enzymes for the food industry. The
Lesaffre, http://www.lesaffre.com) have registered patents for FDA accepts the use of, among others: amyloglucosidase
production of iron, selenium, glutathione, and zinc enriched derived from Rhizopus niveus; carbohydrase from R. oryzae;
yeasts (US Patent 4530846 1985; JP Patent 9248179 1999; JP lactase enzyme preparation from K. lactis and chymosin
Patent 2000279164 2000). Finally, an example of production preparation from K. lactis or A. niger. New generally
of a cultured yeast product for human and animal recognized as a safe (GRAS) fungi and yeasts are
consumption from K. marxianus has been registered by continuously under study with the objective of incorporation
DMV USA (http://www.dmv-international.com/dmvusa) of new fungal enzymes into the food industry (Saxena et al.
subsidiary of Campina International (http://www.campina. 2001; Wolf 1996).
com) (US Patent 5486368 1996; EP0643765B1 2001).

3.2 Inactive Formulations

3 MICROBIAL PROTEIN PRODUCTS AS
FOOD INGREDIENTS Inactivated biomass from yeasts and fungi can be processed
into a variety of different valuable products serving as
A multiple number of special products derived from baker’s ingredients for animal and human consumption. Due to their
and brewer’s yeast have been developed in order to nucleic acid content and cell wall compounds which may
complement the properties of other foods, rather than as cause undesirable effects, the main strategies entail cell lysis
major protein sources. Since the techniques for production of and extraction of the components, mainly proteins, and
yeast are well established (Peppler and Stone 1976; Ward separation and concentration for the production of concen-
1992), most of the new developments deal with post- trates and isolates. These can be used directly (e.g., Beldem
production treatments of the biomass (Halász and Lásztity produces inactive yeast for use in animal feed) or processed in
1991). This aspect will be covered in detail in this chapter, order to get extracts, autolysates, and protein hydrolysates of
therefore. specific functional properties.
Use of yeast protein concentrates and isolates in food
processing is recommended in many scientific papers due to
3.1 Active Formulations their favorable nutritional properties. However, due to the
relative high cost of protein isolate production the use of these
The major microbial products used as feed additives are the formulations is limited to meat products, meat, and milk
two yeast categories approved to maintain their fermentative substitutes.
capacity, “active dry yeast” (96.2) and “yeast culture” (96.8) Yeast and fungi autolysates and hydrolysates are obtained
(Peppler 1983; Official Publication of Association of by hydrolysis of the proteic fraction. These can be used as
American Feed Control Officials 2002). Active dry yeasts natural flavors under the definition of the FDA and Code of
Mycoprotein and Related Microbial Protein Products 253

Federal Regulations (21 CFR 101.22(3)) and to improve the although experience shows that the standard levels fit well
physical properties (texture, emulsifying properties) and below these limits. In products of fungal origin, chemical
nutritional value of food products (Halász and Lásztity 1991). analysis of absence of mycotoxins is considered essential
Thus, yeast autolysates and hydrolysates are used as flavor (Scrimshaw 1985; Stringer 1985). (3) Pathogenicity. The
enhancers in sausage, meat, and cheese manufacture (Halász potential pathogenicity of a microorganism used for feeding,
and Lásztity 1991). Examples of these applications are: the has been evaluated by the injection of the viable organism
production of Aspergillus oryzae extracts by Diamond V, into the body cavity or body fluids of a mammalian species.
used as a livestock feed, the autolyzed yeast flavor In this way the nonpathogenicity of a large number of
ingredients, Provestaw flavors from torula yeast, and Ohlyw microorganisms (S. cerevisiae, C. utilis; C. maltosa,
yeast extracts manufactured by Burns Philp (http://www. C. lipolytica, and Torulopsis) has been evaluated (Stringer
bpfoods.com) for use in different food systems, sauces, 1985). (4) Integrity of the original strain. The maintenance of
soups, marinades, and many other food types. Lallemand the integrity of the original strain and absence of undesirable
Inc, Lesaffre group, and Beldem also produce a complete contaminants has to be proved by specific microbiological
range of yeast extracts for animal feed and as flavor enhancers and biochemical tests (Anonymous 1983c). (5) Continuous
for application in animal and human nutrition. At this monitoring and control of process variables. To ensure quality
moment, the studies are mainly focused on new developments and uniformity of the product the process variables
and more tailored protein formulations whose acceptance by (temperature, pH, aeration, cell concentration) have to be
the consumer will depend on their functional characteristics. carefully monitored, and proof of such monitoring has to be
presented. (6) Nucleic acid content. For animals or humans,
nucleic acids coming from microbial protein sources added to
the daily diet should not exceed 2 g per day (Anonymous
4 SAFETY AND QUALITY CONTROL OF 1975; Scrimshaw 1985). This restriction does not apply for
MICROBIAL PROTEIN PRODUCTS nonprimate mammalian species or other vertebrates (Stringer
1985). (7) Toxicological and biochemical studies in animals.
The safety and acceptance of microbial protein products has The required procedures for testing toxicology of microbial
to be strictly controlled, as in the case of all foods. The protein products are similar irrespective of whether the
concepts underlying the acceptance and safety of novel product is intended for animal and human feeding (Stringer
protein sources including mycoprotein products were 1985). First, the tests include short and long-term classical
developed between 1970 and 1974 by the Protein Advisory animal toxicology tests, with rodents and other target species
Group of the United Nations (PAG), initially based on (e.g. broiler chicken and pig) and the species that will finally
established toxicological experience. During the 70s the consume the product in its diet (Stringer 1985). They consist
experience was accumulating with the incorporation of an of a complete battery of biochemical studies including effects
increasing number of preparations of microbial origin, with on gross pathology, organ weight, and histopathological
more extensive trials in experimental and farm animals and examination of the main organs and tissues (Anonymous
preclinical and clinical testing (Scrimshaw 1985; Stringer 1983a). (8) Tolerance studies in humans. Clinical trials.
1985). The main concepts contemplated in these guidelines Tolerance studies are used to determine the acceptance and
(Anonymous 1975; Anonymous 1983a,b,c; Scrimshaw 1985), the frequency of allergic and other undesirable reactions. The
which define the acceptance and quality control of microbial most common symptoms to be studied are gastrointestinal
protein products are outlined below. intolerance, skin rashes, and presence of other allergic
(1) GRAS status organisms. Generally recognized as safe reactions (Anonymous 1983b). The trials are conducted with
(GRAS) status may be granted to an organism by qualified a number of 50 or 100 individuals randomly assigned to
experts either on the grounds of: (a) scientific studies which experimental and control groups, stratified by sex, with
confirm absence of toxic compounds and non-pathogenicity, careful report of all significant effects, symptoms, changes in
and (b) in the case of a substance used prior to January 1, mood, appetite, libido or sleep patterns, monitored in a diary
1958, through experience based on common use in food along two sets of four-week study periods (Scrimshaw 1985;
(Code of Federal Regulations, 21 CFR 170.30, 182, 184, and Udall et al. 1984). The official recommendations and
186). (2) Absence of health hazards and toxic compounds in guidelines of the PAG, part of them which have been briefly
the product. No living cells derived from the fermentation described here, are made specific in the form of official
process (original strain or contaminants) can be present in the specifications for each microbial protein product, which are
final product. Medium components that may be health hazards finally referred in the Code of Federal Regulations, Codex
must also be absent. In case antifoams, detergents, or Alimentarius (http://www.codexalimentarius.net), AAFCO
flocculants are used in the fermentation process, they have to and other official publications, which are updated regularly.
comply minimum safety requirements or be removed The final control of the quality of the food product is
completely (Anonymous 1983c; Scrimshaw 1985). Con- regulated by specific official organizations and state food
straints are placed upon the heavy metal content of the final agencies through their respective directives. An example
product. Typical maximum values are: fluoride (F) 150 ppm, of this is the Food and Drug Administration (FDA,
lead (Pb) 5 ppm, arsenic (As) 2 ppm, mercury (Hg) 0.1 ppm, http://www.fda.gov) in collaboration with the Association
254 Castrillo and Ugalde

of American Feed Control Officials (AAFCO) and Centre installations, the inherent safety of the fermentation
for Food Safety and Applied Nutrition (CFSAN, technique, and the recycling potential of the by-products.
http://www.cfsan.fda.gov). The food agencies have to The use of genetically modified fungi for the production of
establish among others, the Compliance Policy Guides, foods is an inspiring subject, given the massive potential that
current Good Manufacturing Practices (cGMP), official DNA technologies have provided for the generation of new
methods and mechanisms of inspection and control to be products. However, the introduction of this technology into
used to evaluate the quality and safety of food products. traditional industries is currently limited by public perception.
As an example of the AAFCO and FDA policy, a An increasing number of research studies are focusing on the
direct-fed microbial product listed by the AAFCO development of genetically modified fungi for application in
Official Publication and not labeled or promoted with the food industries (Dequin 2001; MacCabe et al. 2002), but a
any therapeutic function will be regulated as a “food” very limited number of these studies have emerged in the
and usually will not require FDA regulatory attention. form of new products or patents. Recently, a method of
However, a direct-fed microbial product with claims for production of a fermented beverage using genetically
disease cure, mitigation, treatment or prevention (e.g. a modified yeast strains has been registered (US Patent
dietary product containing a specific bioactive compound) 6326184 2001). However, the majority of food companies
will be considered an “unapproved drug” and required avoid the use of genetically modified raw materials or strains,
for regulatory action, the initial proceeding being a and traditional processes dominate the market. The
warning letter (FDA, Compliance Policy Guide CPG application of these techniques into real products is still
7126.41, Directed-Fed Microbial products). In a global considered as a long term process, which will depend on
perspective, taking into account the existing regulations public acceptance (Uzogara 2000).
and quality control procedures, the compactness and high Our view is that there is a market for protein products of
degree of control achieved in production of microbial fungal origin, aimed at animal and human consumption, with
protein processes, which can be manufactured under the mycoprotein process (Quorn products) as a remarkable
controlled factory conditions in compliance with GMP example. The success of the Quorn myco-protein process is
requirements, may provide a high degree of safety to the determined by the product concept, where the business idea
consumer, against the uncertainties which regularly determines the design and development of process and
surround other food products. product. Similar examples, applied to the production of new
protein products from wild type and recombinant GRAS
strains (Wolf 1996) will result in new processes and high
5 CONCLUSIONS value food products in the future.

Interest in mycoprotein and yeast biomass derived proteins

has shifted since the mid twentieth century from a source of ACKNOWLEDGEMENTS
basic food, to specialty products which act as meat
substitutes. This has been successfully achieved, and appears
to be an expanding line. In addition, other products derived We thank Dr. P. D. Collins (Marlow Foods Ltd, UK) for the
from biomass have been developed as food additives, which provided information and comments on the first drafts of this
enhance the functional properties of other food-base review. All our experience in this field was obtained through
formulations, such as flavor, emulsifying properties, and the development of research and industrial projects which
texture. This tendency is likely to be reinforced in the were funded by Gipuzkoako Foru Aldundia/Diputación Foral
foreseeable future, as the risk of new food episodes emerging de Gipuzkoa, and the Basque Government, to whom we are
from uncontrolled industrial production practices, such deeply grateful.
as Bovine Spongiform Encephalopathy (BSE), variant
Creutzfeldt-Jakob Disease (vCJD) or dioxin contaminations
appear. In addition, some appreciated food stocks such as fish, REFERENCES
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23
Genetic Variability of Yeast in Wine Fermentation

Amparo Querol / M. Teresa Fernández-Espinar / Eladio Barrio University of València,

València, Spain, Instituto de Agroquı́mica y Tecnologı́a de Alimentos, CSIC, València, Spain

1 INTRODUCTION pulcherrima is often present, followed by a group of film-

forming yeasts (Pichia anomala) or pigmented species
The first use of yeast for winemaking is lost in the dawn of the (Rhodotorula sp.). In a general study of yeasts isolated from
first agricultural civilizations. There are reports of wine- grapes, however, it was noticed that the profile of yeast
making as far as 7400 years ago. Until middle of the last species may also vary from region to region (Martini and
millennium, wines were mainly produced around the Vaughan-Martini 1990). Numerous factors affect the total
Mediterranean Sea and the Caucasus. Since then, winemaking yeast population and the relative proportions of individual
spread with the European colonizers throughout the temperate species on the grapes. These factors include climatological
regions of the world (Pretorius 2000). Until 1863, must conditions, the grape variety and the degree of maturity at
fermentation was not well understood from a microbiological harvest, the use of fungicides and the physical damage of
point of view. In that year, Pasteur showed that a living the grapes (Fleet and Heard 1992). The yeast diversity
microorganism, the yeast, was responsible for the biotrans- found in wine-producing regions is strongly related to the
formation of the sugar present in the must into ethanol and quality and organoleptic characteristics of the wine
CO2. Although many genera and species of yeasts are found produced from one year to another. However, the most
in the musts, the genus Saccharomyces, and mainly the significant finding was that S. cerevisiae is practically
species S. cerevisiae, is responsible for its biotransformation. absent from grapes and vineyard soils (Martini and
Because of this, S. cerevisiae is referred as “the wine yeast” Vaughan-Martini 1990). The presence or absence of
(Pretorius 2000). S. cerevisiae on grapes is the subject of some debate.
Some authors propose that this species is a “natural”
organism present in plant fruits (Mortimer and Polsinelli
2 YEAST BIODIVERSITY IN WINERIES 1999; Sniegowski et al. 2002). Others argue that it is an
artificial species that originated from the hybridization of
2.1 Yeast Species Diversity During Vinification other Saccharomyces species, and which then evolved over
several centuries to become fully adapted to man-made
All the investigations carried out on grape surfaces by direct environments such as wineries (Martini 1993). Finally,
isolation (without enrichment) have constantly shown that some other authors postulate that S. cerevisiae is a
Hanseniaspora uvarum (anamorph: Kloeckera apiculata) is domesticated species that originated from its closest
always the predominant inhabitant of the grape surface (75% relative, S. paradoxus, a wild species found all around the
of the cells); this numerical supremacy may explain its world that is associated with insects, tree exudates, and
initial domination in natural fermentations. Metschnikowia fermenting plant extracts (Naumov 1996).

257
258 Querol et al.

2.2 Enzyme Activities of Non-Saccharomyces cases could lead to an incorrect classification of a species or a
Wine Yeasts false identification of strains. Methods based on the analysis
of total cell proteins (Guillamón et al. 1993) and long-chain
With respect to the role played by these non-Saccharomyces fatty acids using gas chromatography have been used
species in wine quality, they are known to have the capability (Augustyn et al. 1991), but their reproducibility is question-
to improve the wine aroma (Charoenchai et al. 1997; Esteve- able because they depend on the physiological state of the
Zarzoso et al. 1998). The available aromas in the grape impart yeast cells (Golden et al. 1994).
and define the characteristics and the final quality of wine. Recent progress in molecular biology has led to the
Terpenic compounds account for most of these aromas. Grape development of new techniques for yeast identification and
processing liberates small quantities of aromatic terpenols; characterization (see Table 2). Most of these techniques are
however, odorless precursors in the grape present a large, useless for the routine identification of yeasts and, most
untapped reserve for wine aromas. Not only is the aroma an importantly, there is no available database to identify a large
important quality factor in wine but also the intensity of the number of species. In addition, most of these techniques were
color is another very important quality factor in red wine, applied to characterize only a few genera, and cannot be
where anthocyanins are the main pigments. Various enzyme considered as a general method for yeast identification. In the
activities can improve the process of winemaking and next sections, we review those methods that have been
enhance wine quality. The yeasts involved in wine making preferably applied in recent years and their application to the
could be important producers of these enzymes (for a review rapid identification or specific characterization of wine yeasts
see Table 1). in industrial practice.

3.1 Methods for Yeast Identification

3 MOLECULAR METHODS: INTER- AND
INTRA-SPECIFIC VARIABILITIES The specificity of nucleic acid sequences has prompted the
development of several methods for rapid species identifi-
The use of active dry yeasts is of particular interest to the wine cation. The comparison of RNA (rRNA) and its template
industry, since the sensory properties of the final product vary ribosomal DNA (rDNA) has been used extensively in recent
considerably from one year to another depending of the years to assess both close and distant relationships among
microbial flora present on the grapes (Querol et al. 1990). It is many kinds of organisms as a result of the existence of
generally assumed that indigenous yeasts are suppressed by conserved sequences within this region, and the concerted
the starter; however, different studies show that indigenous evolution among units (Kurtzman and Robnett 1998). Using
yeasts can still participate in the fermentation (Schütz and the information from this region, different methods have been
Gafner 1993; Querol et al. 1992a), although an implantation developed for yeast species identification.
of only 50% was observed when fermentations were
conducted by some commercial strains (Esteve-Zarzoso 3.1.1 Ribosomal RNA Gene Sequencing
et al. 2000). For these reasons, rapid and simple methods for
the routine verification of yeast strain present in fermentations The determination and comparison of nucleotide sequences
would be useful to check the implantation of the starter. for the purpose of recognizing similarities or differences
Classical taxonomy is based predominantly on phenotypic between organisms is at the heart of any molecular approach.
characteristics, such as metabolic and/or cell morphology Therefore, choosing the appropriate gene is paramount to the
(Kurtzman and Fell 1998). However, nutritional character- success or failure of any such sequence analysis between
istics have been shown to be highly variable as well as species. The 26S, 18S, 5.8S, and 5S ribosomal genes have
mutable, and genetic crosses have linked the characteristics to undergone a relatively slow evolution, allowing for their use
one or only a few genes (Petersen et al. 2000), which in some in the comparison of distantly related organisms. Work has

Table 1 Main enzymatic activities described in non-Saccharomyces wine yeasts

Enzymatic activity Yeasts References

Protease Candida, Kloeckera, Pichia Charoenchai et al. 1997
b-Glucosidase Candida, Debaryomyces, Hanseniaspora, Hansenula, Kloeckera, Grossmann, et al. 1987; Rosi et al. 1994;
Kluyveromyces, Metschnikowia, Pichia, Saccharomycodes, Manzanares et al. 2000
Schizosaccharomyces, Zygosaccharomyces
Esterase Brettanomyces, Debaryomyces, Rhodotorula Besançon et al. 1995
Pectinase Candida, Cryptococcus, Kluyveromyces, Rhodotorula McKay 1990
Lipase Candida Charoenchai et al. 1997
Genetic Variability of Yeast in Wine Fermentation 259

Table 2 Studies about wine yeast identification using molecular biology techniques

Methodology Genus References

AFLP Saccharomyces de Barros Lopes de et al. (1999)
d elements Saccharomyces Ness et al. (1993)
Intron splice site Saccharomyces de Barros Lopes de et al. (1999)
Karyotype Saccharomyces Cardinali and Martini, 1994; Guillamón et al (1996);
Ibeas et al. (1997); Martı́nez et al. (1995)
Hanseniaspora Schütz and Gafner (1994)
Zygosaccharomyces Török et al. (1993)
Microsatellite Saccharomyces Baleiras Couto et al. (1996)
Nested-PCR Brettanomyces Ibeas et al. (1996)
Plasmids Saccharomyces Pearson and Mckee (1992)
Zygosaccharomyces Pearson and Mckee (1992)
RAPDs Saccharomyces Baleiras Couto, (1996); Quesada and Cenis (1995)
Metschnikowia Lopandic et al. (1996)
Rhodotorula Quesada and Cenis (1995)
Zygosaccharomyces Baleiras Couto et al. (1994)
Candida Quesada and Cenis (1995)
Picchia Quesada and Cenis (1995)
Torulaspora Quesada and Cenis (1995)
Hansenula Quesada and Cenis (1995)
RFLP-karyotype Candida Versaud and Hallet (1995)
Kloeckera Versaud and Hallet (1995)
Schizosaccharomyces Versaud and Hallet (1995)
RFLP-mtDNA Saccharomyces Constantı́ et al. (1997); Guillamón et al. (1996);
Ibeas et al. (1997); Martı́nez et al. (1995);
Querol et al. (1992a); Querol et al. (1994)
Kluyveromyces Belloch et al. (1997)
Zygosaccharomyces Guillamón et al. (1997)
Brettanomyces Ibeas et al. (1996)
RFLP-ITS/5.8S rRNA gene Candida Constantı́ et al (1997); Guillamón et al. (1998)
Hanseniaspora Guillamón et al. (1998)
Saccharomyces Guillamón et al. (1998)
25 different genera Esteve-Zarzoso et al. (1999)

focussed primarily on the D1/D2 domain of the 26S rDNA database has been improved to identify 300 yeast species
(Kurtzman and Robnett 1998) and on the 18S subunit (James (http.//motor.edinfo.es/iata). Using this method, it is possible
et al. 1997). The techniques have gained substantially from to identify yeasts from isolated colonies or directly from food
the introduction of the polymerase chain reaction, direct samples (see Figure 1). In addition, the anamorph and
sequencing, and the availability of numerous nucleotide teleomorph forms yielded the same patterns and all the strains
databases, which contain sequence information on a diverse of the same species also exhibit the same pattern. Guillamón
range of organisms. et al. (1998) used this method to identify 33 wine yeast
species and Fernández-Espinar et al. (2000) identified all the
3.1.2 Restriction Fragment Length Polymorphism species of the genus Saccharomyces, including the flor yeast
of RDNA responsible for biological ageing in the process of making
“fino” sherry wine. The flor S. cerevisiae strains exhibited
Recently, Esteve-Zarzoso et al. (1999) proposed a rapid and restriction patterns different from those typical of the species
easy method for routine yeast identification, based on RFLPs S. cerevisiae, due to the presence of a 24-bp deletion located
of the 5.8S rRNA gene and the internal transcribed spacers in the ITS1 region. Esteve-Zarzoso and Peris-Torán (2001)
(ITS1 and 2). They presented an initial database for the detected this specific “flor” pattern in all the strains isolated
identification of more than 132 yeast species belonging to 25 from the velum of sherry wines from wineries located in
genera, most isolated from food. This is the first available Jerez, Spain.
molecular method that provides information to identify a Using the same methodology, but amplifying a different
large number of yeast species in an easy and quick way. This region (18S rRNA and ITS1) Dlauchy et al. (1999) also
260 Querol et al.

constructed a database of restriction fragment patterns to The amplified fragment length polymorphism (AFLP) was
identify 128 species associated mainly with food, and used to investigate genetic variation in commercial strains and
fermented drinks. winery isolates (de Barros Lopes de et al. 1999). It was
proposed that AFLP is a very useful method for discriminat-
ing yeasts at both species and subspecies levels, and also to
characterize hybrids (de Barros Lopes de et al. 2002).
3.2 Methods for Yeast Characterization Although initially more labor intensive than other PCR
techniques, the reproducibility of the results is the advantage
Methods by which strains of the same species can be over the previous PCR method described.
differentiated have been shown to be very important for yeast The analysis of mitochondrial DNA restriction fragment
strain characterization. In winemaking, several studies have length polymorphisms (mtDNA-RFLP) has long been used as
analyzed the diverse microflora of grapes and musts and a method for characterizing wine yeast strains (Vezinhet et al.
several interesting methods have been developed (Figure 2). 1990). However, due to the complexity associated with the
PCR amplification using intron splicing site (consensus procedure required to isolate a sufficient amount of mtDNA,
sequences that flank introns) displays polymorphism mainly its use for routine analysis was limited until quite recently.
at the within-species level (de Barros Lopes de et al. 1996); The last decade has seen improvements in the process (Querol
PCR amplification of delta elements (repeat sequences that et al. 1992b), removing the need for specialized equipment
flank the TY1 retrotransposons) has been widely used to and reducing the complexity and time scale, while retaining
characterize wine yeast strains of Saccharomyces cerevisiae the discriminatory power and reproducibility (Figure 3). This
(Ness et al. 1993). Another PCR-based approach, known as simplified technique has been used successfully to charac-
random amplified polymorphic DNA (RAPD), consists of the terize yeast strains of species belonging to genera
amplification of random segments of DNA with a single and Brettanomyces, Candida, Debaryomyces, Kluyveromyces,
short (from 5- to 15-mer) primer of an arbitrary nucleotide Saccharomyces, and Zygosaccharomyces (see Table 2).
sequence. The level of differentiation, either interspecific or Chromosomal DNA profile analysis (electrophoresis
intraspecific, depends highly on the primer used. However, karyotyping). The analysis of chromosomal DNA poly-
one of the most important problems with this technique is the morphisms due to chromosome rearrangements has proved to
low stable (nonrepetitive) patterns obtained. be useful for the differentiation of species (and also strains)
belonging to several genera, e.g., Candida, Kluyveromyces,
Saccharomyces, and Zygosaccharomyces. In the case of wine
yeast, it has been used for yeast characterization and was
applied to analyse the dynamics of the yeast populations
(Querol and Ramón 1996) and in the characterization of the
industrial wine yeast (Fernández-Espinar et al. 2001).
In the following section we will discuss the resolution of
some of these techniques and their industrial application for
the study of yeast population dynamics during natural and
inoculated wine fermentations and for the characterization of
S. cerevisiae strains of industrial interest.

3.3 Study of Yeast Population Dynamics During

Natural Wine Fermentations

Using mtDNA RFLP Querol et al. (1994) analyzed the

population dynamics of Saccharomyces strains during
spontaneous wine fermentations They found that despite the
high diversity of strains observed at the beginning of the
fermentation, very few dominated the process. However,
when similarities in the mtDNA restriction pattern of the
different S. cerevisiae strains (measured as the proportion of
shared restriction fragments) were low, a clear sequential
substitution of predominant strains was observed during each
fermentation phase. When the similarity was high, even
Figure 1 Scheme of the yeast identification using Restriction though a sequential substitution could also be observed
fragment length polymorphism of the 5.8S rRNA and the internal between secondary strains, a clearly predominant strain was
transcribed spacers (Esteve-Zarzoso et al. 1999). present during the whole fermentation process.
Genetic Variability of Yeast in Wine Fermentation 261

Figure 2 Molecular methods to yeast characterization: intron splice sites, d sequences, microsatellites and RAPD.

On the basis of chromosomal profiles, Frezier and areas studied and their presence over a number of years
Dubourdieu (1992) found that a small number of constituted evidence for the occurrence of specific native
S. cerevisiae strains were capable of dominating fermenta- strains representative of a particular wine region. Schütz and
tions in the same winery over two vintages, independently of Gafner (1994) proposed that the composition of yeast strain
the grapevine cultivar. Vezinhet et al. (1992) investigated populations differs from must to must and from year to year.
using mtDNA analysis and electrophoretic karyotyping the However, they also found strains isolated from two grape
evolution of S. cerevisiae strains isolated during six musts and from two vintages that presented very similar
consecutive years in the cellars of two vineyards. They chromosome patterns to the commercial strain W27
concluded that the wide distribution of some strains in the (Lallemand) isolated from the same region.

Figure 3 HinfI mtDNA restriction patterns of commercial wine yeast strains analyzed in Fernández-Espinar et al. (2001).
262 Querol et al.

Guillamón et al. (1996) have analyzed the correlation

between genetic distance and ecological/geographical factors
using mtDNA restriction analysis and electrophoretic
karyotyping. This constitutes the first survey to investigate
molecular variation among natural strains from different wine
regions. They found significant correlation between both
ecological factors (e.g., grape varieties) and/or geographical
origin and the molecular relationships among strains. The
analysis of genetic variation in natural years populations often
results in the determination of the particular biological factors
that influence population structure, hence population
variations within and between wine regions have to be
characterized to elucidate those factors that determine the
distribution of variations in natural populations of
S. cerevisiae.

3.4 Study of Yeast Population Dynamics During Figure 4 Chromosomal profiles of commercial wine yeast
Inoculated Wine Fermentations strains analyzed in Fernández-Espinar et al. (2001).

Using mtDNA restriction patterns, Querol et al. (1992a)

electrophoretic karyotyping (Figure 4) and specific PCR of d
conducted the first study of population dynamics and the roles
sequences (Figure 2), were used for this purpose. The
played by the active dry yeast strain and the natural
maximum discriminatory power was obtained when the
Saccharomyces flora during inoculated industrial fermenta-
results of the three techniques were combined. The results
tions. It was demonstrated that the inoculated strains T73
showed evidence of mistakes during production or fraudulent
(Lallemand) compete with the natural strains but do not
practices by yeast producers, since the same strains
completely suppress their growth until three to six days after
commercialized under different names or the same name is
inoculation. However, the predominance of the inoculated
used for different products by different companies.
strain was evident at the end of the fermentations.
Using physiological tests and karyotyping, Schütz and
Gafner (1993) analyzed the succession of three different dried 3.6 Future Methods
yeasts at three different time points of wine fermentation.
Hanseniaspora uvarum was present at the beginning of the
The use of PCR in molecular microbiology has increased to
fermentation, whereas only the inoculated strains of
the point where it is now accepted as the golden standard for
S. cerevisiae were observed in the middle and at the end of
detecting nucleic acids from a number of origins and has
the fermentation. However, as a consequence of the few
become an essential tool in the research laboratory. Real-time
strains, these authors did not detect as much diversity as that
PCR has engendered wider acceptance of PCR, due to its
observed by Querol et al. (1992a).
improved rapidity, sensitivity, reproducibility, and the
The use of active dry yeasts is of particular interest for
reduced risk of carry-over contamination (Mackay et al.
the wine industry. It is generally assumed, as we discussed
2002). Traditional detection of amplified DNA relies upon
previously, that indigenous yeasts are suppressed by the
electrophoresis of the nucleic acids in the presence of
starter. However, some studies showed that native strains are
ethidium bromide and visual observation after irradiation by
better adapted to fermentation conditions than commercial
ultraviolet light. Southern blot detection of the amplicon
strains (Esteve-Zarzoso et al. 2000; Esteve-Zarzoso and
using hybridization with a labeled oligonucleotide probe is
Peris-Torán 2001).
also time consuming. The detection of the amplicon and the
possibility of visualizing it as the amplification progresses is
the foundation of real-time PCR. The monitoring of the
3.5 Characterization of Commercial Wine Yeast accumulation of the amplicon in real time has been made
Strains possible by the labeling of primers with fluorogenic
molecules. Due to the numerous advantages, these methods
Since numerous yeast strains are being produced for the probably are an alternative to the techniques described
commercial wine market, it is interesting to study the degree previously, not only for the detection and identification of
of relatedness between commercial Saccharomyces strains. spoilage yeast or for wine yeast monitoring during the
Fernández-Espinar et al. (2001) characterized 45 selected alcoholic fermentation. Thus, this technology has also been
yeasts from 10 different yeast producers. Three molecular applied in studies of yeast gene expression during the
techniques, namely mtDNA restriction analysis (Figure 3), alcoholic fermentation.
Genetic Variability of Yeast in Wine Fermentation 263

4 STRUCTURAL AND FUNCTIONAL WINE in order to start growing and to carry out the alcoholic
YEAST GENOMICS fermentation. Ethanol stress is related to the progressive
production of this compound throughout vinification. Alcohol
4.1 Genomic Characteristics is highly toxic to yeast metabolism and growth (Ingram and
Buttke 1984) and the cell membrane is the primary target for
Saccharomyces cerevisiae cells are generally ellipsoidal in its action. Also it is important to mention the stress due to
shape, the vegetative reproduction is by multilateral budding glucose starvation, which could take place towards the end of
and its vegetative phase is predominantly diploid, the only the fermentation stage. Supraoptimal temperatures constitute
haploid stage is the ascospore. S. cerevisiae has a relatively another kind of stress conditions that can take place during
small genome, a large number of chromosomes, little fermentation, although it is not very significant for most
repetitive DNA, and few introns. Haploid strains contain vinifications, given the sophisticated temperature control
approximately 12 –13 megabases of nuclear DNA, distributed systems currently used in wineries (Fleet and Heard 1992).
along 16 linear chromosomes whose sizes vary from 250 to Similarly, adverse growth conditions, such as high ethanol
2000 kb (Barre et al. 1992; Pretorius 2000). Most S. cerevisiae and acetaldehyde concentrations and oxidative stress due to
strains used in the laboratory are either haploid or diploid and respiratory metabolism, can be found in the biological ageing
have a defined set of chromosome lengths. However, wine of fino sherry wine. Acetaldehyde is a known inhibitor of a
strains are mainly diploid, aneuploid, or polyploid (Codón wide range of metabolic activities and is more toxic than
and Benı́tez 1995). Aneuploidy and polyploidy may confer ethanol (Jones 1990). Response to stress conditions requires
advantages to adapt to variable external environments or, the activation of signal transduction pathways, which
perhaps, is a way to increase the dosage of some genes involves the synthesis of protective molecules, including
important for fermentation (Bakalinsky and Snow 1990; heat shock proteins. One of these proteins (Hsp104p) has been
Salmon 1997). The meiotic segregants from wine strains shown to be responsible for stress tolerance in laboratory
diploidise with high frequency, indicating a high frequency of yeast strains carrying out respiratory metabolism (Lindquist
homothallism. Heterozygosity has been observed in both and Kim 1996), but not for wine yeast strains growing under
homothallic and heterothallic strains (Barre et al. 1992; fermentation conditions in a glucose-rich medium (Carrasco
Codón and Benı́tez 1995). et al. 2001) or for brewery yeast strains (Brosnan et al. 2000).
Wine yeasts also show a high level of chromosome length Aranda et al. (2002) analyzed the responses of several
polymorphism (Bidenne et al. 1992; Rachidi et al. 1999). This S. cerevisiae strains (some of them isolated during the
polymorphism is generated mainly by illegitimate recombi- production of fino sherry wine) to several adverse conditions.
nation mediated by Ty transposons or subtelomeric repeated In strains that are dominant during biological ageing, there
sequences. This feature has several practical consequences: was a clear correlation between resistance to ethanol and
sporulation ability is very variable, between 0 and 75% ascus acetaldehyde, the high induction of HSP genes by these
formation can be observed on a sporulation medium and spore compounds and its presence as the predominant strain in most
viability is also highly variable, ranging from 0 to 98% levels of several “soleras.” It is interesting to note that the
(Codón and Benı́tez 1995; Mortimer et al. 1994). dominant strains during biological ageing are the less resistant
ones and the strains isolated during the alcoholic fermentation
are more resistant to the osmotic stress.
4.2 Adaptive Evolution of Wine Yeast Strains
4.2.2 Gene Expression Variability in Wine Yeast
Although the origin of S. cerevisiae is a matter of controversy,
its original genome has been subjected to strong selective The molecular basis of the technological properties of wine
pressures since its first unconscious use in controlled yeast strains are still largely unknown. However, the obvious
fermentation processes. Useful phenotypic traits, such as possibility is that the adaptation of these strains to the
fast growth in sugar-rich media, high alcohol production and enological environment is dependent on specific expression
tolerance, and good flavor production, which have been profiles of their genomes (for a review see Pérez-Ortı́n et al.
selected over billions of generations, have had strong 2002). Comparative analyses of gene expression between
influences on the S. cerevisiae genome (Pérez-Ortı́n et al. industrial and nonindustrial strains and between different
2002). We now will analyze some of the molecular industrial strains could lead to the identification of genes
mechanisms that explain this wine yeast adaptation to involved in the fitness of the strains in industrial
vinification. environments. To date, the study of gene expression during
wine fermentation has focused on genes induced in the
4.2.1 Stress Adaptation stationary phase in order to express specific activities at the
end of the process (Puig et al. 1996; Puig et al. 2000; Riou
During the alcoholic fermentation, yeast cells are subject to a et al. 1997).
number of stresses (Attfield 1997), the most important being Due to the different properties of the particular wine
osmotic and ethanol stresses. Osmotic stress is due to the high strains, it seems of great interest to study real commercial
sugar content in the must and yeast cells must resist this stress wine strains. Consequently, Puig et al. (1996); Puig and
264 Querol et al.

Pérez-Ortı́n (2000) determined the levels and expression reduction), mating type switching of the daughter cells and
patterns of several genes during wine microvinifications by conjugation with any of the mothers of the same single-spore
using a commercial strain (T73, Lallemand). Genes such as colony (Mortimer et al. 1994). This process (called “genome
POT1, HSP104, and SSA3, which are expressed late in renewal”) would produce highly homozygous strains that
laboratory culture conditions, are expressed in wine yeast eliminate deleterious mutations by natural selection. Yeast
cells only during the first few days in microvinifications. The strains, however, are usually aneuploid (Bakalinsky and Snow
reason for this could be the very different growth conditions 1990; Guijo et al. 1997) and heterozygous for many loci
used in microvinification (and therefore in real vinifications) (Barre et al. 1992; Kunkee and Bisson 2000), which is not in
compared to those of laboratory conditions. Vinifications are agreement with the genome renewal hypothesis (Puig et al.
characterized by very high contents of glucose and fructose 2000). There are several other ways in which wine strains
(20–25%) compared with the usual laboratory conditions change over time. Apart from spontaneous mutation, which
(1 – 3%), and anaerobic conditions vs highly aerated occurs at comparatively very low rates, Ty-promoted
laboratory conditions (Puig et al. 1996). chromosomal translocations (Rachidi et al. 1999), mitotic
The completion of the sequence of the genome of crossing-over (Seehaus et al. 1985) and gene conversion will
S. cerevisiae allowed the development of tools for the provoke faster adaptation to environmental changes (Puig
evaluation of the expression of the entire complement of et al. 2000). Extra copies of chromosome XIII, which contains
genes encoded in the genome (transcriptome). The DNA the alcohol dehydrogenase genes ADH2and ADH3, are found
microarray hybridization analysis was used to investigate how in flor wine yeasts, these genes are of special interest to
interesting genes change their expression during a biological oxidize ethanol (Guijo et al. 1997). Finally, (Pérez-Ortı́n et al.
process; several attempts have been made with wine yeast 2002) found that the SSU1-R allele, which confers sulfite
(Cavalieri et al. 2000; Backhus et al. 2001; Hauser et al. resistance to yeast cells, is the product of a reciprocal
2001). The knowledge of genetic features as well as the translocation between chromosomes VIII and XVI due to
specific expression profiles wine yeast strains under different unequal crossing-over mediated by microhomology between
growth conditions could help us to understand better the very short sequences on the 50 upstream regions of the SSU1
biological process of fermentation at the molecular level and and ECM34 genes, which put the SSU1 coding region under
how the gene expression is regulated in relation to changes in the control of the ECM34 promoter. They also showed that
the physical and chemical properties of the growth medium. this translocation is only present in wine yeast strains,
It is surprising that the genes involved in sulphur (SUL1-2) suggesting that the use of sulfite as a preservative in wine
and ammonia (MEP2) transport (Cavalieri et al. 2000), or that production over millennia could have favored its selection.
(sulphite efflux, SSU1) involved in sulfite resistance (Hauser This is the first time that a gross chromosomal rearrangement
et al. 2001), were found to be overexpressed in wine yeast has been shown to be involved in the adaptive evolution of
strains. They investigated in great detail the possible S. cerevisiae.
mechanisms for regulation of the expression of the SSU1
gene of the T73 wine yeast strain. A rearrangement of the
promoter of SSU1 was detected, leading to up-regulation in its
ACKNOWLEDGEMENTS
expression. This can be interpreted as being fixed through its
evolution as a result of the selective pressures imposed by
winemaking procedures (Pérez-Ortı́n et al. 2002). We thank all our colleagues and collaborators who
contributed in the different works reviewed and discussed in
this chapter. Thanks are also indebted to S. Petrorius for
4.2.3 Wine Yeast Genome Evolution comments that improved the manuscript.
The flexibility of the yeast genome to adapt to externally
introduced environmental changes has been demonstrated in
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Pearson BM and Mckee RA (1992). Rapid identification of Schütz M and Gafner J (1994). Dynamics of the yeast strain
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and Zygosaccharomyces rouxii. Int J Food Microbiol 16:63– 67. determined by CHEF gel. Lett Appl Microbiol 19:253 – 257.
Genetic Variability of Yeast in Wine Fermentation 267

Seehaus T, Rodicio R, Heinisch J, Aguilera A, Schmitt HD, and tiation of Candida famata, Kloeckera apiculata and Schizo-
Zimmerman FK (1985). Specific gene probes as tools in yeast saccharomyces pombe with chromosome number and size
taxonomy. Curr Genet 10:103 – 110. estimation of the two former. Syst Appl Microbiol 18:303 –309.
Sniegowski PD, Dombrowski PG, and Fingerman E (2002). Vezinhet F, Blondin B, Hallet J-N, and Chromosomal DNA (1990).
Saccharomyces cerevisae and Saccharomyces paradoxus patterns and mitochondrial DNA polymorphism as tools for
coexist in a natural woodland site in North America and identification of enological strains of Saccharomyces
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Török T, Rockhold D, and King ADJ (1993). Use of electrophoretic survey of wine yeast strains by molecular methods of
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24
Yeast in the Dairy Industry

T. K. Hansen / M. Jakobsen The Royal Veterinary and Agricultural University, Frederiksberg, Denmark

1 INTRODUCTION starter cultures; (c) yeast classification and taxonomy based

upon molecular methods. The present review will deal with
Yeast have been isolated from all kinds of dairy products the positive and negative aspects of yeast in the dairy
(Fleet 1990; Fleet and Mian 1987; Jakobsen and Narvhus industry.
1996; Rohm et al. 1992; Tilbury et al. 1974). Yeast are used as A variety of yeast species has been isolated from milk and
important starter cultures in food products such as bread, beer, dairy products (Fleet 1990; Fleet and Mian 1987; Gadaga et al.
wine, traditional fermented foods (Fleet 1990; Fleet and Mian 2001; Rohm et al. 1992; Roostita and Fleet 1996a,b; van den
1987; Jakobsen and Narvhus 1996) and seems to be the most Tempel and Jakobsen 1998; Tilbury et al. 1974; Tudor and
important micro-organism exploited by man. The role of Board 1993; Westall 1998) but a few main species are
yeast as micro-organisms, which could have a positive frequently detected in dairy products. These yeast are
influence on the quality of dairy products, especially cheese, representatives of the genea Debaryomyces, Klyveromyces,
has not been widely accepted (Fleet 1990; Jakobsen and Yarrowia, Candida, and Saccharomyces (Viljoen 2001). The
Narvhus 1996). Instead, yeasts have often been seen as yeast occur both in the teleomorphic and the anamorphic state
spoilage organisms originating from poor hygiene and in dairy products, depending on the yeasts’ ability to produce
sanitation (Fleet and Mian 1987; Jakobsen and Narvhus ascospores. Some of the most common yeast related to dairy
1996). The nutritional requirement of yeast, the ability to products mentioned first by their teleomorphic then by their
grow at low temperature, low pH, low moisture content, and anamorphic state are: Debaryomyces hansenii/Candida
high salt or sugar concentrations together with their famata, Galactomyces geotrichum/Geotrichum candidum,
enzymatic activity make yeast a natural part of the microbiota Yarrowia lipolytic/Candida lipolytica, Klyveromyces
in dairy products. In 1995 the first symposium on “Yeasts in marxianus/Candida Kefyr, Klyveromyces lactis var. lactis/
the dairy industry: Positive and Negative Aspects” was Candida spherical, and Saccharomyces cerevisiae/Candida
organized and held by the International Dairy Federation robusta.
(IDF) expert group F47—Yeast. As reflected in the published To describe yeast occurring in dairy products and select
literature the interest in yeast in the dairy industry has and maintain yeast as a starter culture, identification at
increased since and the knowledge of the positive and subspecies level is a necessity. The taxonomy of yeast is
negative role played by yeast in dairy products has become mainly based on the criteria described in the third and fourth
more detailed. The key areas have been: (a) study of edition of the taxonomical study “The Yeasts” edited by
important technological characteristics of yeast, e.g., Kreger van Rij (1984) and Kurtzman and Fell (1998),
proteolytic and lipolytic activity, aroma formation and respectively. The criteria includes micro- and macro-
fermentation and/or assimilation of residual sugars and morphology in standard media, assimilation, and fermenta-
lactate and citrate and pigment formation especially related to tion profiles of standard carbohydrates, growth at different
different kind of cheese; (b) properties affecting both the temperatures and under defined osmotic pressure, develop-
negative role of yeast as spoilage organism causing quality ment of pseudo or true mycelium, formation of ascospores as
defects as well as the potential of yeast as highly controlled well as other phenotypic criteria. It takes time and experience

269
270 Hansen and Jakobsen

to conduct a correct classical identification. Therefore, easier 1998) that the agar diffusion tests may not offer the
identification systems like API-ZYM systems (Biomerieux, sensitivities required to detect proteolytic activity. Methods
Macy l’Etoile, France) are widely used. The API-test is based such as gelelectrophoresis, high performance liquid chroma-
on assimilation profiles of the yeast. The API-kit’s were tography (HPLC), or capillary electrophoresis (CE) give a
originally made to offer a fast identification of yeast of different and more detailed picture, which also demonstrates
medical importance. Several investigations have found food- that the proteolytic activity of yeast is strain specific (Hansen
related yeasts that were not included in the reading key of the and Jakobsen 2001; van den Tempel 2000). Several studies of
kit (Deák and Beuchat 1988). The enlarged version of the test, strains of D. hansenii have indicated that the examined strains
API-kit 32C, has been found to be reliable in assisting in yeast could not degrade casein (Hansen and Jakobsen 1997).
identification and acceptable for a rapid identification of However, low proteolytic activity was observed in D. hansenii
yeasts associated to cheese, as long as it is used in isolated from Picante cheese (Freitas et al. 1999) and blue
combination with the classical methods, especially when a veined cheese (van den Tempel 2000). Similar variability in
reliable determination of the sexual form is required, for the the proteolytic activity for strains of K. lactis has been
identification (van den Tempel 2000). Other methods based observed (Grieve et al. 1983; Hansen and Jakobsen 1997;
on criteria such as profiles of volatile metabolites of the yeast Roostita and Fleet 1996a). However, Grieve et al. (1983) did
(Magan et al. 2001; Westall 1998), analysis of volatile show that a strain of K. lactis was able to hydrolyze a-, b-,
profiles of yeast by an electronic nose (Magan et al. 2001) and and k-casein, which indicates that some strains are able to
identification based on Fourier-transform of the specific contribute to the maturation of cheese.
infrared spectrum of the biochemical compounds of the dried Gueguen and Lenoir (1975) examined 30 strains of
yeast cell (Kümmerle et al. 1998) have been reported as G. geotrichum and found that they produced extracellular and
methods for fast identification at species level. intracellular proteinases and peptidases. Extracellular activity
The number of yeast genera and yeast species has was present in 66% of the strains, which could be divided into
increased 100% or more since 1970 where the first edition of two groups of 25% showing high proteolytic activity and 75%
“The Yeasts” was published. At the same time yeast species having low proteolytic activity. Optimum pH for the
described as belonging to different genera or species have proteolytic activity was close to 5.5. Other investigations of
been proven to belong to the same species. This shows the commercial strains of G. geotrichum used in cheese
complexity of the yeast taxonomy but also the growing production showed that the proteolytic activity was weak
interest in yeast classification. One of the major reasons for when present.
the changes in yeast classification and taxonomy is the use of Yarrowia lipolytica produces several proteinases and
molecular methods including analyses of whole chromo- many of these are extracellular. The secretion of both alkaline
somes and as with other micro-organisms, the classification of and acid proteinases has been detected (Ogrydziak et al. 1982;
yeast is based more and more on genotypic rather than Vasileva-Tonkova et al. 1996). The activity of the
phenotypic criteria for yeast isolated from dairy products. extracellular acid proteinase at pH 5.2 was optimal at 158C
Several methods have been reported for taxonomical use, for but was significantly reduced at 88C. Many investigations of
e.g., molecular techniques using SDS-gelelectrophoresis of Y. lipolytica have shown that different strains of Y. lipolytica
whole-cell protein pattern, pulse field gelelectrophoresis have proteolytic activity and are able to breakdown all the
(PFGE) (Montrocher et al. 1998; Esteve-Zarzoso et al. 1999; casein components. (Freitas et al. 1999; van den Tempel and
Gente et al. 2002; Jespersen and Kühle 2000), restriction Jakobsen 2000; Wyder and Puhan 1999) and seems to be at
fragment length polymorphism analysis of mitochondrial the same level as a strong proteolytic strains of P. roqueforti
DNA (DNA-RFLP) (Petersen et al. 2002; Romano et al. 1996; (van den Tempel 2000).
van den Tempel and Jakobsen 2000), internal genomic spacer Candida catenulata seems to have a high proteolytic
sequences (IGS), 18S rRNA analysis (Cappa and Cocconcelli activity similar to the level of Y. lipolytica (Roostita and Fleet
2001) and polymerase chain reaction—restriction fragment 1996a), but detailed studies have not been reported.
length polymorphism (PCR-RFLP) analysis of the intergenic Saccharomyces cerevisiae can be proteolytic but the
transcribed spacer region (ITS) (Caggia et al. 2001; Petersen majority of strains do not show proteolytic activity (Hansen
et al. 2001). However, for many of the yeast genera related to and Jakobsen 1997; Hansen and Jakobsen 2001; Roostita and
dairy products the molecular methods are not yet fully Fleet 1996a). The proteolytic activity was studied by Hansen
developed or sensitive enough to separate strains on species or and Jakobsen (2001) using CE for five selected Saccharo-
subspecies level. myces spp. being a commercial starter cultures in blue veined
cheese, an isolats from blue veined cheeses, a type strains of
Saccharomyces spp. and two starter cultures of Saccharo-
2 PROTEOLYTIC ACTIVITY myces spp. used in other food fermentation. The CE results for
the five strains showed that only the commercial blue cheese
Published results on the proteolytic activity of yeast isolated strain of S. cerevisiae could break down casein. The
from dairy products are mainly based upon the ability to CE-profiles showed that S. cerevisiae was able to hydrolyze
produce a clearing zone in casein and skim milk agar. It has some of the as1-and b-casein. Differences in the CE-profiles
been demonstrated (Hansen and Jakobsen 2001; Larsen et al. indicated a synergistic effect in the degradation of casein by
Yeast in the Dairy Industry 271

S. cerevisiae and Penicillium roqueforti, seen as higher from cows with or without mastitis. Yeast were detected in
number and different grouping of peptides when grown more than 50% of samples from quarters with mastitis
together compared to growth of the individual culture. infections, but only in 25% of the samples from the
noninfected quarters (Lagneau et al. 1996). Yeast have been
detected in the number of 103 cfu/ml in pasteurized milk
3 LIPOLYTIC ACTIVITY (Fleet and Mian 1987; Lagneau et al. 1996; van den Tempel
and Jakobsen 1998) and most of the common species in raw
Almost all yeast related to cheese have esterase activity and milk were able to grow to 108 or 109 cfu/ml in UHT milk even
are able to hydrolyze tributyrin (Fleet and Mian 1987; Hansen at low temperatures (Fleet and Mian 1987; Roostita and Fleet
and Jakobsen 1997; Roostita and Fleet 1996a; van den 1996b). Spoilage of raw milk and pasteurized milk by yeast
Tempel and Jakobsen 1998). This applies for D. hansenii, are not reported as a very common problem. The occurrence
K. lactis, S. cerevisiae and Y. lipolytica, C. catenulata and of yeast in raw milk is assumed to be a contamination source
C. geotrichum, all showing esterase activity. of other dairy products. Reported investigations using
The reported results on the esterase activity of D. hansenii, molecular techniques to follow yeast at subspecies level
K. lactis, and S. cerevisiae are contradicting (Besancon through the whole production and to trace the origin of the
et al. 1992; Fleet and Mian 1987; Hansen and Jakobsen yeast are limited.
1997; Roostita and Fleet 1996a; van den Tempel and In sweetened condensed milk the low water activity, due to
Jakobsen 1998). But in general the activity is low. Tested in high concentrations of sugar favor yeast as a potential
API-ZYM systems (Biomerieux, Macy l’Etoile, France), spoilage organism while bacterial growth is inhibited. Yeasty
S. cerevisiae, D. hansenii, and K. lactis could hydrolyze the taste and flavor and gas production are known spoilage by
fatty acid with chain length of C:8, but not the fatty acid with yeast in this product (Walker and Ayers 1970).
a chain length of C:14 (Hansen and Jakobsen 2001). Lipolysis
of long-chain fatty acids has been demonstrated for
Y. lipolytica, C. catenulata, and C. geotrichum and the 4.2 Cream and Butter
activity seems to be at the same level for the three yeasts
(Roostita and Fleet 1996b; van den Tempel 2000). Yeast have been reported to cause spoilage such as yeasty
Isolation of several intra and extracellular lipases from flavor and foamy appearance in all kind of cream products
Y. lipolytica has been reported and the strong lipolytic activity (Fleet and Mian 1987; Walker and Ayers 1970). In an
of Y. lipolytica seems mainly to be due to extracellular lipases Australian study 48% of the examined cream samples
with optimal activity at pH 6 –10 (Destain et al. 1997; Pereira- contained yeast in the range of 103 – 104 cfu/ml while a
Meirelles and Rocha-Leão 1997). Some of the reported number of 104 –105 were found in 14% of the samples (Fleet
lipases are quite stable and are still active after 370 days at and Mian 1987). The yeast identified in the study were mainly
58C (Pereira-Meirelles and Rocha-Leão 1997). To demon- C. famata, Rhodotorula glutinis, C. diffluens, Cryptococcus
strate the extent of the lipolytic activity of Y. lipolytica, five laurentii, and R. rubra. These yeast species all showed high
strains were compared with five different strains of lipolytic activity and were able to grow at 58C.
P. roqueforti with medium to high lipolytic activity. It was An investigation from Italy showed yeast in 4 out of 5
found that the lipolytic activity was twice the activity of the butter samples examined (Minervini et al. 2001) The detected
strongest lipolytic strain of P. roqueforti (van den Tempel and yeast included C. kefyr, C. guilliermondii, and S. cerevisiae in
Jakobsen 2000). Investigations comparing lipolytic activity concentrations of 103 –105 cfu/g (Minervini et al. 2001). In
after growth under different condition showed that the 1987 Fleet and Mian found yeast in 7 out of 16 butter samples.
lipolytic activity was depending on pH, temperature, and the The yeast occured in numbers below 103 per gram and
growth media (Hansen and Jakobsen 1997; van den Tempel belonged mainly to the species R. glutinis and Rhodotorula
2000). rubra. Lipolytic yeast on the butter surface has also been
reported (Walker and Ayers 1970), but all together the
literature on this subject is very limited.
4 YEAST IN DAIRY PRODUCTS
4.1 Farm Milk
4.3 Yoghurt
Often the level of yeast in raw milk is found to be
102 – 104 cells=ml (Lagneau et al. 1996; van den Tempel and Yoghurt is an acidic fermented milk product, which normally
Jakobsen 1998). The dominant yeast species detected in raw has acetaldehyde in the final aroma. It is made of milk
milk in Denmark, Italy, and Belgium were found to be inoculated with thermophilic lactic acid starter cultures.
C. famata, C. lipolytica, and Trichosporon cutanum (Corbo During fermentation about 35% of the lactose is hydrolyzed
et al. 2001; Lagneau et al. 1996; van den Tempel and to glucose and galactose. The glucose is metabolized to lactic
Jakobsen 1998). The study of raw milk in Belgium indicated acid while galactose remains in the environment of the
that yeast count was depending on whether the milk came yoghurt (Goodenough and Klein 1976). Therefore, galactose
272 Hansen and Jakobsen

fermenting yeast strains in particular are known to cause goat milk inoculated with kefyr grains (Motaghi et al. 1997).
spoilage of yoghurt (Caggia et al. 2001; Giudici et al. 1996). Kefyr grains are white, yellowish hard granules with shape as
Yoghurt is often added nuts, honey, preserved or dried cauliflower. They are a result of a symbiotic microflora that
fruit, containing sucrose and is a source of yeast infections. are encapsulated in grains made of a fibrous matrix of
Yeast spoilage of yoghurt is seen as excessive gas production coagulated casein, polysaccharides, fat and lysed cells. The
followed by swelling of the package, unpleasant yeasty odor polysaccharides in the kefyr grains differ from other known
and taste, changes in texture and color, and formation of polysaccharides and are called kefyran (Wyder 1998). The
visible yeast colonies (Caggia et al. 2001; Fleet and Yeasts relative low temperature in the region of Caucasus where
1998). kefyr originated led to a natural selection of mesophilic yeast,
With intervals, studies of yeast in retail yoghurt have been lactic and acetic acid bacteria in the kefyr grains (Motaghi
reported from different countries. It is not unusual to detect et al. 1997; Wyder 1998). Interactions between the micro-
yeast in yoghurt in numbers of 103 cells/g, and yeast count up organisms have been reported (Koroleva 1988). The yeast
to 108 cells/g has been reported. The contamination rate of stimulated the growth of the lactic acid bacteria releasing
yeast in yogurt were in the order of 103 for 20% of the vitamins and amino acids and causing an increase in pH
examined samples in United Kingdom (Davis 1975) and metabolizing lactic acid (Koroleva 1988). Several different
Canada (Arnott et al. 1974). composition of the microbiota in kefyr grains has been
The most common yeast species reported in yoghurt are detected. The composition depends on different cultivating
C. famata/D. hanseniil, Pichia anaomala, C. versalitilis, techniques of the kefyr grain, which is connected to the origin
S. cerevisiae, and K. lactis. These yeast are able to assimilate and temperature of the production site (Berger et al. 1999).
and ferment several of the main carbohydrates in plain and The origin and temperature of the production site also have
fruit yoghurt. They are also able to assimilate lactate and influence on the development of alcohol and CO2 and then the
citrate. They grow at low pH-values and temperatures below final aroma and the viscosity of the kefyr. The main yeast
108C. Further several are able to produce esterases and species detected in Kefyr are K. marxianus, C. kefyr,
lipases, proteinases and peptidases hydrolyzing the milk fat S. cerevisiae, S. delbrueckii (Wouters et al. 2002). In the
and protein. Therefore the occurrence of yeast even in low industrial production of kefyr consistency in quality is a must.
numbers in yogurt will limit the shelf-life (Fleet 1990). Therefore, retail kefyr is seldom produced using kefyr grains;
The occurrence and growth of yeast in yoghurt are closely instead commercial kefyr cultures are based on a mixture of
related to poor hygiene and sanitation (Fleet and Mian 1987). pure cultures isolated from kefyr grains inoculated in milk.
As emphasized by Fleet (1990); Jakobsen and Narvhus (1996) Commercial kefyr cultures contain only yeast in very low
spoilage should be prevented through implementation of concentration if any. Gas production by the yeast causes the
general principles of good manufacturing practice. To extend package to blow and consumers seem to find swelling of the
the shelf-life of yoghurt the use of preservatives are permitted package undesirable in retail kefyr.
in some countries. However, yeast can be very tolerant to
some preservatives. As an example C. famata and K. maxianus
are still able to multiply in the presence of sorbate and 4.5 Other Fermented Milk Products
benzoate at concentrations of 500 mg/l (Fleet and Mian 1987).
Another well-known fermented milk produced with a natural
yeast microflora is koumiss. Koumiss is like a milk wine with
4.4 Kefyr an alcohol percentage around 2 and a final pH about 4. In the
beginning of the 1900 century koumiss was described as the
As a sign of immortality Allah granted a chosen tribe the first greatest of the fermented milks as reviewed by Steinkraus
kefyr grain and therefore the grains were called grains of the (1996). Koumiss originates from Khazakstan and is
prophet. In another version of this tale the grains were given traditionally made from mare’s milk but variants made from
by the prophet Mohammed as a present as a sign of cows milk has been reported (Steinkraus 1996). The
immortality together with the recipe of kefyr, but it was dominating microflora in koumiss is thermophilic lactic acid
forbidden to give away the grains or the recipe otherwise the bacteria and Saccharomyces spp. especially S. unisporus and
grains would lose their healing power. These tales has been K. marxianus (Montanari et al. 1996).
used as an explanation why the preparation of kefyr was kept Yeast are also present in several indigenous African
as a secret for so long. Even though, the history of this product fermented milk products such as amasi/hodzeko from
is centuries old (Wouters et al. 2002) the first production of Zimbabwe (Gadaga et al. 2001), banik from Senegal
retail kefyr was made in the former USSR in the 1930s (Gningue et al. 1991), rob from Sudan (Abdelgadir et al.
(Wyder 1998). 2001), nono from Nigeria (Okagbue and Bankole 1992), and
Kefyr originates from Caucasus in Central Asia, but has ergo and ititu from Ethiopia (Gonfa et al. 2001). All
become popular in many countries especially in the Middle traditional fermented milks are prepared by allowing raw milk
East. Kefyr is an acidic and mildly alcoholic fermented milk to spontaneously ferment at ambient temperature. Lactic acid
beverage with a high level of CO2. Traditional kefyr is bacteria and yeast with S. cerevisiae, K. marxianus, K. lactis,
prepared in bags from goat hides, from either cow, sheep, or and C. kefyr as dominating yeast are part of the natural
Yeast in the Dairy Industry 273

microflora and it is assumed that yeast play an important role Examinations of different kinds of cheese showed that the
in the microbial interactions between the different micro- yeast population often develops from a heterogeneous to a
organisms and development of the characteristic aroma in homogeneous population during the ripening period
these products. Traditional fermented milks are made in many (Bockelmann and Hoppe-seyler 2001; Hansen et al. 2001;
households daily. These milks products represent an Petersen et al. 2002; van den Tempel and Jakobsen 1998).
important source for human of energy, protein, vitamins, Yeast need an energy source to grow, therefore the ability to
and minerals (Loretan et al. 1998). Because of the socio- assimilate or ferment residual carbohydrates and acids is
economic changes that are taking place in Africa some of the important for the yeasts in order to survive in the cheese and
traditional fermentation technologies might be lost together to compete with other micro-organisms during ripening
with the related microflora (Loretan et al. 1998). Therefore, (Roostita and Fleet 1996b). But also the general tolerance
interest in traditional African fermented milk is increasing, towards NaCl of yeasts and the effect of NaCl on the uptake of
especially examination and identification of the high number lactate and other carbohydrates is important (Petersen et al.
of lactic acid bacteria and yeast and exploration of their role in 2002). The assimilation of different carbohydrates seems to
the fermentation process of the milk (Loretan et al. 1998). be affected by the microenvironment in the cheese.
Investigations of D. hansenii showed that the assimilation
of lactate (Petersen et al. 2002; van den Tempel and Jakobsen
2000) and citrate (van den Tempel and Jakobsen 2000) was
4.6 Cheeses strain specific and that inhibitions for some strains occurred at
6% (w/v) NaCl while other strains could assimilate both
Yeast are detected and isolated from all sorts of cheeses citrate and lactate in the presence of 14% (w/v) NaCl (van den
(Bockelmann and Hoppe-Seyler 2001; de Boer and Kuik Tempel and Jakobsen 2000). Similar results were seen for
1987; Nooitgedagt and Hartog 1988; Roostita and Fleet assimilation of lactose and galactose. The same pattern was
1996a; Schmidt and Lenoir 1980; van den Tempel and observed for Y. lipolytica though inhibition already occurred
Jakobsen 1998; Tzanetakis et al. 1998; Vivier et al. 1994; at low levels of salt, i.e., 2 % (w/v) NaCl for some of the
Welthagen and Viljoen 1998). The number of yeast can be in strains examined (van den Tempel and Jakobsen 2000).
the range 104 cfu/g or even higher. The most common yeast
species in cheese are D. hansenii, Y. lipolytica, G. geotrichum, 4.6.1 Yeast in Surface Ripened Cheese
K. lactis, K. maxianus, and S. cerevisiae with D. hansenii as
the most predominant. The development of yeast in cheese Semi-soft and soft cheeses that develop a smear of microbial
occurs spontaneously while controlled use of yeast as starter growth on the surface during maturation are called surface
cultures in cheese is used for production of some kind of mold ripened cheese. These cheeses have maturation times that
ripened cheeses and smear ripened cheeses, but seldom for vary from several days to month and at temperature ranging
other types of cheese. from 10 to 208C. Some examples of these cheeses are
Until recently it was assumed that the yeast in cheese Limburger, Danbo, Brick, and Tilsiter (Fleet 1990;
primarily originated from the cheese brine. Cheeses are often Bockelmann and Hoppe-Seyler 2001; Petersen et al. 2002).
salted in brines containing 22 –25% NaCl (Hansen et al. 2001) The microbial smear on the surface of these cheeses is very
and often the brine is not changed or pasteurized between the important for the maturation process and play a major role for
salting of different batches leading to accumulation of salt the final cheese quality (Bockelmann 2002; Bockelmann and
tolerant yeast (Tudor and Board 1993) in the range of Hoppe-Seyler 2001; Fleet 1990; Jakobsen and Narvhus 1996).
102 –106 cfu/g (Devoyod and Sponem 1970; Rohm et al. The cheese smear is a bio-mass of yeast and bacteria
1992; Seiler and Busse 1990; van den Tempel and Jakobsen (Bockelmann and Hoppe-Seyler 2001; Fleet 1990; Fleet and
1998). It has also been suggested that the yeast in cheese Mian 1987; Masoud and Jakobsen 2003). The yeast species
originated from raw milk because of the survival of yeast D. hansenii seems to be dominant and also the most
through pasteurization (van den Tempel 2000; Vadillo et al. important yeast during the whole maturation time but
1987). In a study carried out by Petersen et al. (2002) the depending on the type of cheese other yeast such as
successions of yeast on the surface of Danbo was followed Trichosporon spp., Y. lipolytica, K. lactis, and Candida
using mtDNA RFLP. The investigation showed that the spp. have been detected during the first day of ripening
dominating flora after 4 days belongs to D. hansenii and that (Bockelmann 2002; Fleet 1990; Petersen et al. 2002). The
the dominating strain did not originate from raw milk, brine, bacterial flora consists of Brevibacterium lines, Arthobacter
or the starter culture, but from the dairy “house microflora,” spp., Corynebacterium spp., Micrococcus spp., and
which also includes yeast from air in the ripening room, Staphylococcus spp. (Bockelmann 2002; Bockelmann and
ripening pads and humans. This indicates that cheese quality Hoppe-Seyler 2001; Fleet 1990).
depends on an intact “house microflora” where the balance The microbial ecology of the smear is very complex, but
easily can be disturbed because the cheese surface in many during the first days of cheese ripening a natural selection
ways is exposed to the environment of the dairy with its takes place (Petersen et al. 2002). The most suitable group of
inherent population of micro-organisms (Bockelmann and yeast in the surface ripened cheese, seems to adapt easily to
Hoppe-Seyler 2001). the microenvironment with high NaCl concentration, low pH,
274 Hansen and Jakobsen

and lactate as a main carbon source. Among the yeast numbers (Hansen et al. 2001). In four-week old Danablu, the
D. hansenii in most cases grow fast and become the dominant most common yeast are D. hansenii (van den Tempel and
species. It has been observed a particular subspecies out range Jakobsen 1998). Examinations of Danablu produced at four
the other strains and become dominant of D. hansenii present different dairies and the Danish blue cheese Mycella showed
(Petersen et al. 2002). that the yeast population developed from a heterogeneous
The growth of D. hansenii on the surface of the cheese flora to a homogeneous flora of D. hansenii during the
enhances the growth of the smear bacteria (Valdés-Staber ripening period (Hansen et al. 2001; van den Tempel and
et al. 1997) by metabolizing the lactate (Le Clercq-Perlat et al. Jakobsen 1998).
1999). After 4 –7 days the pH increases from 5.2 to 5.7 Yeast are considered to play an important role in the
allowing more acid-sensitive bacteria like B. linens to grow ripening of blue mold cheese and seems to contribute
(Bockelmann and Hoppe-Seyler 2001; Petersen et al. 2002; positively to microbial environment by assimilation of the
Valdés-Staber et al. 1997). The number of yeast begin to residual carbohydrates and acids. Yeast are assumed to create
decrease after one or two weeks of ripening while the bacteria a stable microenvironment, which prevent undesired
dominates on the cheese surface during the last part of the microbial growth. The gas produced in the curd during
ripening period (Bockelmann 2002; Petersen et al. 2002). fermentation is likely to create minor fissures and chinks in
Furthermore, yeast appears to support the bacterial growth by the cheese curd, which is assumed to promote the
release of vitamins and amino acids (Viljoen 2001). development of P. roqueforti (Coghill 1979). Positive
Investigations carried out by Masoud and Jakobsen (2003) interactions have been detected between D. hansenii and
showed that D. hansenii had a significant effect on the P. roqueforti under conditions simulating the environment in
intensity of the reddish pigment produced by the bacteria Danablu (van den Tempel and Nielsen 2000) The yeast
flora. Significant differences were observed between the S. cerevisiae is known to have a positive affect on growth and
D. hansenii strains examined but also the NaCl content and sporulation of P. roqueforti (Hansen and Jakobsen 2001;
pH played a important role in the pigment production. Hansen et al. 2001). S. cerevisiae is also found to stimulate the
release of free fatty acids (FFA) by P. roqueforti and a
4.6.2 Yeast in Blue Mold Cheeses synergistic effect between P. roqueforti and S. cerevisiae has
been demonstrated in the degradation of casein (Hansen and
Blue mold cheeses are semi-soft cheeses primarily ripened by Jakobsen 2001; Hansen et al. 2001). The positive interactions
growth of the mold P. roqueforti. Blue cheeses normally have between P. roqueforti and S. cerevisiae were verified in a
a significally higher content of NaCl compared to surface dairy trial. In the cheeses added S. cerevisiae improved
ripened cheeses and white mold cheeses. The NaCl growth and earlier sporulation of P. roqueforti was observed
concentration in blue mold cheeses after brining is 0.2% compared to the reference cheeses. Furthermore, positive
(w/w) in the core and 7% (w/w) in the surface layer. After contribution from S. cerevisae were also found in the aroma
eight weeks of maturation NaCl concentration is approxi- analysis, the degradation of casein, and by sensory analysis.
mately 2.0% (w/w) in the core and 4.0% (w/w) in the surface The observed differences indicate the potential use of
layer. The pH after 24 hours is at the level pH 4.6 –4.7. During S. cerevisiae as an additional starter culture for production
ripening, pH in the core increases to about 6.5 and to 5.9 in the of Mycella (Hansen et al. 2001).
surface layer (Gobbetti et al. 1997; Godinho and Fox 1982; In laboratory studies inhibition of P. roqueforti by
Hansen et al. 2001). Y. lipolytica, G. geotrichum and K. lactis have been observed.
To permit air into enter the cheese center and carbon A negative effect of the yeast on the growth of P. roqueforti
dioxide to escape the cheeses are pierced before maturation has not been verified in cheese, but should be kept in mind if
which also affect yeast growth. In blue veined cheese, the development of P. roqueforti is slow or absent. It should
yeasts are detected in high levels, without affecting also be keep in mind that some of these yeast, e.g., D. hansenii
cheese quality negatively (Fleet 1992; Hansen et al. 2001; and Y. lipolytica are known to produce reddish pigment on the
van den Tempel 2000). The number of yeasts detected in cheese surface primarily from oxidation of tyrosine to
blue mold cheese is in the order of 107 – 108 cfu/g on the melanin (Carreira et al. 1998; van den Tempel and Jakobsen
surface and 105 – 106 cfu/g in the core, but higher 2000).
concentrations have been observed (Hansen et al. 2001;
van den Tempel and Jakobsen 1998). The most common 4.6.3 Yeast in White Mold Cheese
yeasts isolated from raw milk cheeses like Roquefort are
D. hanseniiIC. famata, C. catenulata, Y. lipolytica/ White mold cheeses are semi-soft cheeses with growth of
C. lipolytica, C. kruseii, T. cutaneum, and K. lactis/ P. camemberti creating a white greyish mycelium on the
C. spherica (Besançon et al. 1992; Roostita and Fleet 1996a). surface on the cheese. The most famous variants are Brie and
In Mycella, which is made from pasteurized milk, the Camembert. In these cheeses pH is reduced to about 4.7
predominant yeasts isolated from both the core and the during the first 24 hours by the primary lactic acid starter
surface is D. hanseniilC. famata but in the beginning of culture. P. camemberti metabolizes the lactate to water and
the maturation other yeast like Zygosaccharomyces spp., CO2 resulting in an increasing pH, most pronounced on the
G. geotrichum Y. lipolytica, and C. rugosa are seen, in low surface of the cheese. A pH gradient of decreasing values will
Yeast in the Dairy Industry 275

be established towards the center of the cheese causing lactate (Dieuleveux et al. 1998) and D-3-phenyllactic acids inhibiting
to migrate towards the surface where it is used as a carbon Listeria monocytogenes (Dieuleveux et al. 1998) have been
source for P. camemberti. When all the lactate is depleted, reported. G. geotrichum is known to inhibit contaminating
casein will be degraded into amino acids and ammonia molds on the surface of mold cheese and in studies where
causing pH to increase further and the gradient to become G. geotrichum was cultured together with P. commune,
stronger while the pH is still low in the center. The acid P. caseifulvum, P. verrucosum, P. solitum, and Aspergillus
condition in the center causes soluble calcium phosphate to versicolor; it was found that mycotoxin were produced in
migrate towards the surface where it precipitates as a result significantly lower concentration by the five molds compared
of the higher pH (Karahadian and Linsa 1987; Vassel et al. to growth of the mold as single cultures (Nielsen et al.
1986). The establishment of the pH gradient caused by 1998a,b). All together, it indicates that G. geotrichum plays
P. camemberti indirectly is the key factor in the maturation an important role in the inhibition of undesired micro-
process (Lawrence et al. 1987), but the desired soft texture organisms in mold ripened cheese (Nielsen et al. 1998a,b).
of white mold cheeses is a direct result of the depletion of Many strains of G. geotrichum have been described and the
calcium phosphate in the center and the proteolytic activity diversity among the strains is very pronounced with regard to
of rennet, plasmin, and enzymes from the lactic acid all their technological characteristics (Spinnler et al. 2001).
bacteria and yeast. Yeast has been detected in several types
of white mold cheeses and the positive role of yeast in the 4.6.4 Contribution and Spoilage of Yeast in Other
maturation and aroma formation of white mold cheese has Types of Cheese
been proposed (Schmidt and Lenoir 1980; Siewert 1986).
However, the use of yeast as starter culture is still the Besides the important role yeast play in surface-ripened and
exception rather than the rule in white mold cheeses. mold-ripened cheese, they may also have a desired influence
During ripening the yeast population increases to a level of on the maturation and final quality of hard and semi-hard
105 –107 at the center and 106 –108 at the surface. Several cheeses like Cheddar, Gouda, and Parmesan (Deiana et al.
different yeast species have been isolated from white mold 1984; Viljoen and Greyling 1995). Yeast are mentioned to
cheese and at the surface K. lactis, K. marxianus, and accelerate the ripening process (Deiana et al. 1984), but the
G. geotrichum have been the dominant cultures while role of yeast in these types of cheeses is not clear. A variety of
D. hansenii has been predominant in the center. S. cerevisiae yeast including, e.g., D. hansenii, Y. lipolytica, S. cerevisiae,
and Zygosaccharomyces rouxii are also found, but less K. marxianus, C. catanulata, T. delbruckii, and R. glutinis
frequently (Baroiller and Schmidt 1990). Except from have been isolated from these types of cheese and seems
G. geotrichum the role of yeast in the maturation process is primarily to be connected to spoilage, e.g., uncontrolled
still not clear, but it is assumed that their lipolytic and maturation, flavor defects and blowing of the cheese, and
proteolytic activity and the capability to metabolize lactate undesired pigment formation. On the other hand, D. hansenii
and galactose, glucose and lactose play a role in the has been reported to have an inhibitory effect on the growth of
maturation. Further yeast have been mentioned to have an Clostridium tyrobutyricum and C. butyricum, which is a well
inhibitory effect on the undesired growth of Mucor spp. on known spoilage organism in theses types of cheeses (Deiana
the surface of Camembert cheese (Siewert 1986). et al. 1984; Fatichenti et al. 1983).
In unripened cheese yeast only seems to cause spoilage. In
a. The Role of Galactomyces Geothricum in White Mold cottage cheese, spoilage caused by, e.g., Y. lipolytica/
Cheese. Galactomyces geotrichum is closely related to the C. lipolytica, Candidum sake, C. spherica, and K. maxianus
production of white mold cheeses. G. geotrichum is often has been reported (Brockelhurst and Lund 1985; Fleet 1990).
used as a co-culture together with P. camemberti in the The spoilage appears as visible colonies on the surface of the
production of white mold cheese (Addis et al. 2001; cheese, flavor and aroma defects and undesired production of
Molimard et al. 1995) and in a few variants of these cheeses gas (Brockelhurst and Lund 1985).
G. geotrichum is used as the only culture. G. geotrichum is Several investigations have been reported on yeast in Feta
able to assimilate lactate and it grows faster on the surface of cheese (Kaminarides and Laskos 1992; Tzanetakis et al. 1998;
the cheese than P. camemberti. It seems to contribute Vivier et al. 1994). The dominant yeast in Greek Feta and the
strongly, along with different sulfides, to the characteristic brine of Greek Feta is D. hansenii/C. famata, S. cerevisiae,
aroma profile. Methanethiol and dimethyl is produced from Torulaspora delbrueckii, Pichia farinosa, and Pichia
methionine by two distinct pathways (Demerigny et al. 2000). membranaefaciens (Kaminarides and Laskos 1992;
The formation of dimethyl disulfide, dimethyl trisulfide, and Tzanetakis et al. 1998). In Danish and Sardinian Feta,
S-methyl thioesters are also well known for this yeast (Berger uncontrolled gas production by yeast caused swelling defects
et al. 1999). Further, G. geotrichum is able to produce volatile in the final product (Fadda et al. 2001; Westall and Filtenborg
compounds like methylketones, alcohols, esters, and fatty 1998). In Danish Feta with swelling defects Torulaspora
acids (Jollivet et al. 1994). delbrueckii was predominant while the responsible yeast in
Some strains of G. geotrichum have anti-microbial Sadinien Feta was found to be Dekkera anomala. Several
activities. Production and excretion of 2-hydroxy-3-phenyl- other yeast species such as Y. lipolytica, D. hansenii, C. sake,
propanoic acid, which have a broad anti-bacterial effect K. marxianus, C. bututyri, and G. candidum, Dekkerar
276 Hansen and Jakobsen

bruxellensis, K. lactis were isolated from Feta cheese in these enterobacteria (Brugier and Patte 1975; Polonelli and Morace
investigations. The yeast population in the Sardinian Feta was 1986). Many yeast also produces metabolites, e.g., short-
the same in the cheese from the two dairies investigated while chain fatty acids known to be toxic against undesired
the occurrence of yeast in Danish Feta seems to vary from micro-organisms in the intestinal tract (Gedek 1991).
dairy to dairy. The high concentration of yeast in the Even though most yeast strains are considered to be safe, it
environment of the production area in the dairies indicated is noteworthy that some strains found in dairy products, e.g.,
that the occurrence of yeast in these types of Feta was due to C. catenulata, G. geotrichum, and C. kefyr are seen as
recontamination. opportunistic pathogens (Radosavljevic et al. 1999), and
could represent a risk for human health especially for
immunocompromised persons (Minervini et al. 2001;
5 YEAST AS PROBIOTICS Radosavljevic et al. 1999).

According to the classical definition probiotic organisms

regulate the microbial colonization in the digestive tract with 6 CONCLUSION
a beneficial effect on human health (Gedek 1991) In relation
to current and future probiotic products this traditional The present study has documented the positive as well as the
definition seems to be too narrow (Jakobsen and Narvhus negative aspects of yeast in the dairy industry, and the focus
1996). The definition of a probiotic starter culture should be on yeast in dairy products seems to be more intensive than
added a third-dimension, i.e., the extra nutritional – ever.
physiological values leading to a more general improvement The negative role of yeast as spoilage organisms is still a
of human health (Kurmann 1993; Lambelet et al. 1992). As problem and seen in all kind of dairy products. The most
such, the role of yeast as probioticum in dairy products has effective way to solve this problem is improvement of
been overlooked. Studies of probiotic yeast as starter culture hygiene and sanitation, and tighter specifications for various
in dairy product, so far, has been very limited even though ingredients, e.g., for yoghurt.
yeast occur in many dairy-related products. Historically, yeast The technological characteristics of important yeast and
as a probioticum has been linked with livestock feed and to the complexity of the microbiota of many dairy products have
the genus Saccharomyces. As reviewed by Gedek (1991) been discussed in several recent publications. Especially, the
S. boulardii is a nonpathogenic yeast and is used both as understanding of the positive role yeast play in maturation of
preventive and therapeutic agent for the treatment of different surface-ripened cheese is increasing, but only to a very
diarrhoeal diseases (McFarland and Bernasconi 1993; limited extent brought to the point of active use of yeast, i.e.,
Surawicz et al. 1989). S. boulardii was first isolated in the application of yeast as starter culture. New information on
Indonesia in the 1950s. In the fourth edition of the the technological characteristics of yeast and the diversity
taxonomical study “The Yeasts” edited by Kurtzman and among strains of the same species is providing a better
Fell (1998). S. boulardii is taxonomically classified under the background for the selection and use of yeast as starter
name S. cerevisiae. cultures in dairy products. The advantages of deliberate yeast
Among few studies on the use of probiotic yeast in dairy addition to fermented and matured dairy products are evident.
products to be mentioned is that Lourens-Hattingh and The advantages are mainly related to the proteolytic and
Viljoen (2001) investigated the potential of adding lipolytic activity of yeast, their influence on aroma formation,
S. boulardii to yoghurt. The result showed that S. boulardii and their acceleration of the ripening process. Further, yeast
was able to survive four weeks at 48C and even multiply to play an important role in microbial interactions resulting in
a level of about 106 in plain and fruit yoghurt. Gas and improved performance of starter cultures and inhibition of
alcohol production were not observed, but it might be a undesired micro-organisms. Yeast also have probiotic
constraint for incorporating S. boulardii into retail properties.
bio-yoghurt (Lourens-Hattingh and Viljoen 2001). The taxonomy for yeast is very complex. New methods,
Several specific interactions between S. cerevisiae and including molecular techniques are used increasingly, but for
enteric pathogens, e.g., Escherichia coli, Salmonella, and many of the yeast genera related to dairy products the
Shigella have been reported. Further, S. cerevisia is reported molecular methods are not yet fully developed or sensitive
to bind enterotoxin from enterobacteria to the surface of the enough to distinguish yeast strains at species or subspecies
yeast through a mannose-specific reaction (Gedek 1991). It level. However, these techniques are a valuable supplement to
has been reported that S. cerevisiae is able to survive the the traditional methods of identification and powerful tools in
passage through the intestinal tract, with live cells detectable ecological studies including the microbiota from indigenous
in the small intestine, which emphasize its potential as a fermented dairy products and typing of starter cultures. New
probiotic. approaches for developing methods for typing yeast with
Exploitation of killer factors as anti-microbial compounds potential as starter culture and characterization of yeast
could lead to another possibility for using yeast as probiotic. causing spoilage in dairy product should be given high
Killer toxins are proteins or glycoproteins secreted by the priority in future research together with development of yeast
yeast cell and toxic to specific range of micro-organisms, e.g., starter cultures and the use of yeast as probiotics.
Yeast in the Dairy Industry 277

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Microbiol 37:193– 199. J 9:117 –124.
25
Flavors and Aromas

Renu Agrawal Central Food Technological Research Institute, Mysore, India

1 INTRODUCTION 2.1 Terpenoids

The demand for natural flavoring substances has increased Terpenoids, constitute one of the largest groups of natural
sharply in recent years, and tremendous interest has been products and impart a wide variety of pleasant and floral
shown in their production by microorganisms (Berge and scents. These compounds contain one, or more, basic isoprene
Evenhuis 1998; Carina 1995; Schindler and Schmid 1982; units which are joined head to tail. Depending on the number
Schreiber et al. 1997). Fungal biotransformation of low- of units, terpenes are classified as monoterpenes (2 units),
cost substrates to flavor and aroma compounds having a sesquiterpenes (3 units), diterpenes (4 units), triterpenes (6
high value appears to have great commercial potential. units), and polyterpenes (higher units). Terpenoids, produced
Such compounds have been accepted by the United States from filamentous fungi are used by various food industries.
Drug Administration (USDA) and by the European Flavour Readily available monoterpenes, such as a-pinene and
Commission (88/388/EWG 1988; 91/71 EWa and 91/2 limonene, are used as substrates for conversion into flavoring
EWG 1991) as being natural and falling under the compounds (Berger et al. 1992; Van der Werf et al. 1997).
generally regarded as safe (GRAS) category (Janssens et al.
1992; Taylor and Mottram 1996). The flavor products 2.1.1 Monoterpenes
obtained by microbial routes are optically pure, extra-
cellular, and suitable for commercial exploitation because a. Limonene Conversion. Limonene can be converted to
of easy down-stream processing and high yields. Extensive major products like carveol and carvone by Penicillium
reviews on this subject have been published by italicum and P. digitatum (Bowen 1975). When limonene
Demyttenaere (2001); Hagedorn and Kaphammer (1994); concentration was increased there was a decrease in the
Mestri (1994); and Van der Werf et al. (1997). This quantity of end-products produced. Addition of sucrose
chapter will deal with the description and discussion on increased microbial growth but the conversion was low. The
fungal flavors and aromas. conversion of limonene to carveol seems to be a single-step
reaction, involving addition of a hydroxyl group at C-3
(Figure 1). Limonene biotransformation was first carried out
by Rama Devi and Bhattacharyya (1978) who studied the
2 PRODUCTION OF MICROBIAL FLAVORS oxygenative and prototropic molecular rearrangements during
terpene transformation by A. niger.
Flavoring chemicals are produced either by biotrans- Abraham et al. (1985) investigated the biotransformation
formation of precursor compounds to flavour end-products, of (R)-(þ) limonene to a-terpineol by P. digitatum. DSM
or by de novo synthesis. Fungi play an important role in 62840. Abraham et al. (1986) studied microbial trans-
this field, as they can transform abundantly available formation of terpenoids with 1-p-menthene; they used
substrates such as terpenes, and thereby make the process Corynespora cassiicola and Diplodia gossypina to convert
economical. (S)-(2) and (R)-(þ) limonene, a-terpinene, g-terpinene, and

281
282 Agrawal

with the problems related to commercialization of terpenoid

products. Rensburg et al. (1997) have shown the possibility of
limonene biotransformation in yeasts.

b. Pinenes. The biotransformation of a-pinene has con-

siderable commercial importance. Hydroxylation of a-pinene
by A. niger has been reported by several workers
(Bhattacharyya et al. 1960); as also the references in their
papers. Verbenol, verbenone, and sobrerol were obtained on
biotransformation of a-pinene (Bhattacharyya et al. 1960).
Verbenone was probably a product of auto-oxidation, whereas
verbenol was formed by microbial oxidation (Figure 2;
Bhattacharyya and Ganapathy 1965). Rama Devi and
Bhattacharyya (1978) studied the conversion of a-pinene to
1-p-menthane, involving rupture of the cyclobutane ring in
the bicyclic system.
Agrawal (1999) used an UV auxotroph of A. niger to
increase the yield of verbenol from 10% obtained with a wild
type to 25%. The enzyme a-pinene hydroxylase involved in
a-pinene conversion to verbenol was NADPH dependent, and
Figure 1 Biotransformation of limonene. could be stabilized for 3 days using sorbitol along with DTT
(Nazhat-ul-Ainn and Agrawal 2002). No biotransformed
products from a-pinene were found under nitrate reducing
terpinolene to 1,2-trans-diols. They found that the inter- conditions (Pavlostathis and Misra 1999). Optimization of
mediary epoxide could be cleaved by the hydroxyl groups growth conditions and media conditions can enhance the yield
present in the substrates. Noma et al. (1992) employed of verbenone (Agrawal and Joseph (2000a)) from a-pinene
Aspergillus cellulosae to transform limonene to major using Penicillium sp. Major biotransformations of some
products like carveol, perillyl alcohol, and a-terpineol. terpene substrates to flavoring compounds are given in
They reported the possibility of introducing an oxygen Table 1.
functional group into limonene at the C-3 position, utilizing
citrus peel oil as C source. Demyttenaere et al. (2001) used a 2.1.2 Other Compounds
solid phase micro-extraction technique to study the conver-
sion of limonene by P. digitatum, to obtain a-terpineol as the Many commercially important flavouring constituents, like
major product. For details refer to the review by Van der Werf esters, lactones, aldehydes, ketones, tobacco flavorings, and
et al. (1997), which deals with terpene biotransformation, and alcohols are produced with the help of fungi.

Figure 2 Biotransformation of pinenes.

Flavors and Aromas 283

Table 1 Fungal biotransformation of terpene substrates to high valued flavoring compounds

Fungi Substrate Products References

P. italicum Limonene Carveol, carvone Bowen (1975)
A. cellulosae Limonene Carveol, perillyl alcohol, and a-terpineol. Noma et al. (1992)
Y. lipolytica Limonene Perillic acid, 7-hydroxy piperitone Rensburg et al. (1997)
P. digitatum Limonene a-Terpineol Demyttenaere et al. (2001)
A. niger a-Pinene Verbenol, verbenone Prema and Bhattacharyya (1962)
Penicillium sp. a-Pinene Verbenol Agrawal et al. (1999)

c. Ester. Various aliphatic esters could be obtained from the formation of g-d- and d-hydroxy acids in Trichoderma
lyophilized, whole cells of Rhizopus oryzae; such cells can harzianum. Sporobolomyces odorus culture was able to
tolerate high substrate concentrations, and hence allow the produce an intense peach (g-decalactone) and mutton (cis-6-
production of large amounts by semi-continuous or dodecen-4-olide) odors (Lee and Chou 1994), P. roqueforti
continuous addition of the substrate, e.g., geranyl butyrate was employed for producing lactones, mainly 4-dodecanolide
(Molinari et al. 1995). Lyophilized whole cells of R. Delemer from hydrolyzed oils like soybean and copra (Chalier and
were utilized to catalyze direct esterification of primary Crouzet 1992). The compound, 4-dodecanolide is formed by
alcohols (n-hexanol) to give a very high yield (98%) of hexyl g-hydroxylation of the corresponding saturated acid, or by
caprylate (Molinari et al. 1998). Agrawal et al. (2000) have b-oxidation of oleic acid into 3-dodecanoic acid, followed by
demonstrated the production of dihydrocarvyl acetate from lactonization of the mentioned acids. The biosynthetic
nerol, using Mucor sp. Although the metabolic pathway was pathway in S. odorus, which results in the formation of
not studied, it appears that ring closure through geranyl 4-hexanolide as an oxidation product of linoleic acid, was
pyrophosphate led to the formation of dihydrocarvone, which studied by Taylor and Mottram 1996. It is possible that
may then be reduced to form dihydrocarveol and then lactone is the result of b-oxidation of linoleic acid to
acetylated to form dihydrocarvyl acetate. Regio-specific 3,6-dodecadienoic acid, which is later hydrated and
esters were obtained by using A. niger to form acetates of lactonized.
citronellol, geraniol, and linalool (Madhyastha 1988). Patel Production of 6-pentyl-a-pyrone by Trichoderma viride
et al. (1992) described a simple method of utilizing has been reported (Prapulla and Karanth 1992). The addition
Geotrichum candidum to improve the optical purity of of amberlite XAD-2 resin to the medium overcomes product
(S)-(2 )-4-chloro-3-hydroxy butanoic acid methyl ester by inhibition. Kalyani et al. (2000) studied the formation of
converting it to 4-chloro-3-oxobutanoic acid methyl ester. 6-pentyl-a-pyrone in surface cultures of T. harzianum, under
Cell extracts contained a single enzyme that catalyzed the submerged conditions. Characterization of 6-pentyl-a-pyrone
reduction to the hydroxy product. Farbood et al. (1987) also has been done using T. koningii (Benoni et al. 1990).
demonstrated the synthesis of terpene esters through an Aldehydes are usually formed via a Strecker degradation
amino acid precursor in G. fragrans. Gatfield (1988) of amino acids. This involves oxidative deamination and
worked out the possibility of ester synthesis from lipase decarboxylation of a-amino acids, leading to the formation of
enzyme in Mucor michei; this process could improve upon an aldehyde containing one carbon atom less than the original
the isolation and purification steps, when compared to the amino acid (Mottram 1994). Fungi have shown great potential
aqueous fermentation systems. in producing high yields of these flavoring compounds.
Among aldehydes, benzaldehyde (almond aroma) and
d. Lactones, Aldehydes, and Ketones. Biogeneration of vanillin, are widely used by food industries. Cis/trans
volatile lactones from fungi has proved to be industrially
successful (Cardillo et al. 1990). Lactone formation appears
to be the result of a metabolic overflow. When the regular
3-hydroxylation of fatty acids is expanded to 4- or
5-hydroxylations, lactone formation takes place. These
compounds impart fruit-like, buttery, sweet, or nutty odors.
Macrocyclic musks were produced from Ustilago zeae,
using ustilagic acids as precursors (Gatfield 1988). Using
octanoic acid, Gregory and Eilerman (1989) accomplished the
bioconversion in Mucor sp. to delta-gamma octalactone.
Farbood et al. (1990) obtained a mixture of saturated and
unsaturated g-decalactones using g-keto acid as the substrate;
the acid is reduced to g-hydroxy acid, which ultimately
cyclizes to g-lactones. Serrano-Carreon et al. (1992) studied Figure 3 Biotransformation of isoeugenol.
284 Agrawal

isoeugenol (1:4) was converted by A. niger into vanillin Larroche et al. (1995) have reported the formation of
(Figure 3; Robenhorst and Hopp 1991). hydroxy and exo derivatives from b-ionone by A. niger. The
Berger et al. (1987) studied the formation of a methoxy recovery of the products was 100%, after 230 h of cultivation.
benzaldehyde in Ischnoderma benzoicum. Casey and Dobb This work paved the way for a possible fed-batch procedure,
(1992) reported the formation of aromatic aldehydes from without replacement of the medium. When b-ionone was the
aromatic amino acids, via a phenylpyruvic acid like only carbon source in the medium it stopped fungal growth,
benzaldehyde, using Trichosporon beiglii. Ketones are and was converted into hydroxy metabolites, probably by the
characterized by the presence of a carbonyl group, and are action of a hydroxylase system. Grivel et al. (1999) made a
classified as aliphatic, aromatic, or phenol derivatives. They dynamic model for biotransformation of b-ionone in A. niger.
are synthesized by fungi in response to the presence of short- As the precursor is less soluble in water, it gave rise to a two-
chain fatty acids, or as a means of recycling of COA. Mestri phase liquid system with high volatility and poor chemical
(1994) has discussed the production of nootkatone from stability. The products were 5,6-epoxy-5,6-dihydro-b-ionone,
valencene by an oxidation process involving P. camemberti dihydro actinidiolide, and 4-oxo-b-ionone, with a molar yield
and A. niger (Figure 4). of 32%; a high loss by stripping is a serious drawback of this
Biotransformation of nerol, geraniol, and citral, in surface process.
cultures of A. niger and P. digitatum, to 6-methyl-hept-5-en-
2-one (92–99%) has been studied by Demyttenaere and
DePooter (1996) and Demyttenaere et al. (2000). These f. Acid Formation. The yields of acid produced by fungi
workers indicated an oxidative pathway wherein the alcohol are found to be commercially feasible, and are being utilized
is oxidized to aldehyde, and then to 6-methyl-hept-5-en-2- by industries. Armstrong et al. (1989) reported high yields of
one, with no intermediary products. The pathway of this citric acid, which is perhaps the best known flavor
biotransformation of geraniol into 6-methylhept-5-en-2-one compound produced by A. niger. Fabritius et al. (1998)
by P. digitatum has been elucidated recently by Wolken and studied the conversion of palmitic acid to (R)-(Z)-3-
Van’ der Werf (2001), who also point out that citral is hydroxy-9-octadecendioic acid in Candida tropicalis; when
converted into geranic acid in this process. Furthermore, they ricinoleic acid was utilized as the sole carbon, optically pure
also detected a novel enzymatic activity, wherein citral lyase (R)-(Z)-7-hydroxy-9-octadecenedioic acid was obtained. As
converts citral to methylheptenone and acetaldehyde, only one regio-isomer was obtained, the hydroxylation was
independently of cofactors. regiospecific. Fatty acids are, therefore of great interest to
the chemical industry, as they provide a new avenue for
e. Ionone. Tobacco flavor is obtained by the transform- commercial exploitation.
ation of ionone compounds. These compounds are widely
distributed in nature, and are the constituents of many g. Alcohols. Various alcohols, which are utilized widely
essential oils. Fungi, such as A. niger, converted ionone to as flavors are produced by fungal conversions. The formation
a-cyclohomo-geraniol, 3-oxo-a-cyclohomo-geraniol, and
of sclareol, a labdoane diterpene used in foods, has been
benzofuran, as depicted in Figure 5 (Krasnobajew and
studied in C. albidus (Farbood et al. 1986). Bioconversion of
Helmlinger 1982).
citronellol, leading to the formation of 2,6-dimethyl-1,8-
octanediol and (E)-2,6-dimethyl-2-octene-1,8-diol was stu-
died with four strains of Botrytis cinerea in grape must
(Brunerie et al. 1987). The substrate was metabolized to the
v-hydroxylation product (E)-2,6 dimethyl-2-octene-1,8-diol
and 2,6-dimethyl-1,8-octanediol. Regiospecific esters were
produced by A. niger from acetates of citronellol, geraniol
and, linalool (Madhyastha 1988).The major product formed
from citronellol acetate, was citronellol. Geraniol was
produced from geranyl acetate while linallol and 8-hydroxy
linalool were the major compounds from linalyl acetate. A
bioreactor, based on an aqueous/organic two-phase system,
was designed by Doig et al. (1998) for the biotransformation
of baker’s yeast to geraniol. Uzura et al. (2001), using resting
cells of Fusarium moniliforme, and propyl benzene as
substrate, was able to form 1-phenylpropenol with high regio-
and stereo-specificity. Lomascolo et al. (2001) worked out the
possibility of producing 2-phenylethanol (rose flavor) from
A. niger, using as precursor phenyl alanine, which was
synthesized as the sole aromatic product. Some examples of
Figure 4 Biotransformation of valencene. nonterpene fungal flavoring compounds are given in Table 2.
Flavors and Aromas 285

Figure 5 Biotransformation of ionone.

2.2 De Novo Synthesis strong coconut aroma of 6-pentyl-2 pyrone (Welsh et al.
1989).
Unlike in the case of biotransformation, in de novo synthesis Composition of the medium and age of culture generally
no specific precursors are needed in the medium. Products influence the formation of flavoring compounds in de novo
formed by using different micro-organisms include lactones, synthesis. For instance, an intense banana aroma was formed
esters, and oxygenated terpenes (Table 3; Schindler and by Ceratocystis fimbriata when the growth medium was
Schmid 1982; Scharpf et al. 1986). supplemented with several nutrients (Christen and Rainbault
The fruity flavor and aroma compounds produced by 1991). Similarly, Lee et al. (1999) also reported formation of
de novo synthesis depend on the media constituents like g-decalactone, in an optimized immobilized culture of
glucose, amino acids, and salts (Quehl and Ruttloff 1992). In Sporidiobolus salmonicolor.
the de novo synthesis category most of the experiments
carried out till now, were only on a laboratory scale, and the
product yields were found to be low. However, they have
helped in the study of the enzyme systems involved, and of 2.3 Complex Flavors
their reaction pathways during the synthesis. Sarris and
Latrasse (1985) demonstrated when Fusarium poae was Sharpell (1985) has discussed in detail some complex
grown on a solid malt medium till sporulation, it produced a mixtures of flavors and fragrances, which are associated with
lactone with a peach-like aroma; but under insufficient natural products. Microbial processes appear to be very
aeration 2-methylbutanol and 3-methylbutanol were pro- promising for the production of complex dairy and mushroom
duced. T. viride on a simple growth medium generated a like flavors. Screening of different organisms made it possible

Table 2 Nonterpene fungal flavoring constituents

Compound Fungi Products References

Ester R. delemer Aliphatic esters Molinari et al. (1998)
Lactones S. odorus 4-Decalactone Lee and Chou (1994)
T. viride 6-Pentyl-a-pyrone Prapulla and Karanth (1992)
Aldehyde I. benzoicum Methoxy benzaldehyde Berger et al. (1987)
T. beigelii Benzaldehyde Casey and Dobb (1992)
Acid C. tropicalis 3-Hydroxydioic acid Fabritius et al. (1998)
Alcohol B. cinerea Octanediol Brunerie et al. (1987)
A. niger 2-Phenylethanol Lomascolo et al. (2001)
286 Agrawal

Table 3 De novo synthesis of fungal flavoring constituents

Fungi Odour attribute Products References

T. viride Coconut 6-Pentyl-2-pyrone Prapulla and Karanth (1992)
G. candidum Quince-like Ethyl ester Latrasse et al. (1987)
F. poae Coconut g-Lactone Sarris and Latrasse (1985)
A. niger Sour Citric acid Armstrong et al. (1989)
A. oryzae Pine-like 1-Octen-1-ol Scharpf et al. (1986)
A. terreus Fruity Ethyl acetate Schindler and Schmid (1982)

to identify some of the organisms, which produced complex Unilever Chemical Company UK; ethyl butyrate by Hercules,
flavors (Gatfield 1988). USA (Dziezak 1986); menthol by Nippon Mining Company,
Japan (Watanabe and Inagaki 1978); and macrocyclic musk
by Quest International (Jeffcoat and Willis 1988).
2.4 Dairy Flavors

Dairy flavors, primarily cheese flavors, are widely used in the

4 CONCLUSIONS
food industry. Kristofferson (1973) has studied the biogenesis
of cheese flavor. Maga (1974) investigated the formation of
flavor constituents by using Penicillium sp. on milk and bread. In view of the increasing demand for natural flavors, their
Similarly, formation of cheese flavor, either by the enzymatic production by microbial means seems to be a good
or the chemical route, has been studied by Law and preposition. The method is efficient, effective, and also
Mulloholland (1991) who used fungal cultures. economical. Monoterpene substrates like limonene and alpha-
pinene, which are produced in large quantities in nature, can
be used for the production of flavoring compounds of high
2.5 Mushroom Flavors value. The product yields obtained are high enough for
industrial production, and many microbial processes are
already operating. Newer genetic engineering techniques may
Sugihara and Humfeld (1954) who found that mushroom
still help in enhancing the product yield. There is also a need
flavor could be produced by Lepiota rhacodes when grown
for metabolic pathways to be studied in detail so that high
under submerged conditions. Gilbert (1960); Litchfield et al.
product yields can be obtained by its regulation. There is also
(1963); and Le Duy et al. (1974) studied the production of
need to investigate the effects of different parameters on a
mushroom flavor by Morchella crassipes. Lentinus edodes
pilot plant scale which will lead to many more successful
produced 1-octen-3-ol, 50 -amp, an intense mushroom flavor
commercial technologies.
when the medium was supplemented with ethanol (Sugimori
et al. 1971). Hamid et al. (1972) investigated the production
of mushroom flavor from Trichoderma nudum under
submerged conditions, while Van Eybergen and Scheffers ACKNOWLEDGEMENTS
(1972) reported its production in the mycelium of Boletus
edulis. Dijkstra (1976), Pyssalo and Honkanen (1976), and
I express my gratitude to V. Prakash. I thank M.S. Prasad,
Card and Avisse (1977) found that the fermentation
Lonsane, Demyttenaere, M.V. Patwardhan for discussions
conditions as well as media constituents play a vital role in
and M.S. Divyashree for assistance.
the production yield. Mushroom flavors have also been
obtained from A. oryzae (Scharpf et al. 1986), Caprinus
micaceus, Merulins rufus, and Poria vaillantu (Schindler and
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26
Antifungal Food Additives

Purbita Ray / Michael B. Liewen General Mills, Inc., Minneapolis, Minnesota, USA

1 INTRODUCTION Fungi are generally easily inactivated by heat treatments

such as pasteurization, although some heat-resistant molds
The problem of food spoilage has plagued humans throughout associated with fruits and fruit products can survive rather
history. As early humans evolved from a gathering and severe heat treatments and spoil products such as pasteurized
hunting life style to raising crops and keeping animals, they juices and canned fruits. In addition, molds can grow over a
were forced to store their own food. Early attempts to wide range of temperature. Some mold can grow at
preserve foods involved use of sugars, spices, salts, and wood temperatures less than 08C while other can grow at 50–558C.
smoke. Today, however, preservation has used such factors as Molds have an absolute requirement for oxygen. Many
temperature, water activity, pH, gases, organic acids, salts, species, however, are efficient oxygen scavengers and can
antibiotics, irradiation, packaging, and various combinations grow in atmospheres containing less that 1.0% O2 (Cerny
of these factors. No matter which factors are selected, use of 1979). From a practical standpoint, it can be difficult to inhibit
the proper antimicrobial is dependant on the chemical mold growth in foods solely by exclusion of oxygen from the
properties of the antimicrobial; properties and composition of package. Foods often contain dissolved oxygen, which slowly
the food product; type of preservation system, other than the equilibrates with the package headspace, and oxygen can
chemical, used in the food; type, characteristics, and number leach through all but the most impermeable packages. While
of microorganism; safety of the antimicrobial and cost mold growth can be delayed, it is not inhibited over long-term
effectiveness of the antimicrobial (Ray 2001). storage. Yeast have no requirement for oxygen and can grow
Fungi are among the most challenging organisms to inhibit in its complete absence.
in foods given their ability to grow under a diverse range of Antifungal food additives are an efficient, cost-effective,
environmental conditions. Environmental conditions such as and often the only successful way to control fungal growth in
water activity (aw), pH, temperature, and atmosphere can be foods. Antifungal food additives are basically chemicals that
manipulated to control fungal growth. However, these prevent or interfere with mold growth. These chemicals may
conditions often need to be taken to extremes to control be found naturally occurring in certain foods, such as some
fungi, since subgroups exist that have become adapted to organic acids and essential oils, or may be added to food
extreme environmental conditions. Using water activity as an during processing (Naidiu 2000; Thompkin and Singh 2000).
example, most molds are inhibited by an aw of 0.80 or lower, The various antifungal food additives are briefly overviewed
although some xerophilic molds can grow at aw values as low in this chapter.
as 0.65. Most yeast are inhibited by aw values of 0.87, but
some osmophilic yeasts can grow at aw values as low as 0.60
(Farkas 1997; ICMSF 1980). 2 ORGANIC ACIDS
Most fungi are little affected by pH over broad range,
commonly 3–8. Some molds can grow at pH 2.0, and yeasts Organic acids have been used for years to control fungal
at pH 1.5 (ICMSF 1980; Rahman and Labuza 1999). spoilage of foods. They find wide use because of solubility,
However, as pH moves away from an organism’s optimum taste, and low toxicity. The mode of action of organic acids is
growth range, typically about 5.0 for fungi, the effect of other attributed to depression of intracellular pH by ionization of
growth-limiting factors becomes more apparent. the undissociated acid molecule or disruption of substrate

291
292 Ray and Liewen

transport, by alteration of cell membrane permeability. In include baked goods (cakes and cake mixes, pies and pie
addition to inhibiting substrate transport, organic acids may fillings, doughnuts, baking mixes, fudges, and icing), dairy
inhibit NADH oxidation, thus eliminating supplies of products (natural and processed cheese, cottage cheese, and
reducing agents to electron transport systems (Doors 1993; sour cream), fruit product (artificially sweetened confections,
Liewen and Marth 1985). dried fruit, fruit drinks, jams, jellies, and wine) vegetable
Since, the undissociated portion of the acid molecule is products (olives, pickles, and relishes salads), and other
primarily responsible for antifungal activity, effectiveness is miscellaneous products (certain fish and meat products,
dependant upon the dissociation constant of the acid and pH mayonnaise, margarine, and salad dressings) (Sofos and
of the food to be preserved. Because the dissociation constant Busta 1993).
of most organic acids is between pH 3 and 5, organic acids are Environmental factors such as pH, water activity,
generally most effective at low pH values. This along with temperature, atmosphere, type of microbial flora, initial
solubility properties determines the foods in which organic microbial load, and certain food components, singly or in
acids may be effectively used. combination can influence the activity of sorbate. Together
A few fungal species possess mechanisms of resistance to with preservatives such as sorbic acid, they often act to
organic acid preservatives. Saccharomyces baili is resistant broaden antimicrobial action or increase it synergistically.
to high concentrations of sorbic and benzoic acids (Warth Use of other preservatives in combination with sorbate can
1977). Some molds in the genus Penicillium can grow in broaden or intensify antimicrobial action. If growth of
the presence of high concentrations of sorbic acid and spoilage or pathogenic organisms is inhibited, but the
decarboxyated sorbic acid to 1,3-pentadiene, a volatile microorganism is not killed, growth will eventually resume
compound with an extremely strong kerosene-like odor under proper conditions. The length of inhibition will vary
(Liewen and Marth 1984; 1985; Tsai et al. 1988). When with storage temperatures as well as with any of the other
resistance to or metabolism of an organic acid is a problem, factors discussed.
other preservative systems must be used. Sorbic acid is a broad-spectrum antimycotic that is
effective against yeast and molds. The antifungal effect of
sorbate is greater at pH less than 5.0. Sorbic acid has little
2.1 Sorbic Acid antifungal activity at pH values higher that 5.6. Above this
pH, little of the acid is in the antimicrobially active
Sorbic acid and its potassium salt are the most widely used dissociated form. However, sorbic acid has a relatively high
forms of this compound and are collectively known as dissociation constant compared to benzoic or propionic acids
sorbates. The salt forms are highly soluble in water, as is true and is, therefore, usually the most effective of the organic
for all organic acids. Their most common use is preservation acids at pH levels of 5.0 or higher. This is presented in Table 2
of food, animal feed, cosmetic, and pharmaceutical products, (Luck and Jager 1997; Ray and Bullerman 1982).
as well as technical preparations that come in contact with the
human body. Methods of application include direct addition
into the product; dipping, spraying, or dusting the product; or 2.2 Benzoic Acid
incorporation into the wrapper (Ranun 1999).
Typical use levels in foods range for 0.02% in wine and
Benzoic acid also has widespread use in the food industry. It
dried fruits to 0.3% in some cheeses (Table 1). Food in which
occurs naturally in raspberries, cranberries, plums prunes,
sorbate has commercially useful antimicrobial activity
cinnamon, and cloves (Doors 1993). As an antifungal food
additives, the water-soluble sodium and potassium salts and
the fat-soluble acid form are suitable for food and beverages
Table 1 Typical concentration (%) of sorbic acid used in
with a pH below 4.5. Benzoates have little effect at neutral pH
various food products
values. They are not as effective as sorbates at pH 5.0
Cheeses 0.2– 3.0 (Table 2), but their effectiveness increases at lower pH values.
Beverages 0.03– 0.10
Cakes and pies 0.05– 0.10
Dried fruits 0.02– 0.05 Table 2 Minimum concentration (%) of preservative required
Margarine 0.05– 0.10 for inhibition of mold growth at pH 5.0
Mayonaise 0.10
Species
Fermented vegetables 0.05– 0.20
Jams and jellies 0.05 Compound A. soini P. citrinum A. niger
Fish 0.03– 0.15
Semimoist pet food 0.1– 0.30 Benzoic acid 0.15 0.20 0.20
Wine 0.02– 0.20 Propionic acid 0.06 0.08 0.08
Fruit juice 0.05– 0.20 Sorbic acid 0.02 0.08 0.08

Source: Liewen and Marth (1985). Source: Liewen and Marth (1984).
Antifungal Food Additives 293

Benzoic acid is active against yeasts and molds, including Marshall and Bullerman (1986) for antifungal properties.
aflatoxin-forming microorganisms (Ray and Bullerman Growth of Aspergillus, Penicillium, Cladosporium, and
1982). The acid form is often added to the fat phase and the Alternaria spp. were inhibited in media containing 1% of
sodium salt to the water phase of product such as salad the sucrose esters.
dressings, mayonnaise, pickled vegetable, fruit products, and
fruit drinks. Because benzoate can impart a fairly strong bitter
off-flavor, it is frequently used in combination with sorbate.
3 ANTIBIOTICS
This mixture is often more effective in inhibiting yeast and 3.1 Natamycin
molds than a comparable level of either preservative alone
(Luck and Jager 1997). In addition, the mixture is less Natamycin, formerly called pimaricin is an antibiotic that
offensive organoleptically than benzoate alone. possesses strong antifungal properties, yet is not active
against bacteria. Its use is currently allowed in several
2.3 Propionic Acid countries. Researchers have demonstrated that natamycin is
active at very low concentrations against many of the fungi
known to cause food spoilage. Levels of 10 ppm have been
This organic acid inhibits molds but not yeasts. It occurs in reported to be effective in Swiss cheese to control the growth
some foods as a result of natural processing. It is present in of Penicillium citrinum while solutions of 1000–2000 ppm
Swiss cheese at concentration up to 1%, where it is produced are effective as dips for cheese (Jay 1982; Pugazhenthi et al.
by the bacterium Propionibacterim shermanii (Beuchat 1999). Natamycin inhibits aflatoxin formation by molds only
2000). Since, yeast are typically unaffected, the acid can be when growth was completely inhibited (Ray and Bullerman
added to bread dough without interfering with leavening 1982).
(Ranun 1999).
In the food industry, propionic acid is often used as a
sodium or calcium salt (Ray 2001). Propionates are used 3.2 Nisin
primarily to inhibit molds in bakery goods. In addition to their
antimycotic properties, propionates will inhibit Bacillus Nisin is active against gram-positive bacteria but has not been
mesntericus, the rope causing bacterium. Propionates are also reported to be effective against yeasts and molds (Luchansky
used in a limited extent to inhibit mold growth in processed 1999).
cheese.
The antifungal activity of propionic acid is weak compared
to the other organic acids. Therefore, propionates must be 4 METABOLITES FROM LACTIC ACID
used in relatively high concentrations to be effective. As with BACTERIA
other organic acids, the pH value of the food to be preserved
affects antimicrobial activity. Because of its low dissociation A wide variety of raw foods are preserved by lactic acid
constant, propionic acid is active in a pH range similar to that fermentation, including milk, meat, fruits, and vegetables.
of sorbic acid (Eckland 1990). Reduction of pH and removal of large amount of
carbohydrates by fermentation are the primary preservation
actions that lactic acid bacteria (LAB) provide to a fermented
2.4 Medium-Chain Fatty Acids food. These actions are largely ineffective in preventing the
growth of fungi in foods. However, it has also been
Generally, fatty acids are most effective as inhibitors of gram- recognized that LAB can produce inhibitory substances other
positive bacteria and yeasts, although some fatty acids exhibit than organic acid (acetate and acetate) that are antagonistic
antimycotic activity. Chipley et al. (1981) observed that fatty toward other microorganisms (Batish et al. 1997). The
acid derivatives reduced growth and aflatoxin production by antibacterial properties of LAB are well documented. Several
Aspergillus spp. LAB, typically of the genera Lactococcus and Lactobacillus
Polyhydric alcohol fatty acid esters have great potential for produce antibacterial substances. Antifungal properties of
use as emulsifiers in food formulations (Razani-Rohani and LAB have received little attention, however, several
Griffiths 1994). They also possess antifungal properties and, metabolites of LAB have been reported to have antifungal
therefore, may exert a preservative effect in foods. Kato and activity.
Shibasaki (1975) demonstrated strong fungistatic activity of Batish et al. (1989) screened different lactic starter
glycerol monocaprate and glycerol monolaurate toward cultures for their antifungal activity with the goal of
Aspergillus niger, Penicillum citrinum, Candida utilis, and commercially exploiting their antifungal potentials. They
Saccharomyces cervisiae. Sucrose monocaparte and sucrose found several strains of Streptococcus that inhibited a wide
monolaurate were found to be slightly inhibitory to a spoilage variety of molds. While the antifungal substances produced
film-forming yeast inoculated into a soy sauce substrate (Kato by the LAB were not identified of characterized, maximum
1981). Six sucrose esters substituted to different degrees with production occurred at 308C and pH 6.8. Several specific LAB
a mixture of palmitic and stearic acids were examined by metabolites have been reported to have antifungal activity.
294 Ray and Liewen

4.1 Diacetyl Aspergillus and Penicillium species, and concluded that

spices used in amounts as employed normally for ordinary
Diacetyl is a metabolic end product produced by some species food were insufficient as preservatives. However, when used
of LAB. It is best known for the buttery aroma that it imparts in larger amount, cinnamon, cloves, and all spices retarded
to cultured dairy products. Its antimicrobial action has been mold growth. Bullerman (1974) reported that cinnamon in
investigated by Jay who reported that a concentration of concentrations as low as 0.02% inhibited mold growth and
200 ppm was inhibitory to yeast and 300 ppm was inhibitory aflatoxin production in culture media and cinnamon bread.
to molds (Jay 1992). Acidity of the growth medium was Combinations of different levels of potassium sorbate with
shown to have a direct effect on the antimicrobial activity of cloves showed enhanced or possibly synergistic inhibitory
diacetyl. The compound was clearly more effective as an effect on the growth of molds, indicating the possibility of
antifungal agent below 7.0 than above this value. Reasons for using spices and commercial antifungal agents together in
pH associated antifungal activity is not clear. small amount to obtain antifungal activity (Azzouz and
Since, effective concentration of diacetyl imparts a sharp Bullerman 1982).
odor of butter, potential for use in foods as an antifungal agent In most instances, herbs and spices are not effective
is limited. However, its use as a utensil sanitizer and in wash antifungal agents when used in amounts normally added to
or rinse water for certain products is feasible. foods. However, when used in combination with other
preservative systems, they can be valuable contributors to an
antifungal system consisting of interacting physical and
4.2 Microgard chemical preservatives (Naidiu 2000).

Microgard is grade A skim milk that has been fermented by 6 ESSENTIAL OILS
Propionibacterium shermanii and then pasteurized (Hoover
2000). The product prolongs the shelf life of cottage cheese
The antimicrobial activities of extracts from several types of
by inhibiting psychotropic spoilage bacteria (Lyon et al.
plants and plant parts used as flavoring agents in foods and
1993). The product is also antagonistic toward some yeast and
beverages have been recognized for many years. Some of
molds. Microgard consists of proionic acid, diacetyl, acetic
these essential oils have antifungal properties. Conner and
acid, and lactic acid (Al-Zoreky 1991).
Beuchat (1984) documented the effects of garlic and onion
against yeasts and other investigators have shown these
extracts to be inhibitory to molds. Alderman and Marth
4.3 Reuterin
(1976) examined the effects of lemon and orange oils on
Aspergillus flavus and found when the citrus oils were added
Reuterin is a low molecular weight nonproteinaceous, to grapefruit juice or glucose yeast extract medium at
highly soluble, pH neutral metabolite produced by concentrations of 3000 –3500 ppm, growth and aflatoxin
Lactoabacillus reuterii. The compound is a broad-spectrum production was suppressed. When orange oil was added to
antimicrobial with activity that encompasses yeast and either medium at concentrations up to 7000 ppm, growth and
molds as well as bacteria. It may have application in aflatoxin production were greatly reduced although still
preservation of food by reducing populations of pathogenic evident. Recent publications have reported that the essential
and spoilage microorganisms (Daeshel and Penner 1992). oils of anise, coriander, Roman chamomile, basil, and
oregano were inhibitory to food and industrial yeasts (Chao
et al. 2000; Elgayyar et al. 2001).
5 HERBS AND SPICES

Herbs and spices are widely used to impart flavor to foods. It 7 PHENOLIC ACIDS
is generally accepted that certain herbs and spices have
antimicrobial activity and may influence the keeping quality Phenolic antioxidants have been shown by several researchers
of food to which they have been added. However, they are not to possess antifungal activity. Chang and Branen (1975)
currently used with the primary purpose of providing a demonstrated that in a glucose salt medium, 1000 ppm
preservative effect. butylated hydroxanisole (BHA) inhibited growth and
Hoffman and Evans (1911) were among the earliest to aflatoxin production of Aspergillus parasticus spores, and
describe the preservative action of cinnamon, cloves, .250 ppm inhibited growth and aflatoxin production of
mustard, allspice, nutmeg, ginger, black pepper, and cayenne A. parasiticus mycelia. However, they found that at 10 ppm of
pepper. They found that cinnamon, cloves, and mustard were BHA, total aflatoxin production was more than twice that of
most effective and ginger, black pepper, and cayenne pepper the control, with virtually no effect on mycelial weight. These
were least effective. results indicate that at high levels, BHA may serve as an
Bachman (1982) studied the effect of spices and their effective antifungal agent, however, at low levels BHA
essential oils on growth of several test organisms, including may actually stimulate aflatoxin production. The BHA has
Antifungal Food Additives 295

been documented as inhibiting A. flavus, A. parasiticus, sensitive to the gas than bacteria. Propylene oxide has been
Penicillium, Geotrichum, Byssochlamys species, and used as a fumigant for control of microorganisms and insects
S. cerevisiae (Davidson and Naidu 2000). in bulk quantities of goods such as cocoa, gums, processed
Since, the primary use of these compounds in foods is as spice, starch, and processed nutmeats (ICMSF 1980).
antioxidants, their effectiveness as antifungal agents in food Ozone (O3) is a strong antimicrobial agent with numerous
systems has not been adequately studied. While results of applications in the food industry. It has been used for decades
experiments in growth media indicate that these compounds in many countries and was recently given GRAS status in the
exert antifungal effects, extrapolation of these resulting to United States. Ozone in the aqueous or gaseous phase is active
food systems should be done with caution. Interaction of these against a wide range of bacteria, molds, and yeasts. Most
compounds with food components will undoubtedly affect applications are targeted to decontamination of fruit and
their antifungal properties. vegetable surfaces by washing in ozonated water (Xu 1999).
A second application is fruit and vegetable storage. Barth et al.
(1995) assessed ozone exposure on storage of blackberries
8 GASES AND MODIFIED ATMOSPHERES stored at 28C in air with 0.3 ppm ozone. Fungal development
was suppressed while 20% of the control fruits showed decay.
Elimination of oxygen is often used as a control measure for The effectiveness of ozone is influenced by the intrinsic
inhibiting the growth of molds. Exclusion of oxygen will not factors of a food. It also oxidizes food surfaces when used at
prevent growth of yeasts. Studies on bakery products have high levels. Further research may reduce some of these
demonstrated that atmospheric O2 levels must be reduced to concerns so ozone can be used in broader food applications.
0.1–1% to effectively inhibit growth of molds. In studies on
toasted bread, Cerny (1979) demonstrated that visible mold
would occur in 3 days in air; 5 days in 99% N2 –1%O2; and 9 INTERACTION OF FACTORS
. 100 days in 99.9% N2 –0.1% O2, 99% CO2 –1.0% O2,
99.8% CO2 – 0.2% O 2, and 100% CO2 . This study Many of the antifungal agents reviewed in this chapter need to
demonstrates that although molds are considered to be be used at extreme concentrations or levels in a food to be
aerobic organisms, certain species have the ability to grow at effective when used alone. However, a variety of factors can
very low levels of O2 concentrations. Effectively controlling prevent growth of fungi. While fungi tend to be more tolerant
mold by simple gas flushing can be difficult in practice. to adverse environmental conditions than bacteria, combining
Chemical oxygen scavenger can be used in place of or to inhibitory factors such as temperature, water activity, or pH
supplement gas flushing (Farber 1991). Oxygen scavengers with antifungal agents can result in considerable improve-
will also give protection against package leaks and ment of the microbial stability of foods. Suitable combi-
infiltrations of O2 through the package. nations of growth-limiting factors at subinhibitory levels can
Carbon dioxide exerts antifungal action that supplements be devised so that certain microorganisms can no longer
simple exclusion of O2 and, thus is more effective than inert proliferate.
gas such as nitrogen. The gas probably exerts antifungal Sorbic acid at 1000 ppm and pH 7.0 will not inhibit mold
activity by altering intracellular pH levels (Gorris 1994). growth. However, if the pH is lowered to 5.0, growth of most
Recent research has shown CO2 to have potential use with molds will be inhibited (Liewen and Marth 1985).
food. Carbon monoxide inhibits yeast and molds that causes Antioxidants such as BHA and BHT have been shown to
postharvest decay in fruits and vegetables (Wagner and potentiate the action of sorbic acid (Scott 1989). In general,
Moberg 1989). The potential toxicity of this compound to antifungal food additives become more effective as
workers requires special handling procedures. environmental conditions move away from the optimum for
Sulfur dioxide is broadly effective against yeasts and a particular organism.
molds. It is used extensively to control growth of undesirable The level of a single growth-limiting factor that will
microorganism in fruits, fruit drinks, wines, sausages, fresh inhibit a microorganism is usually determined under
shrimp, and pickles. The antimicrobial activity of SO2 is conditions in which all other factors are optimum. In
associated with the unionized form of the molecule. preserving foods more that one factor is usually relied upon to
Therefore, it is most effective at pH values , 4.0, where control microbial growth. Addition of a substance, which in
this form predominates (Weidzicha 2000). itself does not give full inhibition, can effectively preserve
Ethylene oxide has been widely used to reduce microbial products in the presence of other subinhibitory factors. The
contamination and to kill insects in various dried foods. The effect of superimposing limiting factors is known as the
gas has been used to treat gums, spice, dried fruits, corn, “hurdles concept” (Leistner 1999).
wheat, barley, dried egg, and gelatin (ICMSF 1980). Concern Little information is currently available on combining
over the toxicity of residues has limited the use of this gas in subinhibitory factors to preserve food. It is very time
recent years. consuming and expensive to design preservative systems
Propylene oxide has been less studied than ethylene oxide. using the hurdles concept by random design. Predicative
However, it appears that its antifungal effects are similar modeling can be used to test the consequences of a number of
(Wagner and Moberg 1989). Yeasts and molds are more factors changing at the same time. With proper design and
296 Ray and Liewen

interpretation, preservative systems can be designed rapidly Chipley JR, Story LD, and Kabara JJ (1981). Inhibition of
and efficiently (Whiting and Buchanan 1994). However, Aspergillus growth and extracellular aflatoxin accumulation
product challenge studies should be conducted to verify the by sorbic acid and derivatives of fatty acids. J Food Saf
effectiveness of a combination of subinhibitory factors 2:109 – 115.
Conner DE and Beuchat LR (1984). Effects of essential oils from
(Labuza and Taoukis 1992).
plants on growth of spoilage yeasts. J Food Sci 49:429 – 434.
Daeshel MA and Penner MH (1992). Hydrogen peroxide,
lactoperoxide systems, and reuterine. In: Ray B, Daeschel
10 CONCLUSIONS MA eds. Food Biopreservatives of Microbial Origin. Boca
Raton: CRC Press. p 155.
Davidson PM and Naidu AS (2000). Phyto-phenols. In: Naidu AS ed.
Although antifungal food additives are, in general, the only Natural Food Antimicrobial Systems. Boca Raton: CRC Press.
successful way to control fungal growth in foods, they should pp 265 –294.
never be used as a substitute for good manufacturing practices Doors S (1993). Organic acid. In: Davidson PM, Branen AL eds.
or proper sanitation procedures. Their proper use is as a Antimicrobials in Foods. New York: Marcel Dekker Inc. p 95.
processing aid. Obviously, antifungal food additive must be Eckland T (1990). Inhibition of growth and uptake process in
safe for human consumption, and their use is limited by law in bacteria by some chemical food preservatives. J Appl Bacteriol
most countries to relatively low levels and to specific foods. 48:423.
In addition, many of the traditional antifungal food additives Elgayyar M, Draughon FA, Golden DA, and Mount JR (2001).
are active only at high levels that adversely affect the taste of Antimicrobial activity of essential oils from plants against
food and cannot be used commercially. However, with greater selected pathogenic and saprophytic microorganisms. J Food
Prot 64:1019 – 1024.
emphasis on the development and marketing of refrigerated
Farber JM (1991). Microbiological aspects of modified atmospheric
foods by the food industry, some new preservation methods packaging technology. J Food Prot: 54 –58.
will become widely used and accepted. Farkas J (1997). Physical methods of food preservation. In: Doyle
MP, Beuchat LR, Montville TJ eds. Food Microbiology:
Fundamentals and Frontiers. Washington, DC: ASM Press.
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Batish VK, Roy U, Lal R, and Grover S (1997). Antifungal attributes Modern Food Microbiology. New York: Chapman & Hall.
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Bullerman LB (1974). Inhibition of aflatoxin production by activities of fatty acids and their esters. J Ferment Technol
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Cerny G (1979). Retardation of toast bread by gassing. Chem Labuza TP, Fu B, and Taoukis PS (1992). Prediction of shelf-life and
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hydrosianisole (BHA) and butylated hydrotoluene (BHT). Leistner L (1999). Combined methods for food preservation. In:
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27
Molecular Detection of Fungi in Foods and Feeds

János Varga University of Szeged, Szeged, Hungary

1 INTRODUCTION will discuss two basic types of detection methods, those based
on DNA hybridization, and those based on DNA amplifica-
Spoilage is a serious problem for the food industry because it tion. In the second part of the chapter, the possible nucleic
renders products unacceptable for consumption. Spoilage of acid targets of direct detection methods will be discussed.
foods and feeds is often the result of microbial activity from a
variety of organisms. The microbial flora that will develop
depends very much on both intrinsic and extrinsic parameters, 2 METHODS USED FOR DETECTION OF
modes of processing and preservation, and implicit FUNGI
parameters (Deak 1991; Van der Vossen and Hofstra 1996). 2.1 Hybridization-Based Methods
Yeasts and moulds can be found in a variety of environments
because they can utilize a variety of substrates, and are
In its basic application, DNA or RNA is fixed to a solid phase
relatively tolerant to low pH, low water activity, low
and a labeled probe is added and allowed to react with its
temperature and preservatives. Consequently, although only a complementary sequence (Southern 1975). The direct
limited range of fungal species are responsible for the hybridization techniques used for the detection of fungi
spoilage of a given food (Filtenborg et al. 1996), include in situ hybridization and colony or dot blot
contamination of foods and feeds by yeasts and moulds has hybridization methods (Geisen 1998; Sterflinger et al.
been extensively reported (Huis in’t Veld 1996). Fungi can 1998). Besides DNA probes, peptide nucleic acid (PNA)
contaminate foods and feeds at different stages including probes have also been developed. Peptide nucleic acids are
harvesting, processing and handling. Changes induced by pseudopeptides in which the sugar phosphate backbone of
spoilage of yeasts and moulds can be of a sensory nature, e.g., DNA is replaced by a polyamide backbone. Due to the
production of slime, pigmented growth, discoloration, rotting, uncharged backbone, PNA probes have unique properties
development of off-odors and off-flavors. The most important relative to oligonucleotides. These characteristics include
aspect of food spoilage is, however, the formation of faster hybridization kinetics, and the ability to form a stable
mycotoxins that may cause food poisonings. Although PNA/NA hybrid even at low ion concentrations, necessary to
traditional morphological and physiological characters are disrupt the secondary structure of nucleic acids such as rRNA
fundamental parameters contributing to the identification of (Perry-O’Keefe et al. 2001a,b; Stender et al. 2001).
microorganisms in foods and feeds, these criteria may be A recent development of hybridization-based techniques is
influenced by environmental conditions. To supplement the microarray technology. DNA microarrays are glass slides
classical methods, a number of nucleic acid-based methods containing an ordered mosaic of the entire genome as a
have been developed in the past few years. These methods collection of either oligonucleotides (oligonucleotide micro-
have the advantage over phenotypic methods of not being arrays) or PCR products representing individual genes (cDNA
influenced by environmental conditions of the cells since the microarrays). The development of microarrays has been
nucleotide sequence of DNA remains constant during growth. fuelled by the application of robotic technology to routine
This review focuses on molecular methods developed for molecular biology. This technique allows a single hybridiz-
direct detection of fungi in different foods and feeds. First, we ation to be performed against multiple replicates of a single

299
300 Varga

microbial genome, or against copies of several unrelated specificity of PCR reactions using other methods, including
genomes on a single glass slide. Microarrays could be used to post-PCR hybridization (Sandhu et al. 1995), PCR-ELISA
identify virulence genes as PCR products in food-borne (enzyme linked immunosorbent assay) or ELOSA (enzyme
bacteria (Chizhikov et al. 2001). Similarly, they could also be linked oligosorbent assay) reactions (Grimm and Geisen
used for the direct detection of a variety of fungi 1998; Schnerr et al. 2001), RFLP (restriction fragment length
contaminating foods and feeds. polymorphism) analysis of the PCR products (Yamagishi et al.
Hybridization techniques have only rarely been used for 1999), denaturing gradient gel electrophoresis (DGGE,
direct detection of fungi in food matrices due to the lack of Cocolin et al. 2001), fluorescent capillary electrophoresis
demanded sensitivity (Scheu et al. 1998). Kosse et al. (1997) (Turenne et al. 1999), or nested PCR, where one set of primers
developed 18 S rDNA-based probes for the detection of is used to amplify DNA fragments from target DNA, and a
yogurt spoilage yeasts, while Geisen (1998) developed second set of primers complementary to an internal sequence
suitable probes for the detection of fumonisin producing of the product of the first PCR reaction is used to score and
Fusarium species based on randomly selected sequences. confirm the results (Ibeas 1997). In case of closely related
Chemiluminescent DNA probe kits are available for the species, single nucleotide differences can be visualized by
detection of some medically important fungi including using single strand conformation polymorphism (SSCP)
Blastomyces dermatitidis, Histoplasma capsulatum, Cocci- analysis (Kumeda and Asao 1996), heteroduplex mobility
dioides immitis, and Cryptococcus neoformans (GenProbe, assay (Olicio et al. 1999), heteroduplex panel analysis
San Diego, USA). However, to our knowledge, such kits have (Kumeda and Asao 2001), or by sequence analysis (Cappa
not yet been developed for food-borne fungi. and Cocconcelli 2001).

2.2.2 Nucleic Acid Sequence-Based Amplification

2.2 Amplification-Based Methods
An alternative to PCR analysis, NASBA has also been applied
Sensitivity of detection can be greatly improved through the to detect fungi (Löffler et al. 2001; Widjojoatmodjo et al.
use of the different in vitro amplification methods 1999). The NASBA is an isothermal nucleic acid amplifi-
[polymerase chain reaction (PCR), ligase chain reaction or cation technology that specifically amplifies RNA sequences
self-sustained sequence replication, nucleic acid sequence- using T7 RNA polymerase (Compton 1991). Major
based amplification (NASBA)]. With the exception of PCR advantages of NASBA over PCR is that it is performed
and NASBA, the above-mentioned in vitro amplification isothermally at 418C, no separate reverse transcription step is
methods have had only limited practical relevance in food required for RNA amplification, and since RNA is less stable
monitoring (e.g., Stubbs et al. 1994). than DNA–provides a better estimate of living cells in the
sample analyzed.
2.2.1 Polymerase Chain Reaction
2.2.3 Quantification of Results
Since its discovery, a considerable number of PCR-based
assays have been developed, but they have been applied most Besides qualitative detection of spoilage microbes, it is also
often to clinical and environmental samples and more rarely often desirable to know their abundance in foods and feeds.
for the detection of food-borne microorganisms. The PCR Quantification of the PCR results can be carried out by
technique allows rapid and selective identification and/or different approaches including limiting dilution of the DNA
detection of microorganisms in different matrices by samples, densitometric measurement of PCR products, by
amplifying specific gene fragments. The reaction cycle HPLC with a UV detector (Katz 1996), by quantitative
consists of three steps: (a) denaturation of the double-stranded competitive PCR (QC-PCR), or by real-time PCR (Cross
DNA (dsDNA), (b) annealing of short DNA fragments 1995). Two of them, QC-PCR and real-time PCR are the most
(primers) to single DNA strands, and (c) extension of the promising tools for quantifying fungi in foods and feeds.
primers with a thermostable DNA polymerase. Following the The QC-PCR using internal DNA standard provide the
completion of one cycle, the sample is denatured for the next means for determining relative amounts of target DNA. The
annealing and extension steps during which not only the principle of QC-PCR is the coamplification of standard DNA
original target region is amplified, but also the amplification together with target DNA. The competitor DNA is of known
product of the first cycle, leading to an exponential increase of sequence (typically identical with the target DNA with added
the number of copies of the target DNA. The detection of deletions or insertions) and has the same primer binding sites
amplification products is possible through gel electrophoresis, as the target DNA (Baek and Kenerley 1998; Haugland et al.
ethidium bromide staining and visual examination of the gel 1999).
using ultraviolet light. To increase the sensitivity and to During conventional PCR, an endpoint analysis is carried
confirm the identity of the amplification product, Southern out by examining the fluorescence of ethidium bromide
blotting and hybridization with a specific probe can also be stained amplification products separated by gel electro-
carried out (Hendolin et al. 2000; Löffler et al. 2000; Sandhu phoresis. With real-time PCR, the continuous analysis of
et al. 1995). Attempts have been made to increase the amplification-associated fluorescence during the whole PCR
Molecular Detection of Fungi 301

reaction gives a graphic display of the time course of the hairpin probe concept has been employed to develop
amplification of the PCR product of interest. Both direct and unimolecular probe systems such as the Sunset or Scorpion
indirect methods are used for generating the fluorescence primers, which are incorporated into the amplified sequence
monitored during the PCR cycle (Walker 2001 and references during the PCR reaction. A variety of PNA-based probes have
therein; Figure 1). Indirect assays employ a system that yields also been developed for real-time PCR applications (Stender
fluorescence generated during the process of primer extension et al. 2002).
during amplification. This is employed in the Taqman system. The LightCycler hybridization probe system consists of a
Taqman probes utilize the intrinsic 50 nuclease activity of Taq labeled donor and acceptor probe. Fluorescence from the
DNA polymerase to digest a probe that has annealed to the acceptor will only be generated when both probes are
specific gene of interest. The probe consists of quencher and annealed to the product (Figure 1). The level of fluorescence
reporter fluorochromes separated by a specific DNA is proportional to the amount of DNA generated during the
sequence. Taq DNA polymerase-associated 50 nuclease PCR reaction (Löffler et al. 2000). The PCR reaction can be
digestion of the probe results in degradation of the probe carried out in small volumes in a glass capillary that speeds up
and separation of the fluorochromes, so loss of quenching the heating process. The LightCycler technology combines
results in amplification-associated production of fluorescence. rapid in vitro amplification in glass capillaries with real-time
Direct methods refer to those systems in which fluorescence is determination and quantification of DNA, enabling a 35 cycle
a direct result of some binding of a fluorescence molecule to PCR with 32 samples to be completed in 45 min.
the amplified product or a direct incorporation of a
fluorescence interference probe into the amplified product. 2.2.4 Limits of PCR
A simple direct method involves the use of a fluorescent dye
such as SYBR Green. This dye possesses selective affinity to One of the main drawbacks of PCR-based detection methods
dsDNA. Binding of the dye to dsDNA enhances fluorescence is that besides living cells, dead cells with relatively intact
at 530 nm proportionally with dsDNA concentration. Specific DNA are also detected, thus leading to false positive results.
hybridization probes can also be used that fluoresce only This limitation can be circumvented by including a
when bound to the gene of interest. All these probes are based propagation step prior to PCR analysis, or by using RNA as
on fluorescent resonance energy transfer (FRET; Walker template in reverse transcription coupled with PCR, or
2001). Molecular beacons are essentially hairpin probes that NASBA reactions (Vaitilingom et al. 1998; Widjojoatmodjo
employ fluorescence interference (Figure 1). A variation on et al. 1999). Although the detection of dead cells is a

Figure 1 Fluorescence systems used in real-time PCR systems. S, SYBR Green; R, reporter fluorochrome; Q, quencher fluorochrome.
Separation of the quencher from proximity to the reporter enables the fluorescence of the reporter to be measured [from Walker (2001);
reprinted by permission of John Wiley & Sons, Inc.].
302 Varga

disadvantage for spoilage bacteria and yeasts, Geisen (1998) regions the rRNA gene cluster are targeted. Other targets
suggested that it is advantageous in the case of mycotoxin- could also be used, including genes of the ergosterol
producing fungi. Mycotoxins are stable molecules, and biosynthesis (Morace et al. 1997), translation elongation
detection of the producing fungi in a sample could be used as factor genes (Vaitilingom et al. 1998), and the chitin synthase
prediction for the presence of mycotoxins. gene (Jordan 1994). For specific detection of a single genus or
Much of the difficulty in implementing PCR for the species, more variable regions of the genome, e.g., spacer
analysis of food samples lies in the problems encountered regions of the rRNA gene cluster, or sequence characterized
during the preparation of template DNAs from food matrices. amplified region (SCAR) markers should be targeted. For
The DNA extraction method used must achieve two aims: the detection of mycotoxin producing fungi, sequences of
efficiently purify fungal DNA with minimal loss, and remove the mycotoxin biosynthetic genes are the best targets. In the
compounds inhibiting the PCR reaction. Foods are complex following, the targets used for molecular detection of fungi
matrices and may contain several compounds that interfere are dealt with, with special emphasis on mycotoxin producing
with the PCR reaction leading to false negative results. These fungi.
compounds include nucleases, chelating agents, or inhibitors
of the polymerase itself. Cheese was found to be an extremely
problematic matrix (Scheu et al. 1998). Several attempts have 3.1 Targets Based on the Ribosomal RNA Gene
been made to develop a DNA extraction protocol to remove Cluster
inhibitors from the samples (Dickinson et al. 1995; Lantz et al.
1999; Rossen et al. 1991; Rossen et al. 1992). The use of
The ribosomal RNA gene cluster occurs in fungi as tandem
additional reagents during DNA extraction was suggested by
repeats of a structured unit comprising three ribosomal
different authors (e.g., DNA binding agents such as
RNA subunit genes, internal transcribes spacers (ITS) and
hexadecyl-trimethylammonium bromide, or polyvinyl pyrro-
intergenic spacers (IGS) (Figure 2). The DNA sequences
lidon for elimination of polyphenols). Additionally, DNA
within the subunits in general contain some extremely
samples can be further purified by dialysis, gel filtration, or by
conserved sequences useful for the development of broad-
other chromatographic methods. Besides careful extraction,
spectrum primers that allow amplification of fungal sequences
other methods have also been suggested, including extensive
from mixed DNA samples. The resulting fragments can be
dilution of the contaminating substances, or application of
further fractionated to obtain species or genus specific profiles
internal standard DNA in the PCR reaction (Geisen 1998).
by other techniques, e.g., nested PCR, SSCP, hybridization,
Separation of the microbes from the food matrix can be
ELOSA, RFLP analysis, or sequencing. The more variable
carried out by subculturing, or by using magnetic beads
spacer regions can be used for genus- or species-specific
coated with specific binding proteins (lectins, antibodies;
detection approaches.
Patel et al. 1993).

3 MOLECULAR TARGETS USED FOR THE 3.1.1 18 S rRNA Gene As Target

DETECTION OF FUNGI There are several reports where 18S rDNA sequences have
been used for the detection and identification of fungi. Kappe
The choice of molecular target depends on the aim wished to et al. (1996), Smit et al. (1999), and Borneman and Hartin
be achieved. For panfungal detection, usually the conserved (2000) developed primer pairs based on this region for the

Figure 2 Organization of the ribosomal RNA gene cluster in filamentous fungi. ETS, external transcribed spacer; NTS, nontranscribed
spacer; IGS, intergenic spacer; ITS1 and ITS2, internal transcribed spacer regions.
Molecular Detection of Fungi 303

detection of a wide range of fungi. Mayer (2002) also based on sequences of an intron located within the 18 S rRNA
developed a primer pair and a Taqman probe based on 18 S gene for the detection of Monilinia fructicola. Montone and
rDNA sequences, which was used successfully for real-time Litzky (1995) targeted the 5 S rRNA gene for the detection of
PCR detection of fungi in black pepper, red pepper, corn, and different Aspergillus species.
cereal samples. Makimura et al. (1994) developed a PCR
detection system based on 18S rDNA sequences for
detection of Aspergillus and Penicillium species. Cappa and 3.2 Mycotoxin Biosynthetic Genes As Targets for
Cocconcelli (2001) developed an 18 S rRNA-based assay for Detection of Fungi
the detection of fungi in food samples.
Mycotoxins are a chemically diverse group of fungal
3.1.2 28 S rRNA Gene As Target secondary metabolites that are harmful to animals and
humans. Several hundreds of different mycotoxins have been
Although the DNA coding for the large (28S) ribosomal identified, but only about 20 of them are relevant to human
rRNA subunit is relatively conserved and is more commonly health. Most of the mycotoxin-producing species are
used for work at higher taxonomic levels, certain portions of filamentous ascomycetes or deuteromycetes, Aspergillus,
it, particularly the eukaryotic D1 and D2 divergent domains Fusarium and Penicillium are considered as being the most
near the 50 end are variable enough to detect species-specific important mycotoxin-producing genera. Most mycotoxins are
differences. The D1 – D2 region has extensively been used for very resistant to physical or chemical treatments. Although
phylogenetic studies of aspergilli, penicillia, and yeasts as well-documented cases of mycotoxicoses are rare, the
well as other fungi (Peterson 2000; Rigó et al. 2001). constant uptake of small amounts of mycotoxins, especially
Universal 28S rDNA-based primers were developed by those with carcinogenic activity, can have profound effects on
Sandhu et al. (1995). These authors used species-specific human health. Apart from the use of specific rDNA gene-
probes in post-PCR hybridization reactions to detect the based or RAPD-based probes, mycotoxin biosynthetic genes
presence of fungi of interest even in situations containing can serve as ideal targets for the detection of the producing
mixed fungal species. fungi. To date, a number of biosynthetic genes of mycotoxins
have been isolated and characterized. These gene sequences
3.1.3 The ITS Region As Target could serve as targets for molecular detection of the
producing fungi as detailed below.
The ITS region has been most frequently used as target
for species-specific detection of fungi in foods and feeds. 3.2.1 Aflatoxins
This region was targeted for the detection of spoilage
yeasts including Zygosaccharomyces sp. and Torulaspora Aflatoxins (ATs) are among the most carcinogenic naturally
delbrueckii (Sancho et al. 2000), Saccharomyces cerevisiae occurring compounds known. Aflatoxins are produced mainly
(Arlorio et al. 1999), Alternaria sp. (Zur et al. 1999), by species of Aspergillus section Flavi, e.g., by A. flavus,
Penicillia (Pedersen et al. 1997; Boysen et al. 2000), and A. parasiticus, A. nomius, and A. bombycis. Sterigmatocystin,
Fusarium avenaceum (Schilling 1995). Olsson (2000) applied an intermediate of AT biosynthesis is also produced by other
a QC-PCR approach using ITS-based primers for the fungal species including, e.g., Aspergillus nidulans and
detection of Penicillium species including Penicillium A. versicolor. A. flavus and A. parasiticus are closely related
roquefortii in cereals. Grimm and Geisen (1998) developed to the nonaflatoxigenic A. oryzae and A. sojae species,
ITS-based primer pairs for the detection of fumonisin respectively, which are used in food industry for producing
producing Fusarium species. The sensitivity of the assay soy sauce and frequently applied as hosts for the expression of
was increased using PCR-ELISA. Hendolin et al. (2000) heterologous proteins. Since these species are both
developed a panfungal PCR technique coupled with multiplex morphologically and physiologically very similar to the
liquid hybridization based on ITS specific primers for the AT-producing species, molecular methods have been
detection of a number of fungi in clinical specimens. extensively surveyed for the differentiation of these species,
and AT-producing and nonproducing A. flavus isolates.
3.1.4 Other rDNA Regions Used As Targets The biosynthetic pathway leading to AT production is one
of the best known secondary metabolite pathways in fungi
Although the ITS region allows for discrimination of closely- (Brown et al. 1996; 1999). Criseo et al. (2001), Färber et al.
related species, it may not be sufficiently variable to (1997), Geisen (1996; 1998), and Shapira et al. (1996) applied
distinguish sibling biological species or isolates. The multiplex PCR targeting 3 –4 genes of the gene cluster to
intergenic spacer region of the nuclear rDNA (IGS) however, identify AT-producing fungi. Bagnara et al. (2000) applied
has been used for species determination when ITS regions the real-time PCR system for the detection of an AT
lacked sufficient variation (Spreadbury et al. 1993). producing A. flavus isolate in black pepper. Mayer et al.
Ribosomal RNAs are also encoded in the mitochondria and (2003) developed a Taqman probe based on sequences of the
have been used for molecular identification (Wakefield et al. norsolorinic acid reductase (nor-1) gene for quantitative
1990). Fulton and Brown (1997) developed a primer pair detection of AT-producing fungi in foods including black
304 Varga

pepper, red pepper, corn, and cereals. All authors found that gene for distinguishing between deoxynivalenol and
some nonaflatoxigenic isolates gave false positive results. nivalenol producing isolates of Gibberella zeae (Fusarium
Even the AT nonproducing A. sojae and A. oryzae isolates graminearum).
were found to carry AT biosynthetic genes (Kusumoto et al.
2000; Matsushima et al. 2001). However, mRNA of the aflR, 3.2.3 Patulin
the regulatory gene of AT biosynthesis was not observed in
A. sojae (Matsushima et al. 2001). These results indicate that Patulin is an unsaturated lactone produced by a number of
a (multiplex) RT-PCR technique developed by Sweeney et al. Aspergillus, Penicillium, and Byssochlamys (Paecilomyces)
(2000) for monitoring AT production in A. parasiticus could species. Representatives of other fungal genera, such as
be used more efficiently for detection of AT producing Mucor, Mortierella, Alternaria, Chrysosporium, Fusarium,
fungi. Mayer et al. (2001) used Taqman-based real-time PCR and Trichoderma were also found to produce this mycotoxin
for monitoring the expression of the nor-1 gene providing a (Steiman et al. 1989). The economically most important
possible mRNA-based method for the detection of producer of patulin is Penicillium expansum, the causative
AT-producing fungi in foods and feeds. agent of soft rot of apples and other pomaceous fruits. Patulin
is receiving worldwide attention because of its occurrence in
3.2.2 Trichothecenes unfermented apple juice. The biosynthesis of patulin is well
known (Paterson et al. 2000). Two genes of the biosynthetic
Trichothecenes are sesquiterpenoid mycotoxins produced by pathway, the polyketide synthase gene, and an iso-epoxydon
several fungal genera including Fusarium, Trichothecium, dehydrogenase (IDH) gene have been cloned and character-
Acremonium, Gliocladium, Myrothecium, Trichoderma and ized to date (Beck et al. 1990; Wang et al. 1991; Gaucher GM,
Stachybotrys. More than 30 structurally related tetracyclic and Fedeschko RW, unpublished results). The IDH gene
trichothecenes are known, among which T-2 toxin, product catalyzes the epoxydon– phyllostine oxidation step of
diacetoxyscirpenol, deoxynivalenol, and nivalenol are patulin biosynthesis (Sekiguchi and Gaucher 1979). Recently,
economically the most important mycotoxins. The main a primer pair specific for the IDH gene (GenBank accession
producers of these toxins are in the genus Fusarium (e.g., number AF006680) has been developed and used successfully
F. acuminatum, F. culmorum, F. graminearum, F. poae, to detect patulin-producing abilities of penicillia (Paterson
F. solani, and F. sporotrichioides). Trichothecenes have et al. 2000). Patulin production of Aspergillus species was
dermatotoxic, cytotoxic, and phytotoxic properties, and have also analyzed using analytical procedures (thin layer
been suggested to play a role in plant pathogenesis as chromatography and HPLC), agar diffusion test, and a
virulence factors. Trichothecene biosynthesis genes were PCR-based approach using the primer pair developed for the
found to comprise a 25 kbp gene cluster including at least 10 detection of the IDH gene (Varga et al. 2003). The analytical-,
genes in F. sporotrichioides and F. graminearum (Brown et al. biological-, and PCR-based approaches used for patulin
2001; Hohn et al. 1993). Similar clustering of the detection gave highly similar results indicating that the
trichothecene biosynthesis genes was found in Myrothecium primers developed for a Penicillium IDH gene could also be
roridum, which produces macrocyclic trichothecenes (Trapp used to detect patulin producing aspergilli in natural
et al. 1998). substrates. A quantitative PCR approach is also being tested
Fekete et al. (1997) determined the sequence of the Tri5 for the detection of patulin producing organisms in foods
gene of Fusarium poae, and used a 378 bp fragment of it as a including apple products and malt.
hybridization probe to detect similar sequences by dot blot
hybridization in other fungi. Strong hybridization was 3.2.4 Other Mycotoxin Biosynthetic Genes
observed to trichothecene producing Fusarium, Myrothecium
and Stachybotrys species, but not to Trichoderma and The PR-toxin is a sesquiterpenoid mycotoxin produced by
Trichothecium DNAs. Niessen and Vogel (1998) developed a P. roqueforti strains isolated from cheese and silage (Geisen
PCR method based on the amplification of Tri5 sequences for 1998). This mycotoxin has been implicated in incidences of
the detection of trichothecene producing Fusarium species in mycotoxicoses resulting from the consumption of contami-
wheat samples. The authors also applied the LightCycler nated grains (Proctor and Hohn 1993). The gene sequence of
system with Sybr Green I for quantitation of their results. For aristolochene synthase (a sesquiterpene cyclase), the key
distinguishing nonspecific products such as primer dimers, enzyme of PR-toxin biosynthesis is known (Proctor and Hohn
melting point analysis was carried out (Schnerr et al. 2001). 1993). Geisen (1998) developed a primer pair based on the
Tri5 gene sequences were chosen as targets too in a sequence of aristolochene synthase, which was used
quantitative competitive PCR approach developed for the successfully for the detection of PR-toxin producing fungi
detection and quantification of trichothecene producing in cereals and cheese.
fusaria in cereals by Edwards et al. (2001), and by Birzele The ergot alkaloids are produced mainly by Claviceps
et al. (2000). Doohan et al. (1999) developed a reverse- purpurea and some other Claviceps species (e.g., C. paspali,
transcription-based PCR assay to quantify the expression C. fusiformis), although other species, e.g., Aspergillus
of the Tri5 gene in Fusarium species. Lee et al. (2001) fumigatus, A. clavatus, Penicillium species, and plants have
developed primer pairs based on sequences of the Tri7 also been described as producers of these mycotoxins. Since
Molecular Detection of Fungi 305

Claviceps strains parasitize not only cereals but also different particularly fumonisin-producing fusaria (Geisen 1998), and
kinds of grasses, ergot poisoning of grazing animals is still an for Aspergillus fumigatus (Brandt et al. 1998; Varga et al.
economic problem. Sequences of some of the biosynthetic 2002, unpublished results). Murillo et al. (1998) developed a
genes responsible for ergot alkaloid production are known primer pair based on the sequence of a random genomic clone
(Tudzynski et al. 1999). Boichenko et al. (2001) developed a for the detection of Fusarium moniliforme.
primer pair based on sequences of 4-(g,g-dimethylallyl)- For the detection of Fusarium graminearum, another
tryptophane (DMAT) synthase, the key enzyme of the ergot target, the galactose oxidase (gaoA) gene has been used
alkaloid biosynthesis, and used successfully in PCR reactions (Niessen and Vogel 1997). This enzyme is produced by
to detect ergot alkaloid producing fungi. only a limited number of fungi including F. graminearum,
Fumonisin B1 was discovered in 1988 in Fusarium Gibberella fujikuroi, and Beltraniella portoricensis. Niessen
moniliforme (F. verticillioides) isolates. Fumonisins are most et al. (1998) also developed a solid phase PCR with detection
frequently encountered from stored corn and other cereals, of immobilized amplified product in a one-phase system
and are the causal agents of leucoencephalomalacia, a fatal (DIAPOPS) to specifically amplify and quantify a DNA
brain disease of horses, pulmonary edema in pigs and fragment of the gaoA gene from F. graminearum.
suspected to be responsible for high incidences of esophageal Microsatellite-based probes were developed for the
cancer in South Africa and China. Fumonisins are synthesized detection of Epichloë species in different grasses (Groppe
through the polyketide biosynthetic route. Seo et al. (2001) and Boller 1997). Mayer (2002) developed a Penicillium
identified four coregulated genes associated with fumonisin nordicum specific primer pair and a Taqman probe based on a
production in F. verticillioides. These data indicate that these polyketide synthase gene sequence for the detection of this
gene sequences could be used for the development of gene species in cereal samples. Pearson and McKee (1992)
probes for the identification of potential fumonisin producing developed a multiplex PCR method based on plasmid
species in foods and feed products. sequences for the detection and discrimination of
Since biosynthetic genes of other mycotoxins, e.g., those S. cerevisiae, Zygosaccharomyces bailii, and Z. rouxii in
of fumonisins (Proctor et al. 1999; Seo et al. 2001) and the foods. Pearson et al. (1995) designed a PCR-based assay to
tremorgenic mycotoxin paxilline (Young et al. 2001) have detect retrotransposon long terminal repeat elements for the
also been characterized, molecular detection methods can detection of Pichia membranefaciens.
potentially be applied in these cases as well. Apart from the
mycotoxins mentioned earlier, the occurrence of the
nephrotoxic ochratoxins in cereals, coffee beans, and other 4 CONCLUSIONS
agricultural commodities also poses serious hazard to human
and animal health (Varga et al. 2001a,b). Experiments are in Fungi responsible for food spoilage cause significant
progress in some European laboratories to isolate and economic losses for food manufacturers. In order to minimize
characterize genes responsible for ochratoxin biosynthesis food spoilage and be able to predict the quality and shelf-life
in Penicillium and Aspergillus species, and to identify of a particular food, a better understanding of the mechanisms
suitable molecular probes for the detection of ochratoxin- underlying food spoilage is essential. To achieve this goal,
producing fungi in foods and feeds (Quality of life and there is a need for the development of direct detection
management of living resources project number QLK1-CT- methods for spoilage organisms including fungi. The main
1999-00433: Prevention of ochratoxin A in cereals; QLK1- aim being to develop a rapid, sensitive, robust, and specific
CT-1999-01380: Early detection of toxigenic Fusarium method to use directly on the sample for the detection of
species and ochratoxigenic fungi in plant products). food spoilage organisms. Rapid identification of spoilage
organisms is of profound importance to the food industry.
This will enable intervention with appropriate measures to
3.3 Other Sequences As Targets prevent serious economic losses. Rapid molecular techniques
are valuable tools for screening foods and feeds for fungi.
One approach used for developing suitable species- or strain- Among these, hybridization techniques have only rarely been
specific probes for the detection of fungi is based on the used for direct detection of fungi, although novel develop-
random amplified polymorphic DNA technique (RAPD) ments in this field, including the application of PNA probes,
(Williams et al. 1990). The RAPD is a variation of and microarray techniques are encouraging. Amplification-
conventional PCR where one primer of arbitrary sequence based methods are frequently applied for direct detection of
is used, and the annealing temperature is low (usually 358C). spoilage organisms. Most PCR-based detection methods
Species- or strain-specific RAPD fragments are selected, target part of the ribosomal RNA gene cluster, although
sequenced, and suitable primers are devised to amplify the SCAR markers are also widely used. For the detection of
specific fragment in conventional PCR reactions. Such SCAR mycotoxin producing fungi, the most direct procedure is
markers have been successfully used to develop species- targeting of the mycotoxin biosynthetic genes. Such
specific probes for a number of Fusarium species approaches have been used recently for the detection of a
(Chelkowski et al. 1999; Nicholson et al. 1996; Nicholson number of fungi-producing mycotoxins such as ATs, patulin,
et al. 1998; Schilling et al. 1996; Young et al. 2001), PR toxin, and trichothecenes, and projects for the detection of
306 Varga

fungi producing ochratoxins and fumonisins are in progress. Penicillium patulum. Its gene structure relative to that of
The recent development of real-time PCR methodology has other polyketide synthases. Eur J Biochem 192:487 – 498.
made it possible to quantify the amount of organisms in foods Birzele B, Prange A, and Kramer J (2000). Deoxynivalenol and
and feeds. ochratoxin A in German wheat and changes of level in relation
to storage conditions. Food Addit Contam 17:1027 – 1035.
Sensitivity of a molecular method depends not only on the
Boichenko LV, Boichenko DM, Vinokurova NG, Reshetilova TA,
detection system, but also to a great extent on the food matrix. and Arinbasarov MU (2001). Use of polymerase chain reaction
In order to increase sensitivity, adequate protocols have to be for searching for producers of ergot alkaloids from among
established in order to discern potential PCR inhibitors. The microscopic fungi. Mikrobiologiia 70:360 –364.
speed, accuracy, and reliability of detection methods can Borneman J and Hartin RJ (2000). PCR primers that amplify fungal
greatly be enhanced by enrichment procedures including the rRNA genes from environmental samples. Appl Environ
use of magnetic particles coated with specific binding proteins Microbiol 66:4356 – 4360.
(antibodies or lectins); by optimizing the DNA extraction Boysen ME, Jacobsson KG, and Schnurer J (2000). Molecular
protocol; or by using internal standard DNA in the PCR identification of species from the Penicillium roqueforti group
reaction. One of the main drawbacks of PCR-based detection associated with spoiled animal feed. Appl Environ Microbiol
66:1523 – 1526.
methods is that DNA from dead cells can also be amplified
Brandt ME, Padhye AA, Mayer LW, and Holloway BP (1998).
leading to false positive results. This limitation can be Utility of random amplified polymorphic DNA PCR and
addressed by including a propagation step prior to PCR, by Taqman automated detection in molecular identification of
using mRNA as template in NASBA or RT-PCR reactions. Aspergillus fumigatus. J Clin Microbiol 36:2057 – 2062.
On the other hand, this disadvantage is only relative, since Brown DW, Yu JH, Kelkar HS, Fernandes M, Nesbitt TC, Keller NP,
detection of dead mycotoxin producing fungi in a food matrix Adams TH, and Leonard TJ (1996). Twenty-five coregulated
can serve as a prediction for mycotoxin contamination of the transcripts define a sterigmatocystin gene cluster in Aspergillus
food. nidulans. Proc Natl Acad Sci USA 93:1418 –1422.
The detection of microorganisms in food is still a time- Brown MP, Brown-Jenco CS, and Payne GA (1999). Genetic and
molecular analysis of aflatoxin biosynthesis. Fungal Genet Biol
consuming method particularly in the case of large-scale
26:81 – 98.
testing. The increasing availability of molecular kits for the
Brown DW, McCormick SP, Alexander NJ, Proctor RH, and
detection of food-borne pathogenic bacteria is a step toward Desjardins AE (2001). A genetic and biochemical approach to
standardization of the molecular techniques. The develop- study trichothecene diversity in Fusarium sporotrichioides and
ment of similar kits for fungi causing food spoilage would be Fusarium graminearum. Fungal Genet Biol 32:121 –133.
a further necessary step for the authorization of molecular Cappa F and Cocconcelli PS (2001). Identification of fungi from
methods as accepted detection methods in food microbiology. dairy products by means of 18S rRNA analysis. Int J Food
Microbiol 69:157 –160.
Chelkowski J, Bateman GL, and Mirocha CHJ (1999). Identification
of toxigenic Fusarium species using PCR assays. J Phytopathol
ACKNOWLEDGEMENTS 147:307 – 311.
Chizhikov V, Rasooly A, Chumakov K, and Levy DD (2001).
Microarray analysis of microbial virulence factors. Appl
We are indebted to Zofia Lawrence (IMI, Egham, UK) for Environ Microbiol 67:3258 –3263.
critical reading of the manuscript. This research was Cocolin L, Heisey A, and Mills DA (2001). Direct identification of
supported by a grant from the Hungarian Scientific Research the indigenous yeasts in commercial wine fermentations. Am J
Fund (OTKA grant No. T037217). J. Varga received a Enol Viticult 52:49 – 53.
Széchenyi Research Fellowship grant. Compton J (1991). Nucleic acid sequence based amplification.
Nature 350:91– 92.
Criseo AG, Bagnara A, and Bisignano G (2001). Differentiation of
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28
The Role of Spoilage Fungi in Seed Deterioration

Naresh Magan / David Aldred Cranfield University, Bedford, United Kingdom

Vicente Sanchis Universitat de Lleida, Lleida, Spain

1 INTRODUCTION paddy in southern India exceeded 15 –25% in only 9 days,

while Rohani et al. (1985) found storage losses of paddy in
Microorganisms are ubiquitously present in terrestrial West Malaysia of only 1%.
ecosystems from which they are disseminated and contami- Deterioration of grain by microorganisms is determined by
nate plant communities. The ripening seed is no exception and several factors which can be classified into four main groups:
is contaminated by a wide range of bacteria, yeasts, and intrinsic factors (those which depend on the characteristic of
filamentous fungi via the air, insects, rain splash, equipment, the growth substrate), extrinsic factors (those imposed from
and agronomic practices. When seeds/cereal grains are the outside), processing factors (those resulting from the
harvested they thus carry a wide range of microbial agronomic practices and food processing, which primarily
contaminants. Postharvest treatment of such seeds and the modify the composition of the microflora), and implicit
prevailing environmental factors are key determinants of the factors (those depending on the particular dominant microbial
impact that they may have on the quality of the seeds, flora that initially develop in response to the intrinsic,
including germinability. It is important to remember that both processing, and extrinsic factors) (Sinha 1995). Figure 1
the harvested seeds/grains and contaminating microorganisms summarizes the factors affecting fungal colonization of the
are alive and respiring slowly under dry, safe storage grain.
conditions. Wallace and Sinha (1981) in the 1970s were the first to
Poor postharvest management can lead to rapid deterio- consider stored seeds as a man-made ecosystem which needed
ration in quality characteristics and severely decrease to be examined in a more holistic and ecological manner to
germinability and nutritional value of the seeds. Microbial enable a proper understanding of the processes occurring and
activity can cause undesirable effects in grains including to improve postharvest management of stored seeds of all
discoloration, contribute to heating and losses in dry matter types. This in many respects enabled prevention strategies to
through the utilization of carbohydrates as energy sources, be developed and implemented to avoid microbial and pest
degrade lipids and proteins or alter their digestibility, produce infestation from damaging seeds. Generally, since most seeds
volatile metabolites giving off-odors, cause loss of germina- are stored dry, bacteria seldom cause biodeterioration
tion and baking and malting quality, affect use as animal feed problems. At intermediate moisture content levels fungal
or as seed, and filamentous fungal spoilage organisms may spoilage and pests are of major concern. This Chapter will
also produce mycotoxins that are highly toxic or carcinogenic endeavor to examine some of these important abiotic and
or cause feed refusal and emesis (Christensen 1973). The biotic factors and their interactions that determine whether
spores of some fungi can also cause respiratory disease deterioration will occur and the dominant fungal species
hazards to exposed workers (Lacey and Crook 1988). which may be involved. This is important as the fungal
Estimated losses of seeds, especially staple cereal grains in community structure influences the type of deterioration and
store from all causes varies widely. They may amount to 10% whether mycotoxins are produced. Cereal grains particularly
worldwide (Anon 1979) but can reach 50% in tropical regions wheat, barley, maize, and rice are used as examples of seed
(Hall 1970). Vassan (1980) estimated losses of high moisture systems, since they represent the key staple seeds worldwide.

311
312 Magan et al.

Figure 1 Integration of the most important abiotic and biotic factors impacting on grain spoilage.

(Cooke and Whipps 1993; Griffin 1981; Scott 1957). Most

2 ENVIRONMENTAL FACTORS WHICH
stored products such as seeds and grain are hygroscopic in
INFLUENCE FUNGAL ACTIVITY IN THE nature, i.e., they will either take up or release moisture until
STORED SEED ECOSYSTEM they reach a dynamic equilibrium with the surrounding
2.1 Water Availability—The Concept environment. It is straightforward to express this water in
terms of the percentage moisture content, based on the ratio of
2.1.1 Fundamental Principles the dry weight to the wet weight (expressed either on a wet
weight or dry weight basis). This, however, implies that the
The extent to which seeds and grain are susceptible to stored material consists of dry matter plus a certain amount of
spoilage fungi depends, above all, on the presence of water in “attached” liquid water. In fact the situation is more complex
the system. In fact, many authors have written about the than this. This is principally because the water in these
fundamental requirement for water in microbial growth substrates is not all equivalent, but held in a number of
Role of Spoilage Fungi in Seed Deterioration 313

different “states.” Broadly speaking, three main states of availability in terms of the thermodynamics of systems,
water can be identified in a hygroscopic material: (a) and considers the movement of water along gradients of
constitutive water; water that is chemically linked to the potential energy. The term “water potential” is actually an
substrate material and forms part of its structural makeup, (b) abbreviation of “potential energy of water” and is defined
adsorbed water, which is closely linked with the substrate as “the free energy of water in a system relative to the
surface by physical interactions, and (c) absorbed water, free energy of a reference pool of pure free water having
which is loosely associated with the substrate surface (Pixton a specified mass or volume,” and is measured in J/m3 or
1967). In practice, probably there is no clear demarcation Pa (Papendick and Mulla 1985). The reference state of
between these different states; the relative proportions of each pure free water is assigned zero. Water which is
type will depend on the overall moisture content of the “constrained” in a system, i.e., the constitutive, adsorbed
material. The absorbed water fraction will become more and (to a lesser extent) absorbed water is, therefore, at a lower
weakly bound as further layers of water molecules (negative) water potential, and any microorganism must
accumulate on the surfaces (i.e., as the substrate becomes expend energy to lower internal water potential values
wetter). It is the loosely held water that provides the “free” or relative to the exterior to make water available. The numerical
“available” water, since it is the fraction that is most readily value of water potential may be related to water activity using
available for microbial growth, although its overall the formula:
availability will be influenced by the presence of solutes.
The important implication here is that microorganisms, c ¼ ðRT=V w Þ ln aw ðPapendick and Mulla 1985Þ:
including spoilage fungi, respond not to water content but to
water availability. Therefore, the concept of water availability where R is gas constant, T, absolute temperature (K), and Vw
is of fundamental importance in relation to microbial growth is molecular weight of water.
and spoilage. Although c and aw are interconvertible by this expression,
measurement of the separate components that make up total
2.1.2 Water Activity water potential cannot be carried out using water activity
measurement, since water activity is not a sufficiently
Water availability itself can be expressed in a number of sensitive parameter.
ways, one of the most convenient being water activity (aw).
The free water in a system is the proportion of the total water
that is immediately available to reach equilibrium with the
surrounding atmosphere. This will be reflected in the 2.1.4 Moisture Sorption Isotherms
equilibrium vapor pressure or equilibrium relative humidity
(ERH) exerted by the system. Water activity is defined as the The relationship between water content and aw in a given
ratio of the vapor pressure of the water in a substrate to that substrate may be usefully expressed by moisture sorption
of pure water at the same temperature and pressure (Scott isotherms. These are curves produced by plotting water
1957). It is, therefore, directly linked to the ERH by the content against water activity, or ERH at constant
expression: temperature (hence, the term “isotherm”). They are highly
specific to substrate type and condition but generally
aw ¼ p=p0 ¼ ERH ð%Þ=100 ðLabuza 1974Þ
sigmoidal in shape show a steep rise in the curve above
where p is the vapor pressure of water in solution or solid 80% ERH. The importance of moisture sorption isotherms is
substrate, p0, vapor pressure of pure water at experimental that they establish the relationship between moisture content
temperature and pressure, and ERH (%) is equilibrium and water availability for specific product types and,
relative humidity at which a solution or solid substrate therefore, allow water availability to be set to safe levels
neither gains nor loses moisture to the atmosphere. from knowledge of moisture content alone. For most stored
Water activity can, therefore, be seen to be a measure of seed products safe moisture levels will typically correspond to
the ability of water to evaporate from a substrate and humidify water activity levels of around 0.7 water activity or 70% ERH.
the immediate environment, and is measured in the range The actual moisture content which it corresponds to will vary
0–1.0 with 1.0 representing the aw of pure water. Most greatly between seed types, and particularly between starchy
importantly it should be noted that aw is a function of and oily types. Moisture sorption isotherms are complicated
temperature, and for a given substrate and moisture content, by a hysteresis effect that influences the position of the
aw will increase with increasing temperature. This is primarily curve depending on whether the material is being wetted or
the consequence of the general increase in thermal motion dried as each equilibrium point is plotted. This effect may
(Multon 1988). account for a difference of as much as 0.5% (Pixton 1967)
and always shows a higher water activity for a given
2.1.3 Water Potential moisture content where water is being sequentially added.
This indicates that the “history” of the material is important
Water availability may also be expressed as water potential and probably reflects irreversible and permanent changes
(c). Water potential approaches the concept of water occurring after wetting.
314 Magan et al.

2.2 Temperature fungal community can be very different colonization of grain

pre- and postharvest are described separately.
Fungi differ widely in the range of temperature over which
growth can occur and those conditions optimum for growth
(Magan 1997). For example, Penicillium species such 3 POSTHARVEST FUNGAL COLONIZATION
P. aurantiogriseum and P. verrucosum can grow over the OF CEREAL GRAINS
range 24 to þ358C while Aspergillus fumigatus has a very
wide tolerance of 10 –558C and Humicola lanuginosa in the 3.1 Factors
range 30 – 608C. The majority of fungi involved in
deterioration in stored grain ecosystems thrive over the Conditions in the stored grain are more stable and controlled
range 10–408C with optima in the range 25–358C. Lowering than those in the field. Stored cereal grain offers a good
temperature reduces the metabolic activity of fungi and the ecological niche for fungi. The aw, temperature, O2 level,
grain, enabling longer mold-free storage periods. However, interactions with other organisms like insects, chemical
moist grain can be prone to slow deterioration by the genera preservatives, and storage time are considered the most
Alternaria, Penicillium, and Fusarium, with species in each important factors that control the growth of fungi in grains but
able to produce mycotoxins. Temperature of grain is a good cannot be considered in isolation. All these factors interact
indicator of quality during storage. Pockets of moisture can with each other during storage. For instance, fluctuations in
allow initiation of fungal activity that produces metabolic temperature can cause condensation of water that increases
heat resulting in a succession of fungi becoming dominant the water activity in pockets of stored grain. Damage to the
and ending with spontaneous heating and dominance by kernels not only increases susceptibility to fungal invasion,
thermotolerant/thermophilic fungi and actinomycetes (Lacey especially if this occurs near the embryo, but also increases
and Magan 1991). the likelihood of visible molding (Perez et al. 1982; Tuite et al.
1985; Lacey et al. 1991).

2.3 Gas Composition 3.2 Mycoflora

While fungi involved in biodeterioration of grain are The number of fungal species that can be found colonizing
considered to be obligate aerobes, many are actually stored grain is very wide. These represent a high number of
microaerophilic, being able to survive and grow in niches genera and include psychrotolerant, mesophilic, thermophilic,
where other species cannot grow and, thus dominate xerophilic, and hydrophilic species (Hill and Lacey 1983;
specialized grain ecosystems. In many cases, decreasing O2 Lacey et al. 1980; Magan and Lacey 1984a,b,c). The most
to ,0.14% is required before growth can be substantially characteristic of these are species belonging to the genus
reduced. Increasing CO2 to . 50% is required for inhibition of Aspergillus and Penicillium. In addition, it is possible to find
growth (Magan and Lacey 1984a). Some species, e.g., species of Eurotium, Fusarium, Rhizopus, and thermophilics
P. roqueforti, are able to grow and infect grain at . 80% CO2 like Talaromyces thermophilus, Rhizomucor pusillus, and
provided at least 4% O2 is available. The use of integrated Thermomyces lanuginosus (Figure 2). All these species are
postharvest systems for prevention of deterioration entails widespread in their occurrence throughout the world and they
modifying O2 and CO2 simultaneously and the use of (O2 cover almost the whole range of environmental conditions
free) N2. The tolerance to low O2 and high CO2 is also likely to be found in stored grain.
influenced by interactions with grain water availability. The The aw and temperature are very important factors that
treatment is more effective if the grain is dry. Controlled determine the mycoflora present in stored grains. The fungi
atmosphere storage is used for both control of molds and present in the field such as Alternaria species that need high
insects in moist stored seed systems. Regimes sufficient for levels of aw to grow, decrease in importance when the grain is
molds may not, however, be effective against some storage stored and subjected to processing practices directed to
insects that can survive and grow over a wide ERH range. decrease the water activity. On the other hand, genera such as
In summary, from the whole range of factors that can Aspergillus, Penicillium, and Eurotium that have a low
affect the colonization of seeds by microorganisms, aw is the importance in the field, become very important during the
most important single factor limiting their activity. Each storage of the grains. Another factor that determines the
microorganism has a specific range over which they can mycoflora in grain is the oxygen concentration, because
develop. Fungi are the most important group of micro- the fungi are generally very sensitive to low oxygen
organisms with capacity to colonize seeds because special- concentrations. Under these conditions Magan and Lacey
ized groups can grow at intermediate and low aw levels. Fungi (1984) reported that Fusarium culmorum, P. roqueforti, and
contribute greatly to seed losses, either alone or together with A. candidus were the most tolerant, which is consistent with
insects. Because the environment and other factors can the fungal community found on cereals under airtight
change during seed/grain filling, harvest, and storage the storage.
Role of Spoilage Fungi in Seed Deterioration 315

Figure 2 Succession of dominant fungi found in stored grain depending on it initial water activity and temperature [adopted from Lacey
et al. (1980)].

Both climatic conditions and geographical location can Penicillium, Aspergillus, and Fusarium spp. isolated from
explain the differences found in the mycoflora of different maize grain have been found to be significantly influenced by
grains in the field. During storage, however, the mycoflora is aw, temperature, and their interactions. The effects of aw and
very similar for all grains (Pelhate 1988). Many of the species temperature on germination can be quantified in two ways: (a)
found on maize, sorghum, rice, and other cereals in tropical the minimum aw for germination and (b) the lag time before
climates are the same as those found in temperate regions germination.
except for a small number of additional species and, perhaps, Fungi normally found in stored grain can germinate at
a greater frequency of Aspergillus and fewer or different lower aw levels than the fungi present in the field, especially
Penicillium spp. in temperate grains (Table 1). Changes in tolerance to low aw
occur when changing temperature level. A. niger germinates
only down to 0.95 at 108C, but down to 0.90 at 158C, and to
4 ECOLOGY OF GERMINATION, GROWTH, 0.80 at 258C. P. hordei germinates down to 0.90 at 58C, but
AND MYCOTOXIN PRODUCTION down to 0.85 at 258C. F. verticillioides germinates only down
to 0.96 at 58C, but down to 0.88 at 258C (Marin et al. 1996,
To colonize grain, fungi need to be capable of germinating 1998a).
and growing under the prevailing environmental conditions In general, the lag time before germination increases with
found in the substrate. If conditions are appropriate, decreasing aw. With water readily available (. 0.98) the lag
establishment can also result in the accumulation of time can range from a few hours to a few days, while at very
mycotoxins. Consequently, it is very important to understand low aw it can extend for months or even years (Magan and
the interacting ecological conditions that can avoid fungal Lacey 1984a; Marin et al. 1998a; Pitt 1975). For example, the
colonization of the grain. The main environmental factors that shortest lag phases for species of Aspergillus, Penicillium,
affect germination, growth, and mycotoxin production by and Fusarium are at 0.994 and 0.95 aw over a wide range of
fungi are aw, temperature, and intergranular atmosphere. temperatures (Marin et al. 1996, 1998a) (Figure 3). At
Species differ in their ecological requirements and in their marginal temperatures the lag phases increases, more
tolerance of low aw and marginal temperatures. Germination markedly for P. aurantiogriseum and P. hordei at . 308C
of conidia and mycelial growth of isolates of some and , 158C (Marin et al. 1998a). The capacity to germinate
316 Magan et al.

Table 1 The minimum water activity for germination and Successful spore germination results in the formation of
growth of some field and storage fungi on wheat extract agar at extending hyphae that are able to colonize the substrate.
258C Hydrolytic enzymes are produced to utilize the rich
nutritional substrate. Water activity and temperature are
Fungal species Germination Growth very important factors impacting on the level of infection of
the grain and the extent of colonization. The interaction
A. alternata 0.85 0.88
between these factors is critical in developing an under-
C. cladosporioides 0.86 0.88
standing of the ecology of individual species, their
C. herbarum 0.85 0.90
interrelationship with one another, and their role in initiation
E. purpurascens 0.88 0.99
of molding.
F. culmorum 0.87 0.90
The range of aw/temperature conditions for germination is
P. brevicompactum 0.80 0.82
generally found to be wider than that for mycelial growth, as
P. aurantiogriseum 0.80 0.83
recently shown for fumonisin-producing species of Fusarium
P. hordei 0.80 0.83
from maize, and for other Aspergillus and Penicillium spp.
P. roquefortii 0.83 0.83
(Magan and Lacey 1984a; Marin et al. 1996). Changing aw at
E. amstelodami 0.72 0.73
different steady-state temperatures affected growth rates of all
E. repens 0.72 0.75
the species examined (Marin et al. 1996, 1998a). Fusarium
A. fumigatus 0.94 0.94
species grow faster on irradiated maize at 258C than at 158C.
A. versicolor 0.76 0.78
Maximum growth rates of about 3.6, 3.5, and 10.4 mm/d
Source: Magan and Lacey (1984a). were obtained for F. verticillioides, F. proliferatum, and
F. graminearum, respectively, at 0.98 aw and 258C. However,
these three species had a growth rate of about 0.4, 0.5, and
very fast can be an advantage in competing effectively with
1.2 mm/d at 0.93 aw and 158C, respectively (Marin et al.
other fungi for nutrients in the grain and exclude other
competitors. Also, conidia can survive for long periods of 1998b).
time depending on the relative humidity and temperature. All While most fungi grow optimally close to 1.00 aw at their
optimum growth temperature, others grow better at lower aw.
of this information gives us an idea about the potential
capability to colonize the stored grain ecosystem if the For instance, Eurotium amstelodami, E. repens, A. niger and
conditions change and become favorable for fungal A. versicolor and A. ochraceus all grow faster at 0.90–0.95 aw
at optimum temperatures (Ayerst 1965; Magan and Lacey
development.
1984a; Ramos et al. 1999a,b). Some Penicillium spp. also
grow better nearer to 0.98 than at 1.00 aw (Hocking and Pitt
1979). However, at marginal temperatures for growth, optima
are almost always close to 1.00 aw. The effects of aw and
temperature on fungal growth and other activities in stored
grain may be further modified by changes in the concentration
of O2 and CO2 in the intergranular atmosphere resulting from
respiration (Magan and Lacey 1984b), and the addition of
preservatives (Marin et al. 1999b).
The production of mycotoxins has been studied mostly at
optimum aw and temperature levels on either rich laboratory
media or on autoclaved wheat, barley, or rice. Few studies
have considered the effects of environmental factors,
especially water stress on mycotoxin production. The
conditions of aw/temperature that permit mycotoxin pro-
duction differ from those for the fungal growth (Table 2). For
instance, F. verticillioides and F. proliferatum, at minimum
aw/temperature for growth (e.g., 0.89– 0.91 aw) did not
produce detectable levels of FB1 (Marin et al. 1999a). The
minimum aw allowing growth of 12 species of fungi was
usually lower and the temperature range wider than those
permitting aflatoxin, patulin, penicillic acid, ochratoxin, or
fumonisins production (Marin et al 1999a,c; Northolt 1979).
Figure 3 Effect of water activity and temperature on the Moreover, the effect of these factors can differ for two toxins
germination rate of A. flavus, A. niger, P. aurantiogriseum, and produced by the same species. For instance, ochratoxin
P. hordei on MMEA. Water activity levels are 0.994 (W), 0.95 production by A. ochraceus on poultry feed was greatest at
(K), 0.90 (A), 0.85 (X), and 0.80 (O). Error bars show standard 308C and 0.95 aw while penicillic acid production was
error of estimated parameters [from Marin et al. (1996)]. favored by 228C and 0.90 aw.
Role of Spoilage Fungi in Seed Deterioration 317

Table 2 Minimum water activity for growth and toxin production by some toxigenic fungi

Minimum aw

Fungal species Mycotoxin Growth Toxin production

A. flavus Aflatoxin 0.78– 0.84 0.84
A. parasiticus 0.84 0.87
A. ochraceus Ochratoxin 0.77 0.85
P. aurantiogriseum 0.82– 0.85 0.87 – 0.90
P. viridicatum 0.80– 0.81 0.83 – 0.86
A. ochraceus Penicillic acid 0.77 0.88
P. aurantiogriseum 0.82– 0.85 0.97
P. patulum Patulin 0.81 0.95
P. expansum 0.82– 0.84 0.99
A. clavatus — 0.99
F. verticillioides Fumonisins 0.88 0.93
F. proliferatum 0.88 0.93

4.1 Interaction Between Grain Fungi, Decreasing the aw conditions increased competitiveness of
Environmental Factors, and Niche P. brevicompactum. Only F. culmorum could compete with
Occupation storage molds, at .0.93 –0.95 aw. They also found that rate of
growth was not directly related to dominance. Previously,
Ayerst (1965) had suggested that speed of germination and
Fungi seldom occur on grains in isolation, but usually as a
growth were key determinants of colonization of nutrient rich
mixed consortium of bacteria, yeasts, and filamentous fungi.
matrices such as grain. The ID approach has been adapted over
It is thus inevitable that interspecific and intraspecific
the years for many food-based ecosystems.
interactions will occur depending on the nutritional substrate
More recently, alternative approaches have been utilized
of the grain. Furthermore, environmental factors may exert a
to try and understand the relative competitiveness of different
selective pressure influencing community structure and
species within fungal communities colonizing grain. It was
dominance of individual species. It is important to understand
suggested by Wilson and Lindow (1994a,b) that the
the type of interactions that occur between fungi under coexistence of microorganisms particularly on plant surfaces
different environmental regimes in grain to enable better may be mediated by nutritional resource partitioning. Thus in
prediction of not just dominance by key spoilage fungi, but vitro carbon utilization patterns could be used to determine
also the potential for production of mycotoxins. Wicklow niche overlap indices (NOI) and, thus the level of ecological
(1988) used the in vitro interactions between hyphae of similarity. Based on the range of similar c-sources utilized
different fungi based on: (a) intermingling of hyphae, (b) and those unique to an individual isolate of species they
inhibition on contact, (c) inhibition at a distance, and suggested that NOI values of . 0.9 were indicative of
(d) dominance by one species over another on contact, and coexistence between species in an ecological niche, while
(e) inhibition by one species at a distance with the dominant scores of ,0.9 represented occupation of separate niches.
species continuing to grow. They used these categories to This approach was modified by Lee and Magan (1999a,b) and
develop an index of antagonism by giving numerical scores to Marin et al. (1998c) to include a multifactorial approach by
each interaction type. This enabled antagonistic interactions including water availability and temperature into the system.
between A. flavus and a range of species to be identified. This approach demonstrated that based on utilization of maize
However, these studies did not examine the dynamics of c-sources the NOIs for F. verticillioides and F. proliferatum
interacting environmental factors and dominance of species. were .0.90 at . 0.96 aw at 25 and 308C, indicative of
Subsequent studies by Magan and Lacey (1984c, 1985) coexistence with other species such as Penicillium species,
modified this scoring system to give a higher numerical score A. flavus and A. ochraceus. However, for some species pairing
to fungi able to dominate in vitro than antagonism and with F. verticillioides resulted in NOI values , 0.80
developed an index of dominance (ID) to assist with indicating occupation of different niches. Interestingly, no
interpreting patterns of colonization and dominance in grain correlation could be found between ID and NOI methods. The
ecosystems. The ID was found to significantly change with aw results suggested that niche overlap was in a state of flux and
and temperature and also with nutritional grain substrate. Of significantly influenced by both temperature and water
the 15 species, the most competitive species in wheat grain in availability. The nutrient status is very important as Lee and
United Kingdom were found to be P. brevicompactum, Magan (1999) demonstrate that comparison of c-sources in a
P. hordei, P. roqueforti, A. fumigatus, and A. nidulans. standard BIOLOG test plate with only those relevant to maize
318 Magan et al.

grain gave very different results in terms of niche size and 6 RESPIRATION AND DRY MATTER
NOI under different environmental conditions. This approach LOSSES
confirms that interactions and dominance are dynamic and not
static and emphasizes the importance of taking account of Harvested grain carries a wide range of bacterial and fungal
such fluxes in any integrated approach to control the activity contaminants. Depending on effectiveness of storage
of spoilage molds in the stored seed ecosystem. conditions, and the climatic region of the world the level
and type of contamination will vary. Grain itself and the
microbial contaminants respire slowly when stored dry.
5 RELATIONSHIP BETWEEN MOLD However, if the water availability is increased to 15 –19%
DETERIORATION OF CEREAL GRAIN moisture content (¼0.75– 0.85 aw) predominantly spoilage
fungi, particularly Eurotium spp., Aspergillus, and Peni-
AND CALORIFIC LOSSES
cillium species grow resulting in a significant increase in
respiratory activity, resulting in increased temperature and
It is surprising to note that few studies have been conducted if sometimes spontaneous heating that results in colonization by
poor postharvest practices can result in the nutritional quality thermophilic fungi and actinomycetes (Fleurat-Lessard 2002;
loss of grain due to the activity of spoilage fungi (Sinha 1982). Lacey and Magan 1991). The chemical process involved in
Different grains have different levels of intrinsic calorific heat generation is predominantly aerobic oxidation of
values and, thus deterioration by different spoilage fungi will carbohydrates such as starch. The energy is released by the
affect these values to different extents. Elegant studies by following equation:
Demenyk and Sinha (1988), Sinha (1995), and Sinha et al.
(1986) demonstrated that the bioenergetics of insects feeding C6 H12 þ 6O2 ! 6CO2 þ 6H2 O þ 2835 kJ
on different grain types enables energy budgets to be
established and provided useful information on the impact Heating occurs when this energy is released faster than it can
that pests have on actual calorific value of stored cereals. escape from the cereal substrate. In contrast, little energy is
However, in stored grain ecosystems factors such as aw, released in anaerobic respiration and little or no heating
temperature, gas composition, and level of fungal contami- occurs in the absence of oxygen. The requirement for oxygen
nation will all have a significant impact on calorific losses and increases with temperature to a maximum of 408C but does
provide a useful link with quantifiable levels of dry matter not decrease greatly until the temperature exceeds 658C. At
losses for different cereals. this temperature, microbial growth is largely inhibited and
Studies by Prasada and Prasad (1982) reported changes in heating results from exothermic chemical oxidation. Thus, the
the calorific value of linseed (Linum usitatissimum; respiratory quotient may be 0.7 to 0.9 up to 658C but less than
L ¼ 1:896 kJ=g) due to seed-borne infection by spoilage 0.5 at higher temperatures.
fungi. They found that A. niger infection resulted in maximal By utilizing the respiratory quotient CO2 production can
calorific losses of 25.2% within 15 days, and almost 50% in be translated into dry matter loss. Typically, complete
30 days at 288C on surface sterilized seeds. Losses from respiration of carbohydrates gives a respiratory quotient, i.e.,
autoclaved and inoculated seeds were higher. However, water ratio of O2 consumed to CO2 produced of 1.0, and it has been
availability was not monitored or controlled. More recent calculated that 14.7 g CO2/kg grain will be released for every
studies where both aw and temperature were carefully 1% loss of grain dry matter. During anaerobic fermentation,
controlled have examined the effects caused by the mycotoxin only about 0.493 g CO2 is evolved from a kilogram grain for
producing species of F. verticillioides and F. proliferatum and every 1% dry matter loss. Alternatively, a respiratory quotient
A. ochraceus (Marin et al. 1999c; Ramos et al. 1999a,b). The below 1.0 may result from lipid or protein metabolism. For
Fusarium spp. were found to maximally cause calorific losses example, tripalmitim has a quotient of 0.7. The higher the
of maize based substrates (¼19.69 kJ/g) at 0.98 aw (17–64%) CO2 production, the shorter the safe storage period without
after 4 weeks depending on temperature of incubation, but loss of dry matter. Studies by Jonsson et al. (2000) utilized
negligible at 0.92 aw (0 –9%). An inverse correlation was respiratory rates over a wide range of aw levels and
found between calorific losses and fungal biomass and temperatures to examine development of molds such as
fumonisin production. For A. ochraceus calorific losses were P. verrucosum in stored grain and also effects on
maximal at 0.95 aw and 20– 308C on similar maize-based germinability, fungal biomass, and maximum safe storage
substrates. Losses were in the region of 10 –17% over 4 weeks times in days. They suggested that the maximum storage time
with much lower losses at 0.85 aw (0–7%). There was a direct without mold growth was probably halved if moisture content
relationship between increase in fungal biomass and calorific at harvest was increased by 1 –3% (¼0.05 aw) or if storage
losses. As each grain type has a different intrinsic calorific temperature was increased by 58C. Fleurat-Lessard (2002) in
value it is critical that these studies are expanded to include his excellent review has suggested that for the modeling and
rice and other staple cereals to enable the relationship prediction of global quality changes the rate of CO2
between the activity of spoilage fungi in stored cereals to be production can be used as a “storability risk factor.”
quantified both on the basis of the significance of nutritional The ratio of contribution of spoilage molds and grain to
losses and in terms of dry matter losses. total respiration has been argued for many years. A range of
Role of Spoilage Fungi in Seed Deterioration 319

studies has demonstrated that grain deterioration and by et al. 1991). Cahagnier and Richard Mollard (1991) suggested
implication dry matter loss is predominantly determined by that the ergosterol content in storage fungi was not
fungal activity. Wheat deterioration has been measured and significantly affected by environmental factors such as aw.
models developed based on germination rates, visible mold They thus suggested that ergosterol could be used as an
growth, or respiration of grain and microorganisms (Fleurat- “index” of the level of fungal biomass and the length of
Lessard 2002). Kreyger (1972) pointed out, from previous storage of the grain. Tothill et al. (1993) showed that there
work with wheat that dry matter loss was unimportant, was a significant positive correlation between ergosterol
provided there was no visible molding. However, his own content and total CFUs at 0.95 aw, while in grain of 0.85 aw
work showed that barley of 24% water content (¼0.94 aw) there was no significant correlation. Grain inoculated with
stored at 168C for 10 weeks lost 2% dry matter with visible individual species (Alternaria alternata, E. amstelodami, and
molding. With maize showed that fungal invasion and P. aurantiogriseum) at 0.95/0.85 aw and 258C showed a
aflatoxin content could be unacceptable before the grain had significant correlation between CFUs and ergosterol although
lost 0.5% dry matter and mold growth became visible (Seitz the content for an individual species varied considerably.
et al. 1982). Latif and Lissik (1986) proposed a model for Overall, levels of , 5–6 mg/g can be found in fresh wheat
respiration based on the rate of dry matter breakdown, but was grain, with that having microscopic growth containing about
not related to important environmental factors such as aw and 7.5–12 mg/g. This correlates with a threshold of 105 CFUs/g
temperature. This worked suggested that Kreyger’s assump- grain as a spoilage threshold indicator. Fleurat-Lessard (2002)
tions were not completely accurate. White et al. (1982) noted has suggested that perhaps modeling of ergosterol production
that 0.1% was unacceptable for first grade wheat and rates under different environmental conditions using sigmoid
proposed an absolute level of 0.04%. However, when 55 days curves similar to those used for insect population dynamics
safe storage was predicted for grain stored at 18.4% mc may enable the use of an ergosterol index in the future when
(¼0.86 aw) visible molding occurred after 23 days of storage. correlation models become available. It may also be possible
Recently, Karunakaran et al. (2001) determined deterioration to use both ergosterol and the production of mycotoxins in
rates in wheat stored at constant or step decreases in predicting potential environmental factors over which
temperature. Deterioration rates at 17% mc for wheat were 5, spoilage/mycotoxins may be produced. Two-dimensional
7, and 15 days at 35, 30, and 258C, respectively. Interestingly, models for growth and fumonisin production have already
they found that respiration rates of 17 –19% mc wheat at 258C been developed (Marin et al. 1999a,b,c) and such information
increased while germination percentages decreased with may be useful in further development of predictive models for
storage time. Dry matter losses were about 0.05% and visible risk assessment of spoilage and toxin contamination. Perhaps,
mold was observed when dry matter loss was about 0.1% at modeling of cumulative ergosterol production by spoilage
these w.c.s. fungi and associated mycotoxins in relation to aw ,
There are problems with the use of visible molding as a temperature, time, gas composition (modified atmosphere
criterion of deterioration (Hamer et al. 1991; Lacey et al. storage), and time may allow more effective and precise risk
1994). While Kreyger (1972) used this extensively, a clear assessment of mold contamination and mycotoxin occurrence
definition was not produced. Many workers have questioned to the consumer.
this subjective index of the safe storability of grain (Hamer
et al. 1991; Lacey and Magan 1991). Magan (1993) pointed
out as an early indicator, microscopic growth may be a more
effective measurement of fungal activity than visible 8 ENZYME CHANGES AS AN INDICATOR
molding. OF DETERIORATION

Changes in grain enzyme concentrations, e.g., amylases, due

7 BIOCHEMICAL TESTS to fungal deterioration are important as they have an impact
on processing and bread making quality of flour and dough.
A wide variety of methods have been used to quantify the However, studies which examined a-amylase, b-amylase,
fungal activity in stored grain. Chitin, ergosterol, adenine and total amylases of wheat found no correlation with the time
triphosphate, immunofluorescence, immunoassays, and DNA to microscopic/visible molding (Magan 1993). Fleurat-
probes have all been developed (Fleurat-Lessard 2002; Lessard (2002) suggested that for both wheat and malting
Magan 1993). Since, ergosterol is the predominant sterol in barley enzyme changes are too small and occur to late as
most spoilage fungi (ascomycetes and deuteromycetes) and functions of storage conditions and duration, especially with
not found in insect pests it has been utilized extensively as an regard to incorporation in a model for decision support
indicator of whether deterioration has occurred in grain. The systems.
method was first described by Seitz et al. (1977) and can now However, there are other studies which suggests the
be performed relatively quickly and routinely using simple opposite. Fungi colonizing the rich grain substrate under
extraction and HPLC. It has thus been used extensively for the conducive environmental conditions produce a battery of
in vitro quantification of biomass of spoilage fungi which hydrolytic enzymes for degrading the grains and causing the
demonstrated that this does change with culture age (Marfleet dry matter losses discussed earlier in this Chapter. Flannigan
320 Magan et al.

and Bana (1980) and Magan (1993) showed that aw and 9.1 Carbohydrate Degradation
temperature affect the production of enzymes by fungi during
grain colonization, including cellulases, polygacturonase, Starchy cereal grains contain 75% carbohydrates that are
pectin methyl esterase, 1-4-b-glucanase, b-glucosidase, hydrolyzed during respiration by amylases. As indicated
b-xylosidase, and lipases. Jain et al. (1991) were the first to earlier, while fungal invasion does lead to increases in
demonstrate that by using chromogenic 4-nitrophenol reducing sugars these changes are not early enough as an early
substrates in an ELISA well format, rapid quantification indicator of deterioration (Magan 1993).
could be carried out for a range of hydrolytic enzymes,
provided that substrates were available for them. They
demonstrated that in both barley and wheat grain at different 9.2 Protein Degradation
aw levels (0.85, 0.90, and 0.95) significant increases in
N-acetyl-b-D -glucosaminidase were produced when com-
Total proteins represents about 14.5% of the grain dry matter
pared to nonmolded dry harvested grain. Grain inoculated
in wheat and quality can vary considerably (Aspinall and
with the xerophile E. amstelodami also showed marked
Greenwood 1965). Protein type, quality, and amount are all
increases in a-D -galactosidase.
important for bread and baking quality. It is thus surprising
Magan (1993) extended this and examined stored dry grain
that, although protein quality is important to human nutrition
with that at different aw levels and temperatures of incubation.
few studies have attempted to analyze in detail the impact that
This showed that a significant change in the production of
fungal activity might have on this component. No changes in
some enzymes was evident at times of microscopic and
total protein during spoilage and heating, while others found
visible molding. Of seven enzymes examined significant
an increase in total protein. Colonization of wheat grain by
changes in b-D -glucoaminidase, b-D -glactosidase, and
fungi such as A. tenuis, A. flavus, A. niger, and F. solani
b-D -glucosidase were observed by the time microscopic
resulted in an increase in total protein although aw or
growth had occurred. Similar results were obtained with
temperature considerations were not considered. More
fumonisin producing Fusaria (F. verticillioides,
research is needed on the potential changes, particularly in
F. proliferatum) by Marin et al. (1998d). Changes could be
storage proteins that may occur due to fungal invasion of
monitored within 72 h of storage. They also suggested that
grain.
these enzymes could be used as an early indicator of infection
of maize grain by such species and that these enzymes were
important in enabling rapid colonization over a wide range of
9.3 Lipid Degradation
environmental factors. Recent work by Keshri and Magan
(1998) and Keshri et al. (2002) have also suggested similar
hydrolytic enzymes are an early indicator of fungal activity Starchy grains contain about 2.5% lipids while oil rich seeds
in vitro on wheat flour-based media and in bread substrates. contain much more (Aspinall and Greenwood 1965). Lipids
Thus, potential does now exist for the use of such relatively can be degraded endogenously and via fungal invasion, both
simple enzyme assays to be used as a possible tool for early by oxidation and by hydrolysis, with lipoxygenases and
detection of fungal activity in cereal grain. lipases, respectively (Zeleny and Coleman 1938). Usually an
increase in free fatty acids in the seeds is indicative of
utilization by spoilage fungi. Indeed Magan et al. (1993)
demonstrated that free fatty acid values for different spoilage
9 VIABILITY AND DEGRADATION OF fungi varied considerably with aw and temperature in in vitro
GRAIN studies with rape seed oil and with rape seed. Farag et al.
(1985a,b) had earlier showed that fungal lipases degraded
Fungal activity inevitably leads to deterioration and loss in different classes of lipids by degrading triglycerides to
viability of grain to be used as seed. Whether seed death mono/diglycerides and free fatty acids.
occurs directly due to fungal activity or due to degradation of
key grain nutritional components is, however, unclear. Under
some circumstances fungi have been shown to be key 10 FUNGAL VOLATILES AS AN EARLY
components in deterioration of viability. The grain embryo is INDICATOR OF DETERIORATION
sometimes preferentially invaded by some fungi, especially
species of Aspergillus and Penicillium. In most early classical The production of volatiles by fungi when colonizing grain
work it was demonstrated that spoilage of stored grain has been quantified and the key components identified in vitro
resulted in seed viability being rapidly reduced to practically and in situ (Kaminski et al 1974; Magan and Evans 2000;
zero. Hill and Lacey (1983) found a linear relationship Schnurer et al. 1999). Key volatiles indicative of spoilage
between initial moisture content and percentage viability. include 3-octanone, methylheptanone, 2-methyl-1-propanol,
However, few studies in the last decade have examined the cyclohexanone, trimethylbenzene, undecane, naphthalene,
detailed changes in nutritional components of grain. Thus, we and dodecane. Previous in situ studies have demonstrated that
are reliant on other earlier work for such information. measurement of such volatiles show promise in early
Role of Spoilage Fungi in Seed Deterioration 321

detection of deterioration of grain in stores (Tuma et al. 1989). seeds has been achieved in the last decade. Certainly, the key
The rapid development of electronic nose technology, which role of spoilage molds, the conditions under which they may
uses a variety of sensor arrays for giving qualitative produce mycotoxins, early detection systems and predictions
information on changes in patterns of volatile production, of dry matter losses, and an understanding of the complex
has enabled this technology to be examined in detail for interactions that occur in the stored seed ecosystem are better
practical applications in the grain commodity chain. Evans understood. In certain areas, particularly with regard to
et al. (2000) demonstrated that it was possible to classify grain quantifying nutritional losses generally and quantifying
samples in real time into categories of good quality, molded, degradation of grain components needs to be moved forward
or suspect grain using an automated computer controlled in the coming years. It is critical that such studies are linked to
system coupled with radial based models. The potential for and correlated with other measures of quality loss of stored
using odor classification from grain as a spoilage indicator has seeds, especially staple grains. This would enable us to realize
also been demonstrated (Magan and Evans 2000). Work has the goal of developing realistic and accurate decision support
also shown that e.nose technology could be used for systems for effective conservation of seeds postharvest. In the
differentiating between different isolates of the same spoilage coming years we will need to build on this sound foundation
species on the basis of qualitative volatile production patterns, in developing strategies for the prevention of spoiled grain
which may differ due to specific biosynthetic pathways for and mycotoxins entering the human and animal food chain.
isolates producing mycotoxins in Fusarium section Liseola
species (Keshri and Magan 2001); and in naturally
contaminated grain samples (Olssen et al. 2002). REFERENCES

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29
Mycotoxins

Fun S. Chu University of Wisconsin, Madison, Wisconsin, USA

Deepak Bhatnagar U.S. Department of Agriculture– Agricultural Research Service,

New Orleans, Louisiana, USA

1 INTRODUCTION role in their toxic actions (Chu 2002; Hussein and Brasel
2001). Recent studies on the effect of mycotoxins on
1.1 Mycotoxins, Mycotoxicosis, and apoptosis have further revealed their mode of action at the
Mycotoxicology cellular level (Chu 2002). The complexity of the biological
effects of mycotoxins has led to a scientific discipline named
Mycotoxin is a convenient generic term describing the toxic “mycotoxicology” for the study of various issues that are
secondary metabolites produced by fungi. “Myco” means encountered with this group of toxins.
fungal (mold) and “toxin” represents poison. They encompass Mycotoxins have been the subject of many reviews, and
a considerable variety of low molecular weight compounds recently several comprehensive reviews have discussed
with diverse chemical structures and biological activities. various aspects of this topic (Bhatnagar et al. 2002; CAST
Some mycotoxins could also be toxic to plants or other 2003; Chu 2002; Scudmore 2000). Due to a limitation of
microorganisms; but these compounds are not classified as space, in this chapter we have tried to cover, as much as
antibiotics of fungal origin. Like most microbial secondary possible, the most recent advances achieved in the field, and
metabolites, the benefit of mycotoxins for the fungi refered primarily to reviews listing all the previous landmark
themselves is still not clearly defined. In considering the references in the field.
effects of mycotoxins on animals, it is important to
distinguish between “mycotoxicosis” and “mycosis.” Myco-
toxicosis is used to describe the action of mycotoxin(s) and is 1.2 Historical Background
frequently mediated through a number of organs, notably the
liver, kidney, lungs, and the nervous, endocrine, and immune Although mycotoxicoses have been known for a long time,
systems. On the other hand, “mycosis” refers to a generalized identification of a specific mycotoxin as a causative agent for
invasion of living tissue(s) by growing fungi (CAST these illnesses was unknown in most of the incidences
2003; Chu 1998). Due to their diverse chemical structures, [reviewed in Bhatnagar et al. (2002)]. For example, at least
mycotoxins may exhibit a number of biological effects, one of the 10 plagues in ancient Egypt recorded in Exodus
including both acute and chronic toxic effects as well as (and as early as 430 B.C.) could have been associated with
carcinogenic, mutagenic /genotoxic, teratogenic, and mycotoxin-contamination of food. Since the ninth century,
immunotoxic effects (Bhatnagar et al. 2002; Chu 1998; ergot-infected rye has (ergotism) afflicted large-populations
2002; Wogan 1992). Modulation of the animal immuno- in Europe, when ergotism was called ignis sacer (sacred fire)
system, either immunosuppressive (most often) or immuno- or St. Anthony’s fire, because it was believed that a
stimulatory, also plays an important role for the overall pilgrimage to the shrine of St. Anthony would bring relief
toxic effects (Bondy and Pestka 2000). The interaction of from the intense burning sensation caused by the mycotoxin
mycotoxins with cellular macromolecules plays a dominant (Van Dongen and DeGroot 1995).

325
326 Chu and Bhatnagar

Outbreaks of toxicoses associated with the ingestion of 2 PRODUCTION OF MYCOTOXINS BY

moldy foods and feeds by humans and animals have also TOXICOGENIC FUNGI
been recorded in last century. Deaths of livestock were
reported earlier from consumption of moldy corn in feed 2.1 General Consideration
of horses in Illinois, Russia and swine in Southeastern
United States (Christiansen and Kauffman 1969) in the Invasion by fungi and production of mycotoxins in
1930s. A well-documented example is the disease called commodities can occur under favorable conditions in the
alimentary toxic aleukia (ATA) that resulted in more than field (preharvest), at harvest, and during processing,
5000 deaths in humans in the Orenberg district of the transportation and storage (Bhatnagar et al. 2002). Fungi that
USSR during World War II, and the cause of later was are frequently found in the field include A. flavus, Alternaria
found to be trichothecene mycotoxins. Modern myco- longipes, A. alternata, Claviceps purpura, Fusarium verti-
toxicology was not developed until the discovery of cillioides (previously called moniliforme), F. graminearum,
aflatoxins in the early 1960s as the causative agent in the and a number of other Fusarium spp. Species most likely
peanut meal causing the “Turkey X” disease that killed introduced at harvest include F. sporotrichioides, Stachy-
more than 10,000 turkeys fed with the contaminated meal. botrys atra, Cladosporium sp., Myrothecium verrucaria,
Because aflatoxins are a series of highly potent carcino- Trichothecium roseum, as well as A. alternata. Most
gens produced by commonly occurring Aspergillus flavus penicillia are storage fungi. These include Penicillium
and A. parasiticus, research has focused new attention on citrinum, P. cyclopium, P. citreoviride, P. islandicum,
mycotoxins. In the last 40 years, many new mycotoxins P. rubrum, P. viridicatum, P. urticae, P. verruculosum,
have been identified and characterized, and their P. palitans, P. puberulum, P. expansum, and P. roqueforti, all
biosynthetic origin in various fungi elucidated. It has of which are capable of producing mycotoxins in grains
been estimated that at least 25% of the world’s agricultural and foods. Other toxicogenic storage fungi are:
product is contaminated with mycotoxins and certain A. parasiticus, A. flavus, A. versicolor, A. ochraceus,
diseases have been linked to ingestion of food and feed A. clavatus, A. fumigatus, A. rubrum, A. chevallieri,
contaminated with mycotoxins. Recent evidence that F. verticillioides, F. tricinctum, F. nivale, and several other
indoor air pollution from toxigenic fungi may play a role Fusarium spp. It is apparent, most of the mycotoxin-
in illnesses may implicate mycotoxins as having a more producing fungi belong to three genera: Aspergillus,
widespread role in chronic disease than was previously Fusarium, and Penicillium. However, not all species in
thought possible (CAST 2003). these genera are toxicogenic.

1.3 Economic Impact of Mycotoxin 2.2 Factors Affecting Mycotoxin Production

Contamination
Genetics and environmental and nutritional factors greatly
The most obvious negative economic impact of myco- affect the formation of mycotoxins. Depending on the
toxins is an outright loss of crops and affected animals, susceptibility of the crop, geographic and seasonal factors,
particularly when a severe outbreak occurs. However, even as well as cultivation, harvesting, storage, and transpor-
lower mycotoxin levels in feed affect animal health by tation practices, mycotoxins are found worldwide (D’Mello
causing feed refusal and an increased susceptibility to and MacDonald 1997). In the field, weather conditions,
infection. Crops contaminated with mycotoxins at certain plant stress, invertebrate vectors, species and spore load of
levels cannot be sold for human/animal consumption in infective fungi, variations within plant and fungal species,
countries, which have rigorous regulatory requirement for and microbial competition all significantly affect myco-
acceptable level of these compounds. However, humans toxin production. Physical factors such as time of
and animals may encounter severe health hazard or high exposure, temperature during exposure, humidity, and
mortality rates in countries with less regulation or extent of insect or other damage to the commodity prior to
monitoring programs as result of exposure to higher levels exposure determine mycotoxin contamination in the field
of toxins in foods and feeds. In addition, costs of chemical or during storage. Chemical factors including the
analyses, quality control and regulatory programs, research nutritional status of the crops or chemicals (such as
development, extension services, lawsuits, and the cost of fungicides) used in crop management could affect fungal
treatment of human illness must be considered in the populations, and consequently toxin production. The
overall economic burden of mycotoxins on the economy temperature and relative humidity range for optimal
worldwide. Thus, the negative economic impact (crop boss) mycotoxin production may differ from that supporting
resulting from mycotoxin contamination is certainly very fungal growth. In general, mycotoxins are optimally
significant (CAST 2003), and estimated to be $932 million produced at 24–288C, but some toxins such as T-2 toxin
annually from aflatoxins, fumonisins, and deoxynivalenol is maximally produced at 158C. Contamination during crop
contamination of crops. storage may be affected by changes in temperature and
Mycotoxins 327

water activity, that allow ecological succession of different 3 NATURAL OCCURRENCE AND TOXIC
fungi as water activity and temperature of stored grain EFFECTS OF SELECTED MYCOTOXINS
changes. During storage and transportation, water activity
(aw), temperature, crop damage, time, blending with moldy 3.1 Aflatoxins
components, and a number of physical and chemical 3.1.1 General Considerations
factors, such as aeration (O2, CO2 levels), types of grains,
pH, and presence or absence of specific nutrients and Aflatoxins (AF), a group of polyketide-derived bisfurans
inhibitors are important. containing dihydrofuranofuran and tetrahydrofinan (rings)

Figure 1 Chemical structure of aflatoxins. (A) The B-type aflatoxins are characterized by a cyclopentane E-ring. These compounds
have a blue fluorescence under long-wavelength ultraviolet light. (B) The G-type aflatoxins, with a green fluorescence, have a xanthone
ring in place of the cyclopentane. (C) Aflatoxins of the B2 and G2 type have a saturated bis-furanyl ring. Only the bis-furan is shown.
(D) Aflatoxin of the B1a and G1a type have a hydrated bis-furanyl structure.
328 Chu and Bhatnagar

fused with substituted coumarin (Figure 1) are the most 3.1.3 Toxic Effects and Mode of Action
characterized class of mycotoxins (Cole and Cox 1981; Eaton
and Groopman 1994). At least 16 structurally related toxins in Aflatoxins are mutagenic, teratogenic, and hepatocarcino-
this group are produced by A. flavus and A. parasiticus genic in experimental animals [reviewed in Eaton and
(Bhatnagar et al. 1992; 1993) and infrequently by Groopman (1994)]. Since AFB1 is one of the most potent
A. pseudotamarii and A. nominus (Goto et al. 1996; Ito et al. naturally occurring carcinogen, extensive research was
2001). A. ochraceoroseus has also been found to produce primarily done on this toxin. The main target organ of AF
aflatoxins (Klich et al. 2000). The optimal temperatures and is the liver. Typical symptoms of aflatoxicoses in animals
aw for the growth of A. flavus and A. parasiticus are around include proliferation of the bile duct, centrilobular necrosis,
35 – 378C (range from 6 – 548C) and 0.95 (range from and fatty infiltration of the liver, generalized hepatic lesions,
0.78– 1.0), respectively; whereas for aflatoxin production, and hepatomas. AFB1 also affects other organs and tissues
they are 28 –338C and 0.90– 0.95 (0.83 –0.97), respectively. including the lungs and the entire respiratory system. The
Aflatoxin B1 is most toxic in this group and is one of the most susceptibility of animals to AFB1 varies considerably with
potent naturally occurring carcinogens; other AFs are less species in the following order: rabbits (most), ducklings,
toxic (B1 . G1 . B2 . G2). Other significant members of mink, cats, pigs, trout, dogs, guinea pigs, sheep, monkeys,
the aflatoxin family, such as M1 and M2, are metabolites of chickens, rats, mice, and hamsters (least). For the
AFB1 and AFB2, respectively, and originally isolated from carcinogenic effects, rats, rainbow trout, monkeys, and
bovine milk [reviewed in Cary et al. (2000); Spahr et al. ducks are most susceptible and mice are relatively resistant.
(1999)]. Consumption of AFB1-contaminated feed by dairy cows
The major steps and the corresponding genes of the AFB1 results in the excretion of AFM1 in milk. AFM1, a
biosynthesis have been elucidated [extensively reviewed in hydroxylated metabolite of AFB1, is about 10 times less
Bhatnagar et al. (2003)]. Starting with the polyketide toxic than AFB1; but its presence in milk is of concern for
precursor, acetate, there are at least 23 enzymatic steps in human health (Chu 2002).
the AFB1 biosynthetic pathway. AFB2, G1, and G2 are Metabolism plays a key role for the toxic action of
synthesized from pathways that diverge from the B1 pathway. aflatoxins. AFB1 must first be activated by mixed-function
The genes for almost all the enzymes have been cloned and a oxidases to a putative short-lived AFB-8.9-exo-epoxide
regulatory gene (aflR) coding for a DNA-binding, Gal 4-type [reviewed in Eaton and Groopman (1994); Sinha and
47 kDa protein has been shown to be required for Bhatnagar (1998)]. This intermediate can either be converted
transcriptional activation of all the structural genes. A defect to hydroxylated metabolites, conjugated to glutathione or
in aflR expression in the koji mold A. sojae causes to turn-off glucuronic acid etc; and then be excreted, or it can bind to
the expression of AF genes that produce AF (Matsushima DNA, RNA, and protein to exert its carcinogenic, mutagenic,
et al. 2001). It has also been shown by restriction mapping and toxic effects. Glutathione S-transferase serves as a key
of cosmid and phage DNA libraries of A. flavus and enzyme in the detoxification process for AFB1. Formation of
A. parasiticus that all the AFB1 pathway genes are clustered AF dialdehyde by aldo-keto reductases and subsequent
within a 75-kb region of the fungal genome (Bhatnagar et al. reaction with proteins also plays a role against AFB1 toxicity
2003). G-protein signaling has recently been found to be (Guengerich et al. 2001). Adduct formation of this
involved in the biosynthesis of AF (Tag et al. 2000). intermediate with DNA occurs at the N-7 guanine position
leading G-C to T-A or to A-T nucleotide substitutions, which
results in defective repair and DNA damage, mutations and
3.1.2 Natural Occurrence ultimately carcinomas in many animal species (Chu 2002;
Aflatoxins have been found in corn, peanuts and peanut Eaton and Gallagher 1994; Wild and Hall 2000; Wogan
products, cotton seeds, peppers, rice, pistachios, tree nuts 2000). Aflatoxin-induced G:C mutations, both G to T and G to
(Brazil nuts, almonds, pecans), pumpkin seeds, sunflower A, have been implicated in the inactivation of human p53
seeds and other oil seeds, copra, spices, and dried fruits (figs, tumor suppressor gene at third position of codon 249; and the
raisins), and yams [see CAST (2003) for a detailed list]. identification of mutations/inactivation of p53 at this site, has
Among these products, frequent contamination with high been used as a biomarker for AFB-induced liver cancers in
levels of AF in peanuts, corn, and cottonseed, mostly due to humans (Groopman et al. 1995; Harris 1996). Activated
infestation with fungi in the field, are of most concern. AFB1 and AFG1 also formed adducts with albumin (Skipper
Soybeans, beans, pulses, cassava, sorghum, millet, wheat, and Tannenbaum 1990) excreted in urine, which has been
oats, barley, and rice are resistant or only moderately used as an index of human exposure to AFs (Sabbioni and
susceptible to AF contamination in the field. It should be Wild 1991; Turner et al. 2000).
reiterated that resistance to AF contamination in the field does
not guarantee that the commodities are free of AF 3.1.4 Impact on Human Health
contamination during storage. Inadequate storage conditions,
such as high moisture and warm temperatures (25 –308C), can Whereas AFB1 has been found to be a potent carcinogen in
create conditions favorable for the growth of fungus and many animal species, the role of AF in carcinogenesis in
production of AF. humans is complicated by hepatitis B virus (HBV) infections
Mycotoxins 329

in humans (Hsieh 1989; Wild et al. 1992). Epidemiological

studies have shown a strong positive correlation between AF
levels in the diet and primary hepatocellular carcinoma (PHC)
incidence in some parts of the world, including certain regions
of the People’s Republic of China, Kenya, Mozambique,
Philippines, Swaziland, Thailand, and Transkei of South
Africa [reviewed in Eaton and Groopman (1994)]. Adducts of
AF, i.e., AFB1-DNA and AF-albumin as well as several AF
metabolites, mainly AFM1 have been detected in serum,
milk, and urine of humans in these regions. However, the
prevalence of HBV infection is also correlated to liver cancer
incidence in these regions. Since multiple factors are
important in carcinogenesis (Wogan 2000) and environmental
contaminants such as AFs and other mycotoxins may, either
in combination with HBV or independently, be important
etiological factors. Recent data on the enhancement of
mutation of p53 gene suggest the synergistic effect of these
two risk factors for PHC in humans (Jackson et al. 2001;
Smela et al. 2001).

3.2 Ochratoxins
3.2.1 General Considerations
Ochratoxins, a group of dihydroisocoumarin-containing
mycotoxins (Figure 2), are produced by a number of fungi
in the genera Aspergillus and Penicillium [reviewed in
Bhatnagar et al. 2002; Chu 2002; Ono et al. 1995; Petzinger
and Ziegler 2000). The largest amounts ochratoxins are
made by A. ochraceus and P. cyclopium. A. ochraceus and
P. viridicatum (reclassified as P. verrucosum), two species Figure 2 Structure of the ochratoxins. These metabolites form
that were first reported as ochratoxin A (OA) producers, different classes depending on the nature of the amide group
occur most frequently in nature. Other fungi, such as (a– c), and the presence or absence of a chlorine moiety at R2 in
Petromyces alliceus, A. citricus, and A. fonsecaeus (both in the phenyl group.
A. niger group), have also been found to produce OA. Most
of the OA producers are storage fungi and preharvest fungal 3.2.2 Toxicological Effects and Mode of Action
infection and OA production is not a serious problem.
Although most OA producers can grow in a range from 48C Ochratoxin A, the most toxic member of this group of
to 378C and at aw as low as 0.78, optimal conditions for mycotoxins, has been found to be a potent nephrotoxin
toxin production are narrower with temperature at 24–258C causing kidney damage as well as liver necrosis and enteritis
and aw values . 0.97 (minimum aw for OA production is in many animal species [reviewed in Bhatnagar et al. 2002;
about 0.85). Ochratoxins are produced primarily in cereal Chu 2002). Although OA is primarily considered a weak
grains (barley, oats, rye, corn, wheat) and mixed feed nephro-carcinogen because a high level of toxin and an
during storage in temperate climatic conditions, with levels extended period of exposure are necessary to induce the
higher than 1 ppm being reported. OA has been found in tumors, it also causes liver tumors in rats (Boorman et al.
other commodities, including beans, coffee, nuts, olives, 1992; Schlatter et al. 1996). The OA is also an
raisin, cheese, fish, pork, milk powder, fruit juices wine immunosuppessor (Bondy and Pestka 2000; Boorman et al.
(Filali et al. 2001; Pietri et al. 2001), beer, peppers 1992; Simon 1996) and has teratogenic, mutagenic, weak
(Thirumala et al. 2001), and bread (Van Egmond and genotoxic properties (Dirheimer 1996; deGroene et al. 1996).
Speijers 1994). OA can be carried through the food chain Several mechanisms have been postulated for the mode of
because of the presence of OA residues in animal products action for OA (Chu 2002). Whereas OA–DNA adducts have
as result of its binding with serum albumin. Natural been found in kidney, liver, and several other organs of mice
occurrence of OA in kidneys, blood serum, blood sausage, and rats, their structures and functions have not been defined
and other sausage made from pork has been reported (Grosse et al. 1995; Pfohl-Leszkowicz et al. 1993). Formation
[reviewed in CAST (2003)]. of a reactive intermediate such as of those for AFB1 is less
330 Chu and Bhatnagar

likely to be involved in the action of OA (Gautier et al. 2001a,

b; Zepnik et al. 2001). The OA inhibits carboxypeptidase A,
renal phosphoenolpyruvate carboxykinase, phenylalanine-
tRNA synthetase, and phenylalanine hydroxylase activity.
Formation of free radicals has been considered as one of the
mechanisms for the carcinogenic/toxic effects of OA
(Baudrimont et al. 1994; Gautier et al. 2001a; Hoehler and
Marquardt 1996; Rahimtula et al. 1988). Studies on the OA
induced apoptosis revealed that it interacts with distinct
members of the mitogen-activated protein kinase family, e.g.,
c-jun amino-terminal-kinase, which can lead to direct or
indirect caspase 3 activation and subsequently to APT (Gekle
et al. 2000; Schwerdt et al. 1999). OA may impair cellular
Ca2þ cAMP homeostasis. In the renal epithelial cells, OA
interferes with hormonal Ca2þ signaling, leading to altered
cell proliferation.

3.2.3 OA and Human Health

Although the role of ochratoxins in human pathogenesis is
still speculative, the pathological lesions of nephropathy in
humans were reported to be similar to those observed in
porcine nephropathy (Krogh 1976). Outbreaks of kidney
disease (Balkan endemic nephropathy) in rural populations in
Bulgaria, Romania, Tunisia, and the former Yugoslavia has
been associated with OA (Maaroufi et al. 1996; Wolff et al.
2000).
Figure 3 Chemical structures of different classes of fumoni-
sins. The R residue is 3-hydroxypidinium in fumonisins, B1 – B3
3.3 Fumonisins while the others are tricarballyl esters.
3.3.1 General Considerations
Fumonisins (Fm) are a group of toxic metabolites produced from negligible to more than 100 ppm; but is generally
primarily by F. verticillioides, F. proliferatum and other reported to be between 1 and 2 ppm. FmB1 is the most
related species readily colonize corn all over the world common Fm in naturally contaminated samples; FmB2
(Dutton 1996; Jackson et al. 1996; Marasas 2001). Although generally accounts for 1/3 or less of the total. Although
F. anthophilum, F. nupiforme, and F. nygamai are capable of production of the toxin generally occurs in the field, continued
producing Fms, they are not commonly isolated from food production of toxin during postharvest storage also con-
and feed. Other related fusaria, including F. subglutinans, tributes to the overall levels. (Chu 2001; 2002; 2001 May
F. annulatum, F. succisae, and F. beomiforme, are not Fm issues of Environmental Health Prospective, Vol. 109).
producers (Nelson et al. 1993). More than 11 structurally The biosynthesis of the 20-carbon chain backbone of Fms
related Fms (B1, B2, B3, B4, C1, C4, A1, A2, etc.), have resembles those for fatty acids and linear polyketides
been found since the discovery of FmB1 (diester of [reviewed in Desjardins et al. 1996; Proctor 2000). Five loci
propane-1,2,3-tricarboxylic acid of 2 amino-12, 16-dimethyl- (designated as fum 1, fum 2, fum 3, and fum 4 and fum 5)
3,5,10,14,15-pentahydroxyicosane) in 1987 (Figure 3) have been identified by classical genetic analyses utilizing
[reviewed in Bhatnagar et al. (2002); Chu (2002)]. Several Gibberella fuijikuroi, the sexual stage of F. monififorme
hydrolyzed derivatives of Fms, resulting from removal of the (Proctor 2000). At least 15 genes are associated with the
tricarballylic acid and other ester groups, have also been fumonisin production and these genes are clustered together
found in nature. Fumonisins are chemically similar in on chromosone “1” (Proctor 2000; Xu and Leslie 1996)
structure to toxins (AAL) produced by A. alternata. including a polyketide synthase gene ( fum5). Gene Fum2 and
Production of furnonisins by Alternaria has also been Fum3 are associated with the interconversion of different
reported, and some fumonisin-producing Fusaria have been fumonisins (Proctor 2000).
known to produce AAL toxins.
Fumonisins are most frequently found in corn, corn-based 3.3.2 Toxicologic Effects
foods, and other grains (such as sorghum and rice) but peanuts
and soybeans are poor substrate. The level of contamination Fumonisin B1 is primarily a hepatotoxin and carcinogen in
varies considerably with different regions and year, ranging rats (Class 2B carcinogen). Feeding culture material from
Mycotoxins 331

F. verticillioides or pure FmB1 to rats resulted in cirrhosis and Fumonisin B1 was identified as an etiological agent
hepatic nodules, adenofibrosis, hepatocellular carcinoma- responsible for ELEM in horses and other Equidae (donkeys
ductular carcinoma, and cholangiocarcinoma (Gelderblom and ponies) and for porcine pulmonary edema (PPE) [Jackson
et al. 2001; Haschek et al. 2001). Kidney is also a target organ, et al. 1996; reviewed in Summerell et al. (2001)]. ELEM is
and tubular nephrosis was found both in rats and in horses of characterized primarily by neurotoxic effects, including
field cases associated with equine leukoencephalomalacia uncoordinated movements and apparent blindness showing
(ELEM). In addition to FmB1, which was originally found to as violent blundering into stalls and walls. The levels of
be a potent cancer-causing agent, FmB2 and FmB3 have also FmB1 and FmB2 in feeds associated with confirmed cases of
been found to be carcinogens and have cancer initiation and ELEM ranged from 1.3 to 27 ppm. In pigs, PPE occurs only at
promoting activities in rats. The effective dose of FmB1 for high FmB levels (175 ppm), while liver damage occurs at
cancer initiation in rat liver depends both on the levels and on much lower concentrations with a NOAEL of ,12 ppm.
the duration of exposure. In cell culture systems, FmB1 has FmB1 resulting in left-sided heart failure alters cardio-
been demonstrated to be mitogenic and cytotoxic, without vascular function. In the cattle, renal injury, hepatic lesions,
genotoxic effects (Gelderblom et al. 2001). Kidney cells have and alteration of sphingolipid in various organs was observed
also been shown to be targeted by these toxins. (Haschek et al. 2001). Similar to AAL toxin, Fms are also

Figure 4 Chemical structure of different trichothecenes. MC ¼ Macrocyclic; ISV ¼ isovalerate; OH-ISV ¼ hydroxyisovalerate.
332 Chu and Bhatnagar

toxic to some plants such as, jimsonweed, black nightshade, generally classified as macrocyclic (Type C) or nonmacro-
duckweed, and tomatoes (Abbas and Riley 1996). cyclic (Types A and B) (Figure 4). Although more than 100
TCTCs have been identified, only a few frequently found in
3.3.3 Mode of Action foods and feeds are potentially hazardous to human and
animal health. Other fungal genera are Myrothecium,
Mechanistically, Fms are inhibitors of ceramide synthase Trichoderma, Trichothecium, Cephalosporium, Verticimono-
(sphinganine/sphingosine N-acyltransferase), a key enzyme sporium, and Stachybotrys. In addition to fungi, extracts from
involved in the biosynthesis of sphingolipids, which are a Brazilian shrub, Baccharis megapotamica, also contain
heavily involved in cellular regulation, including cell macrocyclic TCTCs. The term TCTCs is derived from
differentiation, mitogenesis and apoptosis (Merrill et al. trichothecin, the first compound isolated in this group (Chu
2001; Riley et al. 2001). The primary amino group of Fms is 1997; Jarvis et al. 1995; Miller and Trenholm 1994; Vesonder
essential for its inhibitory and toxic effects (Norred et al. and Golinski 1989).
2001). The inhibitory effect of Fms on ceramide synthase Similar to other sesquiterpenes, TCTCs are biosynthesized
appears to be related to interference with sphingolipid via the mevalonate pathway: the TCTC skeleton is formed by
biosynthesis in multiple organs, such as brain, lung, liver, and cyclization of farnesyl pyrophosphate via the intermediate
kidney of the susceptible animals (Garren et al. 2001). trichodiene by an enzyme trichodiene synthase. The
Increased lipid peroxidation has also been considered as a isovaleroxy side chain in T-2 toxin is derived from leucine.
mode for its effect on initiation of cancer (Gelderblom et al. Several key enzymes in the TCTC biosynthetic pathway have
2001). The mode of action of FmB becomes more clear from been identified. At least six genes (Tri1 to Tri6) involved in
recent data of its effects on apoptosis (Zhang et al. 2001) and the biosynthesis of these mycotoxins have been cloned, and
the tumor necrosis factor (TNF) pathway is important in these genes are clustered together on a chromosome. Genes
inducing tumors (He et al. 2001; Zhang et al. 2001; Sharma Tri3, Tri4, and Tri5, which encode a transacetylase
et al. 2001), but the tumor suppressor gene p53 was not (15-O-acetyltransferase), a cytochrome P-450 monooxy-
required (Jones et al. 2001). The ability of FmB1 to alter gene genase, and trichodiene synthase, respectively, are contained
expression and signal transduction pathways are considered within a 9-kb region, while Tri5 and Tri6 (a regulatory gene)
necessary for its carcinogenic and toxic effects. FmB1 is a are in a 5.7 kb region (Proctor 2000).
good example of an apparently nongenotoxic (non-DNA The structural diversity of TCTCs results in a variety of
reactive) agent producing tumors through the regulation of toxic effects in animals and humans [reviewed in
apoptosis (Dragan et al. 2001). Bhatnagar et al. (2002)]. The TCTC mycotoxicoses affect
many organs, including the gastrointestinal tract, hemato-
3.3.4 Impact on Human and Animal Health poietic, nervous, immune, hepatobiliary, and cardiovascular
systems. Outbreaks of several types of mycotoxicoses in
While Fms are commonly detected in corn-based foods and humans and animals, including moldy corn toxicosis,
feeds, the impact of low levels of Fms in human foods is not scabby wheat toxicosis (or red-mold, or akakbi-byo
clear. Although several reports have indicated a possible role disease, or scabby barley poisoning), feed refusal and
of FmB1 in the etiology of human esophageal cancer in the emetic syndrome (swine), fusaritoxicoses, hemorrhagic
regions of South Africa, China, and northeastern Italy where syndrome, and alimentary toxic aleukia (ATA) have been
Fusarium species are common contaminants, more data are reported. Mechanistically, inhibition of protein synthesis is
necessary to sustain this hypothesis (Groves et al. 1999). The one of the earlier events in manifestation of TCTC toxic
co-occurrence of Fms with carcinogenic mycoroxins such as effects and they act at different steps in the translation
AFB (da-Silva et al. 2000; Vargas et al. 2001) or nitrosamines process. Inhibitory effects of these mycotoxins vary
may play an important role in carcinogenesis in humans. considerably with the chemical structure of the side
Current data suggest that Fms may have greater effect on the chain [see review in Chu (2002)].
health of farm animals than on humans [Bhatnagar et al.
(2002) for review].
3.4.2 T-2 Toxin and Related Type A TCTCs
T-2 toxin, a highly toxic type A TCTC, is produced by
3.4 Selected Trichothecenes (TCTCs) F. tricinctum, F. sporotrichioides (major), F. poae,
3.4.1 General Considerations F. sulphureum, F. acuminatum, and F. sambucinum. Unlike
most mycotoxins, which are usually synthesized near 258C,
Several species of Fusaria infect corn, wheat, barley, and rice. the optimal temperature for T-2 toxin production is around
Under favorable conditions, they elaborate a number of 158C. Higher temperatures (20 – 258C) are needed for the
different types of tetracyclic sesquiterpenoid mycotoxins that production of related metabolites, such as H-T2 toxin.
are composed of the epoxytrichothecene skeleton and an Although T-2 toxin occurs naturally in cereal grains,
olefinic bond with different side chain substitutions (Figure 4). including barley, corn, corn stalk, oats, wheat, and mixed
Based on the presence of a macrocyclic ester or ester –ether feeds, contamination with T-2 toxin is less frequent than
bridge between C-4 and C-15, trichothecenes (TCTCs) are with deoxynivalenol (DON). However, T-2 toxin (LD50 in
Mycotoxins 333

mice: 2–4 mg/kg mice) is much more toxic to animals, perhaps with DON was sufficient to induce these mRNA levels in
also to humans, than DON (LD50 in mice: 50–70 mg/kg). Peyer’s patches and spleen (Bondy and Pestka 2000).
Almost all the major TCTCs, including T-2 toxin, are
cytotoxic and cause hemorrhage, edema, and necrosis of skin
3.4.4 The Impact of TCTC on Human and Animal
tissues. Inflammatory reactions near the nose and mouth of
animals are similar to some lesions found in humans suffering Health
from ATA disease. The severity of lesions is also related to Because of their toxicity and their frequent presence in foods
chemical structure. Compared with other types of TCTCs, and feeds, TCTCs are potentially hazardous to human and
group A toxins (T-2 toxin) are less toxic than macrocyclic animal health. Among the many types of TCTC myco-
toxins but more toxic than type B toxins. Neurologic toxicoses, only ATA and scabby wheat toxicosis have been
dysfunctions, including emesis, tachycardia, diarrhea, refusal demonstrated in human populations. ATA, which has
of feed/anorexia, and depression have also been observed. T-2 symptoms including skin inflammation, vomiting, damage
toxin and some TCTCs also induce pathological lesions in the to hematopoietic tissues, leukocytosis, and leukopenia, was
gastrointestinal system. However, the major lesion of T-2 attributed to the consumption of overwintered cereal grains
toxin is its devastating effect on the hematopoietic system in colonized by F. sporotrichioides and F. poae; it caused the
many mammals, including humans. T-2 toxin and related deaths of hundreds of people in the USSR between 1942 and
TCTCs are the most potent immunosuppressants of the known 1947 (Ueno 1986). TCTCs may also be involved in the
mycotoxins and cause significant lesions in lymph nodes, so-called “sick building syndrome (SBS)” in humans
spleens, thymus, and the bursa of Fabricius (Bondy and (Mahmoudi and Gershwin 2000; Vesper et al. 2000). S. atra
Pestka 2000). The heart and pancreas are other target organs and S. chartarum were isolated from a badly water-damaged
for T-2 toxin intoxication. Urinary and hepatobiliary lesions home or building where the occupants complained about
induced by T-2 toxin and DAS are secondary. headaches, sore throats, hair loss, flu symptoms, diarrhea,
fatigue, dermatitis, and general malaise as well as pulmonary
3.4.3 Deoxynivalenol (DON) hemorrhage cases. Several TCTCs were found in these cases,
but other molds and mycotoxins were also found in the mold
The DON is a major type B TCTC mycotoxin produced by damaged buildings (Toumi et al. 1993).
F. graminearum (major) and other related fungi such as
F. culmorum and F. crookwellense. Because DON causes
feed refusal and emesis in swine, the name “vomitoxin” is 3.5 Other Selected Mycotoxins
also used for this mycotoxin. Although DON is considerably
less toxic than most other TCTC mycotoxins, the level of
contamination of this toxin in corn and wheat is generally In addition to the mycotoxins discussed above, a number of
high, usually above 1 ppm, sometimes greater than 20 ppm. other mycotoxins occur naturally. The impacts of some of
Contamination of DON in other commodities, including these mycotoxins on human and animal health are discussed
barley, oats, sorghum, rye, safflower seeds, and mixed feeds in the following sections.
has also been reported. Worldwide frequent natural
occurrence of DON in cereal grains has been reported (Miller 3.5.1 Other Mycotoxins Produced by Aspergillus
and Trenholm 1994). Although inadequate storage may lead
to the production of some TCTC mycotoxins, infestation of Sterigmatocystin (ST) is a naturally occurring hepatotoxic
fusaria in wheat and corn in the field is of most concern for the and carcinogenic mycotoxin produced by fungi in the genera
DON problem. With wet and cold weather during maturation, Aspergillus, Bipolaris, and Chaetomium as well as P. luteum
grains are especially susceptible to F. graminearum infection, [see Bhatnagar et al. (2002)]. Structurally related to AFB1
which causes so-called “scabby wheat” and simultaneously (Figure 5), ST is known to be a precursor of AFB1 (Bhatnagar
produces the toxin. The optimal temperature for DON
production is about 248C. Outbreaks of DON in winter wheat
in the United States, Finland, and Canada usually occur when
continental chilly and humid weather favoring the fungal
infection is followed by a humid summer favorable for toxin
production.
Toxicologically, DON induces anorexia and emesis both
in humans and animals [reviewed in Bhatnagar et al. (2002);
Chu (2002)]. Swine are most sensitive to feed contaminated
with DON. Whereas most TCTCs are immunosuppressors,
DON is a hyperinducer of cytokines and IgA. Induction of
expression of mRNA of Ils-2, 4, 5, 6 in a T-cell model Figure 5 Structure of sterigmatocystin. The bis-furanyl
EL4.IL-2 by DON was found at levels required for partial or structure is similar to that of the aflatoxins except that the
maximal protein synthesis inhibition. A single oral gavage E-ring is a substituted phenol.
334 Chu and Bhatnagar

et al. 2003). Although the carcinogenicity of ST is less teratogenic. Patulin is frequently found in damaged apples,
(10–100 times) than that of AFB1 in test animals (van der apple juice, apple cider and sometimes in other fruit juices
Watt 1977), ST is a mutagen and genotoxin and has been and feed. PA has been detected in “blue eye corn” beans and
found in cereal grains (barley, rice, and corn), coffee beans, meat. Due to its highly reactive double bonds that readily
and cheese (Chu 2002). A. terreus and several other fungi react with sulfhydryl groups in foods, patulin is not very
(e.g., A. flavus and A. fumigatus and some Penicillia) have stable in foods containing these groups (Scott 1975). As a
been found to produce the tremorgenic toxins, territrems, hepatotoxin but not known as a carcinogen, PT is considered a
aflatrem, and fumitremorgin. These mycotoxins contain both health hazard to humans (CAST 2003).
the indole ring of tryptophan and a dioxopiperazine ring Frequently associated with the natural occurrence of OA,
formed by condensation of two amino acids. A. terreus, citrinin, also a nephrotoxin, is produced by P. citrinum and
A. fumigatus, and Trichoderma viride also produce gliotoxin, several other penicillia, aspergilli (Cole and Cox 1981) and
an epipolythiopiperazines -3,6-diones-sulfur containing Monocuus ruber and M. purpureus (Pastrana et al. 1996). The
piperazine antibiotic, that may have immunosuppressive presence of citrinin in the diet with low quality corn could
effects in animals (Waring and Beaver 1996). In addition, lead to chronic, hard to diagnose kidney disease in susceptible
A. flavus, A. wentii, A. oryzae, and P. atraovenetum are individuals and animals (CAST 2003). One of the mycotoxins
capable of producing nitropropionic acid (NPA), a mycotoxin closely associated with the natural occurrence of AF in
causing apnea, convulsions, congestion in lungs and peanuts is CPA, which causes hyperesthesia and convulsions
subcutaneous vessels, and liver damage in test animals as well as liver, spleen, pancreas, kidney, salivary gland, and
(Burdock et al. 2001). Production of NPA in sugarcanes by myocardial damage (CAST 2003). CPA inhibits the calcium-
Arthrinium sacchari, Arth. saccharicola, and Arth. phaeo- dependent ATPase (Chu 2002; Petr et al. 1999). The toxin is
spermum has been found to be involved in fatal food produced by several species of the genus Penicillium,
poisoning in humans (Liu et al. 1988). including P. cyclopium, P. crustosum, P. griseofulvin,
P. puberulum, P. camemberti, and Aspergilli including
A. versicolor, A. flavus but not by A. parasiticus and A. tamarii
3.5.2 Other Mycotoxins Produced by Penicillium [in Bhatnagar et al. (2002)]. Other than peanuts, CPA has
Other than OA, Penicillia produce many mycotoxins with been found in corn, cheese and fermented meat sausage, and
diverse toxic effects. Cyclochlorotine, luteoskyrin (LS), and sometimes along with aflatoxin (Fernandez-Pinto et al. 2001;
rugulosin (RS) have long been considered to be possibly Lopez-Diaz et al. 2001).
involved in the yellow rice disease during the Second World Penicillium rubrum and P. purpurogenum produce two
War. They are hepatotoxins and also produce hepatomas in highly toxic hepatotoxins (LD50, 3.0 mg/kg mice, IP) called
test animals. However, incidents of food contamination with rubratoxins A (RA, minor) and B (RB, major), which are
these toxins have not been well documented. Several other complex nonadrides fused with anhydrides and lactone rings.
mycotoxins, including patulin (PT, Figure 6), penicillic acid Rubratoxin B has synergistic effects with AFB1 (CAST
(PA, Figure 7), citrinin (CT), cyclopiazonic acid (CPA, 2003). In addition, penicillia produce many neurotoxic
Figure 8), citreoviridin, and xanthomegnin, which are mycotoxins. In many cases these mycotoxins do not cause
produced primarily by several species of Penicillia, have noticeable toxicity, but in some cases have strong tremorgenic
attracted some attention because of their frequent occurrence activity. Animals will also refuse food, and will have lowered
in foods. PT and PA are produced by many species in the resistance to disease. For example, P. crustosum and
genera Aspergillus and Penicillium. Byssochlamys nivea also P. cyclopium produce tremorgenic indolediterpenes called
produces PT (Tournas 1994). Both toxins are hepatotoxic and penitrems A-F. Penitrem A, the major toxin in this group,
causes tremorgenic effects in mice. Roquefortines A-C (C is

Figure 7 Chemical structure of penicillic acid showing both

Figure 6 Structure of patulin showing the closed-ring tautomeric forms. The open-chain form is in equilibrium with the
tautomeric form. closed form shown on the right in the figure.
Mycotoxins 335

in conjunction with DON may result in severe economic

losses to the swine industry.
Fusarium verticillioides and related species, in addition to
Fms, also produce several other mycotoxins, including
fusarins A-F, moniliformin, fusarioic, and fusaric acid,
fusaproliferin and beauvericin (CAST 2003; Chu 2002).
Although the impact of these mycotoxins on human health is
still not known, fusarin C (FC) has been identified as a
potent mutagen and is also produced by F. subglutinans,
F. graminearium and several other Fusaria. Moniliformin,
which causes cardiomyopathy in test animals, may be
involved in the Keshan disease in humans in regions where
dietary selenium deficiency is also a problem (Liu 1996). In
comparing their ability to form DNA-adducts, beauvericin
forms a more stable complex with DNA than fusaproliferin
Figure 8 Chemical structure of cyclopiazonic acid.
(Pocsfalvi et al. 2000). Among many fungi, F. verticilioides is
also most capable of reducing nitrates to form potent
most toxic), which are produced by P. roqueforti and several carcinogenic nitrosamines. These observations further
other penicillia, have neurotoxic effects in animals and have suggest that the contamination of foods with this fungus
been found in cheese. Tremorgens in the paspalitrem group could be one of the etiological factors involved in human
(paspalicine, paspalinine, paspalitrem A and B, paspaline and carcinogenesis in certain regions of the world.
paxilline) are produced by C. paspali and some penicillia
(Plumlee and Galey 1994; Steyn 1995; Yamaguchi et al. 3.5.4 Mycotoxins Produced by Alternaria Species
1993).
Alternaria has been known for centuries to cause various
plant diseases. Species of this fungus are widely distributed in
3.5.3 Other Mycotoxins Produced by Fusarium soil and on aerial plant parts. More than 20 species of
Alternaria are known to produce about 70 secondary
Some fusaria are capable of producing mycotoxins other than
metabolites belonging to a diverse chemical group, including
TCTCs and Fm. Zearalenone (ZE) (Figure 9) [6-(10-hydroxy-
dibenzo-[a]-pyrones, tetramic acids, lactones, quinones,
6-oxo-trans-1-undecenyl)-b(beta)-resorcyclic acid lactone], a
cyclic peptides. However, only alternariol (Figure 10),
mycotoxin produced by the scabby wheat fungus,
tenuazonic acid, altertoxin-I, alternariol monomethyl ether
F. graminearum (roseum), is of most concern. Also called
(AME), altenuene are common contaminants in consumable
F-2, ZE is a phytoestrogen causing hyperestrogenic effects
items like fruits (apples), vegetables (tomato), cereals
and reproductive problems such as premature onset of puberty
(sorghum, barely, oat), and other plant parts (such as leaves)
in female animals, especially swine. ZE has been shown to
(Jelinek et al. 1989). Natural occurrence of isoaltenuene,
bind with the estrogen and steroid receptors, and stimulates
altenuisol, altertoxins II and III (also called stemphyltoxin)
protein synthesis by mimicking hormonal action (Zepnik et al.
are less common. The most common species of Alternaria,
2001). Zearalenone can be toxic to plants; it can inhibit seed
A. alternata (formerly known as A. kikuchiana) produces all
germination and embryo growth at low concentrations.
important Alternaria toxins including the five mentioned
Natural contamination with ZE primarily occurs in cereal
above and tentoxin, alteniusol, alternaric acid, altenusin,
grains such as corn and wheat. Contamination of feed with ZE
dehydroaltenusin (Bottalico and Logrieco 1998; Chelkowski

Figure 9 Stucture of zearalenone. Figure 10 Structure of alternariol.

336 Chu and Bhatnagar

1992). As mentioned previously, A. alternata f sp. lycopersici Use of atoxigenic biocompetitive, native A. flavus strains
produces a group of host-specific toxins named AAL toxins to out-compete the toxigenic isolates has been effective in
with structure and functions similar to Fms. Although most of significantly reducing preharvest contamination with afla-
the compounds produced by Alternaria are generally toxin in cotton and peanuts (Cotty and Bhatnagar 1994;
nontoxic, AME has been shown to be mutagenic in Ames Dorner et al. 1992). However, the aflatoxin contamination
test (Woody and Chu 1992). Tenuazonic acid is a protein process is so complex (Payne 1998) that a combination of
synthesis inhibitor and is capable of chelating metal ions and approaches will be required to eliminate or even control the
forming nitrosamines. This mycotoxin is also produced by preharvest toxin contamination problem (Bhatnagar et al.
Phoma sorghina and Pyricularia oryzae and may be related to 1995).
“Onyalai,” a hematological disorder in humans living south of
the Sahara in Africa.
4.1.2 Postharvest Control
After harvest, crop should not be allowed to overwinter in the
3.5.5 Mycotoxins Produced by Other Fungi field as well as subjected to bird and insect damage or
Sporidesmines, a group of hepatotoxins discovered in the mechanical damage. Grains should be cleaned and dried
1960s, are also worthy of mention. These mycotoxins, quickly to less than 10 –13% moisture and stored in a clean
causing facial eczema in animals, are produced by area to avoid insect and rodent infestation (Trenholm et al.
Pithomyces chartarum and Sporidesmium chartarum and 1988). Postharvest mycotoxin contamination is prevalent in
are very important economically to the sheep industry. most tropical countries due to a hot, wet climate coupled with
Slaframine, a significant mycotoxin produced by Rhizoctonia subadequate methods of harvesting, handling, and storage
leguminicola (in infested legume forage crops), causes practices, which often lead to severe fungal growth and
excessive salivation or slobbering in ruminants as a result mycotoxin contamination of food and feed (Birzele et al.
of blocking acetylcholine receptor sites (CAST 2003). 2000; Phillips et al. 1994). Sometimes contaminated food has
been diverted to animal feed to prevent economic losses and
health concerns. However, this is not a solution to the
contamination problem. Irradiation has been suggested as a
4 PREVENTIVE MEASURES possible means of controlling insect and microbial popu-
4.1 Management of Mycotoxin Contamination lations in stored food, and consequently, reducing the hazard
of mycotoxin production under these conditions [reviewed in
Sharma (1998)]. Significant emphasis has been placed on
The economic implications of the mycotoxin problem and its
detoxification methods to eliminate the toxins from the
potential health threat to humans have clearly created a need
contaminated lots or at least reduce the toxin hazards by
to eliminate or at least minimize mycotoxin contamination of
bringing down the mycotoxin levels under the acceptable
food and feed. While an association between mycotoxin
limits.
contamination and inadequate storage conditions has long
been recognized, studies have revealed that seeds are
a. Removal or Elimination of Mycotoxins. Since most of
contaminated with mycotoxins prior to harvest (Lisker and
the mycotoxin burden in contaminated commodities is
Lillehoj 1991). Therefore, management of mycotoxin
localized to a relatively small number or seeds or kernels
contamination in commodities must include both pre- and
[reviewed in Dickens (1977)], removal of these contaminated
postharvest control measures (CAST 2003).
seeds/kernels is effective in detoxifying the commodity.
Methods currently used include: (a) physical separation by
4.1.1 Preharvest Control identification and removal of damaged seed; mechanical or
electronic sorting; flotation and density separation of
Mycotoxin contamination can be reduced somewhat by using damaged or contaminated seed; physical screening and
of resistant varieties (most effective, but not all are subsequent removal of damaged kernels by air blowing;
successful) and earlier harvest varieties, crop rotation, washing with water or use of specific gravity methods have
adequate irrigation, control of insect pests, etc [reviewed in shown some effect for some mycotoxins, including DON,
Bacon et al. (2001); Chen et al. (2002); Duvick (2001); Sinha FmB, and AFB1 (Trenholm et al. 1992), (b) removal by
and Bhatnagar (1998)]. Significant control of toxin contami- filtration and adsorption onto filter pads, clays, activated
nation is expected to be dependent on a detailed under- charcoal, etc., (c) removal of the toxin by milling processes,
standing of the physiological and environmental factors that (d) removal of the mycotoxin by solvent extraction (CAST
affect the biosynthesis of the toxin, the biology and ecology of 2003; DeVries et al. 2002).
the fungus, and the parameters of the host plant –fungal
interactions. Efforts are underway to study these parameters b. Inactivation of Mycotoxins. When removal or elimin-
primarily for the most agriculturally significant toxins, ation of mycotoxins is not possible, mycotoxins can be
namely AFs, Fms, and TCTCs [reviewed in Brown et al. inactivated by (a) physical methods such as thermal
(1998); Duvick (2001); Sinha and Bhatnagar (1988)]. inactivation, photochemical or gamma irradiation, (b)
Mycotoxins 337

chemical methods such a treatment of commodities with breeding stock (ruminant, poultry, and mink), and others
acids, alkalies, aldehydes, oxidizing agents, and gases like (dogs and cats), respectively. Among 77 countries which have
chlorine, sulfur dioxide, NaNO2, ozone and ammonia, (c) regulations for different mycotoxins, eight have specific
biological methods such as fermentations and enzymatic regulations for OA, with limits ranging from 1 to 20 mg/kg in
digestion that cause the breakdown of mycotoxins. The different foods. Regulatory guidelines to limit the presence of
commercial application of some of these detoxifying PT to 50 mg/kg in various foods and juices have been
mechanisms is not feasible because, in a number of cases, established by at least ten countries worldwide. Details on
the methods will be limited by factors such as the toxicity of worldwide regulatory issues and permissive levels of
the detoxifying agent, nutritional or aesthetic losses of mycotoxins in foods and feeds have appeared in a number
commodities during treatment, and the cost of the of recent reviews (Park and Troxell 2002; Van Egmond 2002;
sophisticated treatment [reviewed in Sinha and Bhatnagar Chu 2002).
1998]. Although several detoxification methods have been
established for aflatoxins, only the ammoniation process is an 4.2.2 Detection and Screening of Mycotoxins
effective and practical method (Piva et al. 1995). Other
chemicals such as ozone, chlorine, and bisulfite have been Because of the diverse chemical structures of mycotoxins, the
tested and some effect for some mycotoxins was shown in it presence of trace amounts of toxins in very complicated
(Doyle et al. 1982). Solvent extractions have been shown to matrices that interfere with analysis, and the uneven
be effective but are not economically feasible. distribution of the toxins in the sample, analysis of
mycotoxins is a difficult task (Chu 1995; Coker 1998;
c. Removal of Mycotoxins During Food Pro- Richard et al. 1993; Wilson et al. 1998). Because many steps
cessing. While cooking generally does not destroy myco- are involved in the analysis, it is not uncommon that the
toxins, some mycotoxins can be detoxified or removed by analytical error can amount to 20 –30% (Horwitz et al. 1993).
certain kinds of food processing. For example, extrusion To obtain reliable analytical data, an adequate sampling
cooking appears to be effective for detoxifying DON but not program and an accurate analytical method are both important
AFB. FmB1 can form Schiff’s bases with reducing sugars (Whitaker et al. 1994; 1995; Wilson et al. 1998). To minimize
such as fructose under certain conditions (Murphy et al. 1995) the errors, studies have led to many improved and innovative
and lose its hepato-carcinogenicity (Liu et al. 2001); but the analytical methods for mycotoxin analysis over the years
hydrolyzed FmB1 was found to be still toxic (Voss et al. (Chu 1991; Gilbert 1999; Maragos 1997). New, more
1996). Loss of FmB1 occurs during extrusion and baking of sensitive TLC, HPLC, and GC techniques are now available.
corn-base foods with sugars and nixtamalization (alkaline Sensitive and versatile high resolution MS and GC/tandem
cooking) and rinsing in the preparation of tortilla chip and MS/MS are coming to the market. The MS methods have also
masa (Dombrink-Kurtzman et al. 2000; Voss et al. 2001). PT been incorporated into HPLC systems. New chemical
can be removed from apple juice by treatment with certain methods, including capillary electrophoresis and biosensors
types of active carbons (Leggott et al. 2001). The effect of are emerging and have gained application for mycotoxin
food processing on various mycotoxins has been recently analysis (Maragos 1997).
reviewed by several authors in an ACS symposium (DeVries After a number of years of research, immunoassays have
et al. 2002). gained wide acceptance as analytical tools for mycotoxins in
the last decade (Chu 2001). Antibodies against almost all the
mycotoxins are now available. Some quantitative and
4.2 Avoiding Human Exposure qualitative immunoassays have been approved as AOAC
4.2.1 Role of Rigorous Monitoring Programs methods. Many immunoscreening kits, which require less
than 15 min. per test, are commercially available (Trucksess
While it is impossible to remove mycotoxins completely from and Wood 1997). Rather than analysis of toxin, PCR methods,
foods and feeds, effective measures to decrease the risk of based on the primers of key enzymes involved in the
exposure depend on a rigorous program of monitoring biosynthesis of mycotoxins, have been introduced for the
mycotoxins in foods and feeds. Consequently, governments in determination of toxicogenic fungi present in foods (Birzele
many countries have set limits for permissible levels or et al. 2000; Edwards et al. 2001). Detailed protocols for
tolerance levels for a number of mycotoxins in foods and mycotoxin analysis can be seen in several of the most recent
feeds. Over 50 countries of the world have developed such reviews and books and the most recent edition of AOAC
guidelines (Van Egmond 2002). For example, levels varying (Gilbert 1999; Trucksess and Pohland 2001; Van Dolah and
from zero tolerance to 50 ppb have been set for total AFs. Richard 1999).
Other than AFs, a tolerance level of 1 ppm for DON in grains
for human consumption has been set by a number of 4.2.3 Dietary Modifications
countries, including the United States. The FmB1 levels
established by FDA in 2000 are limited to 5, 20, 60 100, 30, Dietary modification greatly affects the absorption, distri-
and 10 ppm, in corn and corn by-products to be used for horse bution, and metabolism of mycotoxin and subsequently affect
and rabbit, catfish and swine, ruminants and mink, poultry, its toxicity [reviewed in Bhatnagar et al. (2002)]. For
338 Chu and Bhatnagar

example, the carcinogenic effect of AFB1 is affected by and international levels to remove the contaminated
nutritional factors, dietary additives, and anticarcinogenic commodities. This is because mycotoxins are highly stable
substances. Diet containing chemoprotective agents and compounds, the producing fungi are ubiquitous, and food
antioxidants such as ascorbic acid, BHA, BHT, ethoxyquin, contamination can occur both before and after harvest.
oltipraz, penta-acetyl geniposide, Kolaviron biflavonoids, and Nevertheless, good farm management practices and adequate
even green tea, have also been found to inhibit carcinogenesis storage facilities minimize the toxin contamination problems.
caused by AFB1 in test animals. The toxic effect of OA and A combination of natural biocontrol competition fungi and
FmB to test animals was minimized when antioxidants such enhancement of host-resistance against fungal growth or toxin
as vitamins C and E are added to the diet. Ascorbic acid also production could prevent toxin formation to a very significant
provided protective effect against AFs. Aspartame, which is extent. Rigorous programs for reducing the risk of human and
partially effective in decreasing the nephrotoxic and animal exposure to contaminated food and feed also include
genotoxic effects of OA, competes with OA for binding to economically feasible and safe detoxification processes and
serum albumin. L-phenylalanine was found to have some dietary modifications. Although risk assessment has been
protective effect for the toxic effects of OA because it made for some mycotoxins (Coker 1998; DeVries et al. 2002),
diminishing OA’s inhibitory effect to some of enzymes additional, systematic epidemiological data for human
discussed earlier. exposure is needed for establishing toxicological parameters
Most mycotoxins have a high affinity for hydrated for mycotoxins and the safe dose for humans. It is
sodium calcium aluminasilicate (HSCAS or NovaSil) and unreasonable to expect complete elimination of the
other related products. Diets containing NovaSil and related mycotoxin problem. But multiple approaches will be needed
absorbers have been found effective in preventing absorption to minimize the negative economic impact of the toxins on the
of AFB1 and several other mycotoxins in test animals, thus entire agriculture industry as well as their harmful effects on
decreasing their toxicity [(see Bhatnagar et al. 2002; Huwig human and animal health.
et al. 2001; Philips et al. 2002)]. Likewise, several other
adsorbants such as zeolite, bentonite, and superactive
charcoal have been found to be effective in decreasing the
toxicity of other mycotoxins such as T-2 toxin.
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30
Genetics and Biochemistry of Mycotoxin Synthesis

Jiujiang Yu U.S. Department of Agriculture – Agricultural Research Service,

New Orleans, Louisiana, USA

1 INTRODUCTION comprehensive reviews on this subject (Bhatnagar et al. 1988;

2002).
Mycotoxins are low-molecular weight, nonproteinaceous,
organic secondary metabolites produced by fungi from amino
acids, shikimic acid, or malyonyl CoA. These compounds are
2 AFLATOXINS AND
toxic, mutagenic, teratogenic, and carcinogenic to animals
and humans (Bennett 1987; Bhatnagar et al. 2002; Eaton and
STERIGMATOCYSTINS
Groopman 1994; Hall and Wild 1994; Squire 1989). Over 300 2.1 Occurrence and Toxicology
mycotoxins have been identified, but only those implicated in
mycotoxicoses involving humans have been studied in detail Aflatoxins are toxic and carcinogenic secondary metabolites
with respect to the biochemistry and genetics of their produced primarily by Aspergillus flavus and A. parasiticus as
biosynthesis. Research on the natural occurrence, identifi- well as A. nomius. One isolate of each of A. tamarii (Goto et al.
cation and characterization, biosynthesis and genetic 1996) and A. ochraceoroseus (Frisvad and Samson 1999)
regulatory control of mycotoxins, as well as prevention and have been reported to produce aflatoxins (Klich et al. 2000).
control of mycotoxin contamination of food and feed gained The aflatoxins consist of a group of at least 16 structurally
momentum after the incidence of “Turkey-X” disease in 1960 related toxins (Goldblatt 1969) characterized so far. Amongst
when 10,000 turkeys died due to aflatoxin contamination in these, aflatoxins, B1, B2, G1 and G2 (AFB1, AFB2, AFG1 and
the peanut-meal feed. Due to the risk of mycotoxin AFG2) are the major toxins. Aflatoxins are polyketide-
contamination of foods and feed on human health and derived, bis-furan-containing dihydrofuranofuran and tetra-
livestock productivity (Brown et al. 1998; Eaton and hydrofuran moieties (rings) fused with a substituted coumarin
Groopman 1994), regulations have been established in over (Figure 1). A. flavus produces aflatoxins, B1 and B2; while
50 countries for acceptable levels of mycotoxins in A. parasiticus produces the four major aflatoxins, B1, B2, G1
commodities for commerce or food and feed for human and and G2. The aflatoxins, M1 and M2, are modified forms of
animal consumption (Bhatnagar et al. 2002; Sharma and aflatoxin B1 found in bovine milk. Aflatoxin B1 (AFB1) is the
Salunkhe 1991). Within the last decade, significant progress most potent naturally occurring toxin and carcinogen known
toward the understanding of several mycotoxins in the world (Squire 1989). The toxicity of the major aflatoxins has been
has been made. In this chapter, the genetics and biochemistry established in the following order: B1 . G1 . B2 . G2.
of only the most economically significant mycotoxins, Aflatoxin M1 is 10-fold less toxic than AFB1, but its presence
aflatoxins, sterigmatocystin (ST), trichothecenes, and fumo- in milk is of concern in human health (Cullen et al. 1987;
nisins, are summarized and discussed. For other mycotoxins Galvano et al. 1996; Van Egmond 1989b). Due to the toxic
such as alternaria toxins AAL toxins, AK-toxin, ochratoxins, and carcinogenic properties of aflatoxins (Busby and Wogan
citrinin, cyclopiazonic acid (CPA), patulin, paxilline, 1981; Eaton and Groopman 1994), these compounds are the
zearalenone, ergot alkaloids and related toxins, and other most thoroughly studied of all the mycotoxins, and significant
neurotropic mycotoxins, kindly refer to the most recent research has been conducted on their biosynthetic pathway

343
344 Yu
Genetics and Biochemistry of Mycotoxin Synthesis 345

and genetic control of its regulation (Bhatnagar et al. 2002; It has been long proposed that the aflatoxin pathway genes
Chang et al. 1996; Payne and Brown 1998; Yu et al. 1995a). may be clustered with a common regulator (Cleveland and
Sterigmatocystin and dihydrosterigmatocystin (DHST) are Bhatnagar 1991). The first experimental evidence showing
produced by various Aspergilli including A. nidulans; several the potential clustering of aflatoxin pathway genes was,
genera Bipolaris and Chaetomium, and Penicillium luteum however, demonstrated by Trail et al. (1995b) when they
are also reported to produce ST (Cole and Cox (1987)). found that the nor-1 and ver-1 genes were linked in a cosmid
Aspergillus nidulans is an industrial fungus and has been the clone (NorA) with the regulatory gene aflR and a putative
model organism for the study of ST biosynthesis. ST and aflatoxin pathway gene, uvm8 (now named fas-1) in between.
DHST are the penultimate precursors of AFB1, AFB2, AFG1 This observation provided evidence that at least the early
and AFG2, respectively (Figure 1; Betina 1989; CAST 1989; stages of the pathway may be linked. By mapping overlapping
Chu 1991). Like aflatoxins, ST and DHST are hepatotoxic and cosmid clones in A. parasiticus and A. flavus (Yu et al.
carcinogenic mycotoxins. However, their carcinogenicity is 1995a), a linkage of genes involved in aflatoxin formation,
far less than that of aflatoxin B1 in animal test (Mori and from early stage nor-1 gene to later stage omtA, was
Kawai 1989; Van der Watt 1977). ST contaminates cereal established indicating that the entire aflatoxin biosynthetic
grains (barley, rice and corn), coffee beans and cheese pathway genes may be clustered. This observation and
(Jelinek et al. 1989) and is considered a health hazard as well. subsequent discoveries of pathway genes led to a consensus
Since ST and DHST are aflatoxin precursors, they share a cluster map consisting of at least nine aflatoxin pathway genes
common biochemical pathway, homologous genes and pksA, nor-1, uvm8 (now named fas-1), aflR, ver-1, omtA and
regulatory mechanism. For these reasons, aflatoxins and ST three additional open reading frames (ORFs) with unknown
are discussed together. function (ord-1, now named avnA; ord-2, now named ordA;
and aad, now named norA) (Yu et al. 1995a). The concept of
aflatoxin pathway gene cluster greatly accelerated the rate of
gene discovery (Cary et al. 1996; Chang et al. 1995a; 2000b;
2.2 The Clustering of Aflatoxin Biosynthetic Chang and Yu 2002; Feng and Leonard 1995; Mahanti
Pathway Genes et al. 1996; Silva et al. 1996; Silva and Townsend 1996; Yu
et al. 1997; 1998; 2000a,b,c). It was identified that at least 23
Significant progress has been made in the last decade in genes (Cary et al. 1996; Chang et al. 1992; 1993; 1995a;
deciphering aflatoxin biosynthetic pathway (Bennett 2000b; Chang and Yu 2002; Cleveland et al. 1997; Meyers
1981; 1987; 1986; 1994; Bhatnagar et al. 1988; 1991; 1992; et al. 1998; Motomura et al. 1999; Prieto and Woloshuk 1997;
1993; 1998; Bhatnagar and Cleveland 1990; Cary et al. Silva et al. 1996; Silva and Townsend 1996; Yu et al. 1993;
2000a,b; Chang et al. 1992; 1993; 1995a,b; 2000b; Cleveland 1995a; 1997; 1998; 2000a,b) or ORFs involved in aflatoxin
and Bhatnagar 1992; 2002; Dutton 1988; Minto and biosynthesis including the regulatory genes, aflR and aflJ,
Townsend 1997; Papa 1982; 1984; Payne 1998; Payne and were clustered together within approximately 80 kb DNA
Brown 1998; Silva and Townsend 1996; Silva et al. 1996; regions in the A. parasiticus and A. flavus genomes
Townsend 1997; Trail et al. 1995a; 1995b; Yu et al. 1993; respectively. Most of these genes on the aflatoxin pathway
1995a; 1997; 1998; 2000a,b). As many as 15 structurally gene cluster have been cloned and characterized. The cloning
defined aflatoxin intermediates have been identified in the of cypX and moxY genes (Yu et al. 2000a), the ordB gene
aflatoxin/ST biosynthetic pathway starting with the acetate, (potentially for an oxidoreductase, Yu, unpublished), and the
the polyketide precursors. At least 23 enzymatic steps sugar utilization gene cluster (Yu et al. 2000c) defined one
involved in the aflatoxin biosynthesis have been characterized end of the aflatoxin pathway gene cluster (Figure 1, panel A).
or proposed (Figure 1). The norB, cypA and aflT might possibly mark the other end of

Figure 1 Proposed and generally accepted pathway for aflatoxin B1, B2, G1 and G2 biosynthesis and the corresponding genes and their
enzymes are presented. The aflatoxin biosynthetic pathway gene cluster in A. parasiticus and A. flavus (panel A), sterigmatocystin
biosynthetic pathway gene cluster in A. nidulans (panel B), and the non-functional, partially duplicated aflatoxin gene cluster in
A. parasiticus (panel C) are shown. The gene names are labeled on the side of the cluster. Arrows indicate the direction of gene
transcription. The homologous genes between the sterigmatocystin pathway gene cluster in A. nidulans and aflatoxin pathway gene cluster
in A. parasiticus and A. flavus is connected by line. The four sugar utilization genes linked to the aflatoxin pathway gene cluster are on the
bottom of panel A. Abbreviations for the intermediates are: norsolorinic acid (NOR), averantin (AVN), 50 -hydroxyaverantin (HAVN),
averufanin (AVNN), averufin (AVF), versiconal hemiacetal acetate (VHA), versiconal (VAL), versicolorin B (VER B), versicolorin A
(VER A), demethylsterigmatocystin (DMST), sterigmatocystin (ST), O-methylsterigmatocystin (OMST), aflatoxin B1 (AFB1), aflatoxin
G1 (AFG1), DMDHST, dihydrosterigmatocystin (DHST), dihydro-O-methylsterigmatocystin (DHOMST), aflatoxin B2 (AFB2), and
aflatoxin G2 (AFG2), Methyltransferase (M-transferase).
346 Yu

this cluster (Yu et al., unpublished; Chang et al., unpublished; aflatoxin biosynthetic pathway gene cluster (Figure 1, panel
Figure 1, panel A). Using a similar strategy, Brown et al. A). Sequence analyses have shown that there are two large
(1996b) defined ST biosynthetic pathway gene cluster genes, fas-1A and fas-2A, encoding for beta- and alpha-
consisting of 25 coregulated transcripts in A. nidulans. The subunit of FAS, respectively (Mahanti et al. 1996,
ST gene cluster contains probably a complete set of ST genes unpublished, personal communication). The gene fas-1A
within approximately 60-kb DNA fragment (Brown et al. and fas-2A were renamed fas-1 and fas-2 in the aflatoxin
1996b). The aflatoxin pathway gene clusters between A. flavus pathway gene cluster encoding for FAS-1 (FASa) and FAS-2
and A. parasiticus are identical in term of the sequential order (FASb), respectively (Payne and Brown 1998). Brown et al.
of the genes, the sizes of these genes and the direction of gene (1996a) proposed the involvement of FAS in ST biosynthesis
transcription. The nucleotide and amino acid sequences of the in A. nidulans. They identified two genes, stcJ and stcK in the
aflatoxin pathway genes are also highly conserved (.95%) ST cluster, that are homologous to FASs, fas-2 and fas-1 of
between A. parasiticus and A. flavus (Yu et al. 1995a). aflatoxin pathway genes, respectively. Disruption of stcJ and
Analyzing the corresponding genes and their enzymes for a stcK encoding FASa and FASb subunits (FAS-2 and FAS-1),
specific pathway conversion step between A. flavus/ respectively in A. nidulans, stopped ST synthesis. Watanabe
A. parasiticus for aflatoxin synthesis and A. nidulans for ST et al. (1996) provided the biochemical evidence for the role of
synthesis, it has been shown a high degree of similarity with a FASs and PKSs in the biosynthesis of aflatoxin. The
respect to the genes, the enzyme encoded by these genes and N-acetylcysteamine thioester of hexanoic acid could be
their enzyme function. However, the order of these genes on incorporated into NOR in a disrupted fas-1 transformant.
the chromosome between the two clusters are totally Mayorga and Timberlake (1992) identified a gene wA
scrambled (Figure 1, panel A & B). encoding the PKS in A. nidulans. Chang et al. (1995a)
In A. parasiticus, duplication of aflatoxin genes ver-1 and confirmed that a PKS is required for aflatoxin biosynthesis by
aflR was first suggested (Mehigh et al., unpublished; Liang cloning the pksA gene encoding a PKS for the synthesis of
and Linz, unpublished) and reported by Liang et al. (1996). polyketide from A. parasiticus. Feng and Leonard (1995) also
This partial duplicated aflatoxin gene cluster consisting of isolated a pksL1 gene for PKS, the equivalent of pksA gene, by
seven duplicated genes, aflR2, aflJ2, adhA2, estA2, norA2, PCR from a pool of 19 A. parasiticus clones. Disruption of the
ver1B, omtB2, has been cloned and characterized by Chang pksL1 gene produced no aflatoxin or any aflatoxin
and Yu (2002). The genes within this partial duplicated intermediates. The pksL1 gene is 99% identical to pksA and
cluster, due possibly to the chromosome location (Chiou et al. they are believed to be the same gene. The 1% sequence
2002; Yu et al., unpublished), were found to be unfunctional discrepancy may possibly be due to strain variation and
under normal conditions even though no apparent defects are sequencing errors. Yu (1995) isolated a gene, pks ST that
identified in some of these genes (aflR2, aflJ2, adhA2, estA2). encodes a PKS from A. nidulans. The nucleotide sequence of
pks ST from A. nidulans is identical to stcA (Brown et al.
1996b). However, no significant sequence homology was
found between wA and stcA ( pks ST). The predicted amino
2.3 The Biosynthetic Pathways of Aflatoxins and
acid sequences of these PKS contains a typical four conserved
Sterigmatocystin domains common to other known PKS proteins: b-ketoacyl
2.3.1 Acetate to Norsolorinic Acid (NOR) Trail et al. synthase (KS), acyltransferase (AT), acyl carrier protein
(1995b) (ACP), and thioesterase (TE). The gene for PKS from
A. parasiticus ( pksA or pksL1) was designated as pksA (Yu
Molecular evidence supports the plausible hypothesis that two et al. 1995a) in the aflatoxin pathway gene cluster and its
fatty acid synthases (FAS) and a polyketide synthase (PKS) homolog in A. nidulans was designated as stcA (Brown et al.
are involved in the synthesis of polyketide from acetate 1996b). NOR is the first stable intermediate in the pathway
(Bhatnagar et al. 1992; Brown et al. 1996a; Townsend et al. (Bennett 1981; Bennett et al. 1997; Papa 1979; 1982).
1984; Trail et al. 1995a). Complementation of an aflatoxin However, the predicted conversion product of PKS in the
blocked UV mutant, UVM8, Mahanti et al. (1996) identified a aflatoxin pathway is believed to be a noranthrone. The
gene named uvm8, which is required for NOR biosynthesis conversion for noranthrone to NOR is poorly defined, but it
and aflatoxin production in A. parasiticus. The predicted has been proposed to occur by a noranthrone oxidase
amino acids of the gene uvm8 shares high degree of similarity (Vederas and Nakashima 1980), a monooxygenase (Bhatnagar
(67%) and identity (48%) to the beta-subunit of FASs (FAS1) et al. 1992), or spontaneously (Dutton 1988).
from Saccharomyces cerevisiae and Yarrowia lipolytica
(Trail et al. 1995a,b). Complementation, metabolite feeding 2.3.2 NOR to Averantin (AVN)
and disruption experiments by Mahanti et al. (1996)
confirmed that the 7.5-kb large transcript of the gene uvm8 Using NOR-accumulating mutants, it was demonstrated by
encodes one subunit of a novel FAS directly involved in the Papa (1979); Papa (1982) in A. flavus and by Bennett (1981),
backbone formation prior to nor-1 of aflatoxin synthesis. For Detroy et al. (1973) in A. parasiticus that NOR was an
the proposed function, the gene uvm8 was renamed fas-1A. intermediate in aflatoxin biosynthetic pathway (Bennett et al.
The fas-1A gene was renamed fas-1 encoding FAS-1 in the 1997). It was found that the NOR-accumulating mutants were
Genetics and Biochemistry of Mycotoxin Synthesis 347

always leaky without completely blocking aflatoxin bio- unclear. Woloshuk and Payne (1994) identified an alcohol
synthesis. Bhatnagar et al. (1992) proposed the involvement dehydrogenase gene, adh1, in A. flavus that is expressed
of dehydrogenase in the conversion of NOR to AVN and concurrently with aflatoxin pathway genes. The adh1 gene in
Chang et al. (1992) identified and cloned the gene, nor-1, A. flavus and adhA gene in A. parasiticus shares no significant
that complemented a NOR-non-producing mutant in homology at both DNA and amino acid level. The
A. parasiticus. Trail et al. (1994) further characterized this involvement of the adh1 from A. flavus in aflatoxin synthesis
gene to encode an enzyme that functions as a ketoreductase is to be investigated.
for the conversion of NOR to AVN. Cary et al. (1996)
characterized a gene, norA encoding an aryl-alcohol 2.3.5 AVF to Versiconal Hemiacetal Acetate
dehydrogenase in the aflatoxin pathway gene cluster, which (VHA)
were involved in the conversion of NOR to AVN (Bennett
et al. 1980; Bennett 1981; Bhatnagar et al. 1992). An The conversion from AVF to VHA is thought to involve an
additional gene, norB, which was also found to be oxidase (Bhatnagar et al. 1992). Yu et al. (2000b) cloned the
homologous to the nor-1 and norA genes in the aflatoxin avfA gene from both A. parasiticus and an AVF-accumulating
pathway gene cluster in A. parasiticus (Yu et al. unpublished). A. flavus strain as well as A. sojae strain that was shown by
The enzymatic function, and coordinated genetic regulation gene complementation experiment to encode for an enzyme
of the three NOR-converting genes are to be further (homologous to oxidase) responsible for the conversion from
investigated. The nor-1 homologous gene in A. nidulans is AVF to VHA. The avfA gene homolog in A. nidulans was
stcE (Brown et al. 1996b). identified to be stcO (Yu et al. 2000b).

2.3.3 AVN to 50 hydroxyaverantin (HAVN) 2.3.6 VHA to Versiconal (VAL)

Earliest evidence for the conversion of AVN to HAVN was The evidence for the involvement of an esterase in the
elucidated via radiotracing by Bennett et al. (1980), conversion of VHA to VAL in aflatoxin biosynthesis was
McCormick et al. (1987). Subsequently, Yabe et al. demonstrated by Bennett et al. (1976), Fitzell et al. (1977),
(1991b), based on the enzymatic studies, proposed three Schroeder et al. (1974), Yabe et al. (1991a,b), and Yao and
enzymatic steps in two possible routes involved in the Hsieh (1974), when the A. parasiticus was treated with the
conversion of aflatoxin intermediates from NOR to AVF: organophosphorus pesticide dichlorvos. The esterase has been
(a) conversion from NOR to AVN catalyzed by a purified in A. parasiticus by Hsieh et al. (1989); Kusumoto
dehydrogenase; (b) conversion from NOR to HAVN by a and Hsieh (1996). Yu et al. (2003) cloned a gene, estA, which
monooxygenase; (c) HAVN then would be converted to AVF could well be the gene for this esterase. The homologous gene
by a second dehydrogenase. Yabe et al. (1991b) also proposed in A. nidulans ST biosynthetic gene cluster is stcI. Attempts to
that this reaction was reversible and that the NADPH was the disrupt the estA or stcI have not resulted in any conclusive
preferred cofactor. Bhatnagar et al. (1992) proposed two evidence for the involvement of these genes in toxin
alternate routes requiring 3 – 4 enzymes for the conversion of synthesis.
NOR to AVF. Yu et al. (1997) cloned and disrupted a gene
that encodes a P-450 monooxygenase (previously named 2.3.7 VAL to Versicolorin B (VER B)
ord-1, Yu et al. 1995a). Using disruption and substrate
feeding it was defined that HAVN and averufanin (AVNN) Yabe and Hamasaki (1993) provided enzymatic evidence for
are the conversion intermediate products from AVN to the conversion of VAL to VER B. Silva et al. (1996), Silva
averufin (AVF). This gene was designated avnA, which has and Townsend (1996), and McGuire et al. (1996) cloned,
high sequence similarity to the stcF gene in the A. nidulans ST characterized and expressed the vbs gene in the aflatoxin
cluster (Brown et al. 1996b). pathway gene cluster in A. parasiticus. It was demonstrated
that the VER B synthase catalyzes the side chain
2.3.4 HAVN to AVNN and AVF cyclodehydration of racemic VHA to VER B. This is a key
step in the aflatoxin formation since it closed the bisfuran ring
In the proposed scheme by Yabe et al. (1991b; 1993), AVNN of aflatoxin for binding to DNA. The homologous gene in
was considered a shunt metabolite and not an aflatoxin A. nidulans ST biosynthetic gene cluster is stcN.
intermediate. Bhatnagar et al. (1992) proposed both AVN and
AVNN were the intermediates from NOR to AVF based on 2.3.8 VER B to Versicolorin A (VER A)
radiolabeling experiment. Chang et al. (2000b) cloned and
characterized a gene, adhA, encoding an alcohol dehydro- In the aflatoxin biosynthetic pathway, the VER B is a critical
genase in A. parasiticus. Gene disruption and feeding branch point (Bhatnagar et al. 1991) leading either to AFB1
experiments demonstrated that AVN might be converted and AFG1 or to AFB2 and AFG2. The conversion of VER B to
directly to AVF or indirectly to AVF through an intermediate VER A was proposed to require a desaturation of the bisfuran
substrate AVNN. The exact nature of the enzymatic function ring of VER B (Bhatnagar et al. 1993; Yabe and Hamasaki
and the possible involvement of additional enzymes are still 1993). Disruption of stcL in A. nidulans by Kelkar et al.
348 Yu

(1997) stopped ST synthesis but resulted in the accumulation sequence from A. parasiticus (initially named omt-1, later
of VER B. The stcL gene, for a P-450 monooxygenase, was renamed as omtA) by reverse genetics. The enzyme was
shown to be required for the conversion of VER B to VER A. expressed in Escherichia coli and its activity to convert ST to
A homologous gene to A. nidulans stcL in the aflatoxin OMST was demonstrated by substrate feeding studies. The
pathway gene cluster, verB, encoding a P-450 mono- genomic DNA sequence of this gene (omtA) was cloned from
oxygenase/desaturase, was cloned from A. parasiticus and A. parasiticus and A. flavus (Yu et al. 1995b). This omtA gene
A. flavus strains (Bhatnagar et al., unpublished, GenBank homologue was also detected in other aflatoxigenic and
accession numbers: AF106958, AF106959, and AF106960 nonaflatoxigenic Aspergillus species (Klich et al. 1995).
respectively).
2.3.12 OMST to Aflatoxin B1 (AFB1) and Aflatoxin
2.3.9 VER a to Demethylsterigmatocystin (DMST) G1 (AFG1) and DMDHST to Aflatoxin B2
and VER B to (AFB2) and Aflatoxin G2 (AFG2)
Demethyldihydrosterigmatocystin
(DMDHST) By feeding experiments, Yabe et al. (1988a) proposed the
relationship between B-Group (AFB1 and AFB2) and
Another cloned gene involved in a key step aflatoxin synthesis G-Group (AFG1 and AFG2) aflatoxin biosynthesis. It was
is the ver-1 gene (Skory et al. 1992) in A. parasiticus, this also predicted by Bhatnagar et al. (1992) that in the late stages
gene was shown by complementing the ver-1 mutant to be of aflatoxin biosynthesis, a NADPH-dependent mono-
required for the conversion of VER A to DMST in oxygenase is required for the conversion of OMST to
A. parasiticus. Keller et al. (1994) identified a gene stcU AFB1. Prieto et al. (1996), Prieto and Woloshuk (1997)
(formerly named verA) in A. nidulans, which is a homolog of reported in A. flavus that a P-450 monooxygenase gene, ord-1,
ver-1 in A. parasiticus for a ketoreductase, required for the is required for this reaction. Yu et al. (1998) cloned the P-450
conversion of VER A to DMST. Double mutation of stcU and monooxygenase gene, ordA, from A. parasiticus and an
stcL resulted accumulation of only VER B. Keller et al. A. flavus mutant strain and demonstrated by expression and
(1995a) also identified stcS(formerly named verB, Keller et al. substrate feeding in yeast system that this gene is responsible
1995b), which is homologous to P-450 monooxygenases, to for the conversion of OMST to AFB1 and AFG1, and
be involved in the conversion of VER A to DMST. Disruption DHOMST to AFB2 and AFG2. In this study, the critical amino
of this gene resulted in the accumulation of VER A. Thus, acids for the enzymatic activity and heme-binding motif were
both stcU and stcS are required for the conversion of VER B identified by site-directed mutagenesis as well. Yu et al.
to DMST. The stcS homolog, named verA in A. parasiticus (1998) also demonstrated that additional enzyme(s) is
strain, was also identified (Yu, unpublished). required for the G-group (AFG1 and AFG2) aflatoxin
synthesis. However, the enzyme(s) and corresponding
gene(s) have not been isolated as yet. It should be noted
2.3.10 DMST to ST and DMDHST to DHST
that the functions of three additional genes in the aflatoxin
Yabe et al. (1989) demonstrated a distinct methyltransferase pathway gene cluster, cypA(Yu, unpublished), cypX
activity in A. parasiticus for the conversion of DMST to ST. (cypX ¼ stcB) and moxY (moxY ¼ stcW) encoding cyto-
This enzyme may also be responsible for the conversion of chrome P-450 monooxygenases and monooxygenase (Yu
DMDHST to DHST. The enzyme was purified and et al. 2000a), have not yet been assigned. Keller et al. (2000)
characterized (Yabe et al. 1998; Yabe et al. 1999). Disruption examined several characterized cytochrome P-450 mono-
of stcP in A. nidulans by Kelkar et al. (1996) showed the oxygenases and proposed the functions for some of the
requirement of this gene for the conversion from DMST to identified genes or ORFs encoding monooxygenases in
ST. The gene responsible for the conversion of DMST to ST aflatoxin/ST synthesis. However, there is a possibility that
and DMDHST to DHST were cloned by Motomura et al. these genes might be involved in the G-group toxin formation.
(1999), named dmtA for O-methyltransferase I in
A. parasiticus and concurrently by Yu et al. (2000b), named
omtB for O-methyltransferase B in A. parasiticus, A. flavus 2.4 Factors Affecting Aflatoxin/ST Biosynthesis
and A. sojae.
2.4.1 Genetic Regulation

2.3.11 ST To O-methylsterigmatocystin (OMST) Since the aflatoxin and ST biosynthetic pathway genes are
and DMST to Dihydro-O- found to be clustered on a single chromosome in both
methylsterigmatocystin (DHOMST) A. parasiticus and A. flavus and in A. nidulans, respectively,
(Brown et al. 1996b; Woloshuk and Prieto 1998; Yu et al.
Several researchers (Bhatnagar et al. 1988; Keller et al. 1995a), these genes are presumably expressed concurrently in
1993; Yabe et al. 1989) reported the presence of an the genome. In both the aflatoxin and ST gene clusters, there
O-methyltransferase for the conversion of ST to OMST and is a positive regulatory gene, aflR, for activating pathway
DHST to DHOMST. Yu et al. (1993) cloned the cDNA gene transcription located in the middle of the gene clusters.
Genetics and Biochemistry of Mycotoxin Synthesis 349

The aflR gene, coding for a sequence specific zinc binuclear lipase gene expression and subsequent aflatoxin production
DNA-binding protein, a Gal 4-type 47 kDa polypeptide, has are induced by lipid substrate. Nonaflatoxin-conducive
been shown to be required for transcriptional activation of peptone medium supplemented with 0.5% soybean oil
most, if not all, the structural genes (Chang et al. 1993; 1995b; induces lipase gene expression and leads to aflatoxin
1999a,b; Ehrlich et al. 1998; Flaherty and Payne 1997; Payne formation (Yu et al. unpublished).
et al. 1993; Yu et al. 1997; Woloshuk et al. 1994) by binding Nitrogen source plays an important role in aflatoxin
to the palindromic sequence 50 -TCGN5CGA-30 in the production. Asparagine, aspartate, alanine, ammonium
promoter region of the structural genes (Ehrlich et al. nitrate, ammonium nitrite, ammonium sulfate, glutamate,
1999a,b; Fernandes et al. 1998) in A parasiticus, A. flavus, and glutamine, and proline containing media support aflatoxin
A. nidulans (Yu et al. 1996). In A. sojae, a nontoxigenic strain production; while sodium nitrate and sodium nitrite
used in industrial fermentations, was found to contain a containing media do not (Davis et al. 1967; Eldridge 1964;
defective aflR gene in addition to potential other defects Payne et al. 1983; Reddy et al. 1971; 1979). Studies by
(Matsushima et al. 2001a,b; Takahashi et al. 2002). Thus, Kachholz and Demain (1983), Niehaus and Jiang (1989)
with the absence of the functional regulatory protein, no suggested that nitrate represses AVF and aflatoxin formation.
induction of aflatoxin can occur in this food grade It was shown that while high temperature and nitrate support
Aspergillus. Additional factors involved in regulation of ST production in A. nidulans, the opposite was seen with
aflatoxin synthesis were evidenced by Flaherty and Payne A. parasiticus where these culture conditions repress
(1997). Adjacent to the aflR gene in the aflatoxin gene cluster, synthesis of aflatoxin (Feng and Leonard 1998). Studies by
a divergently transcribed gene, aflJ, was also found to be Chang et al. (1995b) demonstrated that nitrate has
involved in the regulation of transcription (Meyers et al. 1998; suppressive effect on aflatoxin production and overexpres-
Chang, personal communication). The exact mechanism by sion of aflR gene by additional copies of aflR could be
which aflJ modulates transcription of these pathway genes in required to overcome the negative regulatory effect on
concert with aflR is to be studied. A gene, aflT, encoding a aflatoxin pathway gene transcription in the nitrogen control
membrane bound protein with homology to antibiotic efflux circuit. Chang et al. (1996) cloned a nitrogen utilization gene
genes presumed to be for transporting aflatoxin out of the cluster consisting of two genes, niaD and niiA from
fungal cell, was discovered in A. parasiticus (Chang and Yu, A. parasiticus, A. oryzae, A. niger and A. nidulans and a
unpublished, GenBank accession number: AF268071). The nitrogen regulator, areA (Chang et al. 2000a) from
aflT gene might be involved in someway in aflatoxin A. parasiticus. In the intergenic region between aflR and
secretion. aflJ several AreA binding motifs have been identified
(Chang et al. 2000a). The AreA binding could prevent AflR
2.4.2 Nutritional Control (Carbon and Nitrogen binding. It seems that the aflatoxin formation and nitrogen
Sources) metabolism are closely linked.

The best-known nutritional factors affecting aflatoxin biosyn- 2.4.3 Environmental Influences
thesis are carbon and nitrogen sources (Adye and Mateles 1964;
Bennett et al. 1979; Luchese and Harrigan 1993). It is clear that Aflatoxin production are also affected by many environmental
simple sugars such as glucose, sucrose, fructose, and maltose factors (Bennett and Papa 1988; Demain 1972; Kale et al. 1994;
support aflatoxin formation, while peptone and lactose are not Kale et al. 1996; Yabe et al. 1988b) such as physiological pH,
(Buchanan and Stahl 1984; Payne and Brown 1998). Woloshuk temperature and water activity in the environment, volatile
et al. (1997) reported the connection between alpha amylase metabolites produced in host plant in response to fungal
activity and aflatoxin production in A. flavus. Yu et al. (2000c) invasion, and developmental stages of the fungi.
identified a group of four genes that constitute a well-defined Fungi have the ability to maintain internal pH and respond
gene cluster related to sugar utilization in A. parasiticus next to to the environmental changes. Studies by Cotty (1988)
the aflatoxin pathway gene cluster. The expression of the hxtA demonstrated the pH effect on aflatoxin formation and
gene, encoding a hexose transporter protein, was found to be sclerotia development. Acidic pH condition favors aflatoxin
concurrent with the aflatoxin pathway cluster genes in aflatoxin- biosynthesis in response to pH (Espeso et al. 1993; Keller et al.
conducive medium. This is the first evidence that primary 1997). It is believed that pacC is a major transcriptional
metabolism (sugar metabolism) and secondary metabolism regulatory factor (Keller et al. 1997). In the promotor region of
(aflatoxin biosynthesis) are genetically linked on the chromo- the regulatory gene, aflR, at least one pacC binding site has
some. A close physical linkage between the two gene clusters been identified (Ehrlich et al. 1999a). In the nonaflatoxin-
could point to a relationship between the two clusters in inducing peptone medium, it was shown to be inhibitory to
reference to the processing of carbohydrates leading to the aflatoxin formation (Cotty 1988). The regulatory mechanism
induction of aflatoxin biosynthesis. Lipid substrate was shown might be due to the binding of pacC to the PACC site in the
to be a good carbon source to support aflatoxin production aflR promoter region to repress the transcription of acid-
(Fanelli and Fabbri 1989; Fanelli et al. 1983; 1995). A lipase expressed gene aflR and the aflatoxin formation (Espeso et al.
gene, lipA, was cloned by Yu et al. (unpublished) in 1993) since such medium keeps the medium (pH 7.4) at
A. parasiticus and A. flavus. It was demonstrated that this alkaline condition (Cotty 1988).
350 Yu
Genetics and Biochemistry of Mycotoxin Synthesis 351

High water activity favors spore germination and mycelial Verticimonosporium are also found to produce the trichothe-
growth. However, severe aflatoxin outbreak in corn was cenes. The most potent trichothecenes are contact toxins and
documented under hot weather and drought conditions cause severe blistering and necrosis of the target tissue. Their
(Brown et al. 1998). The mechanism of A. flavus infestation cytotoxicity parallels their acute toxicity in animals with T-2
in corn under these conditions is not well understood. The toxin being more potent than nivalenol. Nivalenol is much
possible scenarios may include a combination of these more potent as an acute toxin than is DON. These
factors: (a) the plant defense mechanism is weakened under trichothecenes are of great concern for food and feed
water stress conditions; (b) higher insect feeding and contamination. DON is of considerable importance to
associated injuries to plant tissues, thus providing entry agricultural economies because animals refuse to eat
opportunities for fungal invasion; and (c) more fungal spores contaminated grain. F. sporotrichioides and F. graminearum
dispersed in the air under drier climate conditions. are the best studied among the trichothecene producing fungi
Plant metabolites also play some role on aflatoxin which are responsible for trichothecene contamination of
formation. Zeringue (1991; 1996; 1993) reported plant grains such as maize, wheat, barley, and rye (Brown et al.
metabolism and aflatoxin production. Wright et al. (2000) 2001; Chu 1997; Proctor 2000).
reported that at certain conditions, n-Decyl aldehyde reduces
not only fungal growth of A. parasiticus but also aflatoxin
production by over 95% compared with control. Octanal
reduces fungal growth by 60%, however, increases aflatoxin 3.2 Clustering of Trichothecene Biosynthetic
production by 500%, while hexanal reduces fungal growth Pathway Genes
by 50%, but shows no effect on aflatoxin production. A
relationship between fungal development (production of The trichothecene biosynthesis involves multiple enzymatic
reproductive spore and survival structure sclerotia) and reactions controlled by multiple genes. Biochemical and
aflatoxin synthesis was studied (Garber and Cotty 1997). genetic studies of trichothecene pathway, initially in
Wilson et al. (2001) reported that the 13(S)-hydroperoxide F. sporotrichioides using overlapping cosmid clones has
derivative of linoleic acid, the reaction product of resulted in establishing the consensus map of the trichothe-
lipoxygenase (encoded by L2 LOX gene from maize), cene pathway gene cluster on a 25 kb region of the
involved in reducing aflatoxin production. The fungal F. sporotrichioides chromosome (Brown et al. 2001; Hohn
morphology was significantly altered and toxin synthesis et al. 1993a; Proctor 2000; Trapp et al. 1998). This gene
was inhibited during strain degeneration studies. The effect cluster consists of ten genes with the designation, in
included the inhibition of expression of the toxin pathway sequential order, TRI8, TRI7, TRI3, TRI4, TRI6, TRI5,
genes, including aflR. TRI10, TRI9, TRI11, and TRI12, respectively (Figure 2,
Brown et al. 2001). In F. graminearum, a similar gene cluster
has been identified except that TRI7 in F. graminearum is
3 TRICHOTHECENES nonfunctional (Brown et al. 2001; Matsumoto et al. 1999;
Proctor et al. 1995a; 1997. The gene organization and
3.1 Occurrence and Toxicology direction of transcription are identical in both
F. sporotrichioides and F. graminearum. In both
The trichothecenes constitute a class of over 80 different F. sporotrichioides and F. graminearum, the eleventh gene
members of sesquiterpenoid compounds produced by a involved in trichothecene biosynthesis, TRI101, was isolated
number of genera of filamentous fungi. Based on the presence outside the established trichothecene pathway gene cluster by
of a macrocyclic ester or ester-ether bridge between C-4 and Kimura et al. (1998a), McCormick et al. (1999). Wuchiyama
C-15, trichothecenes are generally classified as macrocyclic et al. (2000) identified the TRI12 counterpart gene in
(Type C) or non-macrocyclic (Types A and B). The F. graminearum designated, TRI102, which encodes a
trichothecenes, including diacetoxyscirpenol (DAS), deoxy- trichothecene efflux pump. Lee et al. (2001) identified a
nivalenol (DON), and T-2 toxin, are mainly produced by similar trichothecene gene cluster in Gibberella zeae in the
Fusarium species (Bhatnagar et al. 2002; Brown et al. 2001; order of, TRI8, TRI7, TRI3, TRI4, TRI6, TRI5, TRI10, TRI9,
Chu 1997; Proctor 2000; Sharma and Kim 1991). The more TRI11, and TRI102, respectively. The TRI102 in Gibberella
structurally complex macrocyclic trichothecenes are pro- zeae shares significant homology with the TRI12 and TRI102
duced by the fungal genera Myrothecium, Stachybotrys, in F. sporotrichioides and F. graminearum. However, the
and Trichothecium. Trichoderma, Cephalosporium, and TRI102 in Gibberella zeae was found to be nonfunctional

Figure 2 Biochemical pathway and the specific genes and their enzymes involved in trichothecene biosynthesis in Fusarium species.
Note that arrows in the gene cluster indicate the direction of gene transcription. (Figure courtesy Daren W. Brown, with modifications
from Brown et al. 2001).
352 Yu

(Lee et al. 2001). Note that TRI1 and TRI2 were named based By gene disruption experiment, the TRI3 gene was found to
on UV-generated nontrichothecene-producing mutants. So far encode an acetyltransferase catalyzing the conversion of
no corresponding gene or DNA sequences on molecular level 15-decalonectrin to calonectrin via acetylation at the C-15
have been identified yet and the location of these mutated loci position (McCormick et al. 1996). Gene disruption and
are still unclear (Robert H. Proctor, personal communication). precursor feeding studies in F. sporotrichioides by Brown
The functions of most of the identified genes have been et al. (2001) demonstrated that TRI7 might function as C-4
proposed or characterized except TRI9 which encodes the acetylase or acetylesterase involved in the conversion from
smallest peptide consisting of only 43 amino acids with its 3,15-DAS to 3,4,15-triacetoxyscirpenol. However, no func-
function unknown (Figure 2, Brown et al. 2001). tional enzyme encoded by TRI7 was predicted in
F. graminearum due to the lack of initiation codon, six
possible frameshifts, and early stop codon (Brown et al.
3.3 Biosynthetic Pathways of Trichothecenes 2001). Brown et al. (2001) demonstrated that disruption of the
TRI8 gene failed to produce T-2 toxin instead accumulated
From the first trichothecene skeleton, trichodiene, to the final 4,15-DAS. The chemical reaction was presumably involved
product, T-2 toxin, through a series chemical modifications of in the 3-deacetylation of 3,4,15-DAS. Since the predicted
oxidation, isomerization, cyclization, and esterification amino acid sequence is homologous to lipase, the TRI8 might
reactions, at least 16 know stable intermediates, which encode an esterase/oxygenase and be responsible for
constitute the various members of the trichothecenes esterification of the C-8 oxygen for the conversion some-
including the branch products DON and nivalenol, have where between 3,4,15-triacetoxyscirpenol and 3-acetyl T-2
been identified. The biochemical conversion scheme of these toxin. TRI12 encode a toxin efflux pump (Alexander et al.
compounds are trichodiene, 2-hydroxytrichodiene, 12,13 1999). TRI6 is a regulatory gene (Desjardins et al. 1993; Hohn
epoxy-9,10-trichoene-2-ol, isotrichodiol, trichotriol, isotri- et al. 1999; Keller and Hohn 1997; Matsumoto et al. 1999;
chodermol, isotrichodermin, 15-decalonectrin, calonectrin, Proctor et al. 1995b; Proctor 2000; Trapp et al. 1998). TRI10
3,15-DAS, 3,4,15-triacetoxyscirpenol, 3-acetoxyscirpenol, encodes a polypeptide of 420 amino acids for another
3-acetyl T-2 toxin, T-2 toxin. The DON is thought to have regulatory protein (Tag et al. 2001). The regulatory function
branched from 15-decalonectrin and the nivalenol is thought of the TRI6 and TRI10 genes will be discussed in more detail
to have branched from 3,15-DAS (Brown et al. 2001; Proctor in the next section. The enzymes responsible for some of the
2000). DON could be converted directly to nivalenol biosynthetic steps, such as the last step for the synthesis of T-2
(Figure 2). toxin from 3-acetyl T-2 toxin by a deacetylase, have been
The initial step of the trichothecene biosynthesis involves proposed. However, the genes for those enzymes have not yet
the incorporation of three molecules of mevalonate into the been identified (Brown et al. 2001). Most recently, two
trichothecene nucleus of trichothecin with the cyclization of additional new genes, TRI13 (from Gibberrella zeae) and
farnesyl pyrophosphate to produce trichodiene. TRI5 is the TRI14 (from F. sporotrichioides), involved in trichothecene
first characterized pathway gene that encodes a trichodiene biosynthesis, have been isolated and deposited in the GenBank
synthase involved in this initial step of trichothecene database (Daren W. Brown et al., personal communication).
biosynthesis (Hohn and Beremand 1989). Hohn and Synthesis of the structurally more complex macrocyclic
Desjardins (1992) isolated a gene, named Tox5, encoding a trichothecenes such as roridin E, verrucrin, and baccharinoid B7
trichodiene synthases for the synthesis of trichodiene from is most commonly by the genus Myrothecium. The macrocyclic
farnesyl pyrophosphate. The Tox5 gene (renamed TRI5) was trichothecenes exhibit about 10-fold more toxic than the
characterized in great detail by Desjardins et al. (1987), Fusarium trichothecenes. Trapp et al. (1998) identified, in
Desjardins et al. (1993), Cane et al. (1995), and Hohn et al. M. roridum, the homologues of the F. sporotrichioides TRI5
(1993b). The predicted amino acid sequences of the TRI4 and (MrTRI5), TRI4 (MrTRI4), and TRI6 (MrTRI6) genes which
TRI11 gene products were similar to those of cytochrome were located within a region of about 40 kb in M. roridum
P-450 monooxygenases (Alexander et al. 1998; Hohn et al. compared to only 8-kb in the F. sporotrichioides. The deduced
1995). TRI4 was found to catalyze the oxygenation of amino acid sequence of the MrTRI5 product demonstrated a
trichodiene at the C-2 position to yield 2-hydroxytrichocdiene homology of 72% to 75% to trichodiene synthases from four
(Hohn et al. 1995). The most significant oxidation step in Fusarium species. The predicted MrTRI6 gene product was
trichothecene biosynthesis is provided by this cytochrome twice as large as that of the FsTRI6 with low amino acid
P-450 monooxygenase encoded by TRI4 to introduce the homologies except in the C-terminal region of MrTRI6 (78%)
C-12, 13 epoxide. Alexander et al. (1998) demonstrated that where the zinc-finger motifs reside.
the TRI11 is involved in the conversion of isotrichodermin to
15-decalonectrin by oxygenating the C-15 position of
isotrichodermin. Studies by McCormick et al. (1999) 3.4 Regulation of Trichothecene Biosynthesis
indicated that the acetylation of isotrichodermin is catalyzed
by TRI101. This gene may also play a role in self-protection The TRI6 gene is located in the middle of the trichothecene
against the toxic effect of trichothecene to the fungus by gene cluster in both F. sporotrichioides and F. graminearum
modulating trichothecene production (Muhitch et al. 2000). (Proctor et al. 1995b). The TRI6 gene encodes a pathway-
Genetics and Biochemistry of Mycotoxin Synthesis 353

specific regulatory protein required for the expression of self-protection against trichothecenes (Kimura et al. 1998b;
trichothecene biosynthetic pathway genes (Desjardins et al. McCormick et al. 1999).
1993; Hohn et al. 1999; Keller and Hohn 1997; Matsumoto
et al. 1999; Proctor et al. 1995b; 2000; Trapp et al. 1998).
The predicted polypeptides encoded by TRI6 in both 4 FUMONISINS
F. sporotrichioides and F. graminearum consist of 217 and
218 amino acids, respectively. In the C-terminus of this 4.1 Occurrence and Toxicology
protein, the Cys2His2 type zinc-finger motifs, which define
the function of this particular enzyme as a gene expression Investigation of the association of human esophageal cancer
regulator, were identified. TRI6 was shown to bind to the and the consumption of Fusarium verticillioides (formerly
promoter regions of nine pathway genes at the minimum called F. moniliforme) infested maize by South African
consensus site 50 YNAGGCC-30 (Hohn et al. 1999). TRI6 as Researchers suggested that fumonisins were a new group of
a regulatory gene for binding of the protein to promoter mycotoxins (Bezuidenhout et al. 1988; Gelderblom et al.
region was supported by the study using site-directed 1988). Even though the fumonisins were originally identified
mutagenesis in the predicted zinc-finger motif of the C- from F. verticillioides, these toxins have been reported in
terminus of TRI6. Studies by Tag et al. (2001) showed that cultures of F. anthophilum, F. dlamini, F. napiforme,
TRI10 is another novel regulatory gene that regulates F. oxysporum, and F. proliferatum (Desjardins et al. 2000;
trichothecene gene expression and trichothecene synthesis. Munkvold and Desjardins 1997; Musser and Plattner
TRI10 gene encodes a protein consisting of 420 amino 1997; Seo et al. 1996). No other genera have been reported to
acids, which does not match significantly to any proteins of produce fumonisins other than fungi. Production of
known or predicted function or to motifs except a single fumonisins by Alternaria has also been reported (Mirocha
transmembrane domain (Tag et al. 2001). Disruption of et al. 1992; Mirocha et al. 1996. Some fumonisin-producing
TRI10 in F. sporotrichioides significantly decreased the Fusaria have been known to produce AAL toxins, a
transcript accumulation of the five tested genes, TRI4, TRI5, compound with its structure closely related to fumonisins
TRI6, TRI101, and the farnesyl pyrophosphate synthethase (Mirocha et al. 1992). The Alternaria alternata, a tomato
(Fpps) gene, and abolished T-2 toxin production. Com- pathogen, was found to produce AAL toxin (Wang et al.
plementation of TRI10 into the disrupted mutant elevated 1996). The fumonisins produced by F. verticillioides and
the transcript accumulation of the above five genes and F. proliferatum pose the greatest health hazard to human and
significantly increased T-2 toxin production (Tag et al. animals since these species readily colonize corn all over the
2001). Interestingly, disruption of TRI6 decreased transcrip- world (Bhatnagar et al. 2002; Dutton 1996; Proctor 2000;
tion of other trichothecene pathway genes but increased Riley et al. 1996; Scott 1993).
TRI10 transcription. TRI10 is required for the full Nine structurally related fumonisins, including FB1, FB2,
expression of trichothecene pathway genes including the FB3, FB4, FA1, and FA2, have been identified. Fumonisin B1
regulatory gene TRI6 and the expression of primary (FB1) is the most abundant in maize. Chemically, FB1 is a
metabolic pathway genes (Fpps) that precedes the derivative (diester) of propane-1,2,3-tricarboxylic acid of
trichothecene biosynthetic pathway. TRI6 is not required 2-amino-12,16-dimethyl-3,5,10,14,15-pentahydroxyicosane
for TRI10 expression but inhibits TRI10 expression. TRI10 (Gelderblom et al. 1988; 1992a,b; Marasas 1995; Nelson et al.
may also be involved in self protection of trichothecene 1993; Riley et al. 1996; Scott 1993; Shier 1992). Other B series
toxin (Tag et al. 2001). TRI12 encodes a trichothecene fumonisins such as fumonisin B2 (FB2), B3 (FB3), and (FB4),
efflux pump with sequence similarities to known members occur at low levels. Related structurally to B series fumonisins,
of the major toxin transporters. Characterization of the the four other series of fumonisins (A, AK, C, and P) also occur
TRI12 gene by Alexander et al. (1999) showed that at low levels (Proctor 2000). The other fumonisins are resulted
disruption of TRI12 resulted in reduced growth on complex from removal of tricarballylic acid and other ester groups
media and reduced levels of trichothecene biosynthesis. (Musser et al. 1996; Seo et al. 1996).
Expression of TRI12 and TRI3 in yeast mutant (defective in Fumonisins have been shown to have diverse biological
the PDR5 transporter which is believed to be for toxin and toxicological effects. The mechanism of fumonisin
resistance in yeast) indicated that the role of TRI12 in toxicity is not well understood. Studies have shown that
F. sporotrichioides functions as self-protection against fumonisins inhibit ceramide synthase. Fumonisins have been
trichothecenes compared with the expression of TRI3 alone shown to be hepatotoxic and carcinogenic in rats resulting in
in this yeast mutant. An additional gene, TRI101 from liver cirrhosis and hepatic nodules, adenofibrosis, hepato-
F. graminearum, which is located outside the established cellular carcinoma, ductular carcinoma, and cholangio-
trichothecene gene cluster, appears to play a role in self- carcinoma (Gelderblom et al. 1988; 1991; 1992b; 1993;
protection against trichothecenes (Kimura et al. 1998b; 1994; Marasas 1996). Studies on rats by Riley et al. (1996)
McCormick et al. 1999). Disruption of TRI101 in suggested that fumonisin B1 (FB1) might act primarily as a
F. sporotrichioides (FsTRI101) leads to reduced-growth tumor promoter. The tumor promoting activity of fumonisins
on trichothecene-containing media compared to the wild- has also been proposed by Huang et al. (1995), Wattenberg
type strain suggesting a similar role for FsTRI101 in et al. (1996) to have been resulted from the stimulation or
354 Yu

suppression of signal transduction enzymes, the mitogen- (Proctor et al. 1997). Xu and Leslie (1996) mapped fum1 onto
activated protein kinase and protein kinase C. The the chromosome 1 in F. moniliforme and the other fum loci
mechanism of toxicological effect is a subject of intense were also mapped onto chromosome 1. Based on the
study. recombination frequencies, a relative distance and linear
relationship of the fum loci and some other gene loci have
been established (Proctor 2000) to be in sequential order,
4.2 Genetics and Biosynthesis of Fumonisins ald1, fum1, fum3 (uv26), fum2, and OPA16.

Genetic and biochemical studies on fumonisin biosynthesis

are fairly recent. No comprehensive biosynthetic pathway and 5 CONCLUSION
genetic and enzymatic establishments like aflatoxins or
trichothecenes are available currently. Fumonisin biosyn- Within the last decade, significant advances have been made in
thesis has been of significant research interest in the last few mycotoxin research with respect to their identification,
years (Desjardins and Proctor 1999; Proctor et al. 1999a; biochemistry, and genetics of their biosynthesis and regulation
Proctor 2000). A linear 19- or 20-carbon chain is the of toxin formation as well as mycotoxicosis and prevention and
fumonisin backbone that is substituted at various positions control. This is especially true for those mycotoxins such as
with hydroxyl, methyl, and tricarballylic acid moieties and an aflatoxins, ST, fumonisins, and trichothecenes that are
amino group at C-2 (Proctor 2000). Carbon labeling studies economically important in agriculture and pose greatest health
suggested that a PKS, alanine, methionine and other amino hazard for human beings and livestock. The biosynthetic
acids are involved in fumonisin synthesis (Desjardins et al. pathways, the clustering of these genes on the chromosome, the
1996a). Classical genetic analyses do provide some insight function of these genes and enzymes involved in the formation
into the fumonisin biosynthesis and genetic control. of these toxins have been elucidated in great details. Scientists
Desjardins et al. (1996b) established that the genes have also acquired significant knowledge on the gene
responsible for fumonisin biosynthesis are closely linked on expression and regulation of toxin synthesis within the
chromosome 1 when they studied on the FB1-non-producing corresponding gene cluster. A better understanding of the
mutant of G. fujikuroi MP-A. By complementation analysis in nutritional and environmental factors that affect the production
FB1-non-producing mutant of G. fujikuroi MP-A using of these mycotoxins has been examined in greater details.
fum5-containing cosmid clone, Proctor et al. (1999b) There remains, however, a vast gap in our understanding
demonstrated that the fum5 is involved in fumonisin of the coordinated global regulation on toxin formation, the
biosynthesis. The predicted amino acid sequence of fum5 potential existence of signal transduction pathways under-
showed high degree of similarity to fungal type I PKSs. lying primary and secondary metabolisms, the effects of
Disruption and complementation analysis of the cloned environmental factors, biotic and abiotic, on the toxin
G. fujikuroi gene, fum5, encoding a PKS, Proctor et al. formation, the mode and regulation of plant-microbe (crop
(1999b) concluded that fumonisins are synthesized from and fungi) interaction during infection. With the development
polyketide rather than fatty acids. In order to identify and application of Expressed Sequence-Tag (EST) and
additional genes that are potentially involved in fumonisin microarray technologies, we will be able to study the whole
production near fum5, the DNA regions approximately 7-kb organism on the molecular genetic level to address those
downstream and 15-kb upstream of this fum5 gene have been unanswered questions. Only when we have a thorough and
sequenced (Proctor 2000). A total of 8 ORFs, 5 ORFs in the comprehensive understanding of the regulatory mechanisms
upstream and 3 ORFs in the downstream have been identified. of mycotoxin formation, will we be able to develop effective
Based on sequence homologies to known genes and proteins, strategies to control mycotoxin contamination of food and
four of the ORFs appear to encode proteins that would be feed on a consistent basis, resulting in a sustainable,
expected to be involved in fumonisin production. One of these nutritious, and healthy food supply for the entire increasing
ORFs encodes a putative cytochrome P-450 monooxygenase. world population.
Even though none of the fumonisin biosynthetic genes was
isolated except fum5 for the PKS, classic genetic analysis
using Gibberella fujikuroi (sexual stage of F. moniliforme)
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31
Cellulose Degradation by Fungi

Justine N. Niamke / Nam Sun Wang University of Maryland, College Park, Maryland, USA

1 INTRODUCTION 2 BIOMASS AS A SUGAR SOURCE

Cellulose and hemicellulose comprise about 50 and 30%, Cellulose is a long linear polymer ranging from 1000 to
respectively, of the biomass the nature produces through 1000 000 D -glucose units. Glucose monomers are linked
photosynthesis and are by far the most abundant carbon together with b-1,4-glycosidic bonds to form highly stable
polymers on earth. Lignin and minor amount of ash make up chains, and these chains further aggregates together via
the remainder of the biomass. In addition to that which hydrogen bonds to form a rigid crystalline structure that is
already exists in natural settings (especially in trees), water-impermeable, water-insoluble, and resistant to enzy-
cellulose/hemicellulose is a large component of wastes matic hydrolysis (Linko 1987). The boundary and sequence
originating from municipal, agricultural, forestry, and certain structure of the molecules determine the chemical properties
industrial sources. Its abundance alone supports the conten- of cellulose (Alen and Sjostrom 1985). On the other hand,
tion that it is a valuable raw material for food production, hemicellulose, which is alkali-soluble, is composed of short,
energy generation, and chemical feedstock. Although only a highly branched copolymer of both six-carbon and five-
small portion of what the nature produces is presently carbon sugars. The branched structure allows hemicellulose
converted into useful products, this picture will likely change to exist in an amorphous form that is more susceptible to
in the future, as the world population must ultimately depend hydrolysis. Compared to cellulose, which is similar across all
on limited renewal resources for food, energy, and material biomass sources, hemicellulose is quite diverse in structure
goods (Millet et al. 1976). Except for directly burning wood and composition, depending on the source. The hydrolysis
and wastes for energy generation, most cellulose-derived product of hemicellulose is typically a mixture of xylose,
products first require degrading of cellulose to constituent arabinose, galactose, mannose, glucuronic acid, and other
sugars before subsequent conversion, usually through sugars. The ratios of five-carbon and six-carbon sugars
microbial fermentation, of the sugars into a wide range of depend on the source species of the biomass. Compared to
useful products, including ethanol, acetic acid, lactic acid, hardwoods and agricultural residues, softwoods generally
and antibiotics. Besides generating valuable chemical contain more six-carbon sugars (D -glucose, D -galactose, and
products, degradation of cellulose also reduces the waste D -mannose) but less five-carbon sugars (D -xylose and
disposal problems associated with landfills and burning L -arabinose) (Alen and Sjostrom 1985). With cellulose, a
forests (Katzen and Monceaux 1995). After several decades chief engineering challenge is degradation; whereas, with
of research worldwide, there are two main routes to hemicellulose, a major limitation lies in microbial fermenta-
cellulose degradation: chemical (alkaline or acid) and tion of some sugars. Although there exists significant
biological (enzymatic or microbial). Of the latter route, variations in the composition among species, biomass roughly
fungi and anaerobic rumen bacteria are excellent candi- consists of 50% cellulose, 30% hemicellulose, and 20%
dates. In this chapter, we specifically focus on cellulose lignin.
degradation by fungi and compare the economics of Cellulose material obtained from waste sources is
alternative processes. typically inexpensive but difficult to degrade. Certain

363
364 Niamke and Wang

processes require that lignin and other components will first b-glucosidases, which release constituent glucose monomers
be removed from cellulose before the actual processing of the from cellobiose and soluble cellodextrins. Many less suitable
material can proceed. In order to remove this nonbio- fungi also produce cellulases, but not necessarily in an
degradable component and other impurities from cellulose optimal ensemble. Although T. reesei is efficient in degrading
feedstock, we must pretreat the feedstock with mechanical, cellulose, it alone cannot convert cellulose directly to a useful
thermal, or enzymatic means. Of the many possible products final product, unless glucose happens to be the target product.
derived from cellulose, ethanol manufactured via microbial In fact, only a few filamentous fungi are known to convert
fermentation has long captured the attention of many cellulose efficiently into useful final products. The following
researchers in the following areas of applications: (a) potable are among the few filamentous fungi that are capable of
ethanol for beer, wine, and distilled beverages, (b) solvent hydrolyzing cellulose as well as converting the sugars
ethanol for laboratory and pharmaceutical applications, and liberated from hydrolysis to ethanol or acetic acid: Monilia,
(c) as a fuel additive/blend to reduce harmful emissions or as a Neurospora crassa, and Fusarium oxysporum; the following
complete substitute for gasoline. Lactic acid, which is a fungal species are capable of converting xylose to ethanol:
specialty chemical utilized mainly in the food industry as an Fusarium, Mucor, and Paecilomyces (Singh et al. 1992). The
acidulent and preservative, finds use in the chemical disadvantage of direct conversion is the slow conversion rate
processing industry as a metal deliming agent (Iyer and Lee compared to yeast, although conversion efficiency can be as
1999; Schmidt and Padukone 1997). high as yeast (Krishna et al. 2001). Thus, economic
A number of processes have been developed to degrade considerations generally preclude converting cellulose to
cellulose and to produce ethanol, although successful ethanol with these fungi. Technical feasibility does not
commercial units to date remain few in number. The most translate to economic feasibility. Table 1 summarizes the
well known is simultaneous saccharification and fermentation different types of fungi responsible for converting cellulose to
(SSF), where the bioreactor brings together cellulose, either ethanol or acetic acid.
glucose, cellobiose, cellulase enzymes, yeast (but not
fungi), and nutrients to produce ethanol (Philippidis 1992).
This process involves a number of different steps all carried
out in one vessel. To avoid the setback associated with SSF, 2.2 Cultivation Conditions and Nutrient
another process known as mixed culture and fermentation was Requirements
developed, where a consortium of fungi and bacteria, or fungi 2.2.1 Carbon Source
and yeast, rather than simply yeast alone as was in SSF,
convert cellulose into ethanol in one combined step (Wilke Since, substrate represents a major cost item (estimated to be
et al. 1983). We will review cellulose pretreatment and focus anything from 30 to 70%), waste will not be tolerated if the
on fungal conversion of cellulose into ethanol, and finally we process is to have a chance to be economically viable. Since
will touch upon the economic aspects of ethanol production. D -xylose is a major component of cellulosic biomass and
since few organisms can convert it at all—and those who do
usually do so inefficiently, the cellulolytic strains that can
2.1 Fungal Strains convert D -xylose to ethanol represent a great opportunity
(Schneider 1989), and they should be scrutinized further in
Since the early days of civilization, humans have been future studies. Especially notable are the cellulolytic strains of
practicing converting hexose to ethanol with yeast, specifi- Monilia, which are capable of producing ethanol from both
cally Saccharomyces species for alcoholic beverages. Thus, it D -glucose and D -xylose. In a study, this fungus converted
is only natural that this process technology is proven and quite more than 40% of D -xylose into ethanol in 7 days (Kumar
advanced. More recently, many studies have focused on
utilizing bacteria, especially Zymomonas mobilis, to produce
industrial ethanol from hexose where taste is not at all a Table 1 Fungi responsible for cellulose degradation
factor. In comparison, converting pentose and other sugars to
ethanol is a much more recent development and still faces Organism Features
many unresolved challenges. F. oxysporum F3 Direct conversion of cellulose into
The most intensively studied fungus capable of cellulose ethanol
degradation is Trichoderma reesei, which was originally F. oxysporum DSM 841 Efficient direct conversion of waste
named T. viride but later renamed in honor of a pioneer in cellulose to acetic acid
cellulase research, E. T. Reese. This fungus prodigiously Monilia sp. Direct conversion of cellulose to
produces an excellent ensemble of three major classes of ethanol
cellulase enzymes that function synergistically to convert N. crassa Direct conversion of
cellulose all the way to glucose: (a) endoglucanases, which cellulose/hemicellulose into
attack at random locations in the cellulose chain, (b) ethanol
exoglucanases or glucanhydrolases, which liberate glucose
dimer cellobiose from the end of the cellulose chain, and (c) Source: Singh et al. (1992).
Cellulose Degradation by Fungi 365

et al. 1991). Like Monilia, the fungus F. oxysporum also economic feasibility viewpoint, supplementing nitrogen from
successfully converts a variety of carbon sources: glucose, complex sources is undesirable because the practice adds
xylose, cellobiose, with a yield of 5.0 –9.1 g/l of ethanol. significantly to the cost. A future challenge is to eliminate
N. crassa is yet another species that biologically transforms completely the need for nitrogen addition and to satisfy
cellulose all the way to ethanol (Singh et al. 1992). It should fungi’s nitrogen demand entirely from natural lignocellulosic
be noted that many fungal cellulose degradation studies and sources. At least, one would consider cheaper sources of
fungal cellulase production studies employ well-defined nitrogen.
model substrates such as carboxymethyl cellulose (CMC),
Avicel (which is more crystalline), or xylan. The synthetic 2.2.3 Minerals and Vitamins
media are further supplemented with minerals and yeast
extract. Although defined media tend to yield more Minerals, trace elements, vitamins, and growth factors play a
reproducible laboratory results, they do not realistically vital role in the growth of fungi and their biosynthesis of
approximate commercial processes. On a related point, these metabolites. There has been no systematic study on the effect
model compounds also serve as substrates for standardized of minerals and trace elements such as Ca, Mg, Fe, Zn, Cu,
cellulase activity measurements. For example, a common Co, and Mn on growth of fungi and ethanol production by
gauge of endoglucanase activity is decreased CMC viscosity. fungi. The present strategy is to provide sufficient quantity of
Note that a decrease in the CMC viscosity does not linearly these compounds, estimated based on information gathered
correspond to an increase in the reducing sugar level. from other microbial fermentation, such that they do not
become rate limiting. However, it is unclear what constitutes
2.2.2 Nitrogen Source an optimal level. The large number of compounds involved
immediately translates to unpractical number of runs. For
Nitrogen is a major cellular constituent of any organism, and example, if one were to conduct brute-force experiments by
fungi are certainly no exception. Any successful media varying one variable at a time, say, each variable set at three
formulation must carefully balance microorganism’s inherent separate levels (high, medium, and low), n variables yields 3n
need for nitrogen. Nitrogen limitation usually leads to slow or combinations if the variables interact with one another, or 3n
no growth or even death of fungi, slow xylose consumption, combinations if the variables do not interact. To appreciate
and retarded ethanol production. Table 2 summarizes the the magnitude of this undertaking, 10 trace compounds
different sources of nitrogen and the typical concentration translates to 310 ¼ , 300,000 possible combinations! And
levels present in fungal growth media in a laboratory setting. that does not count the unsuccessful runs. Although a sound
In a defined media formulation, typical nitrogen sources are experimental design can reduce the number of runs, the
ammonia, nitrates, and urea. In complex media, peptone, number of runs required remains utterly unpractical if the
tryptone, yeast extract, wheat bran, and an array of protein variables interact.
digests supply other crucial but less understood set of
nutrients. Metabolic pathways trace the fate of carbon rather 2.2.4 Aeration
than nitrogen sources. Nonetheless, nitrogen sources’ effect
on fungal metabolism can be pronounced. For example, Sale Under aerobic conditions, the fungi listed in Table 1 grow
(1967) reports that ammonium ions stimulate glycolysis by well but are unable to produce ethanol. On the other hand,
counteracting ATP’s inhibition of phosphofructokinase, and under anaerobic or microaerobic conditions, they are able to
they stimulate pentose phosphate pathway by derepressing produce ethanol but grow only slowly. Thus, if one is to
glucose-6-phosphate dehydrogenase. However, the effects of pursue cellulose-to-ethanol with a single fungal species, a
nitrogen sources are generally not as well understood. From common strategy is to cultivate fungi first under aerobic
conditions, followed by ethanol production under anaerobic
conditions. For example, in a study utilizing N. crassa, the
Table 2 Nitrogen sources in lignocellulosic conversion mold grew first aerobically for 48 h. Subsequently, the content
processes by fungi of the growth flask was transferred to a special flask that had a
capillary opening at the top to exclude oxygen from entering
Nitrogenous compounds Concentration (g/l) while allowing carbon dioxide to escape as ethanol is
Ammonium nitrate 1.0 produced (Singh et al. 1992).
Ammonium dihydrogen phosphate 2.0 Compared to higher organisms, fungi are more susceptible
Potassium nitrate 2.5 to changes in their surrounding environments. However, the
Sodium nitrate 2.0 cytoplasmic pH of the fungi tends to change very little over a
Urea 2.0 wide range of extracellular pH. In most prior studies, which
Peptone 5.0 have been conducted without active pH control, the initial pH
Yeast extract 0.25 values are within the range of 5 –6. Both the rate of cellulose
Potato protein liquor 40 degradation and product distribution depend on the pH. For
example, Enari and Shihko (1984) report an optimal pH of 5.5
Source: Singh et al. (1992). for ethanol production for F. oxysporum, but acetic acid
366 Niamke and Wang

production increased considerably at pH 6.0. For N. crassa, building material). In order for organisms to digest biomass
maximum ethanol production occurred in the range of efficiently and productively, it must be pretreated by
5.0–6.0, which coincided with maximum cellulase activity mechanical, chemical, or biological means to break down
(Deshpande et al. 1986). the stable polymeric structure of cellulose, hemicellulose, and
In nature, fungi may be exposed to different environmental lignin. For this purpose, there are presently four major
conditions that can inhibit their growth. In general, the upstream processing technology platforms, and many
intrinsic enzyme (including cellulase) reaction rate and its variations exist around these basic technologies: (a)
deactivation rate both increase with temperature. As concentrated acid hydrolysis carried out at low temperature,
temperature increases, the rate of enzyme deactivation (b) dilute acid hydrolysis carried out at high temperature, (c)
surpasses that of enzyme reaction temperature; thus, the enzymatic (cellulase) hydrolysis, and (d) biomass gasification
apparent enzyme reaction rate exhibits a maximum with (pyrolysis). The two acid technologies are old developments;
respect to temperature. Deshpande et al. (1986) show that whereas, the enzymatic process is relatively newer, and the
N. crassa works best at 378C. It converts more than 90% of gasification process is much more recent. Enzymatic
cellulose into ethanol within 4 days at 378C. However, above hydrolysis alone is normally insufficient in degrading
this optimum, the cellulase activity increases but the cellulose; thus, mechanical and chemical pretreatment
production of end product, such as ethanol, decreases. becomes necessary. Finally, the fermentation step normally
Table 3 shows optimum pH and temperature needed for the is further subdivided into two steps: (a) conversion of
conversion of cellulose by different fungi. cellulose to glucose and (b) conversion of glucose to ethanol
Light plays an important role in the growth of filamentous (Blazej and Kosik 1985). A separate organism is usually
fungi. It can hinder or arouse the emergence of mycelia, and responsible for each one of these two steps. Only a few
sensitivity to light is species dependent. In Trichoderma, the microorganisms in nature can perform tasks in both steps.
most efficient wavelengths to induce growth are 310, 430, Physical pretreatments can be either mechanical or
455, and 480 nm. nonmechanical (Fan et al. 1987). In the mechanical process,
the force applied in pretreatment breaks lignocellulosic
materials into small highly digestible particles for enzymatic
3 DEGRADATION OF COMPLEX hydrolysis, where soluble enzyme works on insoluble
MOLECULES cellulose mainly at the liquid – solid interface and the
observed reaction rate is not necessarily limited by the
3.1 Cellulose Degradation intrinsic reaction rate but by mass transfer. Thus, breaking
down particles increases the surface area for reaction. In
In a simplistic summary, there are four major steps in addition, the nonmechanical pretreatment with high tempera-
generating bioethanol and other biomass-derived products: ture steam causes disruption in the fundamental molecular
(a) generate biomass through photosynthesis, (b) process raw and crystalline structure of the cellulose.
biomass to a form suitable for microbial fermentation, (c) Ball Milling: Ball milling is one of the best methods to
ferment biomass into ethanol (or other products), and (d) enhance enzymatic hydrolysis. In this process, shear and
recover product and recycle unfermented residual biomass. mechanical forces break down the particle size in order to
We will not dwell here on the first step that takes place in facilitate the digestion of cellulose. Ball milling, although
nature. We present the remaining steps to place in proper highly efficient, cannot be operated on a large scale because
perspective fungi based cellulose degradation. Pretreatment of the high cost and the lengthy time it takes to break down the
of the cellulosic biomass is necessary in the yeast and bacteria molecular structure of cellulose. Like ball milling, two-roll
based cellulose conversion, because these microorganisms do milling also decreases the particle size.
not easily hydrolyze woods and agricultural residues. If they To facilitate digestion, chemical agents such as alkali or
did, biomass material would not remain for long nor acid solutions have long been utilized to pretreat cellulose.
accumulate (and wood would be totally unsuitable as a Acid treatment increases the reduction power of the reactive
groups along the cellulose polymer chain, and cellulose is
thus destabilized and rendered more susceptible to subsequent
Table 3 Optimum pH and temperature range attacks (Fan et al. 1987). Concentrated acid disrupts the
hydrogen bonds between cellulose chains, thus, it breaks the
Organism Optimum pH Optimum temperature (8C) otherwise highly stable crystalline structure of cellulose and
F. oxysporum 5.5 30 dissolves cellulose into a thick, hydrogel-like solution.
F. oxysporum 5.0 30 Cellulose is now in an amorphous state and is readily
F. oxysporum 6 34 digested. Addition of water dilutes the acid and rapidly breaks
Monilia sp. — 26 the b-1,4-glucosidic linkage, leading to complete hydrolysis
Mucor sp. 5.4 30 of cellulose to glucose (known as saccharification) with an
N. crassa 5.0– 6.0 28 – 37 yield that is close to the theoretical value. At the end of
saccharification, glucose molecules remain intact. Concen-
Source: Singh et al. (1992). trated sulfuric acid is the acid of choice, although other acids
Cellulose Degradation by Fungi 367

(hydrochloric, nitric, and phosphoric) are also employed in pretreatment step that is followed by an enzyme step. Table 4
some laboratory studies. In a typical process, five parts of shows the sugar yields from different alkali pretreated
, 75% sulfuric acid is added to four parts of dried biomass biomass sources after enzymatic digestion.
containing ,10% moisture at 508C. This is followed by Biological degradation of cellulose features prominently
diluting with water to , 25% acid and holding at 1008C for in the nature’s carbon cycle. Cellulase refers generally to the
1 h. The following schematic shows the fate of cellulose in enzymes that degrade cellulose. Most known cellulases are of
concentrated acid. fungal origin. Instead of simply one enzyme, cellulase from
most fungi is actually a collection of several distinct enzymes
cellulose ! acid complex ! oligosaccharides ! glucose that work synergistically to accomplish the overall task of
cellulose degradation. We can classify cellulase-secreting
High temperature dilute acid process works in two stages, fungi into different groups: brown rots, white rots, and red
with the first stage optimized for the more readily hydrolyzed rots. Unlike white and red rots that degrade cellulose and
hemicellulose and the second stage’s conditions specifically lignin, brown rots degrade mainly cellulose (Kirk 1976).
tuned for the tougher cellulose. Typical conditions are 0.7% Several microorganisms are known to produce cellulase
sulfuric acid at 1908C for the first stage and 0.4% at 2158C for that hydrolyzes cellulose and hemicellulose. The best known
the second stage. The residence time for each stage is is the filamentous fungi T. reesei. This microorganism
approximately 3 min. A dilute acid process exposes cellulose generates at least three well-known enzymes known as: (a)
to acid for a shorter time compared to a concentrated acid b-1,4-D -glucan glucanohydrolase (an endo-enzyme),
process, but only about 50% of the glucose is recovered in a (b) b-1-4-D -glucan cellobiohydrolase (an exo-enzyme), and
dilute acid process compared to nearly 100% recovery for a (c) b-glucosidase (Jeffries 1987). The most common
concentrated acid process. In hot dilute acids, hydrolysis of cellulase, b-1,4-D -glucan glucanohydrolase or Cx, has several
cellulose proceeds as follows. constituents. One of these constituents acts as the initiator of
native cellulose ! stable cellulose ðhydrocelluloseÞ cellulose hydrolysis. The second enzyme, b-1,4-D glucan
cellobiohydrolase, has two constituents: b-1,4-glucan gluco-
! soluble polysaccharides ! glucose hydrolase excises one glucose molecule from the nonreducing
end of the cellulose chain, and b-1,4-glucan cellulobio-
Under certain conditions, alkaline solutions can degrade hydrolase removes two glucose units at a time from the end of
cellulose by breaking down its long molecular chain at the cellulose chain. It is further divided into two
random interchain locations and generate a number of shorter subcomponents CBHI and CBHII. The third class of enzyme,
molecules of cellulose. Many alkalis such as metal b-glucosidase, does not act directly on cellulose. It is
hydroxides, salts in a strong alkali solution, inorganic salts, nonetheless important because it hydrolyzes the glucose
a number of amines, and related compounds break the dimer cellobiose, which yeast cannot ferment, to liberate two
hydrogen bonds between cellulose molecules. Alkalis glucose monomers, which yeast can readily utilize.
commonly employed in practice include sodium hydroxide Furthermore, cellobiose inhibits cellulase enzymes much
and ammonia. The cellulose structure resulting from alkaline more strongly than glucose does. The presence of sufficient
treatment is more amorphous. The swollen cellulose structure quantities of b-glucosidase helps reduce feedback product
allows subsequent enzymatic degradation to proceed more inhibition.
readily because enzyme molecules can gain access to its Through pyrolysis, biomass is gasified into synthesis gas.
interior region. Unlike acid treatment, alkaline treatment Above 3008C, cellulose is degraded into volatile, gaseous
alone is usually insufficient in releasing constituent monomer products such as hydrogen, carbon monoxide, and carbon
sugars to theoretical yield. As a result, it is normally only a dioxide. At intermediate temperatures, decomposition is

Table 4 Yield of sugars from enzyme hydrolysis (5 wt% suspension) of acid and base treated solid based on 100 lb of original material

Conversion of available carbohydrate

Material Glucose Poly glucose Xylanose Arabinose G & PG Pentose Sugar

Barley 16 2.4 0.21 0.06 47.7 2.2 36.7
Corn stover 13.8 8.1 0.93 0.07 66.4 35.7 64
Cotton gin trash 6.9 1.8 0.32 0.05 44.8 6.1 35.6
Rice hulls 7.5 1.2 0.01 0.01 27.2 0.3 21.7
Rice straw 15.6 8.3 0.55 0.16 66.9 15.8 60.8
Sorghum straw 13.9 9.7 1.8 0.23 80.8 22.8 67.3
Wheat straw 14.6 3.5 2.07 0.10 57.5 36.5 53.4
Source: Wilkes et al. (1983).
368 Niamke and Wang

rather slow and the products formed are less volatile. viewpoint. Fermenting biomass with the fungus T. reesei to
Atmospheric conditions greatly affect pyrolysis. In the produce the cellulase needed in SSF is perhaps just as
presence of oxygen, dehydrogenation and depolymerization complex and expensive an operation as the cellulose-to-
occur quickly. On the other hand, in the presence of inert ethanol conversion portion.
substances, depolymerization is slow, and unwanted
byproducts appear (Wilke et al. 1983). In subsequently
steps, the synthesis gas is bubbled into a submerged culture,
and anaerobic microorganisms (e.g., bacteria Clostridium 3.2.1 Process Description
ljungdahlii) convert the pyrolysis product into ethanol
(Klason et al. 1990). The chief obstacle of this technology We give the description of a typical SSF process that aims to
is the high cost of energy (mainly electricity) to heat the recycle solid waste into ethanol, which functions as an
biomass, and the low value of the synthesis gas thus produced. alternative fuel. The solid utilized in this process is
Pyrolysis treatment may be economically viable if the approximately two-thirds urban waste and one-third pulp
fermentation product is valuable. mill waste. This solid mixture contains approximately 57% in
cellulose. The waste is pretreated, sterilized, and then
forwarded to three reactors of 2500 gal each. A culture of
mutant fungus T. reesei is inoculated into each reactor. The
3.2 Simultaneous Saccharification and fungus continuously produces a full complement of cellulases
Fermentation that degrade cellulose. The total residence time for each
cellulase production strain is 48 h. Ninety percent of the
In late 1970s, Gulf Oil Company developed a process for cellulose introduced into the reactors is degraded into sugars,
simultaneous saccharification of cellulose and fermentation of such as pentose, xylose, arabinose, and glucose. Sub-
the hydrolysis product to ethanol (Katzen and Monceaux sequently, the degraded cellulose is cooled in a heat
1995). Researchers at the University of Arkansas later exchanger and sent into 12 reactors for fermentation into
improved upon the SSF process, summarized in Figure 1. ethanol. In this system, one can shut down four reactors while
Conceptually, we simply replace the acid hydrolysis step with continuing operating the remaining reactors in full swing. The
an enzymatic one and carry out the combined operations of four reactors have an effective residence time of 48 h and are
hydrolysis and fermentation in a single reactor to convert maintained at 408C. The initial feed to the reactors contains
biomass into ethanol. 8% of intact cellulose, and the remaining 92% is degraded
Most reports on the SSF process cite combining two cellulose, which is hydrolyzed to glucose. The yeast
separate reactors for saccharification and fermentation into simultaneously ferments the glucose generated to produce
one reactor as an advantage; however, this claim is 2–3.6 w/v% ethanol beer slurry. Any unconverted material is
misleading. Perhaps because the researchers may have simply recycled back to the process.
obtained the cellulase enzyme off the shelf for their studies, In Szczodrak’s study (1989), the filamentous fungi
they ignore the fact that the enzyme has to be produced T. reesei produced the cellulase, and a thermotolerant yeast
separately in an economical fashion (Shin et al. 2000). In strain Kluyveromyces fragilis FT 23 carried out the
practice, there is an additional cellulase production step, conversion of sugars to ethanol. Table 5 gives a summary
which is nontrivial from the operational and economic of the percentage of ethanol produced.

Figure 1 The SSF process flow chart. From So and Brown (1999).
Cellulose Degradation by Fungi 369

Table 5 The SSF of chemically modified straw by T. reesei cellulase preparations derived from the parent and bGDase mutant strain
and K. fragilis cells

Activity (U/g straw)

Cellulase source Cellulase bGdase Time (h) Ethanol percent (w/v) Glucose hydrolyzate (mg) Final pH
T. reesei F-522 (parent) 40 14.1 24 1.9 0 5.0
48 2.5 0 4.9
T. reesei F-522-V-7 (mutant) 40 64.2 24 3.4 — 4.7
48 3.4 0 4.7

Source: Szczodrak (1989). One unit of enzyme activity (U) is defined as the amount of the enzyme that liberates one moles of reducing sugars (calculated as
glucose) per minute under the assay conditions. bGdase stands for endo b-1,4-D -glucanase.

3.2.2 Mixed Cultures and Fermentation replaced by lower priced gasoline. With the oil crises in the
1970s and increasing environmental concerns, ethanol has
One disadvantage of the SSF process is the number of steps regained some appeal as an alternative motor fuel (Linko
because of the separate steps needed for cellulase generation. 1987) or as an additive to be blended with gasoline. Because
In the original SSF process, cellulase, yeast, cellulose, and ethanol is an oxygenate that reduces vehicle exhaust
other nutrient supplements are all thrown into one bioreactor. emissions, it is environmentally friendly (Bollok et al.
To reduce the number of steps and cost, a modified process 2000). To remain successful in that role, the cost of
termed mixed cultures and fermentation has been developed. production must be low compared to gasoline. In this part, the
In this process, more than one organism coexist in the same economic aspect of ethanol production will be discussed.
reactor. While one organism degrades cellulose into sugars,
another ferments sugars into ethanol, acetic acid, and lactic
acid. This process is more efficient because it eliminates the 4.1 Economic Analysis of Ethanol Conversion
steps associated with the separation of the enzyme from the Technologies
products. In the process shown in Figure 2, C. thermocellum
saccharifies cellulose into glucose and cellobiose; the same
organism also hydrolyzes hemicellulose to xylose and Several technologies have been developed in order to convert
xylobiose. Furthermore, it ferments glucose and cellobiose efficiently cellulose to ethanol. The best-known processes that
to ethanol, acetic acid, and lactic acid. The second organism we discuss in the earlier section are: (a) SSF, (b) dilute
C. thermosaccharolyticum cannot degrade cellulose, but it sulfuric acid hydrolysis and fermentation, and (c) fast
ferments glucose, cellobiose, xylose, and xylobiose to pyrolysis and fermentation. We will briefly describe the dilute
ethanol, acetic acid, and lactic acid (Wilke et al. 1983). sulfuric acid hydrolysis process and the fast pyrolysis process,
Mixed culture fermentation is performed under anaerobic and then compare the cost and the production of ethanol
conditions, at a temperature of 608C and pH 7. In summary, among the three processes.
C. thermocellum efficiently degrades cellulose and hemi- Figure 3 illustrates the process of ethanol production by
cellulose, and C. thermosaccharolyticum then metabolizes dilute sulfuric acid hydrolysis and fermentation. In the
xylose to generate ethanol and useful byproducts. hydrolysis step, cellulose is pretreated in 0.05 g/l of sulfuric
acid at 1808C. For the purpose of economic comparison, we
consider the concentration of sugars yielded to be 103.7 g/l.
Following hydrolysis, a strain of fungus is responsible for the
4 ECONOMIC ASPECTS OF ETHANOL continuous fermentation of sugars (pentose and hexose) into
PRODUCTION ethanol (So and Brown 1999).
Figure 4 shows the Waterloo fast pyrolysis and
Ethanol has been used as a fuel in the United States since the fermentation process. Its pretreatment step uses 5% hot
beginning of the 20th century. However, it was quickly sulfuric acid. In the fermentation step, fungi degrade cellulose

Figure 2 Mixed culture hydrolysis and fermentation.

370 Niamke and Wang

Figure 3 Dilute sulfuric-acid hydrolysis and fermentation process. From So and Brown (1999).

into sugars while bacteria ferment the different types of 4.2 Costs and Benefits of Ethanol Production
sugars (pentose and hexose) to ethanol.
The capital cost analysis given in Table 6 shows that fast The production cost of ethanol must be low for it to be
hydrolysis, SSF, and acid hydrolysis all have fairly similar competitive with other existing fuels on the market. Presently,
total capital costs; the small differences are probably within the competition comes from methyl tertiary butyl ether
the error of estimates. Likewise, the operating cost analysis (MTBE) in gasoline blend, but in time the target for
presented in Table 7 also shows similar numbers, albeit the comparison will shift. Fluctuation in the prices of other
cost of ethanol is the lowest with the SSF process. energy sources makes it difficult to predict the future of

Figure 4 Waterloo fast pyrolysis and fermentation process. From So and Brown (1999).
Cellulose Degradation by Fungi 371

Table 6 Capital cost investment comparisons

Fast pyrolysis SSF Acid hydrolysis

Capital cost Capital cost

Plant areas (millions dollars) Plant areas (millions dollars) Plant areas Capital cost (millions dollars)
WFPP 12 Pretreatment 16 Hydrolysis 2
Fermentor 22 Pentose fermentor 4 Fermentor 27
Cellulase production 2
SSF 16
Ethanol recovery 11 Ethanol recovery 3 Ethanol recovery 14
Utilities 12 Utilities 12 Utilities 12
Off-site tankage 3 Off-site tankage 3 Off-site tankage 3
Fixed capital 60 Fixed capital 56 Fixed capital 58
Working capital 9 Working capital 8 Working capital 9
Total capital 69 Total capital 64 Total capital 67
Source: So and Brown (1999).

bioethanol. Although from time to time the government may The critical factors that contribute to the cost of ethanol are
provide tax incentives to jump-start an interest in renewable summarized below.
energy resources—which in practice is synonymous with (a) The cost of collected feedstock such as municipal and
ethanol from biomass, the production process must be commercial wastes must be low. Thus, the production
inherently competitive for it to be sustained in the long run. facilities should be located near the source of biomass
Table 8 shows a study of ethanol production in California. It material. Furthermore, we can cross the break-even point
gives the price of different feedstocks at near-term and mid- easier with higher cellulose content in the municipal and
term operation at a large scale. The target price takes into commercial wastes. (b) Ideally, the production site should be
account the operating costs, the debt, and return on close to the consumption site to minimize shipping. Ethanol
investment. The target price decreases from near-term to absorbs moisture very quickly and attacks rust spots in the
mid-term, as the technology improves and forces down the transportation system. This attack provokes corrosion along
production cost. Even when the cellulosic feedstock is the distribution system. (c) Because feedstock constitutes a
inexpensive, conversion into ethanol may be costly. Cellulase major fraction of the overall cost, we must efficiently utilize
enzymes cost 45 cents/gal of ethanol and are, therefore, too not just cellulose but all components in the feedstock,
expensive at the commercial level. especially xylose and lignin. This is especially true for

Table 7 Operating cost comparison with cost figures expressed in millions of 1997 US$

Fast pyrolysis SSF Acid hydrolysis

Total capital 69 64 67
Annual operating cost
Wood 11.09 11.25 11.03
Steam 1.15 24.50 2 2.95
Electricity 1.20 1.37 1.25
Operating labor 0.27 0.58 0.30
Supervisory 0.04 0.09 0.05
Maintenance and repair 3.52 3.36 3.48
Indirect operating costsa 3.67 3.64 3.58
General expensesb 4.40 3.49 3.63
Annual capital Charge 13.80 12.80 1334
Total annual operating costs 39.21 32.09 33.70
Production cost of ethanol ($/gal) 1.57 1.28 1.35

Source: So and Brown (1999).

a
Indirect operating costs include overhead, local taxes, and insurance.
b
General expenses include administrative, distribution, selling, research, and developments costs.
372 Niamke and Wang

Table 8 Assumed feedstock cost, ethanol production yields, CO, NOx, and volatile organic carbons. We also envision a
ethanol prices from cellulose based biomass future where biomass-derived products (ethanol, acetic acid,
acetone, etc.) become significant raw materials for
Time frame Near-term Mid-term chemical processing industries (California Energy
Commission 2001).
Feedstock price ($/BDT)
Forest material 36 38
Agricultural residue 24 26
Waste paper 2 10 2 10 5 CONCLUSION
Ethanol yield (gal/BDT)
Forest material 69.3 77.4 The most important commercial product of cellulose
Agricultural material 62.4 64 degradation is ethanol, which has the potential of filling the
Waste paper 74.4 81.7 national energy and raw material needs. Furthermore, burning
Target ethanol price ($/gal) bioethanol has the advantage that it does not contribute to
Forest material 1.73 1.23 green gas emission from the viewpoint of overall carbon
Agricultural residue 1.69 1.24 cycle. The present technology derives carbon mainly from
Waste paper 1.64 1.39 corn in a two-step process: hydrolysis of starch to sugar
Source: California Energy Commission 2001. followed by fermentation of sugar to ethanol. In the future,
starch is perhaps better saved for food or chemical raw
material. To make the process competitive against existing
sources of energy and raw materials, we must expand the
feedstock derived from forest and agricultural residues. feedstock to include lignocellulosic biomass. Economics play
Furthermore, unutilized residues incur a disposal cost. Strain a major role in deciding which process is ultimately
improvement and recycling of residuals move us closer commercially viable. To this end, future research must
toward fulfilling this goal. (d) For processes that depend on focus on minimizing unfermented residual biomass,
fungi-derived cellulase for cellulose degradation, the cost of especially lignin and hemicellulose. We must attack an old
the enzyme must be dramatically reduced. Other commer- problem systematically with a multipronged approach and
cially viable processes that utilize cellulase (e.g., stone- with newly discovered tools in biotechnology: genetic
washed jean in textile processing) deal with high-valued engineering, site directed mutagenesis, and metabolic
products and require only partial hydrolysis. In contrast, engineering.
ethanol production from cellulose deals with bulk commodity
products and requires nearly complete hydrolysis. Thus,
relative to the price of the product, the cost of cellulase looms
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32
The Importance of Wood-Decay Fungi in Forest Ecosystems

Nia A. White University of Abertay, Dundee, Scotland, United Kingdom

1 INTRODUCTION woodlands provide a habitat for biological diversity. They

contribute substantially to the global carbon cycle and are the
This chapter considers the role of wood-decay fungi in forest natural venue for many derived pharmaceutical, practical, and
ecosystems in the broadest sense, embracing examples of fungi edible products. Woodlands are also an environment for
displaying wood and litter degradative activities, even if only indigenous peoples, and provide much aesthetic and amenity
transiently. The aim is to emphasize the fundamental value. Alarmingly, our forests are being lost at a rate of
significance and pervasiveness of wood-decay activity as an around 14:6 £ 106 ha=yr due to pollution, disease, and
integral process in ecosystem functioning. I therefore present a excessive exploitation (Anon 2001).
coherent text of diverse themes, but not a comprehensive Wood accounts for around 80% of the global organic
treatise of any. Each individual topic presented offers an carbon reservoir. About 70% of woodland net primary
overview of material that has formed the basis of many production is deposited annually as litter, whilst the
individual and extensive texts. I have therefore been selective, remaining 30% accumulates at least transiently as woody
have tried to avoid lists of species and instead attempted to biomass (Boddy 1991; Rayner and Boddy 1988). Below
highlight important ecological activities, features, trends, and ground, persistent lignified roots may contribute between 18
concepts. In examining the current status of knowledge, I have and 22% to the total subterranean biomass (Boddy and
on occasion restated familiar works, thus providing pro- Watkinson 1995). Thus, approximately 2 ton of plant biomass
gression to more recent research. As a result of my own may be deposited annually per hectare of forest in temperate
background and interests examples are mostly drawn from the regions due to the accumulation of leaf litter, fallen branches
Northern Hemisphere and information regarding, for example, and periodically, whole trees. Together with carbon reserves,
important tropical biomes, has largely been omitted. The deadwood litter fall is estimated to retain between 1.1 –3.7
chapter aims to identify some general concepts underpinning and 0.1–0.8 kg/(ha yr) of nitrogen and phosphorus, respect-
wood-decay fungal ecology, and provide guided access to ively, which serves to immobilize essential nutrients until
pertinent literature where possible. I conclude by identifying decay is achieved (Boddy and Watkinson 1995; Dighton and
neglected arenas of study and speculating on future research Boddy 1989). Without decay, nutrient reserves would
trends. After-all, continued investigation concerned with accumulate and eventually halt ecosystem productivity.
wood-decay fungi and their role in forest ecosystems is an Saprotrophic wood-decay fungi play a pivotal role in the
academically challenging and immensely important process in ecology of forests as they are the principal agents of wood and
biological, ecological as well as practical terms. chitin decomposition and hence nutrient and energy fluxes
(Dighton 1997). Unsurprisingly, fungi therefore represent the
dominant microbial biomass of the forest floor and soils of
2 WOODLAND ECOSYSTEMS AS A GLOBAL many forest ecosystems, of which 60% may be due to
RESOURCE decomposer basidiomycetes. Energy flow through woodland
ecosystems is ultimately dependent on a limited mineral
Woodland ecosystems are a major planetary resource, nutrient availability. Consequently, the balanced cycling of
involving about a third of the land surface. Forests and mineral nutrients within woodlands is central to ecosystem

375
376 White

functioning, and fungal participation in mineral cycling is to redress our ignorance on many key aspects of their
extensive (Boddy 1991; Dighton and Boddy 1989; scientific understanding, their distribution and functions.”
Wainwright 1992). Fungi have been shown to be capable of
all major nitrogen transformations except nitrogen fixation,
although certain fungi may sequester nitrogen from nitrogen 3 THE FUNGAL HABIT
fixing bacteria within wood and fungal sporocarps (Barron
1992). Fungi can utilize organic (during decay) and inorganic Filamentous fungi are immobile but may achieve motility by
(solubilized by organic acids and siderophores) phosphorus virtue of apical growth of hyphae from a supporting
sources and many fungi produce siderophores to sequester substratum. The indeterminate and versatile development of
limiting supplies of bioavailable iron (Cromack and Caldwell fungal mycelia is directed by interactions with the dynamic
1992). Mineral resources are transformed into new fungal abiotic and biotic environments, the latter involving both
biomass, organic acids, and humic substances. Indeed inter- and intra- specific interactions between fungi and
significant quantities of nutrients may be immobilized as between fungi and other organisms. As heterotrophs fungi are
fungal biomass, and this immobilization has resulted in the nutritional opportunists, having evolved an array of optionally
adoption of management practices such as burning to programmed developmental pathways to take advantage of
eliminate woody debris from forestry sites prior to changing local circumstances and resource fluxes (Rayner
re-plantation, thereby reducing potential competition for 1996). Fungi often possess a more restricted degradative
mineral nutrients between fungal biomass and tree transplants repertoire compared with many bacteria, but fungal mycelia
(Dighton 1995). Nutrients sequestered as fungal biomass may possess clear advantages over unicellular or short-chained
be released subsequently as a result of processes such as growth morphs for invading and degrading solid substrata.
interactions with other fungal individuals or bacteria, by Fungal mycelia absorb nutrients through a high contact
physical damage or invertebrate grazing and withstanding surface area and may penetrate substrata by generation of
these, by senescence and death of the mycelium (Boddy and mechanical pressure and extracellular degradative enzymatic
Watkinson 1995). and chemical activity. Mycelia are capable of nutrient
translocation to a greater or lesser extent, and therefore have
The biological diversity contained within woodland
the capacity to support exploration from one nutrient rich
ecosystems may be exploited for practical and aesthetic
source to another, thereby transcending regions of nutrient
gain. Traditional methods of exploitation have involved
deprivation.
collection or cultivation of fungi for food, e.g., truffle fungi,
Highly specialized multihyphal structures such as
Lentinula edodes (Shii-take), and Pleurotus ostreatus (oyster
rhizomorphs and mycelial cords are produced by a variety
mushroom) as well as many other edible woodland fungi.
of wood decay, root pathogenic, and ectomycorrhizal fungi,
Wood colonized by certain fungal species may be employed
such as Armillaria spp., Phanerochaete velutina, Hypholoma
to generate valuable timber products. For example, “Brown
fasciculare, Phallus impudicus, Tricholomopsis platyphylla,
oak” veneer timber ( pourriture rouge dur) is produced by and ectomycorrhizal Leccinum scabrum (see Boddy 1992;
Fistulina hepatica colonizing heartwood, Chlorosplenium 1999; Rayner and Boddy 1988 for review). These linear
aeruginascens is used in the commercial production of organs are often evident at the interface of surface litter and
Tunbridge ware, and wood containing interaction zone lines is soil in boreal, deciduous and tropical woodlands, where they
turned to produce decorative artifacts. Novel methods of may contribute to stabilizing woodland litter against weather
exploitation may involve the application of fungal decay erosion particularly on steep slopes (Lodge and Asbury 1988).
systems to convert a range of renewable lignocellulosics into Some may even form networks within the tree canopies of
protein, fermentable sugars, and other products, or to tropical forests (Hedger et al. 1993). Cords are organs of
bioremediate certain recalcitrant pollutants. Appreciation migration and exploration, with more efficient rates of
and understanding of fungal activities, relationships, and biomass deployment and translocation of water and nutrients,
distributions in natural ecological contexts may provide compared to a more diffuse hyphal network, allowing the
insight into methods of searching and screening for useful individual to transcend vast distances of often hostile regions
products and conversion systems, and support the manage- or inhospitable nutrient deserts. Cords also provide resistance
ment and conservation of our valuable global resource. As to the damaging effects produced by the soil microbial and
Hawksworth and Colwell (1992) succinctly expressed as part microfaunal communities. The voracious opportunistic
of the Microbial Diversity 21 statement, saprophytic fungi, in scavenging and persistent biomass involved may also
common with other microbes, “are vital to the function of the facilitate nutrient storage. Cord formers are commonly
Earth’s ecosystems and biosphere. As major contributors in particularly combative and are capable of producing rapid
biogeochemical cycles, they perform unique and indispen- decay in nature, thereby making significant contributions to
sable activities in the circulation of matter in the world, on nutrient cycling (Boddy 1999; Boddy and Watkinson 1995;
which all larger organisms, including humans, depend. They Chapela et al. 1988; Coates and Rayner 1985a).
constitute a genetic resource of great potential for contribut- Cord formation may be promoted by resource depletion
ing to the sustainable development of the planet as well as (low C:N ratios and nutrients such as copper and calcium),
human, animal, and plant health. Urgent attention is required recognition or contact with an antagonist, and reduced water
Wood-Decay Fungi in Forest Ecosystems 377

potential (Rayner and Webber 1984; Watkinson 1999). then target specific hypotheses. However, the indeterminate
Directional growth of cords towards resource baits may nature of the fungal mycelium clouds palpable identification
possibly be mediated by volatiles or soluble diffusates, and of an appropriate scale of study, as interactions occur within a
involves coordinated redeployment of mycelial biomass continuous hierarchy of spatial and temporal scales (see
following detection of localized resource base. Different “Modeling Fungal Decay Communities”). Many patterns
species display different foraging strategies, subject to identified at a small scale may become less evident or
resource quality, quantity and distribution, biotic factors and significant at larger scales and vice versa. Furthermore, such
microclimate (e.g., Boddy 1999; Donnely and Boddy 1998; features may be relatively insignificant under a particular set
Wells et al. 1999; 2001; Zakaria and Boddy 2002). These of environmental conditions, but ecological change or
linear organs can interconnect heterogeneously dispersed disturbance may augment their importance. Thus information
nutrient sources to form coordinated resource distribution derived at a variety of temporal and spatial scales is clearly
networks (Boddy 1999), and may translocate different most likely to contribute to a genuine understanding of fungal
nutrients independently even bi-directionally, e.g., certain ecology. The future challenge will be linking information
ectomycorrhizal cords may translocate carbon from tree roots derived from differently scaled studies in an appropriate
to the growing mycelium, and mineral nutrients and water in manner.
the opposite direction.

5 DESCRIBING THE ROLES OF FUNGI

4 FUNGAL ASSOCIATIONS AND DECAY
Understanding the pattern of occurrence of an organism or
Fungal communities occupying forest ecosystems are population is largely a problem of identifying its realized
dynamic and usually diverse, consisting of assemblages of niche. This task involves the identification of not only the
organisms that interact with their surrounding abiotic and space– time occupied by the organism in nature but also the
biotic environment (Boddy 1992; Rayner and Boddy 1988; role within that space–time during interaction with the biotic
Shearer 1995; Widden 1997; Zak and Rabatin 1997). environment (Cooke and Rayner 1984). In turn, this leads to
Nevertheless, the unit community for each resource type the problem of how best to classify the role of an organism.
often reveals distinct dominants and recurring associations of Apropos, appreciating the ecological (Andrews 1992; Pugh
species during degradation (Dighton 1997; Frankland 1992). and Boddy 1988) and nutritional strategies displayed by
Consequently, prominence has often been given to the individual fungi is a useful first consideration. Here I will
sophistication of the nutritional physiology of individuals consider the latter only.
when attempting to understand or explain fungal community An individual filamentous fungus may achieve an
development and successions. Thus, many explanations have absorptive heterotrophic life-style via biotrophy or necro-
centered around leagues of simple substrate groupings, or trophy, either exclusively or sequentially. Fungal residents of
assemblages of individuals with particular degradative woodland ecosystems exhibiting biotrophy include the
repertoires, simply following changes in resource quality as (neutralistic to mutualistic to parasitic) endophytes and
decay proceeds (e.g., Park 1968). Such a simplistic view (mutualistic to parasitic) mycorrhizae, whereas archetypal
should be considered with caution, as it tends to obscure other necrotrophic representatives include certain (parasitic)
important ecological adaptations. It is also suggested that the pathogens and the decay saprotrophs of dead wood- and
term succession itself should be used with caution when litter-tissues. These broad activity categories represent
describing fungal community development, particularly convenient but sometimes restrictive descriptors for the
within certain resources such as wood. Terms such as ecologist because they may obscure the dynamic nature of the
substratum succession (Park 1968), and resource succession relationship. In the next section I wish to avoid discussion on
(Frankland 1992) may be misleading in suggesting simple the boundaries of classification schemes, but will consider the
order, thereby obscuring the actual complex multi- wood-decay activities displayed by the associations men-
dimensional dynamics involved, which may follow a diverse tioned above, so as to illustrate the spatio-temporal
array of optional pathways (Boddy 1992; Cooke and Rayner complexity and versatility of filamentous fungi. We as
1984; Renvall 1995). However, Frankland (1992) emphasized mycologists often focus on the wood-decay activities of true
that the term succession should “accommodate a multi- wood and litter saprotrophs, sometimes at the expense of
dimensional phenomenon not a simple linear process” so as to recognizing the prevalence of such activities to other
include features such as environmental fluctuation, cycles and association habits. These association categories merely
disturbance, life-cycle changes, and mycoparasitism without represent distinct points within a continuum of overlapping
replacement. activity groups. Therefore, certain individuals may be
But how should fungal ecology be best studied? classified simply as wood or litter inhabitants or as
Synecological studies are valuable in investigating the range mycorrhizae, but others may decay both wood and litter, or
and complexities of fungal communities, particularly when may be mycorrhizal but also cause lignocellulose decay.
underpinned with appropriate sampling techniques and Moreover, the nature of interactions between individuals may
statistical analysis. Considered autecological approaches can alter with life-cycle stage or environmental circumstances. An
378 White

individual may thus commence as an endophyte, then develop other competitor plants, with obvious consequences for the
as a latent pathogen, and subsequently adopt a more plant community, and have therefore been investigated as a
saprotrophic role, following senescence of the host. The potential biological control system (Leuchtmann and Clay
specificity of relationships also varies. Consequently, some 1988).
fungi will be restricted to a particular biome, geographic area, Endophytes may also be involved with initiation of decay
specific resource, host species, part or cell-type within a host following stress or death of host tree tissues. Many
species. Others may be nonselective, ubiquitous generalists, endophytes, such as the Xylariaceae and their anamorphs,
such as the opportunistic decomposers. Furthermore, mycelial Daldinia and Hypoxylon spp. and Nodulisporium, are known
patterns may operate over a range of scales, from an wood-decay species. Some are observed to fruit extensively
individual pine needle (Kendrick and Burges 1962), to an on very recently dead fallen logs, or form extensive primary
entire forest (Smith 1999) or even at a global scale (Arnolds decay columns or strip cankers (e.g., by Eutypa spinosa in
1997). European beech) very rapidly following host tree stress,
Finally, the heart rot and active pathogenic activities of particularly after drought (Hendry et al. 1998). Observation of
fungi causing death and decay of standing trees was described such rapid and massive decay column formation has identified
in the first edition of this text and will therefore not be the role of endophytic morphs or latent invaders, distributed
considered here (Boddy 1991). The structural, chemical, and as somatically compatible or clonal latent propagules,
microclimatic features prevalent in living wood and the dispersed within functional sapwood of standing trees. Such
biology, ecology, and epidemiology of fungi causing its decay propagules may then be rapidly activated due to host stress
have been reviewed elsewhere (Butin 1995; Rayner and such as declining wood water content and the growing spatial
Boddy 1988; Schwarze et al. 2000; Smith et al. 1992; 1999; domains of individual genets may then converge and merge,
Tainter and Baker 1996; Vasiliauskas 2001; Woodward et al. thereby securing early and spatially extensive infection of
1998). dead wood (Boddy 1992; 1994). Latent presence of
propagules in living bark tissues allows rapid and extensive
invasion of dead attached twigs or recently felled logs
5.1 Endophytes (Griffith and Boddy 1990). However, detection of propagules
does not necessarily implicate that species in initiating decay,
Endophytes are defined as organisms that grow within a living as some endophytes have been isolated from host species
host plant but not strictly within living cells, thereby deriving known infrequently to support decay columns of these latent
nutrition without detrimental symptoms for the host. It is colonizers (Boddy 1994; Petrini 1991). This seemingly
believed that the living stems, twigs, and leaves of most destructive activity within stressed and functionally redun-
woody plants contain symptomless endophytes, but because dant tissues may also be of ecological advantage to the tree,
of their clandestine nature their prevalence is ambiguous, and resulting in natural pruning of expensive nutrient sinks and
identification of their presence relies on arduous techniques the subsequent mobilization of nutrient resource.
such as careful isolation from surface sterilized tissues and
histological studies (Petrini 1991). Numerous endophytic
species may be potentially allied with an individual host (e.g., 5.2 Mycorrhizae
85 different fungal taxa have been isolated from root and stem
tissues of Alnus). Moreover, host specificity varies, with some Mycorrhizae are mutually beneficial associations formed
endophytes displaying little host specificity (e.g., Phomopsis between the majority of plant and tree species and certain
has been isolated from Buxus, Ilex, Hedera, Ruscus, Ulex, biotrophic soil fungi. The evolution of such a symbiosis
Pinus, Fagus, Juniperus, Fraxinus, and Quercus; and provides a means through which the mycobiont can acquire
anamorphs of Xylaria isolated from Fagus, Fraxinus, and most of its carbon from the photobiont mainly in the form of
Quercus) whereas others, such as Peniophora and Vuilleminia hexose, whilst donating mineral nutrients, particularly
comedens tend to be more stringent (Griffith and Boddy 1990; phosphorus and nitrogen, and supplementing plant water
Fisher and Petrini 1990). Although resident endophytes may requirements. Plant resource acquisition and uptake is
not produce disease symptoms as such, their occupation is not enhanced by the presence of a massive mycobiont hyphal
without effect on the host. surface-area, the activity of which improves mobile resource
The leaves or needles of many if not most plants contain uptake and is usually solely responsible for uptake of most
fungal endophytes, estimated at a supporting cost of up to immobile resource. Affinity for and uptake of nitrogen as
0.5% of annual needle production in the case of conifer needle ammonia and phosphorus as phosphate is greater for the
endophytes (Carroll 1992). However, the presence of some mycobiont than for the uninfected root. Fungi as mycorrhizal
endophytes, such as Phomposis oblonga in elm stems (Carroll associates may protect against soil pathogens and environ-
1991), may confer a selective advantage on the plant host, mental stress and contribute to the survival of most plants
either due to secondary metabolite production or conceivably within the natural woodland environment (Smith and Reid
due to competitive exclusion, thereby protecting against 1997).
pathogen invasion particularly insect attack or larvae Some mycorrhizal fungi show little host specificity;
development. Some may even be potential pathogens of e.g., Amanita muscaria may associate with conifers such as
Wood-Decay Fungi in Forest Ecosystems 379

Pinus and Picea, and with deciduous species such as Betula. species Phanerochaete velutina. This latter species sub-
Such nebulous specificity may allow formation of connecting sequently accumulated the labeled carbon source (Figure 1;
networks, linking individual plants of the same and different Leake et al. 2001). Other examples of competition between
species, in source –sink relationships via communal mycor- mycorrhizae and saprotrophs have been reported (Shaw et al.
rhizal fungi. Such conducting and communicating networks 1995). However, the extreme mycorrhizal dependency
may be most significant during for e.g., the translocation of exhibited by achlorophyllous plants is testimony to the role
nutrients by a more mature plant community, to support the of fungal associates in translocating carbon to host plants
adjacent growth of stressed or shaded seedling plants (Trappe (Leake 1994). The significance of similar carbon donation by
and Luoma 1992). The ecological significance of such
resource sharing in sustaining primary production in natural
forests, heathlands, and grasslands is currently the focus of
research attention (e.g., Simard et al. 1997). Other
mycorrhizal fungi exhibit more strict host specificity; for
e.g., Alpova diplophloeus will only associate with Alnus spp.
Nevertheless, the development of general successions of
mycorrhizal fungi are often recognized during forest
maturation, the individual occupants of which have been
associated with r- and s-selected strategies and the nature of
the litter resource. The ecological strategies of individual
mycorrhizae have important implications for forestry
practices with respect to choice of mycorrhiza inoculum for
plantation within woodlands at different stages of
development.
Four broad types exist; the vesicular-arbuscular- (VAM) or
arbuscular- (AMF), orchid-, ericoid-, and ecto- mycorrhizae.
The VAM or AMF generally occupy environments where
phosphorus is the main growth limiting nutrient. They
dominate forests in tropical climates, but some temperate
trees such as sycamore, ash, and poplars have AM, and some
such as willow can form both AM and ectomycorrhizal
associations. Ectomycorrhizae, however, are supreme in
forests on moder, mull, or brown earth soils in temperate
regions of moderate latitude and altitude (e.g., Read 1991),
and in relatively infertile soils particularly where nitrogen and
phosphorus uptake is curtailed. The ectomycorrhizal fungal
mycelium sheaths root cortical cells, forming a Hartig net,
and extends through the litter and surface soil layers forming a
foraging, ramifying network. The substantial mycelial
investment involved implies that prolonged associations
tend to occur. The biology, ecology, and biotechnological Figure 1 (a) Localized proliferation of mycelium of Paxillus
application of mycorrhizae have been reviewed and described involutus in mycorrhizal association with Pinus sylvestris on
in many excellent texts, for e.g., Allen and Allen (1992); entering discrete patches of nonsterile forest litter in trays placed
Carroll (1992); Smith and Reid (1997); Podila and Douds in an otherwise homogeneous peat substrate. (b) A digital
(2000). This section will focus only on the saprophytic autoradiograph of the microcosm shown in Figure a, 5 days later.
activity of mycorrhizae in temperate forest soils. The autoradiograph was aquired 48 h after 14C pulse labeling of
Conventional belief has been that the main function of the shoot of the P. sylvestris seedling. Over 50% of the 14C
mycorrhizal fungi is uptake and translocation of mainly detected in the mycorrhizal mycelium (excluding roots) was
immobile mineral nutrients released by free-living sapro- allocated in the recently colonized litter patch labeled T2. (c) A
trophs and that saprotrophy is generally lacking in control 20 £ 20 cm microcosm with mycelium Suillus bovinus
mycorrhizal fungi. Indeed, nutrient flow between plant mycorrhizal with P. sylvestris, forming an almost complete
roots, ectomycorrhizae, and interacting saprotrophic non- covering of the surface of the peat. (d) Similar microcosm in
mycorrhizal species has been demonstrated. For example, the which the mycelium of S. bovinus met and interacted with the
wood-decay species Hypholoma fasciculare evidently lost mycelial cords of the wood-decomposer fungus Phanerochaete
phosphorus to mycorrhizal Suillus variegatus (Lindahl et al. velutina growing from a wood block. Note the difference in
1999), whereas, carbon donation from Pinus sylvestris density of cover and extent of mycorrhizal mycelium compared
seedlings to mycorrhizal partner Suillus bovinus was with (c). From Leake et al. (2001); Reproduced in black and
visualized and declined in the presence of the decomposer white from color images.
380 White

mycobionts to autotrophic plants is now being investigated. woodlands by mycorrhizal species, together with donation of
Thus, it is becoming increasingly evident that, particularly in carbon supply by host plant species, may render the
more acidic woodland environments, mycorrhizal fungi degradative role of mycorrhizal fungi as significant compared
influence the degradation of leaf litter and woody debris. to nonmycorrhizal saprotrophs, at an ecosystem scale.
For example, mycorrhizal activity is stimulated by addition of
litter to microcosms, and is maximal in temperate and boreal
forests when nutrient litter supply peaks (Leake and Reid 5.3 Saprotrophs of Attached and Fallen Wood
1997; Unestam 1991). Furthermore, visual evidence of and Litter
penetration of litter material by ectomycorrhizal hyphae
(Leake et al. 2001; Figure 1; Ponge 1990), and nutrient release Saprotrophic fungi are the principle decomposers of nonliving
measurements following ectomycorrhizal (Suillus bovinus or plant and animal detritus in the natural environment, thus
Thelephora terrestris with Pinus sylvestris) colonization of recycling chemical elements back to the environment in a
litter patches (Bending and Reid 1995), all implicate a form other organisms may utilize. Filamentous fungi usually
saprotrophic role for the mycobiont. Ericoid- and some ecto- dominate wood and litter decomposing communities, but
mycorrhizal fungi, commonly found dominating ecosystems under particular ecological circumstances, for e.g., for wood
where nitrogen and phosphorus reserves often reside within in tropical ecosystems termites may predominate, and under
accumulated organic matter, are known to produce extra- waterlogged conditions bacteria may prevail (Rayner and
cellular enzymes capable of decaying complex carbon Boddy 1988). Other wood and litter residents may include
polymers present in litter and soil, thereby exposing and yeasts, bacteria, Myxomycetes and invertebrates such as
mobilizing otherwise trapped mineral nutrient reserves Insecta, Oligochaeta, Acaria, and Nematoda. These may
(Leake and Reid 1997). Proteins, amino acids, chitin, nucleic influence fungal community dynamics and consequently
acids, phospholipids, and sugar phosphates all serve as affect overall decay rates, either via direct interaction, such as
nitrogen- and phosphorus-rich organic materials. Enzyme antibiosis or grazing of fungal mycelium or spores, or by
capacities so far identified include proteinase, peptidase, indirect interaction through impact on the abiotic environ-
chitinase, acid and alkaline phosphatase, phytase, DNAase ment (Dighton 1997; Rayner and Boddy 1988). Thus,
and RNAase, polygalacturonase, cellulase, xylanase, tyrosi- invertebrate activity can increase the exposed surface area for
nase, peroxidase, polyphenoloxidase (including laccase), and decay activity, may provide channels for mycelial invasion,
ligninolytic activity (see Leake and Reid 1997). The and also improve aeration. Invertebrate waste and remains
hydrolytic capability of different individual ectomycorrhizal will also increase local available nitrogen supplies. Therefore,
fungi is diverse (Hutchinson 1990), some being capable of decay of litter and wood in forest systems are ecologically
degrading holocellulose, lignin, and lignocellulose significant and complex processes, produced by intricate and
(Trojanowski et al. 1984), while others utilize protein for dynamic communities.
carbon and nitrogen and translocate derivatives to host plants
(Abuzinadah et al. 1986). The latter capabilities are of 5.3.1 Leaf-Litter Decomposition at the Substratum
primary importance where successful mycorrhizal associ- Community Scale
ations are established within ecosystems where the carbon
may be supplied to the mycobiont often entirely by the plant Estimates for litter production in temperate woodlands range
symbiont, but available nitrogen and phosphorus supplies are from 3.8 ton/ha/yr for oak (Quercus petraea) and 5.7 ton/ha/yr
growth limiting to the fungus and then ultimately to the plant for Norway spruce (Picea abies), although tropical forest
host. litter production may be twice this. Leaves form the bulk of
It has been estimated that maintenance of the mycorrhizal litter material, representing 65 –75% of the total nonwoody
association costs the host between 9 and 28% of the total input to forest ecosystems. The overall process of leaf
photosynthetic production annually (Finlay and Söderström decomposition involves the activities of fungi, bacteria, and
1992; Leake et al. 2001; Vogt et al. 1992). The benefits of animals, to produce a humic product, which becomes
such a costly relationship to the photobiont are not only incorporated into the mineral soil fraction. Litter decompo-
limited to nutrient scavenging and mobilization, and to the sition may take months (e.g., for Ulmus and Fraxinus
saprotrophic release of otherwise unavailable resource, but excelsior) or years (e.g., for Pinus sylvestris, Quercus, Fagus
extend to the ability of the mycobiont to construct extensive sylvatica), and may accumulate to form layers if litter
and persistent mycelial networks. This allows retention of a production is high and decomposition rates are low. Fungal
mineral budget within a fluctuating and discontinuous successions on leaves as they mature, senesce, die and fall, are
nutritional environment, as the mycorrhizal rootlets undergo well characterized and described in detail elsewhere
dynamic degradation and formation, thereby preventing (Andrews and Hirano 1991; Frankland 1984; 1998; Dix and
leaching of important nutrient reserves to the surrounding Webster 1995; Kinkel 1991; Ponge 1991). Unfortunately,
rhizosphere. Leake and Reid (1997) discuss the ecological investigations tend to focus on the generation of lists of
significance of degradative activities of mycorrhizal and species rather than enquiry into the underlying processes
nonmycorrhizal fungi. They argue that the massive mycelial involved. Exceptions that attempt to explore the fundamental
biomass and localized degradative activities produced within processes driving population and community dynamics
Wood-Decay Fungi in Forest Ecosystems 381

include application of “Life-history strategy” concepts resource quality, particularly nitrogen content and also to
(Andrews and Harris 1986), “Island theory” (Andrews et al. levels of fungi-inhibitory tannins. So, Quercus litter with the
1987; Wildman 1992) and a dynamic “Patch occupancy” highest C:N ratio and tannin content was the slowest litter to
model (Gourbière et al. 1999). Populations of fungi decay, whereas Fraxinius with the lowest, produced the
colonizing needles and leaves are governed by both external shortest decomposition period. In fact, during the decompo-
climatic effects with implicit immigration rates, and by fungal sition of litter collected from a single source, but allowed to
interaction effects such as competition and successional decay in adjacent but environmentally dissimilar habitats,
associations (Gourbière et al. 2001; Kinkel 1991). Coloniza- similar successions were observed but with different temporal
tion by phylloplane filamentous fungi and yeasts may scales (Frankland 1998).
commence immediately following emergence of a new Senescent leaves together with resident mycoflora will
leaf, and some species may already have a prior eventually fall to the litter or soil surface. Some phylloplane
colonization history within the bud itself. Fungal spores fungi (Aurobasidium spp. and Cladosporium spp.) may
deposited from the atmosphere must first survive stressful persist, and some may even complete their (sexual) life cycle
conditions, such as desiccation and rapid wetting, and during this phase. However their consequential net decompo-
intense UV and visible light as well as other environmental sition may be very low. Litter is rapidly colonized by certain
and climatic factors. Subsequently, primary colonizers soil-inhabiting fungi (e.g., Penicillium, Trichoderma, and
overcome leaf inhibitory structures and compounds as Fusarium spp.), which appear to cause little direct litter
well as microbial competition, whilst deriving nutrients via decomposition, but may produce significant indirect effects,
absorption of simple atmospheric nutrient supplies and leaf such as synergistically increasing decay rates with litter-
cell exudates. agarics. The early ruderal strategists are progressively
Phylloplane and litter studies usually involve cultural replaced by saprotrophic communities, which decay leaf
methods: either plating of macerated portions or washings surface waxes, pectins, and the lignocellulose complex itself.
from leaf tissue, or of surface-sterilized portions (to Later significant decay stages are associated with the litter-
distinguish between true colonists and nonviable propagules). basidiomycetes, especially agarics such as Mycena,
However, Frankland (1998) cautioned that plate isolations Marasmius, and Clitocybe, which form a significant portion
might generate misinformation by merely selecting certain
of fungal biomass within litter. Such species are capable of
species from a complex of substratum successions. The
cellulose and hemicellulose hydrolysis and often also
isolation of certain species may better indicate the prevalence
ligninolysis, as well as the detoxification of litter phenolics.
of dormant survival propagules, than their actual activity
Mycena galopus is often prevalent in temperate litter,
within the substratum. Direct observation following brief
displaying little resource specificity and causing typical
incubation in damp chambers is also useful, or following
white-rot, but is never found in bulky woody debris.
staining of leaf surface impressions produced in nail varnish
Evidently, factors other than lignocellulose decay potential
or clear sticky tape, or even via electron microscopy. These
different techniques have revealed that many incidental are significant in determining the part an individual may play
spores are in fact not viable, and that the pattern or succession in litter decomposition. For example, explanation of an
of true colonizers is remarkably consistent for any particular apparent paradox co-existence of two litter saprotrophs
leaf species, despite the diversity of propagules present. Early M. galopus and Marasmius androsaceus both utilizing the
colonizers tend to be weak parasites or epiphytic saprophytes, same resource but possessing different colonizing vigor,
e.g., certain yeasts and the cosmopolitan species Aureobasi- involved microclimatic factors and another trophic level, i.e.,
dium pullulans and Cladosporium herbarium, their activity preferential grazing by a mycophagous collembolan
often being restricted to the phylloplane until the onset of leaf (Frankland 1998). Certain basidiomycetes show preferences
senescence. The status of some species may indeed change for particular litter types, possibly due in part to the
from epiphyte to endophyte or even to weak parasite, stimulatory effect that certain litter flavenoids have on their
depending on the interaction dynamic with the host plant growth. So, for example, the growth of certain Clavaria,
defenses. Endophytes, pathogenic, parasitic or symptomless, Collybia, Marasmius, and Mycena species is stimulated by
may occupy up to 75% of leaves within a site. However, addition of minute quantities of taxifolin glycoside to growth
despite the diversity of endophytic genera involved, which media. However, differences between representative com-
may include Phomopsis, Cryptosporiopsis, and Phoma, the munities occurring on angiosperm and coniferous litter, were
dominant mycoflora appear to be characteristic of the host attributed more to pH than to leaf phenolic properties (see
species, regardless of host geographical distribution, thereby Carlile et al. 2001 for references). Agarics are also most
indicating a close evolutionary relationship between plant and significant within deeper litter layers, where the improved
fungus (Petrini 1991). Frankland (1998) also recognized the moisture regime is conducive to basidiomycete growth and
influence of host substrate on fungal species composition. they are sheltered from fluctuating climatic conditions. The
Comparison of successions on different litter and nonwoody significant involvement of small soil animals in nutrient
plant debris, revealed that the main differences in species cycling, promoting litter decomposition via leaf fragmenta-
composition were the early stage weak parasites, and that the tion, nitrogen input, and detoxification of phenolics is
duration of the decay succession, could be attributed to reviewed elsewhere (Dix and Webster 1995; Ingham 1992).
382 White

5.3.2 Attached and Fallen Wood Decay at the (Chapela 1989; Crane et al. 1996; Crawford et al. 1990;
Substratum Community Scale Lumley et al. 2000; Rayner and Boddy 1988). Host-wood
species, physico-chemical properties, and microclimate
Wood degradation is a relatively slow process that may in govern the basic fungal community dynamics, as does the
temperate woodlands, take tens of years for the decompo- prior history of the substratum (Barron 1992; Butin and
sition of small branches to several hundred years for large Kowalski 1986; Chapela et al. 1988; Griffith and Boddy 1990;
trunks. The heterogeneous and polymeric nature of wood Keizer and Arnolds 1990). Water distribution and its
together with low nitrogen and phosphorus content limits reciprocal relationship with aeration, were identified as
decay of woody resources to certain fungi and less principal determinants of colonization patterns (Rayner and
significantly to a few bacteria. Boddy 1988; Griffith and Boddy 1991b). Thus, the moisture
The 1980s and early 90s saw major advances in our relations of living trees inhibit fungal growth and decay by the
understanding of the ecology of wood-decay fungi. This is majority of fungi, except for certain pathogenic and
comprehensively reviewed in several extensive and excellent endophytic species. However, to overcome this, some fungi
texts (Boddy 1992; Rayner and Boddy 1988; Renvall 1995). apparently employ a strategy of active wood desiccation to
Here I will simply present a brief outline. Investigations of achieve appropriate conditions for establishment and decay
indeterminate fungal growth within most natural environ- (Hendry et al. 1998).
ments present the ecologist with numerous constraints and The decay process often commences in the standing tree,
practical challenges. However, the colonization of wood by in attached lower or stressed branches (Rayner and Boddy
fungi, particularly the higher fungi, provides the ecologist 1988). Fungi may gain access either through wounds, tissues
with an unparalleled opportunity to map the spatial activity following microbial or stress damage or via lenticels or leaf
domain of individuals in situ. Such systems can therefore scars. Studies have indicated that pioneer species such as
provide a superb experimental system and conceptual Stereum gausapatum, Phlebia rufa, Phellinus ferreus, Exidia
framework for mainstream ecology. Competitive interactions glandulosa, and Vuilleminia comedens in oak or Daldinia
dominate fungal communities within wood (Boddy 2000). concentrica, Hypoxylon rubiginosum, and Peniophora
Consequently, distinct territories prevail and are delineated limitata in ash, can colonize living or recently dead wood.
within the wood by the production of antagonistic reactions The host tree may instigate a response to this invasion, by
such as pigmented zone lines, and colored activity domains accelerating localized premature heartwood tissue formation,
indicated by different types of decay. Moreover, careful which contributes to restriction of the invading front. The
examination of wood sections may also reveal relic zone lines identification of massive decay columns comprising a single
indicating the former occupants of woody domain. The individual extending for several meters along branches known
community structures of colonized wood at various stages of to have been dead for a single growing season only, indicated
decay have been mapped and the features affecting the involvement of latent invaders (see “Endophytes”)
community development have been studied, using an astute initially distributed within functional sapwood as “dormant”
but simple method based on these principles (Boddy 1992; hyphal fragments or propagules. Mycelial growth and
Rayner and Boddy 1988). Essentially the technique involved colonization would then activate from multiple inoculum
sectioning wood, and recording the delineated boundaries of sites, following stress alleviation for the fungus (usually
spatial domains occupied by individuals, prior to their drying sapwood), imposed by stress aggravation for the host.
isolation onto agar media. Isolates were identified and Ramets of an individual genet could then grow and unite by
somatic compatibility of Basidiomycota and Ascomycota anastomoses thereby forming extensive decay columns
assessed using pairing techniques to map the presence of apparently occupied by a single individual. Secondary
individuals. Interspecific pairing studies were used to assess invaders such as Coriolus versicolor, Phlebia radiata,
antagonism and to rank species within a hierarchy of Sterium hirsutum, and Peniophora lycii in oak, and
combative ability, to aid inference regarding the order of Radulomyces confluens in ash, could invade and replace
colonization and replacement (succession) of individuals. pioneers in already dead or decaying wood.
Thus, a 3D map may be constructed by aggregating Decomposing dead wood gradually releases sources of
information from serial wood sections. This basic method nutrients (Harmon et al. 1994). Exposed stumps of felled
has also been complemented with other studies involving trees, fallen branches and twigs, or cut timbers may become
manipulation of drying regime, temperature, water potential, rapidly colonized by large numbers of individuals of fairly
and gaseous environment. The approach has been used to nonselective saprotrophs, thereby forming numerous smaller
study the community structure of attached branches and twigs decay columns. Community structure and development is
of oak (Quercus petraea and Q. robur), Ash (Fraxinus affected by the degree of exposure, contact with the ground or
excelsior), beech (Fagus sylvatica), and birch (Betula with other wood, and is influenced by microenvironmental
pendula) (Boddy 1992; Griffith and Boddy 1991a; Rayner conditions and the arrival mode of individuals. Exposed
and Boddy 1988). The majority of wood-decay studies have surfaces may be colonized by established air-borne spores of
been concerned with basidiomycetes from early stages of Coriolus versicolor, Bjerkandera adusta, Stereum hirsutum,
decomposition, and with ascomycetes, moulds, and zygo- Chondrostereum purpureum basidiomycetes or ascomycetes
mycetes particularly from later decomposition stages in the genus Hypoxylon or Xylaria commonly on hardwoods.
Wood-Decay Fungi in Forest Ecosystems 383

Such a mode of establishment often produces slower considered when interpreting survey data. However, careful
expansion of decay columns compared to that from ground experimental design and statistical treatment of such data can
contact, presumably due to the more stressful drying regimes. provide the ecologist with a useful approach to investigating
Buried or ground contact wood may be colonized by soil- factors influencing fungal community dynamics and decay at
derived spores, mycelia, or cords. Later decay stages may the forest ecosystem scale.
involve Mycena galericulata and Pluteus cervinus on Microclimate, substrate quality determined by host species
hardwoods, or Tricholomopsis rutilans and Paxillus and decay stage, and forest history are cited as being the most
atrotomentosus on conifers. On very wet, well-decayed significant determinants of fungal community structure in
wood Dacrymycetales such as Dacrymyces stillatus, or decaying wood (Lindblad 1998; Lumley et al. 2001; Rayner
discomycetes such as Mollisia cinerea may occur. and Boddy 1988; Sippola and Renvall 1999; Vogt et al. 1992;
That different individuals produce different types and rates Zhou and Hyde 2001). Other factors affecting species
of decay is now evident (Worrall et al. 1997). Furthermore, compositions include soil chemical properties (Ruhling and
the spatio-temporal combination of extracellular enzymes Tyler 1990), and vegetation type (Wasterlund and Ingelog
secreted by a fungus is dependent not only on its evolutionary 1981). Furthermore, the initial heterotroph community (bark
heritage, but also on the local environmental and biotic beetles, ambrosia beetles, moulds, or decay fungi) has been
conditions (Griffin 1994; White and Boddy 1992a,b). Few found to influence decomposition or carbon flux in freshly cut
studies have related community structure and development Douglas fir (Progar et al. 2000). Multivariate analysis of
with the decomposition process itself. This seems to be a little survey data, such as detrended correspondence analysis
imprudent as both are obviously intricately linked. Notable (DCA), can indicate the relative importance of these variables
exceptions (Coates and Rayner 1985a,b) have demonstrated for community structure and development within forest
that inoculation via large numbers of individuals produce ecosystems. In dead-wood, decay state and microclimatic
slower net decay rates when compared with that for low spore stress have been identified as most influential with some
loads, probably due to expression of alternative metabolic impact of soil conditions (Heilmann-Clausen 2001). Cluster
pathways related to antagonism between numerous small analysis and ordination of microfungus communities in white
domains. However, several authors have observed enhanced spruce (Picea glauca) and trembling aspen (Populus
decomposition rates by mixtures or sequences of fungi tremuloides) fallen logs in disturbed and undisturbed boreal
(Boddy et al. 1989; Deacon 1985). woodland sites revealed tree species to be most influential,
followed by stage of decomposition and moisture content
5.3.3 Community Studies at the Forest Ecosystem (Lumley et al. 2001). From the foregoing, the importance of
Scale appropriate moisture regime for decay community develop-
ment is evident, indeed particularly wet forests have revealed
Direct examination of twigs, branches, and logs for fruiting relatively depressed respiration and decomposition rates
structures is a traditional method of surveying for (Progar et al. 2000). Community analysis can also suggest
basidiomycete and ascomycete activity. But the flora detected general trends relating to species diversity and community
using this approach is often very different to that isolated by development. Communities develop relatively quickly and
plating wood portions from interior wood regions. Inferences predictably at early decay stages, but become slower and
regarding community dynamics if based on sporophore more diversified at later stages as the impact of fluctuating
surveys should be made with extreme caution as their microclimatic stress become more significant due to
appearance may bear little relationship to the arrival, activity increasing wood porosity (Heilmann-Clausen 2001). Bio-
or decline of the supporting mycelia and the relationship will diversity is greatest in undisturbed deadwood, and tends to
vary for different individuals. Thus, the time taken for the increase as decay proceeds (Lumley et al. 2001; Norden and
mycelium to derive sufficient resource to devote to Paltto 2001; Renvall 1995). Moreover, some late-stage decay
sporophore production may be one or more seasons but may species may be prone to local extinction due to strict
be evident only temporarily and the active mycelium of some successional associations formed between certain species
fungi may not produce sporophores until their final stages of (Niemelä et al. 1995).
colonization. Some, such as Ganoderma and Fomes produce The DCA of sporophore data from decomposing conifer
perennial fruit bodies, which may survive for several years, trunks in northern Finland indicated the development of
whereas others may produce seasonal and/or ephemeral fruit regular successions of wood-decay fungi. Differences
bodies. For example, soft-rot fungi such as Chaetomium spp. observed among successions were due to associations with
are prevalent colonizers frequently only detected via plating the prevailing microclimate and dependent on the resource
techniques. Similarly, the dominant ectomycorrhizal species capture strategies and combative ability of individuals
Tylospora fibrillosa in a Sikta spruce plantation was not (Renvall 1995). In short, environmental stress created
detected in sporophore surveys, and species indicating most succession pathways for specific saprotrophic groups. Such
abundant fruiting represented only a very small abundance in an inference is very much in accordance with the concept
association with sampled roots (Taylor and Alexander 1991). presented by Cooke and Rayner (1984), that community
Therefore the phenology (climate and time of appearance) development pathways are initiated under varying degrees of
and abundance of fruit-bodies for different species must be high abiotic stress and/or low stress conditions following
384 White

disturbance and is subsequently directed by four influences; community has been indicated by production of conspicuous
disturbance, stress aggravation, stress alleviation, and epigeous fruit bodies (Dahlberg et al. 1997; Peter et al. 2001).
intensification of combat (Boddy 1992). Sampling plot size, frequency, and distribution are also
likely to influence the data set, as different ecological groups
will operate over a range of spatio-temporal scales (Newton
and Haigh 1998; Schmit et al. 1999). Indeed, it has been
6 FUNGAL DIVERSITY, ENVIRONMENTAL suggested that different ecological guilds would require
CHANGE, AND CONSERVATION differently sized and spatio-temporally distributed sampling
protocols (Villeneuve et al. 1991). To compound such
Biodiversity is generally considered to be fundamental to problems, Tofts and Orton (1998) declared that 21 years of
ecosystem functioning and stability. Thus, if this tenet is recording species accumulation was insufficient to reliably
extrapolated to fungi in forest ecosystems, fungal biodiversity estimate (extrapolate) the fungal biodiversity of the site, as
is critical to global geochemical cycles. A well-cited global the species accumulation-time curve had not yet started to
estimate for fungal biodiversity is in the range of 1.5-M plateau. At a practical level, the quality of a data set is even
species (Hawksworth 1991; 2001). However, at a local level, dependent on the expertise of the collector (Straatsma et al.
appropriate monitoring of fungal diversity can identify 2001). However, sporophore surveys may be justified as a
ecological and climatic trends, provide a database for spatio- suitable method for warning of species disappearance due to
temporally predictive biodiversity models, and inform detrimental external factors within a site, as evidence
appropriate conservation strategies at both the regional and suggests that fruiting may be more sensitive to environ-
global scale (Arnolds 1997). mental stress than survival of the supporting mycelium
Most local diversity studies are based on survey of itself (Termorshuizen and Schaffers 1991).
conspicuous fruit bodies of higher fungi, either represented as Despite these limitations evidence accrues, particularly
presence, number, or biomass within a plot. Diversity studies from Europe and more recently the United States, suggesting
of ephemeral and sometimes sensitive fruit bodies will only that fungal biodiversity is in decline. The likely causes of
represent a partial account of the resident fungi. Nevertheless, decline are due to habitat loss and/or pollution. Harvesting of
such studies are often valid because of their ease of (usually wild edible mushrooms is believed to have little detrimental
nondestructive) analysis over extensive sampling domains. effect on fungi, except where collection has involved
Fruit body surveys are further justified because of the damaging or exhausting the mycelium, or trampling or raking
ecological significance of spore dissemination for the the soil (Arnolds 1995). Nevertheless, the environmental
establishment of new genets, and due to the involvement of impact of large-scale commercial harvesting remains a
sporocarps in soil mineralization processes owing to contentious issue. Fungal habitat may be lost, with implicit
developmental regulation of extracellular enzyme activities reduction of fungal species diversity, either by deforestation,
(Ohga et al. 1999). Moreover, the fruit bodies of many species or because of commercial forestry management practices,
are a valuable edible resource. However, sporocarp such as the conversion to less-mixed or monoculture
inventories of species with varying resource allocation to plantations, stand felling of a particular age, and the removal
reproduction, especially for comparison of taxonomically and of course woody debris (Fridman and Walheim 2000; Høiland
ecologically different groups, are fallible as a bioindicator of and Bendiksen 1996; Lindblad 1998; Norden and Paltto 2001;
diversity. Thus, ruderal strategists are generally likely to fruit Ohlson et al. 1997; Straatsma et al. 2001). Red-List
more frequently and transiently than combative strategists. (endangered, vulnerable, or rare) species may be particularly
Moreover, sporocarp productivity of different species are sensitive, often showing a preference for large diameter logs
known to vary spatially (clustered or dispersed and associated in late decay stages, of which managed forests are largely
with distributed substrata), temporally (yearly, seasonally or deficient (e.g., Humphrey et al. 2000; Kruys et al. 1999).
weekly), can mature at differing rates and persist for varying Pollutants such as lead, sulfur dioxide, ozone, and oxides
time periods (Egli et al. 1997; Vogt et al. 1992). Fruiting of nitrogen (direct or as acid precipitation) are known to affect
may also be influenced by environmental factors such as biodiversity (Wainwright and Gadd 1997). Ectomycorrhizal
temperature and precipitation, natural and artificial amend- communities are particularly threatened by pollutants such as
ments to soil and by local or nonlocal ecological disturbance sulfur dioxide and NHx, probably due to forest soil
(Straatsma et al. 2001; Watling 1995). In the case of certain acidification and nitrogen input, thereby rendering any
mycorrhizal fungi fruit body development is affected by host potential mycorrhizal association less advantageous to the
genotype, age, provenance, edaphic factors, climatic factors, host plant community (Arnolds 1995). Elevated sulfur
and supporting photosynthetic activity (Dix and Webster dioxide levels are known also to affect the phylloplane
1995). The relationship between individual visible fruiting community, although some pigmented species may be less
structures and mycelium activity within substrata is therefore susceptible, thereby favoring their population growth (Magan
varied and complex, and considerable disparity has been 1993). The resulting impact on community interactions may
identified between the two in numerous studies (Cotter and have implications in the dynamics between pathogens and
Bills 1985; Gardes and Bruns 1996; Yamada and Katsuya saprotrophs and the consequent natural limitation of plant
2001). Notably, only about half of the ectomycorrhizal diseases, and even on the subsequent decay rates of litter and
Wood-Decay Fungi in Forest Ecosystems 385

hence nutrient cycling (Newsham et al. 1992). Similarly, high or processes driving ecological systems. Thus, the biological
levels of radioactivity occurring around the Chernobyl site processes that regulate and generate patterns of organisation
have produced altered fungal community structures in fungal communities may be elucidated through the
(Zhdanova et al. 1994). understanding of the interactive and feedback control steps
Considering the absolute and relative diversity, funda- involved within a working theoretical model. So, as
mental ecological importance, some host-specificity and Moorhead and Reynolds (1992) reasoned, “the process of
-exclusivity, and established and potential biotechnological developing a model tests our understanding of the system,
significance of fungi (Hawksworth and Colwell 1992; identifies areas of uncertainty and, importantly, provides a
Oberwinkler 1992), their limited conservation seems means of examining alternative conceptual frameworks.”
unjustified and surprising. For example, only four fungal Furthermore, mycology may offer important systems to study
species are protected by UK law, comparing pitifully with and test the suitability of general ecological theories and laws
over a hundred flowering plants (Marren 2001). usually developed for determinate organisms, for application
to indeterminate biological behavior. Rare examples of this
approach include consideration of the ecological strategies of
individual fungi (Andrews and Harris 1986; Boddy 1992),
7 MODELING FUNGAL DECAY and testing the relevance of Island theory, and the Species-
COMMUNITIES area curve, to certain fungal communities (Andrews et al.
1987; Newton and Haigh 1998; Wildman 1987).
From the foregoing it is evident that significant limitations The majority of natural environments display both spatial
exist in both field- and laboratory-based studies of and temporal heterogeneity or patchiness in terms of both
wood-decay fungal communities. Moreover, much of the abiotic and biotic factors, which may profoundly effect the
information reported is qualitative and at best semi- functioning and development of fungal communities (Ritz
quantitative. A complete understanding of the natural and Crawford 1999). So models that address environmental
environment based on observation alone is probably heterogeneity are the most likely to produce realistic theory.
unattainable due to its complexity, the appreciation of Certain distinctive biological attributes displayed by the
which may be approached in several ways. Conventional fungal form should also be incorporated into any reasonable
methods often involve an empirical or field based approach to theoretical model. Features such as indeterminacy, inter-
investigate the activities of individual decay fungi and their connectedness, variation, and versatility (genotypic and
temporal relationship with the abiotic and biotic environment. phenotypic plasticity), all contribute to varying degrees and
These may be analyzed statistically or further complemented at different scales, to the success of filamentous fungi within
with laboratory experiments. More rarely, fungal ecological the natural heterogeneous environment. As indeterminate
investigation may involve the development of conceptual and life-forms the growth of filamentous fungi is potentially
experimental models based on general ecological theory unlimited, unlike that of determinate life forms such as
(Carroll and Wicklow 1992). The use of conceptual unicells and animals, which possess genetically programmed
representations or models has been instrumental in expanding limits in both space and time. However this statement may be
the boundaries of our knowledge and understanding of the an oversimplification of the fact, as the longevity of
natural environment. A model in its broadest sense is a partial, indeterminate individuals and species does vary. Ruderals,
simplified version of a real entity or system. The main value for e.g., may persist only briefly for a few weeks, whilst some
of models is that they allow us to first represent and then make such as Ganoderma and Armillaria are renowned to persist
measurements and predictions that would be otherwise for years. Nevertheless, the indeterminacy displayed by
awkward. Theoretical or mathematical models based on eucarpic fungi facilitates success within a heterogeneous
experimentally observed characteristics of fungi are increas- environment, as hyphae have the potential to temporally
ingly being used, for e.g., in formulating hypothesis about sustain or spatially extend great distances, often spanning
their population dynamics and epidemiology (which will not inhospitable conditions in time or space to encounter new
be considered here and the reader is directed to Worrall 1999). resource bases. Indeterminacy may generate versatile
A particular advantage of linked models is the interplay responses to environmental unpredictability in a way that
between quantitative experimental data and theoretical determinate organisms can only engage with by way of
predictions. It is therefore possible to test and validate the evolutionary selection or social behavior.
theoretical model by predicting the result of, for e.g., Indeterminate and modular life forms show some
changing an environmental factor in the experimental system analogies and have sometimes been treated as theoretically
and then observing whether the predicted result occurs. equivalent (Trinci 1978; Prosser 1994). However, modular
The development of theoretical or mathematical models to organisms develop by repeated addition of the same
describe any real ecological system will always be organizational unit to a pre-existing one, whereas mycelia
contentious, given the inevitable simplification or reduction are nonadditive as they operate as dynamic flow systems with
of the system inherently required by such an approach. branching, anastomosing and radiating components com-
However, appropriate theoretical models should allow municating at different scales and to varying degrees (Rayner
inferences to be made regarding the underlying mechanisms 1996). Therefore, the temptation to apply modular models,
386 White

which incorporate iterative steps at a single reference scale, to generic spatially explicit model adopting a process-based
indeterminate systems, should be considered with caution. approach, in which the fungal individual is defined by a set of
The challenge will be to develop modeling frameworks that measurable traits that describe physiological processes such
can link biological processes operating over multiple scales as nutrient uptake, redistribution and growth (Figure 2). Each
and with varying constancy, to large-scale or ecosystem individual may be described by a characteristic set of traits
behavior. Developing and therefore understanding this and therefore community diversity may be accommodated
complexity would make a major contribution to both fungal within the model. Being spatially explicit where biomass is
and general ecoevolutionary theories. located in cells on a discrete spatial grid, the model permits
regions of mycelia to interact within a neighborhood and
change according to the local environment and context. Thus,
7.1 Modeling the Growth of Hyphae and Mycelia the ethos of the modeling framework is that any system can be
described by a set of processes that can approximate the
Many mycological models have attempted to represent and system. By linking these processes to parameters that may be
explain the growth and development of hyphae and mycelia in measured experimentally (physiological traits), a mechanistic
relation to their abiotic environment. The reader is directed to understanding of those parameters influencing the overall
Ritz and Crawford (1999) for a review of some of the organisation of the system can be obtained.
experimental approaches and mathematical models devel-
oped to date, and so these will not be considered in any detail
here. Most have focussed on the foraging and space-filling 7.2 Modeling Fungal Community Dynamics
properties of mycelia developing on resources distributed
discretely or as gradients. Reaction-diffusion models have Modeling approaches for fungal communities, have received
been derived to reflect hyphal growth and branching far less attention, perhaps because this is a most ambitious
(Regalado et al. 1996) and different colony morphologies project, summed-up by Frankland (1998) as “unravelling the
(Davidson et al. 1996). More recently a simple stochastic unpredictable.” Indeed, the work of Halley et al. (1996)
model accommodating some biological processes (inhibition represents one of the few attempts to model interactions
by toxic metabolic products and nutrient uptake) was able to within a multispecies fungal decay community. In this work, a
reproduce a variety of fungal growth patterns observed on computer based simulation model known, as a “cellular
solidified media (Lopez and Jensen 2002). However, there is a automaton” was developed to predict the decomposition of
paucity of theoretical models that accommodate environ- wheat straw by four saprotrophic fungi. The model was based
mental topography, translocational source –sink relationships, on real experimental data obtained by Robinson et al. (1994)
and exploitation vs. exploration growth morphs, either during studies on resource capture by interacting fungal
individually or in combination. In an attempt to address colonisers of straw. Although this model was capable of
these issues, work is currently being undertaken to develop a reproducing some of the behaviors exhibited during the

Figure 2 (a) Image showing the underlying soil pore space derived from real data using a CAT scan—with white to black scale
representing pore to solid respectively. (b) Image showing the resulting fungal biomass gradient distribution obtained from the theoretical
model with the mycelium growing from a single point and resource (bottom right hand corner) with white to black scale representing high
to low biomass, respectively. The model is based on a set of biological processes, intended to characterize any fungal individual. By
linking these processes to parameters that can be measured experimentally, a mechanistic understanding of which parameters are
influencing the overall organization of the system can be obtained (Falconer R, Bown J, Crawford JW, and White NA, unpublished data).
Wood-Decay Fungi in Forest Ecosystems 387

experimental study, validation of the model was only possible because individuals in a group or patch behave differently to
on a qualitative basis. Furthermore, the model admittedly individuals that are isolated. Processes such as modification of
ignored important aspects of the fungal community such as the environment, resource translocation and hyphal network-
sporulation and the coordinated behavior of the mycelium. ing or anastomosis (Rayner 1996) may all influence
More recent modeling studies have attempted to examine community development. Issues relating to scale have been
and accommodate the complex and coordinated behavior a central problem in ecology over the last fifty years.
exhibited by indeterminate mycelia. A stochastic cellular Nevertheless, the importance of appreciating the relationships
automaton for modeling the dynamics of two-species across different scales should be emphasized, as under-
microcosm communities of differing patch size, revealed standing and predicting large-scale ecosystem events, will
the significance of local and nonlocal interactions in have origins in and consequences for fine-scale phenomena
generating the emergent behavior of mycelial systems (Levin 1992). Therefore, in the development of a complete
(Bown et al. 1999). Importantly, the experimental system understanding of fungal ecology it is necessary to address
allowed for detailed quantitative spatial analysis of the issues relating to scale and to adopt a hierarchical framework
community. This revealed that despite the predictability of the (Allen and Hoekstra 1992; Swift 1976).
final interaction outcome irrespective of patch size, the finer- In vitro mycelial interaction studies, particularly those
scale dynamics were highly dependent on non-local evident on agar media, have often formed the basis of
interactions (Bown et al. 1999; Sturrock et al. 2002). understanding or predicting ex situ fungal community
Furthermore, experimental studies indicate that fungi dynamics. Furthermore, these have often centred on the
occupying large domains commonly display a higher pair-wise interaction between a limited number of individuals
combative success compared to those occupying smaller under experimentally defined environmental conditions
domains when challenged by the same species, and in vitro (Boddy 2000). Data produced from combative interaction
that younger mycelia may be less combative than more studies are often used to rank species in order of their
mature growth (Holmer and Stenlid 1993; Stahl and combative ability, and hence to indicate sequences of fungal
Christensen 1992). Consequently, despite the benefits of in colonisation in the field. Correlation between antagonistic
vitro interaction studies in understanding the factors that behavior in artificial culture and in the natural environment is
influence the community dynamics of fungi, a major mixed, and may be related to the spatial or temporal scale at
limitation exists in how such small-scale studies relate to which the data are collected or aggregated. Nevertheless, such
the behavior at larger community scales. At larger scales it is studies represent a valuable approach to understanding fungal
probable that contrasting emergent behaviors may arise ecology, providing that any limitations are recognized,

Figure 3 (a) – (c) Example maps showing the spatial distribution of fungal species in a 3 £ 3 (equal proportion) tessellated agar tile
interaction array at the onset of the experiment. Bold lines denote air-gaps between individual tiles. Dimension of each tile is 1 cm2.
Symbols indicate the species inoculated onto each tile. (d) – (f) Plots of first and second principal components from analysis of interface
classes and state transition classes of tessellated agar tile arrangements (a) – (c). S ¼ 0 week, A ¼ 1 week, K ¼ 3 weeks, W ¼ 5 weeks
incubation at 158C. From Sturrock et al. (2002).
388 White

qualified and considered. According to general ecological have been studied at a range of spatio-temporal scales. The
terminology combative hierarchies may be either transitive future challenge will involve linking information derived at a
(following a strict linear order of hierarchy) or intransitive (no range of scales in an appropriate manner. Theoretical
strict linear hierarchical order) (Boddy 2000). Consequently, modeling approaches have value here, the development of
predicting the interaction outcome of more than two species which can highlight the important biological features driving
may become extremely difficult, if not impossible, if the community dynamics and identify further hypotheses for
species under investigation display an intransitive hierarchy testing. Such models may one day even have predictive value,
(White et al. 1998). Thus, the expansion from two-species an important asset in view of changing management
interactions to three or more species may allow for the practices, land use, climate, and other environmental
emergence of “higher-order interactions” involving greater pressures. However, future experimental studies and theo-
complexity and even different biological phenomena, often retical models should attempt to link fungal diversity,
resulting in shifts in interactive functioning (Culver 1992). community structure, and dynamics to function, either in
Furthermore, the initial spatial configuration of three-species terms of nutrient cycling or biological control. The fungal
communities of equal inoculum and patch size, has been ecologist is faced with challenging and exciting opportunities
shown to impact on community development and reprodu- to study what is one of mankind’s most valuable assets.
cibility, and may contribute to the persistence of individuals
which would otherwise be expected (based on binary
pairings) to be eliminated from the system (Figure 3;
Sturrock et al. 2002). In addition, fungal individuals can often ACKNOWLEDGEMENTS
display a variety of responses when subjected to ostensibly
the same set of conditions (Figure 3; Halley et al. 1996; Thanks to the University of Abertay Dundee and to my
Rayner et al. 1995; White et al. 1998). Such stochasticity may colleagues Jim Bown, Ruth Falconer, and Craig Sturrock
be interpreted as offering the mycelium an ecological (Abertay) and to Lynne Boddy (Cardiff) for reading and
advantage within a dynamic and unpredictable heterogeneous advice on the manuscript.
environment. The complex stochastic and sensitive nature of
community interactions is particularly evident within
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33
The Biodegradation of Lignocellulose by White Rot Fungi

Gary Ward* MIGAL-Galilee Technology Center, Kiryat Shmona, Israel

Yitzhak Hadar The Hebrew University of Jerusalem, Rehovot, Israel

Carlos G. Dosoretz Technion-Israel Institute of Technology, Haifa, Israel

1 INTRODUCTION protected from degradation and as a result their bioavail-

ability is very low. Lignin degradation, therefore, plays a
Lignocelluloses are defined as plant or wood cell walls in central role in the earth’s carbon cycle, since most renewable
which celluloses and hemicelluloses are intimately associated carbon is either in lignin or in cellulose and hemicellulose.
with lignin. The role of lignin is to provide strength, serve as This chapter will focus on the biodegradation of lignin by
barrier against microbial attack, and act as water impermeable fungi, with particular emphasis on white rots, which are the
seal across the cell walls in the xylem tissue (Argyropoulos most efficient degraders and the only known organisms that
and Menachem 1997). Lignin is a phenylpropanoid polymer can completely breakdown lignin to carbon dioxide (CO2)
synthesized from the phenolic precursors, coniferyl, synapyl, and water (H2O) (Kirk and Farrell 1987).
and p-coumaryl alcohols (Sarkanen and Ludwig 1971). Free
radical condensation of these precursors initiated by plant cell
wall peroxidases results in the formation of a heterogeneous, 2 BROWN ROT AND SOFT ROT FUNGI
amorphous, optically inactive, random, and highly branched
polymer with at least 12 different types of linkages such as Only a few organisms are capable of degrading the aromatic
aryl–ether and carbon –carbon bonds connecting the aromatic polymer lignin, the most efficient of which are fungi. Three
nuclei. Such structural features impose unusual restrictions on groups of fungi are capable of lignin degradation (Eriksson
its biodegradability (Hatakka 1994; Higuchi 1990; Kirk and et al. 1990): White rot, brown rot, and soft rot fungi (Table 1).
Farrell 1987). Most biological macromolecules such as Brown rot fungi belonging to the basidiomycetes
cellulose are largely linear polymers whose subunits are extensively degrade cell wall carbohydrates and only modify
linked together by a repeating bond, and thus the mechanism the lignin (Eriksson et al. 1990). Demethylation is the most
of polymer degradation is generally centered around the obvious consequence of attack on lignin by these fungi. The
common bonds. The complexity of the lignin polymer means, brown rot fungi grows mainly in the cell lumen next to the
however, that it is not subject to enzymatic hydrolysis and the secondary wall and cause a generalized, diffuse rot (Blanchette
initial attack must be oxidative, nonspecific, nonhydrolytic, 2000). The residual wood is brown and often cracks into
and extracellular (Hatakka 1994; Higuchi 1990; Kirk and cubical pieces when dry. Brown rot fungi have an obvious
Farrell 1987). Since lignin is particularly complex to preference for coniferous substrates (gymnosperms), which
biodegrade, the cellulose, hemicellulose, and other cell wall are softwoods. A survey of substrate relationships reported that
constituents with which it is intimately associated are 19% of North American basiodiomycetes are brown rot fungi,

*Current affiliation: Technion-Israel Institute of Technology,

Haifa, Israel

393
394 Ward et al.

Table 1 Lignin degrading fungi, their actions, and distribution

Organism Subdivision Examples Actionsa Distribution

White rot Basidiomycetes Phanerochaete sp., Mineralize lignin to CO2 and H2O; Predominantly degrade wood
fungi Pleurotus sp., some species preferentially remove from deciduous trees
Bjerkandera sp., Trametes lignin (selective delignification) (angiosperms), containing
sp., and Phlebia sp. whereas others degrade lignin and hardwood
cellulose simultaneously
Brown Basidiomycetes S. lacrymans P. betulinus, Modify lignin by demethylation, Preference for coniferous
rot fungi G. trabeum, and limited aromatic hydroxylation, and substrates (gymnosperms),
P. placenta ring cleavage which are softwoods
Soft rot Ascomycetes, Chaetomium sp., Some lignin modification Active generally in wet
fungi Deuteromycetes Ceratocystis sp., and environments as well as in plant
Phialophora sp. litter; attack both hardwood and
softwood
a
All groups degrade cellulose and hemicellulose that serve as actual carbon and energy source.

among which 60 out of 71 (85%) occur primarily on conifers, source of carbon and energy by white rot fungi. Degradation
and that they are mainly softwood degraders (Gilbertson of lignin enables them to gain access to cellulose and
1980). They commonly cause decay of timber in buildings hemicellulose, which serve as their actual carbon and energy
(Blanchette 2000). One of the most destructive brown rot source. White rot fungi predominantly degrade wood from
fungi is Serpula lacrymans, which is well adapted to attacking deciduous trees (angiosperms), containing hardwood. In a
timber in service and can spread rapidly on wood and survey of 65 central European wood-decaying basidiomy-
traverse non-nutritional surfaces. Commonly, this type of cetes: four were reported to only attack coniferous wood, 34
decay has been referred to as dry rot. This term, apparently first attacked angiosperms exclusively, and 27 attacked both
used to describe any deterioration of dead wood or wood in (Rypacek 1977).
service is misleading because moisture must be present for the The many species that cause white rots are a hetero-
decay to occur. geneous group that may degrade greater or lesser amounts of a
Soft rot fungi are taxonomically classified in the specific cell wall component. Some species preferentially
subdivisions, Ascomycota and Deuteromycota. They are active remove lignin from wood, leaving pockets of white degraded
in environments that are too severe for white- or brown-rot cells that consist entirely of cellulose. This is referred to as
fungi, generally in wet environments, but they also decompose selective delignification (Blanchette 1995; Eriksson et al.
plant litter in soils (Blanchette 1995). Soft rots are relatively 1990). Other species degrade lignin and cellulose simul-
unspecialized cellulolytic fungi in the genera Chaetomium, taneously which is referred to as nonselective delignification.
Ceratocystis, Phialophora, etc., that readily degrade cellulose Among the best-studied white rot fungi are Phanerochate
and hemicellulose, but only modify lignin. They penetrate the chrysosporium and Phlebia radiata, which degrade lignin
secondary wall of the wood cell, forming cylindrical cavities in selectively, and Trametes versicolor which degrades lignin
which the hyphae propagate. The rot is of limited extent, being nonselectively. There are fungi such as Ganoderma
closely associated with the fungal hyphae, because the applanatum and Heterobasidion annosum, which are capable
cellulase enzymes do not diffuse freely through the wood. of both forms of degradation (Blanchette 1995; Eriksson et al.
Two distinct types of soft rot are currently recognized 1990). The ratio lignin –hemicellulose –cellulose decayed by
(Blanchette 2000). Type I is characterized by longitudinal a selective fungus can differ enormously and even different
cavities formed within the secondary wall of wood cells and strains of the same species, e.g., of P. chrysosporium and
Type 2 results in erosion of the entire secondary wall. The Ceriporiopsis subvermispora, may behave differently on the
middle lamella is not degraded in contrast to cell wall erosion same kind of wood.
by white rot fungi, but may be modified in advanced stages of White rot fungi typically colonize the cell lumen and cause
decay. As decay progresses, extensive carbohydrate loss cell wall erosion. Degradation is usually localized to cells
occurs and lignin concentrations increase in the residual wood. colonized by fungal hyphae and substantial amounts of
undecayed wood remains. Progressive erosion of the cell wall
occurs when components are degraded simultaneously during
3 WHITE ROT FUNGI nonselective delignification and eroded zones coalesce as
decay progresses forming large voids filled with mycelium.
White rot fungi are the only known organisms that can During selective delignification a diffuse attack of lignin
completely break down lignin to CO2 and H2O (Kirk and occurs and white pocket or white-mottled type of rot results
Farrell 1987). However, lignin cannot be degraded as a sole (Blanchette 1991; 2000).
The Biodegradation of Lignocellulose by White Rot Fungi 395

Electron microscopy studies have revealed that lignin is ligninolytic reactions in vivo has been a long-standing
degraded at some distance from the hyphae and is removed difficulty for mechanistic studies (Crawford 1981).
progressively from the lumen towards the middle lamella, Dimeric model compounds that represent the principal
which is also degraded (Blanchette 1984; Blanchette et al. substructures of lignin have been used successfully to
1987). Sheaths, often composed of b-1,3-glucans, appear to characterize the ligninolytic systems of white rot fungi.
be produced during early stages of wood colonization and Models of this type provided some of the first evidence that
facilitate its degradation (Blanchette et al. 1989; Nicole et al. P. chrysosporium and T. versicolor cleave the lignin isopropyl
1995; Ruel and Joseleau 1991). These hyphal sheaths may side chain between Ca and Cb (Crawford 1981). Dimeric
play an important role in transport and presentation of wood- models played a large role in revealing that fungal lignin
degrading enzymes from the hyphae during the decay process, peroxidase (LIP) cleaves lignin between Ca and Cb, which
thus establishing a point of attachment to the site of represent 7% of the linkages in the lignin polymer (Gold et al.
degradation. The association of peroxidases to the glucan 1989; Hammel et al. 1993; Kirk and Farrell 1987).
matrix is in favor of the role of the sheath as a supporting Furthermore, oxidation of a b-O-4 model compound, which
structure (Ruel and Joseleau 1991). Furthermore, the fact that represents 50–60% of the bonds within the lignin molecule
the sheath was hydrolyzed during the attack demonstrated its demonstrated that LIP can cleave the predominance of
active role both in providing the H2O2 necessary for linkages in lignin (Glenn et al. 1983; Tien and Kirk 1983a,b).
peroxidase activity and in providing a mode of transport for The main product is the corresponding benzaldehyde.
fungal enzymes to their substrates at the surface of the wood Dimeric models have also been used to detect LIP activity
cell wall. in situ in fungus-colonized wood, where extraction and
conventional assay of the enzyme is technically difficult
(Srebotnik et al. 1994).
3.1 Screening for Lignin Degradation by White Degradation of a (b-O-4)-(5-50 ) type trimer, arylglycerol-
Rot Fungi b-(dehydrodivanillyl alcohol) ether (I) by LIP showed that
during its degradation by the enzyme, Ca– Cb cleavage,
Lignin-degrading ability is commonly evaluated by measuring b-O-4 bond cleavage, and b-etherified aromatic ring (B-ring)
14
CO2 evolution from 14C-labeled lignin preparations, such as opening products were formed (Umezawa and Higuchi 1989;
14
C-ring-labeled dehydrogenation polymerizate (DHP). The Yokota et al. 1991). The results showed that the B-ring of
measurement of 14CO2 evolution is the most sensitive and substrate (I), which must be sterically hindered more than
accurate method for testing ligninolytic activity (Eriksson et al. those of arylglycerol-b-guaiacyl and arylglycerol-b-(2,6-
1990). The evolution of 14CO2 and the modification of DHP has dimethoxyphenyl) ethers was oxidized by LIP (Umezawa and
been employed for determining ligninolytic activity in many Higuchi 1989; Yokota et al. 1991).
white rot fungi as well as for elucidating the role of different Dimeric lignin model compounds have the disadvantage of
enzymes and other constituents in lignin degradation (Boyle low molecular weight. Unlike lignin, they can be taken up and
et al. 1992; CostaFerreira et al. 1996; Eggert et al. 1997; metabolized intracellularly by microorganisms, which can
Hatakka 1994; Hatakka and Uusi-Rauva 1983; Hatakka et al. make it difficult to determine whether the degradation
1983; Hofrichter et al. 1999; Perez and Jeffries 1992; Reid and products observed really reflect ligninolytic activity
Deschamps 1991; Sethuraman et al. 1999; Silva et al. 1996; (Crawford 1981). Ideally, lignin model compounds should
Umezawa and Higuchi 1989; Yoshida et al. 1998). Methods to be macromolecular like lignin, but to facilitate product
study the degradation of polymeric lignin such as nuclear analysis, they should have simpler structures than those of the
magnetic resonance spectroscopy have been developed (Davis natural polymer. For this reason dimeric lignin compounds
et al. 1994; Gamble et al. 1994), but they are not easily attached to a polymer backbone such as polystyrene and
amenable for detailed physiological studies with microorgan- polyethylene glycol have been developed (Kawai et al. 1995).
isms or biochemical studies with enzymes.
A simple and reliable screening procedure that dis-
tinguishes between fungi that cause decay by selectively
removing lignin and those that degrade both cellulose and 4 LIGNINOLYTIC SYSTEM OF WHITE ROT
lignin simultaneously has been developed involving staining FUNGI
of lignin with astra-blue, which stains cellulose blue only in the
absence of lignin, and safranin, which stains lignin regardless In order to degrade lignin, the white rot fungi have developed
of whether cellulose is present (Srebotnik and Messner 1994). an unspecific ligninolytic system consisting of peroxidases
and laccases (phenol oxidases; LAC), which degrade lignin in
an oxidative process (Hatakka 1994). The peroxidases are
3.2 Metabolic Studies heme-containing enzymes and require the presence of
hydrogen peroxide (H2O2) to oxidize lignin and lignin-
Unfortunately, the complexity of the lignin polymer makes it related compounds. Three types of peroxidases have been
difficult to study microbial ligninolysis. The lack of well- discovered in white rot fungi: LIP, manganese peroxidase
characterized model substrates that can be used to identify (MnP), and more recently versatile peroxidase (VP)
 396

Table 2 Enzymes and their roles in lignin degradation by white rot fungi

Enzyme Cofactors or mediators Role in lignin degradation References

LIP H2O2, 3,4-dimethoxybenzyl alcohol (veratryl Direct and mediated oxidation of phenolic and For review see Tuor et al. (1995)
alcohol), 3,4-dimethoxycinnamic acid, 1,2- nonphenolic lignin structures, respectively. This
dimethoxybenzene, and 2-chloro-1,4- results in cleavage of Ca – Cb, b-O-4 and aryl – Ca
dimethoxybenzene bonds, aromatic ring opening, hydroxylation, and
demethoxylation
MnP H2O2, Mn2þ, organic acids as chelators, and Mn2þ oxidized to Mn3þ; chelated Mn3þ oxidizes For review see Tuor et al. (1995)
unsaturated fatty acids phenolic lignin structures and subsequently,
alkyl – phenyl cleavage, Ca – Cb cleavage, or benzylic
carbinol oxidation may result; nonphenolic moieties
may be co-oxidized when MnP peroxidizes
unsaturated fatty acids
VP H2O2, veratryl alcohol, Mn2þ, organic acids as Still unknown Camarero et al. (1999), Mester and Field
chelators, and unsaturated fatty acids (1998), and Ruiz-Duenas et al. (1999,
2001)
LAC O2, 3-hydroxyanthranillic acid, and Phenolic lignin structures are oxidized to phenoxy For review see Tuor et al. (1995)
hydroxybenzotriazole radicals and subsequently, alkyl – phenyl cleavage or
Ca –Cb cleavage may result. Dimethoxylation of
several lignin model compounds has been witnessed;
nonphenolic lignin structures may be oxidized only in
the presence of mediators
CDH Use electron acceptors including quinones, phenoxy Reduces aromatic radicals preventing For review see Henriksson et al. (2000)
radicals, Fe3þ, and Cu2þ to generate lactones from repolymerization, demethoxylation, or hydroxylation
various substrates of nonphenolic lignin, and reduction of precipitated
MnO2
GLOX Glyoxal and methyl glyoxal Glyoxal oxidized to glyoxylic acid and concomitant Zhao and Janse (1996)
production of H2O2
AO Aromatic alcohols (anisyl and veratryl alcohol) Aromatic alcohols oxidized to aldehydes with Zhao and Janse (1996)
concomitant production of H2O2
Other H2O2 Many organic compounds O2 reduced to H2O2 Urzua et al. (1998) and Zhao and Janse
generating (1996)
enzymes
Ward et al.
The Biodegradation of Lignocellulose by White Rot Fungi 397

Table 3 Selective white rot fungi and their reported ligninolytic enzymes

Enzymes

Microorganism LIP MnP Lac VP References

P. brevispora U U U Arora and Gill, (2001), Perez and Jeffries (1990), and
Ruttimannet al. (1992)
P. radiata U U U Niku-Paavolaet al. (1988) and Vares et al. (1995)
P. tremellosa U U U Bonnarme and Jefferies (1990) and Hatakka et al. (1992, 1993)
I. lacteus U U U Novotny et al. (2000) and Rothschild et al. (2002)
B. adusta U U U U Heinfling et al. (1998a,b), Kimura et al. (1991), and
Nakamura et al. (1999)
Bjerkandera sp. strain BOS55 U U U U Mester and Field (1998), Mester et al. (1995), and
ten Have et al. (1998a)
T. versicolor U U U Dodson et al. (1987), Fahraeus and Reinhammar (1967), and
Johansson and Nyman (1987)
P. chrysoporium U U Couto et al. (1999), Glenn et al. (1983), Leisola et al. 1987, and
Tien and Kirk (1983b)
P. pini U U Bonnarme and Jefferies (1990)
T. pruinosum U U Waldner et al. (1988)
P. ochraceofulva U U Hatakka (1994) and Vares et al. (1993)
J. separabilima U U Vares et al. (1992)
P. ostreatus U U U Cohen et al. (2001), Sannia et al. (1986), and Waldner et al. (1988)
P. eryngii U U U Camarero et al. (1999), Heinfling et al. (1998b), Munoz et al. (1997),
and Ruiz-Duenas et al. (1999)
P. sajor-caju U U Bourbonnais and Paice (1989), Buswell et al. (1996), and
Fukuzumi 1987
D. squalens U U Perie and Gold (1991) and Perie et al. (1998)
L. edodes U U Forrester et al. (1990) and Leatham and Stahmann (1984)
G. lucidum U U D’Souza et al. (1999)
P. tigrinus U U Golovleva et al. (1993) and Leontievsky et al. (1994)
R. lignosus U U Galliano et al. (1991)

(Camarero et al. 1999; Mester and Field 1998; Ruiz-Duenas Bjerkandera adusta (Camarero et al. 1999; Mester and
et al. 1999; 2001). Laccases are multicopper phenol oxidases, Field 1998; Ruiz-Duenas et al. 1999; 2001), warrants
which oxidize phenols and aromatic amines. Rather than re-evaluation of the classification scheme. Selective white
H2O2, these enzymes utilize dioxygen (O2) as an oxidant, rot fungi and the ligninolytic enzymes that they produce are
reducing it by four electrons to water (Call and Mucke 1997). given in Table 3.
The enzymes involved in lignin degradation along with the
roles they play are given in Table 2.
White rot fungi have been classified according to the 4.1 Lignin Peroxidase
ligninolytic enzymes they express. Whereas Hatakka 4.1.1 Properties
suggested that they can be classified into three categories
(Hatakka 1994), Tuor et al. classified them into five categories Lignin peroxidase is secreted as a series of glycosylated
as follows (Tuor et al. 1995): (a) White rot fungi expressing isoenzymes with pIs ranging from 3.2 to 4.0 and molecular
LIP, MnP, and LAC, (b) white rot fungi simultaneously masses ranging from 38 to 43 kDa, with each isoenzyme
producing MnP and LAC, but not LIP, (c) white rot fungi containing 1 mol heme per mole of protein (Farrell et al. 1989;
producing LIP and either MnP or LAC, (d) white rot fungi Gold and Alic 1993; Leisola et al. 1987). It possesses a higher
reported to produce LIP without MnP or LAC, (e) a group redox potential and a lower pH optimum than that of any other
which is incompletely characterized and in which neither of isolated peroxidase or oxidase (Call and Mucke 1997;
the oxidative enzymes have been identified. Hammel et al. 1986; Kersten et al. 1990). Like other
However, the continual discovery of enzymes in different peroxidases, LIP is capable of oxidizing most phenolic
white rot fungi species means that classifications of this type compounds through the generation of phenoxy radicals.
are subject to ongoing changes. In particular, the recent However, due to its exceptionally high redox potential and
discovery of the novel VP in Pleurotus eryngii and low pH optimum, it is able to oxidize nonphenolic aromatic
398 Ward et al.

substrates, typically not oxidized by other peroxidases the physiological roles of which is believed to protect LIP
including the nonphenolic phenylpropanoid units of lignin from H2O2-dependent inactivation by reverting LIPIII to the
(Hammel et al. 1986; Hatakka 1994; ten Have et al. 1998b; native state. This is of particular significance, since during
Kersten et al. 1990). Stable cation centered radicals formed oxidation of certain chemicals such as phenols, LIPIII has
during the oxidation of nonphenolic aromatic nuclei may been shown to accumulate, indicating that they are either poor
serve as redox mediators for LIP-catalyzed oxidations, substrates for LIPII or they lack the ability to revert LIPIII to
effectively extending the substrate range. Reactions catalyzed the native state (Chung and Aust 1995; Harvey and Palmer
by LIP include benzyl alcohol oxidations, side-chain 1990). Thus, oxidation of such chemicals is inefficient at high
cleavages, ring-opening reactions, dimethoxylations, and H2O2 concentrations.
oxidative dechlorinations. The ability of LIP to attack such a
variety of linkages suggests that it plays a key role in lignin
degradation.

Veratryl alcohol has also been shown to act as a charge-

4.1.2 Catalytic Cycle transfer mediator in LIP catalyzed reactions (Goodwin et al.
1995; Harvey et al. 1986). During the catalytic cycle of LIP,
The catalytic cycle of LIP is similar to that of other
VA is oxidized to VA cation radical (VAþ†), which in the
peroxidases (Renganathan and Gold 1986; Tien et al. 1986).
presence of suitable reducing substrate is reduced back to VA
and ready for another LIP catalyzed charge-transfer reaction.
The roles played by VA in the catalytic cycle of LIP are
highlighted.

Reaction of native ferric enzyme [Fe-LIP; Fe3þ, P

(porphyrin)] with H2O2 yields LIP-compound I (LIPI) a Low molecular weight redox mediators such as VA are
complex of high valent oxo-iron and porphyrin cation radical believed to be essential for degradation of lignin, since
(Fe4þ ¼ O; P†þ). One-electron-oxidation of a reducing theoretical and electron microscopic studies have demon-
substrate (SH) by LIPI yields a radical cation (S†) and the strated that enzymes as large as peroxidases and LAC cannot
one-electron-oxidized enzyme intermediate, LIP-compound have direct contact with lignin, since they appear to be too
II (LIPII; Fe4þ ¼ O; P). A single one-electron oxidation of a large for the penetration of the cell wall or the middle lamella
second substrate molecule returns the enzyme to Fe-LIP (Call and Mucke 1997). Pine decayed by wood rotting fungi
completing the catalytic cycle. was infiltrated with a concentrated culture filtrate of the white
However, in the absence of suitable reducing substrate or, rot fungus P. chrysosporium and labeled for LIP by
at high H2O2 concentrations, LIPII is further oxidized by postembedding immunoelectron microscopy. This method
H2O2 to LIP-compound III (LIPIII; Fe3þ ¼ O2†2 ; P), a demonstrated that enzymatic attack on pine by LIP and
species with limited catalytic activity. presumably also other enzymes of the same size is restricted
to the surface of the wood cell wall (Srebotnik and Messner
1988).
Measurements of the stability of VAþ† have led to the
suggestion that it would be unable to act as a diffusable
Lignin peroxidase is unique from other peroxidases in that it oxidant without some form of stabilization (Candeias and
exhibits an unusually high reactivity between LIPII and H2O2 Harvey 1995; Khindaria et al. 1996). It was suggested that a
(Cai and Tien 1989; 1992; Wariishi and Gold 1990; Wariishi LIP– VAþ† complex acts as the redox partner, with the cation
et al. 1990). Since, LIPIII is inactivated rapidly in the radical being stabilized by a protein microenvironment of
presence of excess H2O2, if it is not rapidly reverted to the acidic character (Khindaria et al. 1996).
native state, the enzyme has a “suicidal” tendency. Nonphenolic aromatic compounds other than VA, such as
3,4-dimethoxycinnamic acid, 1,2-dimethoxybenzene, and
4.1.3 Role of Veratryl Alcohol During 2-chloro-1,4-dimethoxybenzene have also been shown to be
LIP-catalyzed Oxidation capable of mediating oxidation (Teunissen and Field 1998;
Ward et al. 2002). The mediation phenomenon appears to be
The LIPIII has been shown to readily return to the native driven by the difference in the oxidation potential (OP) and
ferric state in the presence of H2O2 and veratryl alcohol site-binding affinity of the mediators (possessing higher OP
(3,4-dimethoxybenzyl alcohol; VA) (Barr and Aust 1994; Cai values and higher affinity) and the target substrates
and Tien 1989; 1992; Wariishi and Gold 1990). Ligninolytic (possessing lower OP values and lower affinity) (Ward et al.
cultures of P. chrysosporium normally produce VA, one of 2002).
The Biodegradation of Lignocellulose by White Rot Fungi 399

4.2 Manganese Peroxidase defined medium has been demonstrated (Jensen et al. 1996).
Lipid peroxidation has been suggested as the mechanism
4.2.1 Properties involved in the oxidation of the nonphenolic lignin structures
Manganese peroxidase exists as a series of glycosylated by white rot fungi that do not produce LIP.
isozymes with pIs ranging from 4.2 to 4.9 and molecular
masses ranging from 45 to 47 kDa. Similar to LIP, each
4.3 Versatile Peroxidase
isozyme contains 1 mol of iron per mole of protein (Leisola
et al. 1987; Paszczynski et al. 1986). To date, five isozymes
have been detected in P. chrysosporium MP-1 (Kirk and A heme peroxidase different from other microbial, plant, and
Cullen 1998). animal peroxidases, termed VP has recently been discovered
in Pleurotus and Bjerkandera species (Camarero et al. 1999;
Mester and Field 1998; Ruiz-Duenas et al. 1999; 2001). The
4.2.2 Catalytic Cycle VP is characterized by catalytic properties of MnP and LIP.
The enzyme exhibits high affinity for Mn2þ, hydroquinones,
Manganese peroxidase, like LIP has the same catalytic cycle and dyes, and also oxidizes VA, dimethoxybenzene, and
as other peroxidases, involving a two-electron oxidation of lignin dimers (Ruiz-Duenas et al. 2001). Molecular models
the heme by H2O2, followed by two subsequent one-electron show a Mn2þ-binding site formed by three acidic residues
reductions to the native ferric enzyme. The primary reducing near the heme internal propionate accounting for the ability of
substrate in the MnP catalytic cycle is Mn2þ, which efficiently VP to oxidize Mn2þ (Ruiz-Duenas et al. 1999). Concerning
reduces compound I and compound II, generating Mn3þ, aromatic substrate oxidation, VP shows a putative long-range
which then serves to oxidize phenols to phenoxy radicals electron transfer pathway from an exposed trytophan to heme,
(Hatakka 1994). similar to that postulated in LIP (Ruiz-Duenas et al. 2001).
Whereas it has been shown that cation radicals of aromatic Mutagenesis and chemical modification of this tryptophan
substrates such as that of VA maintain the active form of LIP and the acidic residues forming the Mn2þ-binding site
by oxidatively converting compound III to the native enzyme confirmed their role in catalysis.
and preventing H2O2-dependent inactivation (Barr and Aust
1994), Mn3þ has similarly been shown to convert MnP
compound III to native enzyme (Timofeevski et al. 1998). 4.4 Laccase
Additionally, Mn2þ also reactivated compound III and
although this reaction was slower, it could prevent compound 4.4.1 Properties
III accumulation when excess Mn2þ was present. Laccases are multicopper phenol oxidases that oxidize
phenols and aromatic amines. Rather than H2O2, these
4.2.3 Role of MnP in Lignin Degradation enzymes utilize O2 as an oxidant, reducing it by four electrons
to H2O (Call and Mucke 1997). LACs are generally larger
In many fungi, MnP is thought to play a crucial role in the than peroxidases, having molecular weights of approximately
primary attack on lignin, because it generates Mn3þ, a strong 60 kDa and above (Call and Mucke 1997). As with other
diffusable oxidant able to penetrate the small “molecular extracellular enzymes, LACs are glycosylated.
pores” between cellulose microfibrils, which precludes the
action of LIP because of steric hindrances (Flournoy et al. 4.4.2 Role of LAC in Lignin Degradation
1993). Organic acids, such as oxalate (Kuan and Tien 1993),
and fumarate and malate (Hofrichter et al. 1999), which are Laccase oxidizes phenolic lignin model compounds directly
also produced by white rot fungi, chelate Mn3þ. These stable (Kawai et al. 1988). Although it is a phenoloxidase and its
complexes then deliver the oxidizing power. Although MnP redox potential is too low to directly oxidize the nonphenolic
does not oxidize nonphenolic lignin structures during normal components of lignin, it has been shown to degrade lignin
turnover, these structures have been shown to be slowly efficiently in the white rot Pycnoporus cinnabarinus, which
co-oxidized when MnP peroxidizes unsaturated fatty acids does not produce MnP or LIP (Eggert et al. 1997). To
(Jensen et al. 1996; Kapich et al. 1999a). Bao et al. described overcome the redox potential barrier, P. cinnabarinus
the oxidation of a nonphenolic lignin model by a lipid produces a metabolite, 3-hydroxyanthranilate that can
peroxidation system that consisted of P. chrysosporium MnP, mediate the oxidation of nonphenolic substrates by LAC
Mn2þ, and unsaturated fatty acid esters (Bao et al. 1994). (Eggert et al. 1996). It is believed that natural mediators, such
Substrate oxidation occurred via benzylic hydrogen abstrac- as 3-hydroxyanthranillic acid in the white rot P. cinnabarinus
tion and it was suggested that this process might enable the act as diffusable lignin-oxidizing agents. In the presence of
white rot fungi to accomplish the initial delignification of the mediators 3-hydroxyanthranillic acid, hydroxybenzotria-
wood. The importance of Mn2þ and the involvement of lipid zole, or 2,20 -azinobis(3-ethylbenzthiazoline-6-sulfonate)
peroxidation in depolymerization and mineralization of (ABTS), LAC has been shown to oxidize nonphenolic lignin,
14
C-labeled, polyethylene glycol linked, b-O-4 lignin model VA, and PAH (Bourbonnais et al. 1995; Collins et al. 1996;
compound by C. subvermispora in wood block cultures and Eggert et al. 1996; Majcherczyk et al. 1998; 1999).
400 Ward et al.

4.5 H2O2 Generating Enzymes 1994). The modification of lignin by OH† radicals produced
by CDH may result in hydroxylation of nonphenolic
In addition to the enzymes mentioned earlier, the ligninolytic structures to phenolic ones, facilitating MnP and LAC action
system of white rot fungi includes extracellular H2O2 and suggesting that the enzymes may form a pathway in lignin
generating enzymes, essential for peroxidase activity. To biodegradation (Hilden et al. 2000). It has also been suggested
date, a number of oxidase enzymes have been shown to lead to that CDH may support MnP by reducing precipitated MnO2,
the production of H2O2, including glyoxal oxidase (GLOX), which is common in rotten wood to Mn2þ or Mn3þ. It might
glucose 1-oxidase, methanol oxidase, and aryl-alcohol oxidase also aid MnP by producing cellobionic acid, which should
(AO) (Zhao and Janse 1996). White rot fungi that lack H2O2 complex Mn3þ (Roy et al. 1994).
generating oxidases may rely on the oxidation of physiological
organic acids such as oxalate and glyoxalate, which indirectly
results in H2O2 production (Urzua et al. 1995). 4.7 Reactive Oxygen Species

Reactive oxygen species and the suggested roles that they

play in lignin degradation are given in Table 4.
4.6 Cellobiose Dehydrogenase The production of OH† radicals by white rot fungi is well
documented (Barr et al. 1992; Kutsuki and Gold 1982;
Cellobiose dehydrogenase (CDH) is an extracellular enzyme Tanaka et al. 1999a,b). OH† radicals are very reactive and
produced by many white rot fungi (Henriksson et al. 2000). It can attack the subunits of lignin by both abstracting aliphatic
oxidizes soluble cellodextrins, mannodextrins, and lactose Ca-hydrogens and by adding to aromatic rings (Hammel et al.
efficiently to their corresponding lactones by a ping-pong 2002). Typical reactions of OH† radical with the major
mechanism using a wide spectrum of electron acceptors arylglycerol-b-aryl ether structure of lignin can result in
including quinones, phenoxy radicals, Fe3þ, Cu2þ, and demethoxylation, b-O-4 cleavage, hydroxylation, or
triodide ion. The function of CDH is not obvious, but Ca-oxidation (Hammel et al. 2002). The oxidation of lignin
P. chrysosporium produces relatively high levels of the by OH† radicals, therefore, results in diverse reactions, some
enzyme, approximately 0.5% of the secreted protein on a of which are expected to degrade the polymer. However, it
mass basis, suggesting it plays an important role. Many remains unclear whether any wood decay fungus uses OH†
functions have been suggested for CDH, some of which are radical to oxidize lignin. Hydroxylation of both phenolic and
related to cellulose degradation, but in the current context nonphenolic lignin resulting in new phenolic substructures on
only those that are relevant to lignin biodegradation will be the lignin polymer may make it susceptible to attack by LAC
discussed. It has been suggested that CDH reduces aromatic or MnP (Hilden et al. 2000; Tanaka et al. 1999a).
radicals formed by ligninolytic enzymes, thereby preventing If white rot fungi produce OH† radicals, then it is also
their repolymerization and supporting lignin degradation necessary to consider the effects that peroxyl (ROO†) and
(Ander et al. 1990; Temp and Eggert 1999). In addition, it has hydroperoxyl (HOO†) radicals have on lignin, since both of
been shown that CDH can generate hydroxyl radicals (OH†) these ROS are expected as secondary radicals when OH†
in a fenton type reaction, which can modify cellulose, radicals oxidize wood polymers (Hammel et al. 2002).
hemicellulose, and lignin (Henriksson et al. 1995; Wood Manganese peroxidase of white rot fungi peroxidizes

Table 4 Reactive oxygen species and their role in lignin degradation by white rot fungi

Reactive
oxygen species Role in lignin degradation References
Hydroxyl Demethoxylation, b-O-4 cleavage, hydroxylation, or Ca-oxidation Hammel et al. (2002), Hilden et al. (2000),
radicals (OH†) of nonphenolic structures; hydroxylation of nonphenolic lignin and Tanaka et al. (1999a)
results in the formation of phenolic structures, making it susceptible
to attack by MnP and LAC
Peroxyl Oxidation of nonphenolic lignin. Ca – Cb and b-O-4 cleavage Hammel et al. (2002)
radicals
(ROO†)
Superoxide Production of H2O2 by dismutation; Mn2þ oxidation to Mn3þ; Archibald and Fridovich (1982), Barr et al.
radicals (O†2
2 ) production of OH† radicals through iron-catalyzed Haber – Weiss (1992), and Gierer et al. (1994)
reaction; by reacting with phenoxyl radicals produced from lignin
model compounds, it can result in oxidative degradation being
favored over coupling reactions
The Biodegradation of Lignocellulose by White Rot Fungi 401

unsaturated fatty acids, which results in the formation of ROS reduce the rate of lignin mineralization in ligninolytic cultures
that include ROO† (Moen and Hammel 1994). ROO† of white rot fungi (Akamatsu et al. 1990; Ma et al. 1992;
radical-generating systems including MnP in the presence of Shimada et al. 1994).
unsaturated fatty acids were shown to oxidize a nonphenolic
b-O-4-linked lignin model dimer to products indicative of
hydrogen abstraction (Kapich et al. 1999b). Since, white rot
fungi do produce extracellular lipids (Enoki et al. 1999), the 5 PHYSIOLOGICAL REQUIREMENTS FOR
formation of ROO† radicals by an MnP dependent LIGNIN DEGRADATION BY WHITE ROT
mechanism and their involvement in lignin degradation FUNGI
does seem reasonable. The involvement of ROO† radicals in
the chemical consumption of 1-(30 ,40 -dimethoxyphenyl) Lignin is unable to serve as the sole carbon and energy source
propene (DMPP) by LIP was confirmed by using the well- for white rot fungi. In order for lignin degradation to proceed,
known ROO† radical reductant Mn2þ (ten Have et al. 2000). white rot fungi require an additional more readily utilizable
This metal ion severely inhibited the DMPP consumption rate source of carbon (Ander and Eriksson 1975; Kirk et al. 1976).
under air, but did not affect the lower enzymatic DMPP It has been hypothesized that the normal mode for white rot
consumption rate under N2. In the absence of O2, the Ca–Cb fungi to degrade wood is by simultaneous attack of
cleavage of DMPA to veratryl aldehyde was strongly polysaccharides and lignin (Boominathan and Reddy 1992).
inhibited and side-chain coupling products (dimers) were Degradation of cellulose and hemicellulose provides glucose
formed instead. As a whole, these results suggest that during
for the fungus and when the flow of sugar ceases, the fungus
LIP-catalyzed oxidation of aromatic substrates, O2 is
starves and thereby goes from primary to secondary
responsible for the formation of reactive ROO† intermedi-
metabolism.
ates, which can directly react with other substrate molecules
White rot fungi degrade lignin at the onset of the
and thereby accelerate consumption rates and also prevents
secondary growth phase, when utilizable nutrients are
coupling reactions by lowering the pool of carbon-centered
depleted and primary fungal growth ceases (Bonnarme et al.
radicals accumulating during LIP catalysis.
1991). However, it should be considered that the concepts of
In contrast to OH† and ROO†, superoxide anion radicals
primary and secondary metabolism might be an over-
(O†2
2 ) are unable to oxidize lignin units. However, O2
†2
simplification regarding lignin degradation during natural
produced by white rot fungi can participate in the production
of H2O2 via both dismutation ð2O†2 þ conditions where fungal growth and lignin degradation can
2 þ 2H ¼ H2 O2 þ O2 Þ
and Mn oxidation with concomitant production of Mn3þ
2þ occur at the same time. Carbon, nitrogen, and manganese are
ðO†2 þ 2Hþ ¼ H2 O2 þ Mn3þ Þ (Archibald and Fri- all critical nutritional variables in triggering secondary
2 þ Mn
2þ

dovich 1982). They can also be involved in HO† production metabolism and the production of ligninolytic enzymes
through the iron-catalyzed Haber – Weiss reaction ðO†2 including LIP and MnP by P. chrysosporium and other white
2 þ
H2 O2 ¼ HO† þ HO2 þ O2 Þ (Barr et al. 1992). Furthermore, rot fungi (Bonnarme et al. 1991). Considering that the
by reacting with phenoxyl radicals produced from lignin nitrogen content of wood is very low (C/N 350-500/1)
model compounds, they can result in oxidative (Cowling and Merrill 1966), it is no surprise that nitrogen
degradation being favored over coupling reactions (Gierer plays an important role in growth and metabolism (Buswell
et al. 1994). and Odier 1987; Kirk and Farrell 1987). In P. chrysosporium,
One potential source of O†2
2 evolves from the cleavage of
limitation of nitrogen, carbon, or sulfur (Jeffries et al. 1981)
oxalate via oxalate decarboxylase (ten Have and Teunissen can trigger secondary metabolism and lignin degradation.
2001). Oxalate is produced as a major aliphatic acid by white Fungal degradation of lignin in wheat straw was affected by
rot fungi (Makela et al. 2002). Its decomposition results in the the amount of nitrogen (NH4NO3), which appears to repress
formation of CO2 and the formate anion radical (CO†2 2 ),
lignin degradation, by most fungi on this substrate (Zadrazil
which is further oxidized by O2 to give CO2 and O†2 2 or and Brunnert 1980). However, nutrient nitrogen only had a
HOO†. Subsequent dismutation of O†2 2 resulting in the moderate influence on lignin mineralization by T. versicolor
formation of H2O2 indicates that oxalate may serve as a and almost no influence on P. ostreatus and Lentinus edodes
passive sink for production of the latter. Both LIP and MnP (Leatham and Kirk 1983). In general, nitrogen repression of
are also capable of decomposing oxalate in the presence of lignin degradation in white rot fungi is common but it is not
VA and Mn2þ, respectively (Akamatsu et al. 1990; Shimada always a rule.
et al. 1994) and indeed other organic acids (Hofrichter et al. The ligninolytic activity in P. chrysosporium is also
1998; Urzua et al. 1998). These reactions account for the triggered in cultures where carbon becomes limiting. The
observed oxidation of phenol red and kojic acid by MnP in the activity appears when the carbon source is depleted, and this
presence of Mn2þ, without exogenous addition of H2O2 activity is associated with a decrease of mycelial dry weight.
(Kuan and Tien 1993; Urzua et al. 1995). The reduction of The amount of lignin degraded depends on the amount of
VAþ† þ or Mn3þ by oxalate suggests that as long as oxalate carbohydrate provided, which in turn determines the amount
coexists with LIP and MnP, it would inhibit lignin of mycelium produced during primary growth (Jeffries et al.
degradation. Indeed, oxalate has been shown to strongly 1981).
402 Ward et al.

6 POTENTIAL APPLICATIONS OF WHITE 7 CONCLUSIONS

ROT FUNGI
Lignin degradation plays a central role in the earth’s carbon
6.1 Upgrading Agricultural Wastes for Animal
cycle, since it hinders decomposition of the most renewable
Feed carbon sources, cellulose and hemicellulose. White rot fungi
able to cause complete wood decay are the most efficient
The direct use of lignocellulosic residues as ruminant animal lignin degraders known and they are perhaps nature’s major
feed, or as a component of such feeds, represents one of agents for recycling the carbon of lignified tissues.
its oldest and most widespread applications and, as such, it Considerable research has been devoted to understanding
plays an important role in the ruminant diet. The the chemistry, biochemistry, and genetics underlying the
lignocellulose complex in straw and other plant residues is degradation of lignin by these fungi. Many enzymes and
degraded very slowly by ruminants, because of the physical several small molecules including reactive oxygen species
and chemical barrier imposed by lignin polymers, preventing and organic acids have been described as playing a role in
free access of hydrolytic enzymes, such as cellulases and lignin degradation by white rot fungi in nature. However, a
hemicellulases to their substrates. Normally, the rate of decay major breakthrough is still required to understand the relative
of plant debris is proportional to its lignin content. contribution of the individual components of the ligninolytic
Delignification of straw by white rot fungi seems to be the system and the synergism that exists between them.
most promising way of improving its digestibility (Kamra and Overcoming the difficulties associated with studying the
Zadrazil 1986; Streeter et al. 1982; Zadrazil and Reinger degradation of such a complex polymer could enable this.
1988). The role of fungi in agricultural waste conversion by Obviously, progress in these directions would have far
different fungi species has been recently reviewed (Cohen reaching implications and could be of considerable aid in
and Hadar 2001). developing the many potential biotechnological applications
that have been proposed for the ligninolytic system of white
rot fungi.
6.2 Biopulping

The objective of pulping is to extract cellulose fibers from ACKNOWLEDGEMENTS

plant material, generally hard or soft wood trees. Mechanical
and chemical pulping are usually employed for this process. This work was supported by The Israel Science Foundation of
However, a biological approach involving white rot fungi The Israel Academy of Science and Humanities Grant
could replace environmentally unfriendly chemicals (e.g., (No. 655/99-2), The Fund for the Promotion of Research at
chlorine) save on mechanical pulping energy costs and the Technion, and by The Ministry of Science of Israel
improve the quality of pulp and the properties of paper (Grant No 8867197).
(Breen and Singleton 1999). The ligninolytic enzymes of
white rot fungi selectively remove or alter lignin and allow
cellulose fibers to be obtained. Recent data suggest that
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FEBS Lett 242:325 –329. Wariishi H, Marquez L, Dunford HB, and Gold MH (1990). Lignin
Urzua U, Larrondo LF, Lobos S, Larrain J, and Vicuna R (1995). peroxidase compound-II and compound-III—spectral and
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371:132 – 136. Wood PM (1994). Pathways of production of fenton reagent by
Urzua U, Kersten PJ, and Vicuna R (1998). Manganese peroxidase wood-rotting fungi. FEMS Microbiol Rev 13:313 – 320.
dependent oxidation of glyoxylic and oxalic acids synthesized Wood DA and Smith JF (1987). The cultivation of mushrooms. In:
by Ceriporiopsis subvermispora produces extracellular Norris JR, Pettipher GL eds. Essays in Agricultural and Food
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Vares T, Lundell TK, and Hatakka AI (1992). Novel heme- Yokota S, Umezawa T, Hattori T, and Higuchi T (1991). Degradation
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Microbiol Lett 99:53 – 58. Yoshida S, Chatani A, Honda Y, Watanabe T, and Kuwahara M
Vares T, Lundell TK, and Hatakka AI (1993). Production of (1998). Reaction of manganese peroxidase of Bjerkandera
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Vares T, Kalsi M, and Hatakka A (1995). Lignin peroxidases, Zadrazil F and Brunnert H (1980). The influence of ammonium
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34
Biomineralization of Heavy Metals

T. C. Crusberg / S. S. Mark* / A. Dilorio Worcester Polytechnic Institute, Worcester,

Massachusetts, USA

1 INTRODUCTION concentrations fall into the 1 –10 mg/l range. Table 2 provides
a listing of discharge limits of metal finishing wastewaters in
For well over a century, metal-contaminated industrial the United States, and as such represents goals that must be
wastewaters have been released into the environment by met by any new technology or existing technologies.
industry, agriculture, sewage treatment, and mining opera- Discharge limits for municipal wastewater treatment plants
tions worldwide. Since the WWII era, the nuclear fuel cycle in the United States are much stricter than those listed in
has contributed an additional and unique waste burden of Table 2. Influent levels of Cu2þ in wastewaters arriving at
uranium and other radioactive metals. Metal ions, unlike most municipal sewage treatment plants range from 100 to
organic chemicals, can persist in the environment indefinitely, 250 mg/l, but effluent levels of 6–25 mg/l are expected to be
posing threats to organisms which are exposed to them attained under new U.S. Environmental Protection Agency
(Volesky and Holan 1995). Governmental control of such guidelines (Amer 1998). Regulations governing aqueous
discharges has only been energetically regulated in the past metal discharges in the United Kingdom have been reviewed
two or three decades. Many toxic inorganic chemicals have, by Forster and Wase (1997) who also discussed the toxic
over time, accumulated in soils, sediments, and impound- biological effects of several of the important heavy metals. A
ments throughout the world. Metal-bearing liquid wastes may genuine need now exists for new and certainly more cost-
be of known and predictable composition if generated by a effective technologies to replace or supplement the physico-
single industry, e.g., electroplating wastewaters, or in other chemical approaches currently in use for removing metal
cases may be a heterogeneous mix of many dissolved metal contamination at existing sites and for preventing future
ions and organic compounds at various pH values and ionic contamination of natural waters by heavy metals. There is
strengths, with colloidal and particulate matter present as hope that biotechnology may provide new insights to solving
well. Governments are now regulating this problem by these problems (Crusberg et al. 1994; 1996; Gretsky 1994).
mandating preventative actions and forcing industries and Biotechnology has been successfully exploited as a remedy
laboratories and other waste generators to intercept toxic for many types of discharges of organic wastes and for in situ
metals before they are discharged. Most heavy metal bioremediation of sediments at contaminated sites and in fact is
containing wastewaters are treated using remediation the preferred remedy for many instances (Alexander 1999).
technologies that have been borrowed primarily from the There is hope that some of the physiological processes and
unit operations of the chemical industry which rely on a genetic adaptations that protect organisms against toxic metals
mixture of physical and chemical processes (Table 1) to and other inorganic contaminants can be identified and
render the metal ion contaminants less toxic or more easily exploited for the removal and recovery of those metals from
handled. Unfortunately the chemical form of the converted aqueous waste streams (Crusberg et al. 1991; 1996; Hartley
metal (e.g., a gelatinous precipitate) is itself often in need of et al. 1997; Nies 1999; Ow 1996; 1997). Systems which use
careful and expensive disposal, and conventional treatment renewable biomass to extract metal ions from solutions may be
becomes less efficient and more expensive when metal ion an environmentally friendly alternative to physico-chemical

*Current affiliation: Cornell University, Ithaca, New York, USA

409
410 Crusberg et al.

Table 1 Physicochemical processes for heavy metal toxic metal ions and to alter them in such a way that renders
wastewater treatment those ions less harmful. In fact, genetic adaptation to survival
of microorganisms in heavy metal-rich environments is not at
Process Detrimental factors all unusual in nature (Nies 1999; Ow 1996).
One aim of biotechnology is to put that special physiology
Membrane separation Expensive, durability of membranes
to use and produce a commercial product (cell or cell
Liquid – liquid extraction Limited applications
component) for remediating heavy metal contamination.
Carbon adsorption Expensive adsorbent, requires
Biological materials that can accomplish this mission may be
regeneration
therefore termed biotraps and can be grouped into three major
Ion exchange Expensive adsorbent, requires
categories based on function:
regeneration
Electrolytic treatment Limited applications a. Biosorption or the process by which individual cells
Precipitation Forms sludges, often very or cell components bind or take up metals
gelatinous a. Adsorption of ions onto a surface, including ion
Coagulation/flocculation Forms hydrated sludges exchange and complexing with ligands, and
Chemical reduction Limited applications b. Uptake into the cell and sequestration by
Flotation Forms hydrated materials cytoplasmic components,
Vitrification Very expensive, landfill required b. Transformation by oxidation/reduction into a less
Evaporation Energy intensive toxic or volatile form, and
Crystallization Time dependent, landfill required c. Precipitation by forming insoluble salts, or by a
combination of these processes.
(From U.S.E.P.A., 1991; Smith et al. 1995.)
This review discusses general heavy metal bioremediation
processes rather than have to deal with each group of
processes and will be considered for their ability to serve as organisms individually.
biotraps for metals (chiefly as ions). This discussion will center
on heterotrophic microorganisms, primarily yeast and fungi as
possible candidates for development as heavy metal 3 FUNGAL-MEDIATED HEAVY METAL
biosorbents or “biotraps.” Biotraps in this review are defined REMOVAL FROM AQUEOUS SOLUTIONS
as any organism (living or nonliving) or component of an
organism, which can alter the form of, or bind with a toxic 3.1 Biosorption
metal or metal ion allowing its removal and recovery from a
waste stream, or rendering it harmless. Biosorption is a term that describes the broad range of
processes by which biomass removes metals (and other
substances) from solution, yet it can also be used in a stricter
2 POTENTIAL OF FUNGAL sense to describe uptake by dead (detritus) or living biomass
BIOTECHNOLOGY by purely physico-chemical processes such as adsorption or
ion exchange (White et al. 1995). Metabolic processes
It has been recognized for decades that many micro- inherent in living biomass may contribute to the uptake
organisms have a physiology, which allows them to act on mechanism. Ideally a biosorbent has the ability to be recycled
and the sorbed metal ions recovered for reuse or safer
disposal. Choice of a suitable biotrap is at times made easier if
Table 2 Pretreatment standards based upon Best Practical certain genetic and biochemical characteristics of an
Control Technology (BPT) for existing sources of metal finishing organism are known. That fungi and yeast can serve as
wastewaters. Discharges for releases of 10,000 or more ga biotraps for heavy metals has been the subject of a great deal
(38,000 l) a day in the United States. (40 CFR (Code of Federal of research as evidenced by several prior reviews on the
Regulations of the U.S.) 413) subject (Blackwell et al. 1995; Kapoor and Viraraghavan
1995; 1997).
Maximum for Average daily value for Predicting the effectiveness of a biotrap requires that some
Metal ion any 1 day 30 consecutive days of its chemical composition be understood. For example,
Cu 3.38 mg/l 2.07 mg/l chitin and chitosan are well known metal-ion adsorbers due to
Ni 3.98 2.38 the presence of both carboxyl and amine groups which make
Cr 2.77 1.71 up these biopolymers (Ashkenazy et al. 1997; Cuero 1996;
Zn 2.61 1.48 Fourest and Roux 1992; Juang et al. 1999). In fungi, the
Pb 0.69 0.43 cell walls present a multilamillar architecture where up to
Cd 0.69 0.26 90% of the dry weight consists of amino- or nonamino-
Ag 0.43 0.24 polysaccharides (Farkas 1980). The fungal cell wall is in
essence a two-phase system consisting of a fibrous
Biomineralization of Heavy Metals 411

chitin-based skeletal framework embedded within an by cell growth, with the particles eventually becoming
amorphous polysaccharide matrix (Griffin 1994). Yeast and entrapped within the matrix of the fungal hyphae. Al-Asheh
higher fungi such as the deuteromycetes Trichoderma viride and Duvnjak (1992) demonstrated that living mycelia of
have cell walls composed primarily of chitin and glucan Aspergillus carbonarius were able to adsorb copper and
polymers while the lower fungi such as Rhizopus arrhizus chromium and noted that an increase in uptake correlated with
have cell walls of chitosan and chitin (Morley and Gadd an increase in pH. Sag et al. (1999) recently reported the
1995). Although chitosan does bind metal ions, the more simultaneous adsorption of chromate and divalent copper ions
highly polymerized and cross linked chitin performs better as by living R. arrhizus in packed columns operated in
a metal biotrap. However, Crusberg et al. (1994) reported that continuous mode. Other fungal adsorbents were successfully
chitin, derived from a marine invertebrate source, was capable demonstrated by Suhasini et al. (1999) to remove nickel ion
at best of binding 14.7 mg Cu2þ/g biotrap at pH 4.0, with a from aqueous solutions, achieving uptake capacities of
binding constant of 27 mM, and concluded that this system 214 mg Ni2þ/g dry wt fungus.
was not likely to be developed commercially. There were Yeast similarly have proven to be effective research
better alternatives. models for metal sorption studies. The abundant brewery and
There is virtually no standardization in the literature for bakery yeast Saccharomyces cerevisiae has been the object of
reporting details of metal-binding experiments with biotraps. numerous biosorption studies. Wilhelmi and Duncan (1995)
Concentrations of metal ions are reported in several variations showed that immobilized cells of this yeast had the capability
of mass per unit volume (mg/l, parts per million or ppm, and to adsorb Cu, Co, Cd, Ni, Zn, and Cr with an uptake averaging
mg/l, parts per billion or ppb), as well as in moles per unit around 40 mmol/g in continuous flow packed bed columns
volume, millimolar (mmol/l) and micromolar (mml/l). For through 8 repeated adsorption–desorption cycles. Optimum
example, copper ion (Cu2þ) at 63 mg/l (ppm) may also be chromate removal from electroplating effluents has been
expressed as 1 mM but a uranium concentration of 1 mM recently demonstrated in fixed-bed columns at pH 2.5 using
would be almost four times larger in terms of mass per unit formaldehyde cross-linked S. cerevisiae (Zhao and Duncan
volume, at 238 mg/l. In fact, Cu2þ is often chosen as the 1998). Lead was found to adsorb at pH 5.5, to a nonliving cell
model heavy metal in initial studies on a potential biosorbent mass of Saccharomyces uvarum, up to a maximum of 48.9 mg
because there are many quite sensitive analytical techniques
Pb/g dry wt biotrap. Given that carboxyl and amine groups
available for its analysis. Most regulatory agencies mandate
served as the ligand to which the metal bound, it was assumed
permissible metal ion concentrations in wastewater dis-
that chitin was the most likely provider of these groups
charges in terms of mass per unit volume, e.g., ppm or ppb
(Ashkenazy et al. 1997).
(parts per billion or mg/l). Other issues that should be properly
Several authors now propose that biosorption processes
addressed by investigators, but are often omitted in scientific
utilizing whole cell biomass can realistically be considered as
reports, are effects of pH of an experiment on metal ion
replacement technologies for existing metal-removal pro-
solubility, and ionic strength effects on equilibrium constants.
For example, Cu2þ at 10 mg/l precipitates (as Cu(OH)2) when cesses, or even as an effective polishing unit in place of
the pH rises above 6.3, and precipitates at 1 mg/l when the pH existing treatment (Kapoor and Viraraghavan 1995; Volesky
rises above 6.8, based on a Ksp of 5 £ 10220 (13*, 3*, 24). and Holan 1995). One recent promising report noted that
Ionic strength may affect rate constants which comprise an dried powdered mycelium of Fusarium flocciferum can take
equilibrium constant according to Debey –Huckle theory. up 19.2 mg Cd and 5.2 mg Ni, and between 4 and 6 for Cu, for
Even buffers used to maintain pH during uptake/binding each 100 mg of fungus (Delgado et al. 1998). Rather severe
studies should be chosen to minimize their chelating potential treatment regimens also may produce a biomass more capable
for the metal ions under consideration. of metal ion uptake. For example Brady et al. (1994) showed
that hot alkali treatment of yeast biomass increased
accumulation of divalent cation adsorption but limited
3.2 Heavy Metal Biosorption success in removing chromate was reported. Formaldehyde
treatment also was found to render baker’s yeast more
Fungi and yeasts (Puranik et al. 1995; Sag and Kutsal 1996; efficient in binding Cr(VI) (Zhao and Duncan 1998) but only
Volesky and May-Phillips 1995; Zhao and Duncan 1998) a disappointing 6.3 mg Cr/g treated biomass was achieved.
have in the past received the most attention in connection with However Kapoor et al. (1999) recently reported that A. niger
metal biosorption systems, particularly because plentiful biomass was more effective than biomass treated by boiling in
amounts of fungal biomass are generated as by-products of 0.1 N NaOH for the removal of Ni by biosorption. A number
several types of industrial alcohol and antibiotic fermenta- of questions regarding the behavior of dead vs. living biomass
tions (Omar et al. 1996; Sag et al. 2000; Volesky and Holan in the biosorption process was recently examined by Yetis
1995). One such system consisting of Aspergillus niger waste et al. (2000) in a study of Pb(II) uptake by dead, resting and
from citric acid production was shown to remove zinc (as living Phanerochaete chrysosporium mycelia. Table 3
dust), magnetite, and metal sulfides from wastewater provides a comprehensive listing of a wide variety of biotraps
(Singleton et al. 1990; Wainwright et al. 1990). This process (including some of bacterial origin as a comparison) capable
was shown to be independent of metabolism but was favored of specifically sorbing copper from aqueous solutions.
Table 3 Copper biosorption by various types of microbial biomass 412
Experimental operating conditions
Organism Biosorption
Biomass type Biomass class Capacitya (mg Cu/g) pH T (8C) Cb (mg/l) Bio-mass (g/l) Reference
Z. ramigera Bacterium 270 5.5 0– 500(e) 0.83 Norberg (1984)
B. subtilis Bacterium 152 Brierley and Brierley (1993)
Arthrobacter sp. Bacterium 148 3.5 –6 30 180 (e) 0.4 Veglio et al. (1996)
P. notatum Fungus 80 Siegel et al. (1980)
C. tropicalis Yeast 80 Mattuschka and Straube (1993)
Activated sludge bacteria Bacteria 50 5 25 15– 200 (e) 0.5 Aksu et al. (1992)
C. vulgaris Alga 42.9 4 25 10– 260 (i) Aksu et al. (1992)
B. licheniformis (CWP) Bacterium 32 Beveridge (1986)
Z. ramigera Bacterium 29 4 25 12– 125 (i) Aksu et al. (1992)
P. syringae Bacterium 25.4 22 0– 13 (i) 0.28 Cabral (1992)
C. resinae (MP) Fungus 25.4 5.5 25 1– 320 (i) 1 Gadd and deRome (1988)
G. lucidum Fungus 24 5 5– 50 (e) Venkobachar (1990)
P. chrysosporium Fungus 20.2 6 5– 500(i) dvan Say (2001)
R. arrhizus Fungus 19 5.5 25 1.05 deRome and Gadd (1987)
C. resinae Fungus 18 Gadd et al. (1998)
S. cerevisiae Yeast 17 4 –5 25 190 (e) 1 Volesky and May-Phillips (1995)
R. arrhizus Fungus 16 Tobin et al. (1984)
R. arrhizus Fungus 16 Tobin et al. (1984)
C. resinae Fungus 16 5.5 25 1– 320 (i) 1 Gadd and deRome (1988)
A. oryzae Fungus 13.6 Huang et al. (1990)
P. guilliermondii Yeast 11 Mattuschka and Straube (1993)
S. cerevisiae Yeast 10 Mattuschka et al. (1993)
S. obliquus Alga 10 Mattuschka et al. (1993)
R. arrhizus Fungus 9.5 5.5 25 0.6– 25 (i) Gadd et al. (1998)
P. chrysogenum Fungus 9 Niu et al. (1993)
S. noursei Bacterium 9 5.5 30 06– 65 (i) 3.5 Mattuschka and Straube (1993)
A. pullulans (MP) Fungus 9 5.5 25 1– 320 (i) 1 Gadd and deRome (1988)
A. niger Fungus 7.22 Rao et al. (1993)
S. cerevisiae Yeast 6.3 Brady and Duncan (1993)
A. pullulans Fungus 6 5.5 25 1– 320 (i) 1 Gadd and deRome (1988)
S. noursei Bacterium 5 Mattuschka and Straube (1993)
Bacillus sp. Bacterium 5 Cotoras et al. (1993)
A. niger Fungus 4 5 5– 100 (e) Venkobachar (1990)
P. spinulosum Fungus 3.6 Townsley and Ross (1985)
P. digitatum Fungus 3 5.5 25 10– 50 (e) 6.5 Galun et al. (1987)
A. niger Fungus 1.7 Townsley et al. (1986)
T. viride Fungus 1.2
S. cerevisiae Yeast 0.8 4 25 3.2 (i) 2 Huang et al. (1990)
S. cerevisiae Yeast 0.4 4 25 3.2 (i) 2 Huang et al. (1990)
P. spinulosum Fungus 0.4 –2 Townsley et al. (1986)

Table 3 is a compilation of data reported in previous reviews (Kapoor and Viraraghavan 1995; Veglio and Beolchini 1997; Volesky and Holan 1995). Areas in the table which are not filled imply
that such data were not available to and/or not reviewed by the authors. References to the primary literature are given in the right-most column. CWP ¼ cell wall preparation; MP ¼ melanin
preparation.
a
Metal uptake as reported is not necessarily at maximum.
b
(i) ¼ initial concentration; (e) ¼ equilibrium concentration.
Crusberg et al.
Biomineralization of Heavy Metals 413

3.3 Extracellular Precipitation of Metals binds Cuþ2 and Znþ2 as well as Znþ2 and provides resistance
through detoxification of metal ions encountered in the
Exceeding a solubility product leads to precipitation of an natural environment (Turner and Robinson 1995).
insoluble salt of the reacting species. Sulfide anion (S22) and
oxalate anion ðC2 O22 4 Þ are produced by some species of
micro-organisms and these anions can form very insoluble
3.5 The Special Case for Uranium
salts with heavy metal ions which exhibit very small
solubility products as noted by Veglio and Beolchini (1997). A serious effort has gone into finding biological means to
Copper phosphate precipitation occurring within the matrix of reduce forms of more soluble uranium [U(VI)] compounds
mycelia of the fungus Penicillium ochro-chloron after 4 days from aqueous waste streams, which appear at various stages
of incubation in shake flask cultures at pH 4, was of the nuclear fuel cycle. By exploiting the physiology of
demonstrated using scanning electron microscopy and energy several different groups of microorganisms it has been shown
dispersive x-ray microanalysis (Crusberg et al. 1994). that U(VI) can be adsorbed to microbial surfaces or
Wrinkled 40 – 50 mm dia. spheres of insoluble copper biochemically converted to the very insoluble reduced
phosphate, inferred from EDX analysis, are trapped within [U(IV)] oxidation state, usually the uranite form (UO2).
the mycelia of the fungus grown for 4 days in aerated cultures Several organisms have been reported to carry out reduction
in the presence of 100 mg/l Cu2þ. Penicilllium and of U(VI) to U(IV) anaerobically. A nonviable preparation of a
Aspergillus have been shown to produce extracellular acid thermotolerant ethanol producing strain of Kluyveromyces
phosphatases which correlate with copper removal from marxianus was found to rapidly take up uranium(VI) with an
solution (Haas et al. 1991; Tsekova et al. 2002). It is likely efficiency of up to 150 mg U/g dry wt biomass, but with lower
that the extracellular phosphatases are localized in the binding at lower pH values (Bustard and McHale 1997;
periplasmic space, sandwished between the membrane and Bustard et al. 1997). Likewise, a cross-linked immobilized
cell wall materials. There they can act on extracellular residual biomass from distillery spent wash was found
organophosphates, hydrolyzing the phosphate and organic capable of sorbing over 200 mg U/g dry wt biomass (Bustard
moieties for uptake via specialized transport systems. As a and McHale 1997). A proprietary method using processed
means of detoxification, the free phosphate anions can then granules of the fungus R. arrhizus proved successful for
react with and precipitate heavy metal ions as insoluble heavy uranium ion immobilization (Brierley and Brierely 1993) at
metal phosphates before those ions enter the cell. A. niger has 50 mg U/g biomass dry weight, in batch uptake studies.
also been recently shown to immobilize metals as insoluble Complete uranium removal was demonstrated for dilute
oxalate salts (Cunningham and Kuiack 1992; Sayer and Gadd uranium ore bioleaching solutions (, 300 mg/l) with eluate
1997). In fact, oxalate is a common secondary metabolite of concentrations after desorption approaching 5000 mgU/l.
Penicillium spp. (Sayer and Gadd 1997) and is known to
precipitate calcium oxalate under natural conditions in litter
(Tait et al. 1999). Lead oxalate precipitation between pH 4
3.6 COMMERCIAL APPLICATIONS
and 5 has also been demonstrated as a means of detoxifiction
using living A. niger biomass (Sayer et al. 1999). Fungi Most living cell systems exploited to date have been used
therefore possess two mechanisms to protect themselves for decontamination of effluents containing metals at
against extracellular heavy metals involving the precipitation concentrations below toxic concentrations (Kapoor and
of heavy metals as insoluble salts. Recently, Mukherjee et al. Viraraghavan 1995; White et al. 1995). Some of those
(2001) reported the bioreduction of AuCl2 4 ions by
technologies employ a consortium of undefined micro-
Verticillum sp. In which gold nanoparticles 20 nm dia., organisms as well as higher plants. The “Meander System”
characterized by a vivid purple color over the surface of the used at the Homestake lead mine (MO, USA) passes effluents
fungus growing on culture plates were observed. containing Pb, Cu, Zn, Ni, Fe, and Cd ions through various
ditches or channels containing autotrophic cyanobacteria,
algae, and higher plants. Metal ion removal is achieved with
an efficiency greater than 99% (Erlich and Brierley 1990;
3.4 Metal Biosorption by Specialist Isolated Jennet and Wixson 1983). These complex systems likely
Molecules utilize precipitation and entrapment of particulates, in
addition to biosorption and uptake into cells and plant tissues,
Virtually all biological material has some affinity for toxic in the removal process and concentrate the metals in the
metals (Gadd and White 1993) and several mechanisms by sediment in forms which greatly reduce environmental
which metals interact with microbial cell walls and envelopes mobility and bioavailability (Brierley and Brierely 1993). In
are well established (Beveridge et al. 1997; Lovley et al. order to render sparse minerals in low grade ores soluble,
1991). However some biomolecules function specifically to those involved in biohydrometallurgy have exploited other
bind metals and are even genetically induced by their micro-organisms to facilitate metal recovery (Brombacher
presence (Macaskie and Dean 1990). In S. cerevisiae the low et al. 1997) with the aim of mobilizing precious metals, Au,
molecular weight cysteine-rich polypeptide metalothionein Ag, and sometimes Pt, but Ga, Ge. Patents (described in the
414 Crusberg et al.

above references) outlining various processes employ reactor (Figueira et al. 1997; Schiewer and Volesky 1996; Veglio and
technology and the most simple heap-leach or in situ Beolchini 1997). A mathematical Langmuir model has was
operations to strip metals from their ores. Bioleaching of recently developed for the prediction and performance of the
metals from ores has successfully employed bacteria such as simultaneous biosorption of Cr(VI and Fe(III) by R. arrhizus
Thiobacilli, Sulfobacilli, and Sulfolobus, as well as consortia in a semi-batch reactor. Likewise Sag et al. (2000) have
of organisms and some unidentified indigent species. The recently reported the theoretical approach to and use of a
same practices, which are in use in the mining industry to continuous-flow stirred tank reactor for removing lead, nickel
liberate metals from ores perhaps have applications in the and copper from aqueous solution by nonliving R. arrhizus
remediation of soils contaminated with those same metals by biomass.
mobilization, followed by biosorption. Commercial processes
using biosorbents for metal ion removal from aqueous waste
streams are still in their infancy. 5 LIMITATIONS AND POTENTIAL

The economic threshold for commercial selection of a

4 BIOREACTOR DESIGN biological process to replace a physico-chemical process for
heavy metal removal from a waste stream as assessed by
Bench-scale shake flask experiments rarely mimic the Macaskie (1991) is that a metal-loading capacity greater than
complexities encountered in unit operations where large 15% of the biomass dry wt must be demonstrated. Before the
volumes and short reaction times must be employed to ensure selection of any technology, it is imperative to note the
the economy of scale. Unit operations are preferred in the hierarchy of hazardous waste management options: reduce;
chemical industry since the process can usually be subject to reuse; recycle.
computational modeling and thereby more predictable. Also, The option of last resort is to treat and dispose of the waste
many industrial wastewaters contain several heavy metal in safe landfills, while minimizing the resultant volume, since
contaminants and therefore competitive adsorption to sites on disposal sites are few and space is precious not to mention
the surface of fungi has to be accounted for as described in a expensive. A given bioremediation technology should be able
recent study involving Penicillium chrysogenum and to perform on a large scale in order for it to be commercially
Streptoverticillium cinnamoneum by Puranik and Paknikar viable. The organism or biomaterial selected to accomplish
(1999). Biomass used in a reactor has to be either inexpensive the goal of removing or altering a heavy metal or metal ion
and disposable or preferably in any case reusable after rendering it less toxic must be very efficient in performing its
adsorbed metal ions are eluted. Yeast and fungi present intended function. The literature is rich with reports of
somewhat difficult materials for carrying out column studies attesting to the “potential” of a particular biomass
separation studies as the biomass often forms an impervious or biomaterial to carry out bioremediation of metal-
mat. However, the use of polyurethane foams as supports for contaminated waste streams, but few have actually ventured
biomass may help to overcome this problem (Dias et al. 2002; beyond the laboratory bench scale. What is clear is the
Tsekova and Ilieva 2001). Reaction times of biomass with apparent dearth of genetic engineering reports in the
metals to achieve sorption of metal ions must be short, yet literature. Classical genetic selection methods have proven
many reports in the literature allow incubations to run for days useful, for e.g., for isolating a strain of S. cerevisiae out of 240
to achieve suitable and reportable metal ion uptake. A typical tested, capable of uptake of 3.2 mg chromium (Cr(VI)) per g
experiment requires a 4-day incubation to prepare sufficient dry wt of yeast cells (Liu et al. 2001). Genes which carry out
mycelia of A. niger for study and then another 24 hr for certain functions in micro-organisms that provide them with
adsorption in shake flask following addition of metal ion resistance to heavy metals may be exploited for the
(Price et al. 2001). However there are report of more rapid development of new bioremediation technologies. Hunts for
equilibration requiring from 10 to 30 min of contact (Puranik metal-resistant and metal ion-binding microorganisms
and Paknikar 1999). Immobilizing A. niger biomass within a appear to be very successful, but the investigators nearly
polyurethane foam within an adsorption column increased always neglect to pursue the genetics behind their discovery.
copper uptake threefold compared with free mycelia studied Silver recently reviewed the genetics of metal resistance by
in batch adsorption experiments (Tsekova and Ilieva 2001). bacteria and noted several specific genes involved in metal
Computational methods applied to unit operations permit the uptake (Silver 1998). Many of these genes are located on
economy of a process to be predicted (Al-Asheh and Duvnjak plasmids and although not directly relevant to fungi and yeast
1992; Sag et al. 2001; Schiewer and Wong 1999). However, they may have some characteristics in common with
commercial scale bioreactors must address the problem of Eukaryotes. Ow (1996) has suggested that that employing
how a particular biotrap can render the influent metal Schizosaccharomyces pombe as a model system, and
concentration less harmful as the effluent is discharged. If it is understanding of yeast metal tolerance genes may become
to succeed, new biotrap technology must compete economi- clearer. Newly developed technologies can also be used to
cally with those already at hand in the chemical industry. immobilize or encapsulate an isolated biotrap into gels where
Sophisticated approaches have been reported for mathe- it can function in an in vitro manner if desirable. For example,
matical modeling of metal-biotrap sorption and precipitation gel-immobilized metal-binding proteins could then trap metal
Biomineralization of Heavy Metals 415

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35
Decoloration of Industrial Wastes and Degradation of Dye Water

Kirsten Schliephake / Warren L. Baker / Greg T. Lonergan Environment and Biotechnology

Centre, Swinburne University of Technology, Melbourne, Australia

1 INTRODUCTION 2 ORGANISMS DEGRADING LIGNIN

Large quantities of toxic and intensely colored waste effluents Lignin is an amorphous, highly aromatic heterogenous, 3-D
are produced by a range of industrial processes. These branched polymer (Buswell and Odier 1987; Higuchi 1985)
effluents are treated by processes ranging from aerobic to with its substituents connected by carbon –carbon and ether
anaerobic and physico-chemical techniques. More innovative linkages. There are different lignin structures consisting of
technologies using white-rot fungi have also been studied various amounts of the three cinnamyl alcohol precursors
(Kahmark and Unwin 1996). The use of microorganisms to present: p-coumaryl, coniferyl, and synapyl alcohols. These
detoxify environmental pollutants is generally referred to as alcohols give rise to p-hydroxyphenyl units, guaiacyl units,
bioremediation. Environmental issues are now of increasing and syringyl units, respectively, in lignin (Higuchi 1985;
concern and biological methods are being directed towards Kirk and Farrell 1987). The lignin cinnamyl alcohol
technologies to minimize pollution or remedy it if it occurs. precursors produce phenoxy radicals which randomly
White-rot fungi and their ligninolytic enzymes appear to couple.
have wide industrial potential. Phanerochaete chrysosporium White-rot is characterized by the bleached white color of
and Coriolus versicolor can degrade lignin and a number of advanced wood decay due to fungi (white-rot fungi) and other
related chlorinated compounds. They are also able to organisms. White-rot can totally degrade all major wood
efficiently decolorize and dechlorinate such effluents polymers, including lignin (Crawford and Crawford 1980;
(Archibald et al. 1990; Fukui et al. 1992; Sundmann et al. Kirk 1971). Lignin is significantly de-aromatized. The
1981). Efficient color removal from sulfite pulping effluents degradation follows two general patterns (a) simultaneous
has also been observed with C. versicolor (Bergbauer et al. attack on lignin and polysaccharides or (b) preferential or
1991; Royer et al. 1983). Furthermore, white-rot fungi are selective removal of lignin and/or hemicellulose without
showing significant potential to biodegrade a large number of extensive depletion of cellulose (Buswell 1991). White-rot
xenobiotic compounds and hazardous wastes (Field et al. fungi degrading lignin in preference to polysaccharides are
1993; Glaser 1990; Hammel 1989). Lignin peroxidase, Pycnoporus cinnabarinus, Pleurotus ostreatus, Phlebia
manganese dependent peroxidase, and laccase are the three radiata, and Phlebia (Merulius) tremellosus (Ander and
main ligninolytic enzymes (Leisola and Garcia 1989; Eriksson 1977).
Schoemaker et al. 1989) and have been well-studied but Other organisms degrading lignin include the brown-rot
several other enzymes are involved in further degradation of fungi, the soft-rot fungi, which seem capable of some
the products resulting from their action. demethylation and possibly degradation of side chains and
This chapter describes the rationale and results for three aromatic rings (Ander and Eriksson 1978; Buswell 1991)
related case studies using a white-rot fungus to (a) treat and some bacteria associated with the Actinomycetes and
effluent wastewater to reduce color, (b) to decolorize dye Eubacteria (Kirk 1971). Bacteria may extensively degrade,
solutions used in industrial processes, and (c) to determine the demethoxylate, and modify aromatic rings and side
enzyme involved in the decolorization process. chains.

419
420 Schliephake et al.

3 DECOLORIZATION OF DYES BY appears to be unrelated due to increased laccase secretion

MICROORGANISMS (Masaphy and Levanon 1992).
The utilization of the white-rot fungi and their nonspecific
Synthetic azo, anthraquinone, triarylmethane, and other dyes ligninolytic enzyme system for the biodegradation of other
are extensively used for textile dyeing, paper printing, and industrial dyes has found increased interest, since it has
color photography. A necessary criterion for the application of also become known that P. chrysosporiurm is able to degrade
these dyes is that they resist photodegradation and wash- and decolorize a range of azo, sulfonated dyes, heterocyclic,
ing processes. In addition, these dyes are also resistant and triphenylmethane dyes (Bumpus and Brock 1988; Cripps
to microbial degradation and are not easily removed from et al. 1990; Paszczynski et al. 1992; Spadaro et al. 1992)
wastes during conventional biological treatment (Meyer 1981; (Table 1). The triphenylmethane dye, crystal violet, has been
Shaul et al. 1991). The removal of synthetic dyes from waste- decolorized by P. chrysosporium. Three metabolites were
waters is mainly achieved by elimination of the dye stuffs identified and the dye was N-demethylated by purified lignin
using adsorption, chemical oxidation, and flocculation. peroxidase providing further evidence that the ligninolytic
Azo linkages and aromatic sulfo groups do not occur system is at least involved in oxidative biodegradation
naturally and they resist oxidative degradation. Partial (Bumpus and Brock 1988). Pararosaniline and cresol violet
degradation has been observed for anthraquinone and azo and cresol red were also decolorized.
dyes during aerobic activated sludge treatment, but A number of other azo, sulfonated azo, triphenylmethane,
substantial removal of color from the wastewater was and heterocyclic dyes can also be degraded by various strains
attributed to adsorption of the intact dye onto the sludge of P. chrysosporium (Table 1). This further demonstrates the
(Athanasopoulos 1991). The dyestuffs are generally more usefulness of the ligninolytic system in biodegrading and
susceptible to anaerobic sludge degradation. The anaerobic decolorizing wastewaters contaminated with dyes from textile
reduction of azo dyes to colorless products by bacteria has and other industries (Cripps et al. 1990). Fluorescein acid
been reported (Meyer 1981). The initial step in this (Gogna et al. 1992) and thiazine dyes (Kling and Neto 1991)
transformation is the reductive fission of the azo group, have also been degraded by P. chrysosporium or lignin
resulting in the formation and accumulation of toxic and peroxidase. Many studies have used P. chrysosporium,
possibly carcinogenic aromatic amines. The rapid although a H2O2-dependent enzymatic decolorization by
decolorization of a number of textile dyes and effluents by P. ostreatus has also been shown (Vyas and Molitoris 1995).
an immobilized anaerobic consortium had also been Knapp et al. (1995) suggests that more work on the effect of
demonstrated (Nigam et al. 1996; Oxspring et al. 1996). the decolorizing capacity of other members of the
The ability of P. chrysosporium to decolorize polymeric Basidiomycetes is warranted.
dyes, such as Poly B-411, Poly R-481, and Poly Y-606, has
been correlated with its lignin degrading system (Glenn and
Gold 1983; Gold et al. 1983; 1988). Strains of P. ostreatus
have also been shown to effectively decolorize Poly B-411 4 LIGNINOLYSIS AND DECOLORIZATION
(Platt et al. 1985). P. pulmonarius increased Poly B BY ENZYMES
decolorization, as well as enhanced laccase production.
Laccase is often produced by Pleurotus sp. as part of their A number of white-rot fungi and/or their enzymes have been
ligninolytic system but dye decolorization by this fungus used successfully in detoxification and degradation of

Table 1 Dyes decolorized by Phanerochaete chrysosporium

Organism Dye class Dye References

P. chrysosporium BKM 1667 (ATCC 24725) Sulfonated azo dyes Synthesized dyes Paszczynski et al. (1992)
P. chrysosporium OGC 101 Azo dyes Disperse yellow 3 Spadaro et al. (1993)
Disperse orange 3
Solvent yellow 14
P. chrysosporium BKM-F-1767 Azo dyes Tropabolin O Cripps et al. (1990)
Orange II
Congo red
Heterocyclic dyes Azure B
P. chrysosporium ME-446 Polymeric dyes Poly B-441 Glenn and Gold (1983)
Poly R-481
Poly Y-606
P. chrysosporium BKM-F-1767 Triphenylmethane dyes Crystal violet Bumpus and Brock (1988)
P. chrysosporium BKM-1667 (ATTC 24725) Fluorescein acid dye Rose Bengal Gogna et al. (1992)
Decoloration of Industrial Wastes and Degradation of Dye Water 421

selected xenobiotics. The most studied white-rot fungus is dependent peroxidase oxidation of Mn(II) to Mn(III) was
P. chrysosporium. subsequently demonstrated (Glenn et al. 1986).
Most of the oxidative reactions observed are presumed
to be one-electron oxidation of suitable phenolic substrates
to yield phenoxy radicals. Nonphenolic substrates yield
radical cationic intermediates (Kirk and Farrell 1987). 4.3 Laccase: Role and Function in Lignin
The result is the formation of opened-ring products, small Biodegradation
aromatic fragments, and quinones and these can be
further metabolized. Laccases classified as polyphenoloxidases are now widely
accepted as p-diphenoloxidases (EC 1.10.3.2) (Tuor et al.
1995) and are widespread. The enzyme is found in many
4.1 Lignin Peroxidase plant species (Mayer and Harel 1979) and is widely
distributed in fungi (Mayer 1987) including wood-rotting
fungi where it is often associated with lignin peroxidase,
A lignin-degrading enzyme from P. chrysosporium was
manganese dependent peroxidase, or both (Nerud and
identified in 1983 by two independent research teams (Glenn
Mišurcová 1996; Tuor et al. 1995). They are remarkably
et al. 1983; Tien and Kirk 1983). This enzyme was termed
nonspecific as to reducing substrates and show much in
ligninase, but it is now generally known as lignin
common with another copper-containing oxidase, tyrosinase
peroxidase. The enzyme was shown to partly depolymerize
(monophenol mono-oxygenase; EC 1.14.18.1).
lignin, but can cleave Ca–Cb linkages in side chains
Laccases are blue copper oxidases and participate in
suggesting that the depolymerizing and ring cleavage ability
electron transfer in biological systems by catalyzing the four-
of this enzyme constitutes its main function in lignin
electron reduction of dioxygen to water with the simul-
degradation (Chen and Chang 1985; Eriksson et al. 1990).
taneous oxidation of organic substrates (Reinhammar and
The enzyme has also been found in other fungi including
Malmström 1981; Thurston 1994). A minimum of four
C. versicolor (Dodson et al. 1987; Jönsson et al. 1987;
copper atoms, distributed in three spectroscopically distinct
Waldner et al. 1988), P. radiata (Hatakka et al. 1987;
binding sites, appears necessary to facilitate efficient
Kantelinen et al. 1988; Niku-Paavola 1987; Niku-Paavola
catalysis. The Type I (blue) copper center has a strong
et al. 1988) and P. tremellosus (Biswas-Hawkes et al. 1987).
absorbance near 600 nm and gives rise to the enzyme’s blue
color. The other two copper centers known as the Type II
(normal) and the Type III involve a pair of magnetically
4.2 Manganese Dependent Peroxidase coupled cupric ions (Farver and Pecht 1981; Li et al. 1992;
Morpurgo et al. 1993). Studies into the mechanism of
One year after detection of lignin peroxidase, Kuwahara the reduction of O2 to H2O by laccases have shown that a
et al. (1984) reported the isolation of another enzyme bridge between the coupled binuclear center and the
fraction from P. chrysosporium strain ME 446. This fraction Type II center defines a trinuclear cluster as the active site
showed Mn(II), H2 O 2 and lactate dependency and (Solomon 1988).
stimulation by increased protein concentration in reaction Plant laccase mechanism and function has been investi-
mixtures. The enzyme can oxidize a variety of dyes, gated predominantly on Rhus vernicifera. It is thought that the
including phenol red, o-dianisidine, and Poly R. Purification enzyme has a protective function, possibly causing formation
and characterization of this manganese dependent peroxi- of a natural polyphenolic polymerisate in the case of tree
dase was subsequently carried out (Glenn and Gold 1985). damage (Reinhammar and Malmström 1981). A plant laccase
A similar enzyme, tentatively named vanillylacetone has also been found to be involved in lignification (Bao et al.
peroxidase was purified from P. chrysosporium strain 1993) and the role of laccase in lignification has been
BKM-1767 (Paszczynski et al. 1985). This enzyme oxidized reviewed extensively by O’Malley et al. (1993).
various low-molecular weight phenols and an aromatic amine Fungal laccases oxidize phenols and phenolic sub-
in the presence of Mn(II) and H2O2. It did not oxidize phenol structures of lignin with subsequent polymerization or
red and was not activated by lactate. The enzyme oxidized depolymerization (Higuchi 1989). These pathways are
NADPH and reduced glutathione (GSH) thereby, possibly, considered to proceed via phenoxy radicals of the phenolic
indicating a link with xenobiotic metabolism. Hydrogen units. The role of laccase in lignin biodegradation remains
peroxide was formed in this reaction. Generation of hydrogen largely unresolved, since strains of the extensively studied
peroxide from GSH occurs with a manganese dependent white-rot fungus, P. chrysosporium, and several other
peroxidase from Lentinus edodes (Forrester et al. 1988). Basidiomycetes are active in lignin degradation but do not
Manganese accumulation in the decayed residue of wood produce laccase under the ligninolytic conditions employed in
(Blanchette 1984) supported the hypothesis that manganese laboratory studies (Nerud and Mišurcová 1996; Thurston
dependent peroxidases participated in lignin degradation 1994; Tuor et al. 1995).
(Paszczynski et al. 1985) via oxidation to a higher oxidation In some fungi, laccase has a function seemingly unrelated
state (Glenn et al. 1986; Paszczynski et al. 1986). Manganese to ligninolysis. For example, in Aspergillus nidulans, the
422 Schliephake et al.

enzyme appears to be essential for the synthesis of the spore The strain of P. cinnabarinus was inoculated onto a nylon
pigments (Clutterbuck 1972). web cube support (0.5 cm3 of Scotchbritew). The effluent was
prepared in a defined medium (Tien and Kirk 1988) which
5 CASE 1—DECOLORIZATION OF AN was modified by increasing the final concentration of
diammonium tartrate to 0.8 g/l to give a carbon to nitrogen
INDUSTRIAL WASTE
ratio of 38.3:1. Although it stimulated phenoloxidase activity,
5.1 The Composition of the Waste veratryl alcohol (40 mM) was omitted in this trial because it
retarded mycelial growth, inhibited glucose and nitrogen
The initial problem was to decolorize the complex waste- utilization and delayed decolorization. Addition of veratryl
water from an industrial site. The waste originated in a alcohol at the time of inoculation may interfere with fungal
pigment plant and advice indicated that it contained low metabolism (Tonon and Odier 1988).
molecular weight alkyd (sic) polyester resins derived from The medium was circulated through the reactor at 0.45 l/h
the reaction of triglycerides and anhydrides like phthalic and samples were taken daily and analyzed for glucose
anhydride along with epoxide resins, derived from bisphenol, concentration, available nitrogen, and oxidative enzyme
and epichlorohydrin and amine resins from melamine and activity which at this stage was termed phenoloxidase. A
formaldehyde. Lesser quantities of higher molecular weight control reactor lacking the effluent was also used.
materials, from polymerization of vinyl, acrylate, or
methacrylate monomers were also present. The effluent 5.4 Decolorization in the Reactor
had a total organic carbon content (TOC) of 2770 mg/l and a
total suspended content (TSC) of 7429 mg/l.
The results are summarized as follows for a 15-day reactor
cycle. The desirable characteristics of vigorous growth and
5.2 Selection of Decolorizing Organisms rapid decolorization (Joyce et al. 1984) were achieved by
omitting veratryl alcohol from the medium in the reactor
In view of the numerous reports of involvement in dye cycle. In this case, the nitrogen was consumed in 24 h and
decolorization and lignin degradation (Sections 2 and 3) the glucose in 48 h. The phenoloxidase reached a level of
white-rot fungi were considered the organisms of choice for 4.8 units of ABTS* oxidized/ml on day 9 and then declined to
this work. It was essential that the organism could either grow remain at a relatively steady level of 1.85 U/ml for the rest of
on or metabolize the effluent. Plates of malt extract agar the cycle. This decrease broadly coincided with detection of
(MEA) were prepared in the effluent diluted to contain 695, autolytic activity and reappearance of nitrogen in the medium.
1389, 2083, and 2770 mg/l TOC, respectively. They were The dark effluent was rapidly decolorized during the first 24 h.
adjusted to pH 4.5 to aid fungal growth. Plates were centrally The fungal mycelium was originally dark suggesting the
inoculated with several white-rot fungi that had been collected adsorption of some of the solid by the mycelium. This
in Victoria, Australia, and some standard strains. The plates intensity decreased over the 15-day cycle until the mycelium
were grown under high humidity and examined daily. became indistinguishable from the control. The final effluent
The most appropriate organism was judged to be a strain of was a light yellow.
a Victoria isolate of P. cinnabarinus, identified according to There were differences in the spectra at different stages of
Fuhrer (1985) and Macdonald and Westerman (1979). In the decolorization of the effluent and some of these are shown in
presence of a primary carbon source (and a TOC of the Figure 1. In this increased decolorizing effect three stages are
effluent of 1385 mg/l) this fungus covered the entire plate in suggested. Firstly some of the color adsorbs to the enhanced
96 h. This TOC concentration was also used in bioreactor mycelial mass formed in the absence of veratryl alcohol.
experiments. Secondly, the phenoloxidase acts to decolorize the soluble
dye components of the effluent and thirdly the phenoloxidase
5.3 Conditions in a Packed-Bed Bioreactor acts on the adsorbed color of the mycelium in situ. Evidence
that the phenoloxidase was most probably a laccase is
reported in Section 6.
Most degradation studies employing Phanerochaete have
been carried out in solid cultures or in shallow liquid
stationary or shake cultures (Ander and Eriksson 1977; 6 CASE 2—DECOLORIZATION OF
Hatakka 1985). Packed-bed bioreactor configurations have CHICAGO SKY BLUE
been mainly used to study the ligninolytic enzymes and
degradative abilities of P. chrysosporium (Lewandowski et al. 6.1 Structure of the Dye
1990; Linko 1988). In view of this it was decided to examine
the effect of the fungus on the industrial waste in a 2 l Chicago Sky Blue (Figure 2) is a disazo dye used extensively
packed-bed reactor. in dyeing cotton and cellulosic fiber material. It is often
21
*One unit (U) of enzyme activity is defined as the amount of enzyme causing a change in absorbance of 1.0 min at room temperature
(Bourbonnais and Paice 1990).
Decoloration of Industrial Wastes and Degradation of Dye Water 423

Figure 1 Decolorization of effluent by P. cinnabarinus.

incorporated into inks, and used for dyeing of leather and phenolic rings adjacent to the azo linkage appeared necessary
paper. It belongs to the largest class of commercially for oxidation and phenoxy radical formation. Mass spectral
produced water-soluble dyes that are characterized by up to (MS) and electrospray ionization (EI) –MS analyses con-
three azo linkages which link phenyl and naphthyl groups firmed the release of azo linkages as molecular nitrogen. This
containing combinations of functional groups (sulfonic acid, prevented aromatic amine formation.
sodium salt; amino; chloro; hydroxyl; methyl and nitro) on
the rings. 6.2 Decolorization of CSB Using Pycnoporus
Decolorization and degradation of azo dyes have been
cinnabarinus
demonstrated with P. chrysosporium (Cripps et al. 1990;
Ollikka et al. 1993; Pasti-Grigsby et al. 1992; Paszczynski
et al. 1991; 1992; Spadaro et al. 1992). A lignin peroxidase of The isolate of P. cinnabarinus was examined for its ability
this organism catalyses the decolorization of azo, hetero- to decolorize CBB on plates initially, then in shake flasks
cyclic, triphenylmethane, and polymeric dyes (Ollikka et al. and subsequently in 2 and 10 l packed-bed bioreactors. In
1993). every case the dye was decolorized at relatively high
Much less is known about the role of laccases in the concentrations (500 mg/l).
decolorization of synthetic dyes, although several wood-
rotting fungi produce laccase as well as peroxidases and have 6.3 Enzymes Involved in the Degradation
been shown to decolorize a range of structurally different
dyes (Knapp et al. 1995). A mechanism for degradation of The decolorization of the dye by P. cinnabarinus led to a
phenolic azo dyes by laccase from Pyricularia oryzae search for the responsible enzymes. In the early experiments,
(Chivukula and Renganathan 1995) is similar to the one solutions had been tested in the presence of a catalase from
proposed for degradation of phenolic azo dyes by a lignin Aspergillus niger. Despite the catalase the ABTS was still
peroxidase (Chivukula et al. 1995). In the latter case oxidized. This suggested an involvement of laccase in the
formation of novel sulfophenyl hydroperoxides was postu- decolorization reaction and a search was undertaken to see
lated and the elimination of the azo linkage as molecular whether it acted alone or whether it required other mediators.
nitrogen. However, in the case of laccase, electron-rich
6.4 Production and Purification of Laccase from
Pycnoporus cinnabarinus

In the 10 l packed-bed bioreactor, P. cinnabarinus used

glucose at a constant rate. The ammonia content of the
medium was exhausted by day 4. Laccase activity was first
detected on day 4 and had reached 0.28 U/ml by day 10 when
Figure 2 Structural formula of Chicago sky blue (CSB). the culture broth was harvested for laccase purification.
424 Schliephake et al.

The isolation procedure follows that of Schliephake et al. 0.03 mM for syringaldazine and 0.33 mM guaiacol. Activity
(2000). Laccase was purified from 7.9 l of culture fluid in two was irreversibly inhibited by 0.1 mM sodium azide and by
ultrafiltration steps followed by concentration, dialysis, and ascorbic acid at 1 mM.
column chromatography on Sephadex G 75. Fractions
absorbing at 614 nm were analyzed for laccases activity,
pooled, dialyzed against 20 mM histidine buffer pH 6, and 6.7 Decolorization Using Purified Enzyme
again concentrated. The concentrate was chromatographed on
a 5/5 Mono Q anion exchange column with a sodium chloride In degradation studies, the absorbance of the dye at 618 nm
gradient of 0 –0.5 M over 50 min at 1 ml/min. Fractions (25 mg CSB/l) was monitored against a heat treated blank.
absorbing at 614 nm were pooled and stored at 2 858C in the The rate of decolorization was initially linear (0.82 –25 mg
presence of a protease inhibitor mixture. A 45-fold increase in protein), and proportional to enzyme concentration but
specific activity was obtained on purification. Capillary equilibrium was reached after 2 h. The major CSB peak at
electrophoresis estimated purity at greater than 95% with 618 nm and minor peak at 320 nm disappeared and a new peak
minor peaks (areas 0.079 and 4.596%) at longer elution times. appeared at 550 nm.
The dye had a retention time of 22 min on HPLC (C18
reversed phase column) (Schliephake et al. 2000). After 20 h
6.5 Characterization of Laccase reaction time the 22 min peak disappeared and two new peaks
appeared with retention times of 1.28 and 3.96 min,
The enzyme contained 510 amino acid residues and was a respectively. Their molar absorptivities were an order of
single polypeptide of a molecular size of 63 kDa. It contained magnitude lower than CSB. Thus, the disazo dye CSB had
11% carbohydrate that is consistent with other laccases (Coll been clearly degraded by the activity of the laccase. The
et al. 1993). The first 10 amino acids at the N-terminal were products have not been characterized so far.
A, I, G, P, V, A, B, L, T, and L and differed from the enzyme
from P. cinnabarinus PB, also isolated in Australia, only in
the B for D in residue 7 (Eggert et al. 1996). It also showed 7 CASE 3—DECOLORIZATION OF
considerable homology to the laccase of P. ostreatus (Youn REMAZOL BRILLIANT BLUE
et al. 1995), C. (Trametes) versicolor (Bourbonnais et al.
7.1 Laccase and Remazol Brilliant Blue
1995), and Coriolus hirsutus (Kojima et al. 1990).
A major band and two minor bands were observed at pH 3
and pH 2.85 and 2.65, respectively, on isoelectric focusing Laccase was shown to be a major extracellular enzyme of
(IEF). Each band stained the laccase substrate guaiacol P. cinnabarinus and responsible for decolorization of CSB
(10 mM). One band only, corresponding to laccase activity, and most likely the pigmented wastewater. A number of
was obtained on electrophoresis (4 –20% gradient gel) under successful trials of the strain P. cinnabarinus in decolorizing
nondenaturing conditions. This band, stained with 10 mM the vinyl sulfonyl industrial dye, Remazol Brilliant Blue R
guaiacol, corresponded with the co-electrophoresis protein (RBBR) (Figure 3), were carried out on the laboratory scale
band. (Jones et al. 1993; Lonergan et al. 1993). Remazol Brilliant
Blue R is used for dyeing jeans. Subsequently a purified
laccase from P. cinnabarinus was also shown to be involved
6.6 Properties in the decolorization of industrial grade and purified RBBR
(Lonergan et al. 1996).
The ultra-violet visible (UV/Vis) absorption spectrum of It therefore seemed appropriate to monitor the laccase
laccase was similar to that observed of other fungal laccases development of P. cinnabarinus during a dye decolorization
(Coll et al. 1993; Eggert et al. 1996) with a Type I band at reactor cycle on a pilot scale prior to an industrial scale up. A
614 nm, corresponding to the blue copper of laccases 200 l packed-bed bioreactor was designed for the pilot scale
(Reinhammer and Malström 1981) and a broad band at run (Lonergan et al. 1995a,b). This pilot reactor trial provided
330 nm indicative of a Type III binuclear copper center much larger and more active samples from which laccase
(Malkin et al. 1969; Reinhammer 1984). The EPR spectrum could be purified.
of purified laccase had two superimposed signals of Type I
and Type II copper centers.
The laccase was stable at 608C for1 h and still retained
much activity after 2 h incubation at 808C. It differed from the
laccase of P. cinnabarinus PB (Eggert et al. 1996) which was
inactivated after incubation at 808C for 1 h. The enzyme was
still active in bioreactors run at 378C for 25 days but lost
activity on prolonged treatment at the higher temperatures.
The pH optimum for syringaldazine was between pH 4.4
and 5 and for guaiacol between pH 4 and 4.5. The Km was Figure 3 Structural formula of RBBR.
Decoloration of Industrial Wastes and Degradation of Dye Water 425

7.2 The Pilot Scale Bioreactor

The fermentation was conducted in a 200 l aerated polythene

drum, packed with 2 cm3 nylon (Scotchbritew) cubes
(Lonergan et al. 1995 b). A reactor cycle of 25 days was
used in this instance with the medium continually recycled at
36 ^ 18C. P. cinnabarinus rapidly colonized the packing.
Veratryl alcohol (4 mM) was used as a laccase inducing agent.
The RBBR was added (500 mg/l) to the medium on day 3
after inoculation. Daily samples were analyzed for glucose,
nitrogen, and phenoloxidase (laccase) activity. A sample was
also taken 30 min after addition of the dye and is termed
D3 þ 30.

7.3 Decolorization of Dye in Bioreactor

The dye (595 nm) was decolorized by 80% within 24 h and

almost completely decolorized within 48 h after addition. The
nitrogen in the reactor was exhausted in 7 days and the
glucose exhausted in 12 days whereupon more glucose was
added to the reactor (Lonergan et al. 1995b). There was a
small increase in nitrogen towards the end of the cycle due to
autolysis.
Laccase activity was detected on day 2, peaked at a very
large 60 U/ml on day 8 and fell to a minimum on day 12. It
then rose again towards the end of the cycle as the additional Figure 4 Elution profile of laccases from day 4 (A) and day 23
glucose was added. (B). The 100 ml sample was concentrated to 5 ml, passed through
In this series of experiments laccases were purified from Sephadex G 75 and active fragments pooled. The active solution
100 ml samples removed during the reactor operation on day was concentrated through Centricon ultrafilters and the
3, D3 þ 30, 4, 20, 23, and 25 by a slight modification to the concentrate passed through a Bioscale 5 anion exchange column,
method reported (Schliephake et al. 2000). The elution using the same gradient as for the Mono Q column but spread
profiles for all samples were similar to those of the laccase over 35 min and at a same flow rate of 1 ml/min.
purification carried out on the Mono Q anion exchanger. The
elution pattern for days 4 and 23 are shown in Figure 4. The
anomaly in enzyme elution times (day 4, 18.5 min and day 23, Purified laccases were subjected to IEF and stained for
15.5 min) was examined. activity. The enzymic banding pattern showed significant
variation on days 3, 4, 20, 23, and 25. The activity stain
showed that on day 3, prior to the addition of the dye to the
7.4 Variation in Enzyme reactor, five bands stained for laccase activity, with the
strongest staining bands towards the anode. On day 4, six
The apparent molecular weight of laccase, previously bands were observed and the band closer to the cathode had
estimated at 63 kDa by SDS-PAGE (Schliepake et al. 2000) the stronger stain. A similar pattern was also observed on day
was consistent in the laccase from days 3, D3 þ 30 min, 4, 20 of the reactor cycle. On day 23 and 25, however, there
and 20. The protein bands from samples on days 23 and 25 were only four bands and the band nearest the anode was
migrated further in the gel and gave a molecular weight of again the strongest staining band. Such a pattern of laccase on
54,620. IEF was considered artifacts due to the nature of the gel
On electrophoresis of the purified reactor samples, under (Eggert et al. 1996). The variations, together with the
native conditions, and visualizing laccase active bands with migration on the native PAGE, however, did not exclude
guaiacol, significant variation occurred in the migration of modification of the enzyme over the reactor cycle.
the enzyme through the gel from different days. Electro- The decolorizing activity of laccases during a 7 h
phoretic migration of proteins under nondissociating buffer incubation varied substantially for the different days
conditions occurs on the basis of both size and charge investigated (Figure 5). Day 4 laccase decolorized RBBR at
(Hames 1990). This variation in migration indicated that the fastest rate. This particular enzyme had been isolated from
physical transformation of the enzyme may have occurred the reactor one day after the addition of the dye. The second
during the reactor cycle without loss of the enzyme activity. most active laccase was isolated from day 3, i.e., before dye
426 Schliephake et al.

8 CONCLUSIONS

A strain of P. cinnabarinus was isolated which decolorized

an industrial waste as well as the disazo dye CSB and the
vinyl sulfonyl dye RBBR. On purification the phenoloxidase
of the enzyme showed all the characteristics of a laccase.
The purified laccase from the white-rot fungus rapidly
decolorized both dyes. There appeared to be some charge to
size variations of laccase during the reactor cycle. It may be
hypothesized that the dye may have acted on a proportion of
laccase molecules in such a way that it modulated the
protein. This could give rise to a conformational change,
which may optimize the catalytic site of the enzyme in such
a way that it is more easily accessible. As the dye molecule
is very small ðFW , 625Þ and contains a free aromatic
amino group, there could be a possible reaction occurring
between a possibly free carboxyl groups of an exposed
amino acid of laccase and the amino group of the dye. This
would mean that such amino-containing molecules under
certain circumstances might behave like amino acids. Such a
modulation could possibly explain the presence of an
Figure 5 Decolorization of dye RBBR by laccases from additional acidic band appearing closer to the cathode upon
various days. Laccase preparations (15 mg protein) from day 3, 4, exposure to the dye.
23, and 25 were mixed with 1 ml of RBBR solution (50 mg/l) and
the absorbance at 595 nm monitored for 7 h.
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36
Bioconversion of Distillery Waste

Jozefa Friedrich National Institute of Chemistry, Ljubljana, Slovenia

1 INTRODUCTION of effluents and properties of stillage of different origins have

been reviewed and compared by Wilkie et al. (2000).
Alcoholic beverages containing higher concentrations of The presence of a large amount of organic substances of
alcohol than can be obtained by wine fermentation have to be natural origin opens up the possibility of bioconversion by
prepared by distillation. After the distillation process all the microorganisms. While bacteria participate in anaerobic
ingredients of the original fermented mash or liquid, except processes producing biogas, fungi are suitable for aerobic
volatiles, remain in the waste, called stillage, spent wash, bioconversion. There have been several attempts to use fungi
slop, or vinasse. The composition of this distillery waste for treating distillery wastewaters. The aim of microbiological
depends on the original raw materials used: sugarcane- or treatment is to purify the effluent by consumption of organic
sugar beet molasses, corn, wheat, barley, cassava, potato, rice, substances, thus, reducing its COD and BOD, and at the same
fruits, etc. These materials have to be prepared for alcoholic time to obtain some valuable product, such as fungal biomass
fermentation by dilution or mashing, including eventual for protein-rich animal feed, or some specific fungal
hydrolysis and/or addition of nutrients. Stillages remaining metabolite. The present review deals with the application of
after distilling off the alcohol are troublesome wastewaters fungi for treating distillery stillages or slops. Suitable fungi,
that are either pulpy materials or clear fluids containing the bioconversion process, possible products and effects,
settling particles. The stillages are about 10 –14 times the together with economic considerations are described.
volume of the alcohol produced and have a high content of
organic substances (Shojaosadati et al. 1999). The pollution
load is quantified by biochemical oxygen demand (BOD) and 2 DISTILLERY WASTE
chemical oxygen demand (COD). Distillery stillages are
characterized by a very high BOD, very high COD, and a low Distillery stillages have a complex organic and inorganic
pH value. In addition, with certain types of distillery composition. The most important characteristics, in terms of
wastewater such as fruit slops, the separation of solids from their organic strength and nutrient content are given in
the liquid is almost impossible (Friedrich et al. 1986). There Table 1.
are substantial pressures to develop a proper treatment for As can be seen, the values of BOD and COD may vary
making wastes acceptable. A text on organic waste significantly and may reach extremely high values of over 300
conversion (Bewick 1980), as well as several review articles, and more than 150 g/l, respectively. The pH ranges from 3 to
discusses the problem of distillery wastewater (Costa Ribeiro 5. The waste can contain different amounts of solids up to
and Castello Branco 1980; Sheehan and Greenfield 1980; more than 400 g/l. For cultivating microorganisms, the
Weathers 1995; Wilkie et al. 2000). There are many potential content of carbon and nitrogen sources is the most important
solutions for these problems, including simple disposal, factor. The ratio of C:N varies with the raw material used for
sewage treatment, and recovery of useful ingredients. In alcoholic fermentation. In addition to the components given
addition, many attempts have been made to exploit the in Table 1, there are different amounts of organic and amino
organic compounds from stillages in biotechnological acids, polyols, vitamins, phenolics, etc. Minerals include
processes for the production of commercially valuable potassium, phosphorus, sulfates, and some heavy metals.
products. Industrial and experimental utilization and disposal These components may also have an important influence on

431
432 Friedrich

Table 1 Characteristics and composition of distillery stillages

BOD COD Solids Nitrogen Protein Reducing sugars Total sugars

Origin (g/L) (g/L) pH (g/L) (g/L) (g/L) (g/L) (g/L)
Molasses 16 – 103 32 – 158 4.2– 5.2 31 –119 0.2– 6.0 3.7 –8 2 – 18 6 – 30
Grain 10 – 340 15 3.2– 7.5 20 –75 0.2– 1.9 5 –23 11 – 30 2.9a – 5
Malt 24 – 43 35 – 58 2.9– 3.8 15 –31 6 – 21a
Cassava 19 23 5.0 24 0.4 2.5 6.8
Rice 25 – 84 51 3.5– 4.3 94 19.3 –20 1.1 – 13 2.6– 9
Barley 3.9 31 20 14
Sweet potato 20 4.3 2.0 4.4 2.4 11
SWL 40 – 50 100 1 8
Wine 12 – 18 26 3.6– 4.5 23 –125 0.4– 1.0 0.8– 1.1
Fruit 38 – 156 3.3– 4.5 30 –440 0.4– 1.0
SWL, sulfite waste liquor.
a
Carbohydrate.

the pollution load of the wastewater as well as on the molasses stillage. In screening for a useful yeast, Hansenula,
suitability for bioconversion. Debaromyces, and Rhodotorula were selected out of 203
strains (Akaki et al. 1981). Hansenula spp. (Moriya et al.
3 FUNGI FOR BIOCONVERSION OF 1990; Shojaosadati et al. 1999) and Debaromyces sp. (Selim
et al. 1991) were also successful as were Saccharomyces
DISTILLERY WASTE
cerevisiae (Selim et al. 1991), Kluyveromyces spp. (Braun and
Meyrath 1981; Selim et al. 1991), Pichia tainania (Chang and
The selection of the proper organisms to be used for stillage Yang 1973; Lin et al. 1973) and Phaffia rhodozyma (Fontana
bioconversion is of the utmost importance. The organism has et al. 1997). Rhodotorula glutinis produced biomass for
to be able to utilize the substrate effectively and at the same fodder rich in vitamin B on raisin vinasse (Aran 1977;
time its biomass has to have a high nutritive value, especially Yazicioğlu et al. 1980). A distillery effluent from rice spirit
in its protein content. According to Tomlinson (1976a) the production could be successfully reused by a S. cerevisiae
lower and higher fungi are the most suitable for bioprocessing strain (Yang and Tung 1996).
distillery wastes.

3.2 Filamentous Fungi

3.1 Yeasts
Compared to yeasts, filamentous fungi have been used less
Yeasts have been chosen in the great majority of cases frequently for bioconversion of distillery wastes. The reasons
for cultivation in distillery stillages of different origin. They might be that they are slower growing and more susceptible to
have been commonly used in food preparation for thousands infection. However, filamentous fungi have some advantages
of years so there are no prejudices regarding their use for food as they produce a series of extracellular hydrolytic enzymes
or feed, as is the case with bacteria and molds. The quick and are therefore able to exploit complex carbohydrates
growth of yeasts lead to advantages of time and low cost, as without prior hydrolysis; this property enables them to grow
well as limited possibility of contamination by other in starch and cellulose substrates. The second advantage is in
microorganisms. The fermentation process is easy to handle their filamentous morphology that allows separation of the
and continuous cultivation is possible. The use of yeast is biomass by simple filtration. Compared to yeasts, filamentous
interesting because of its low price and its high protein and fungi have lower sensitivity to variations in temperature, pH,
vitamin content. However, some facts must be taken into nutrients, and aeration, and they also have a lower nucleic
account (Huyard et al. 1986): (a) the yeast should not contain acid content in the biomass (Araújo et al. 1977). Various
high levels of contaminants, if it is to be used for feed, (b) species of fungi were used for distillery stillage fermentations,
energy is needed for separating the yeast from the substrate, among them Aspergillus spp. being the most popular ones.
(c) effective aeration is necessary, and (d) the remaining Aspergillus oryzae (Araújo et al. 1977; De Lamo and De
effluent still has a high COD and needs to be treated in a Menezes 1978) and Aspergillus niger (De Lamo and De
sewage treatment facility. Menezes 1978) were selected out of several filamentous fungi
Candida (Torula) is the fungal organism most used for due to their high biomass yield, rapid filtration, and
molasses stillages and to a lesser extent for other distillery substantial COD reduction in cane molasses stillage.
effluents. Other yeasts are also suitable for bioconversion of A. niger was also suitable for bioconversion of rice spirit
Bioconversion of Distillery Waste 433

(Yang and Lin 1998). Aspergillus awamori var. kawachi and in certain kinds of food. Falanghe (1962) investigated the
Aspergillus usami mut. shirousami were used for bioconver- suitability of stillage for growing mushroom mycelia as a
sion of distillery waste of a Japanese alcohol beverage, source of protein and fat. Among ten strains cultivated in
shochu (Kida et al. 1995; Morimura et al. 1991; 1992; 1994a). submerged conditions, Agaricus campestris and Boletus
Filamentous fungi were chosen for conversion of stillage, due indecisus were chosen as the two most suitable. A. campestris
to their texture, protein profile, and easy harvesting, and was more effective in producing mycelial protein content.
Aspergillus phoenicis was the most promising among isolates B. indecisus, however, exhibited a greater ability for mycelial
from contaminated stillage (De Gonzáles and De Murphy production. The dried mycelia had a pleasant slight flavor and
1979). Aspergillus species were also suitable for converting they seemed to have adequate characteristics for improving
fruit based distillery stillages, A. niger biomass produced in foods; their growth in pellet form enabled easy separation
vinasse from raisin brandy has a good amino acid content from the medium. In apple distillery slop, Phanerochaete
(Aran 1977; Yazicioğlu et al. 1980), while cellulolytic chrysosporium grew successfully; it reduced the amount of
Aspergillus species were very effective in improving filtration fiber and improved protein content in the biomass (Friedrich
and reducing COD of apple brandy slops (Friedrich et al. et al. 1986). Coriolus versicolor and P. chrysosporium could
1983; 1986; 1987; Gunde-Cimerman et al. 1986; Perdih et al. be used for reducing pollution and decolorizing the diluted
1991). Many other fungi proved promising in treating and molasses spent wash (Fitzgibbon et al. 1995; 1998). Other
converting different kinds of stillages. For example, white rot fungi, Funalia trogii and Trametes versicolor were
Penicillium species were chosen for utilization of white used for producing plant growth hormones in vinasse from
wine vinasse, due to their good adaptation, utilization of the molasses and, in addition, partial COD and color were
medium, and development at low pH values (Magny et al. removed (Yürekli et al. 1999).
1977). Penicillium oxalicum produced the highest biomass
amount with high protein content in raisin brandy vinasse
(Aran 1977; Yazicioğlu et al. 1980). Geotrichum candidum 3.3 Mixed Cultures
was selected from a variety of soil microorganisms for whisky
stillage conversion (Quinn and Marchant 1979a,b; 1980) and Some research was done on the use of mixed cultures of
for decolorizing molasses stillage, since it was able to reduce different microorganisms to improve the utilization of
the phenolic compounds (Fitzgibbon et al. 1995; 1998). nutrients in stillage and to obtain better yields of biomass.
Compared to several yeasts, molds, and bacteria, Paecilo- Combinations of different yeasts, yeasts and bacteria, yeasts
myces varioti was very effective in aerobic treatment (Nudel and molds or different molds were studied. Biomass yield of
et al. 1987) and for producing single cell protein (SCP) from the mixed cultures can be substantially higher than that of
cane molasses stillage (Azzam and Heikel 1989; Cabib et al. pure cultures. A two-step fermentation proved to be very
1983). Gliocladium deliquescens performed best among effective. Yeast was used in the first step to produce large
different fungi in rum distillery wastewater (Rolz et al. 1975). amounts of biomass, and a filamentous fungus in the second
Acremonium fusidioides was isolated from soil, treated with step markedly reduced the COD of the effluent (Azzam and
stillage, and it clarified and decolorized the effluent and Heikel 1989; Bottaro Castilla et al. 1984; Nudel et al. 1987).
produced the highest biomass amount (Rosalem et al. 1985). In malt whisky spent wash a three-membered mixed culture
Trichoderma species were successfully utilized in aerobic of G. candidum, Hansenula anomala, and Candida krusei
treatment of cane molasses stillage (Nudel et al. 1987) and for constituted a stable population. The relative proportions of the
bioconversion of fruit distillery slops; the fungi degraded raw three organisms were dependent on the dilution rate during
fibers, exhibited high cellulolytic activity, and produced continuous cultivation (Barker et al. 1982). Thirteen different
high protein content in the biomass (Friedrich et al. 1986; yeast species, mainly Candia spp. and Trichosporon spp.,
Perdih et al. 1991). Fusarium moniliforme grown in vinasse were used in a mixed culture for treating the vinasse of beet
from raisin brandy produced high lipid biomass (Aran 1977; molasses (Malnou et al. 1987). Mixed cultures of two
Yazicioğlu et al. 1980). Myrothecium verrucaria was ascomycetous fungi, A. awamori and Trichoderma reesei,
effective in reducing organic substances in apple distillery were tested in apple slops; they expressed the positive
slop as well as in improving its filterability (Friedrich et al. properties of both genera: Aspergillus improved the filtration
1986). Aureobasidium sp. could produce a biopolymer time and COD reduction whereas Trichoderma decreased
pullulan from corn condensed distiller’s solubles (Leathers fiber and increased protein content in the biomass (Friedrich
and Gupta 1994). Among the microorganisms tested for the et al. 1987).
treatment and bioconversion of shochu, a Japanese distilled
spirit, zygomycetous fungi, Absidia atrospora and Gongro-
nella butleri strains were selected and used in a cost effective 4 BIOCONVERSION PROCESS
procedure for chitosan production in the wastewater (Yokoi
et al. 1998). The stillage may be used as a fermentation medium directly
The development of basidiomycetous fungi (mushrooms) after distillation, or it may be pretreated by clarification,
in submerged culture is interesting because of their probable dilution, pH adjustment, and sterilization. In certain cases,
suitability as cheap substitutes for mushroom fruiting bodies supplementation with additional sources of nitrogen and/or
 434

Table 2 Products and effects of bioconversion of distillery waste by yeasts

Cultivation Yeast species Stillage Biomass (g/L) Protein (%) COD reduction (%) References
Batch C. utilis CMS 9 – 18 28 – 50 46 Several references
C. utilis CMS suppl. 4 – 25 30 – 52 16 – 52 Several references
C. arborea CMS 7 – 13 33 – 43 12 – 22 Matsuo et al. (1965)
C. brumptii CMS suppl. 10 53 20 Kim et al. (1976)
C. krusei CMS suppl. 3–8 31 – 48 Tauk (1982) and Tauk and Gambale (1981)
C. membranaefaciens CMS suppl. 3.6 45 Tauk and Gambale (1981)
C. rugosa BMS suppl. 14.7 45.5 Lee and Baerwald (1991)
C. steatolytica HHS 12 Zhang et al. (1982)
C. stellatoidea CMS suppl. 3.5 48 Tauk and Gambale (1981)
C. tropicalis WV 48 Karova et al. (1976)
P. tainania CMS suppl. 19 – 20 42 – 49 Lin et al. (1973)
S. cerevisiae MS suppl. 12.7 Selim et al. (1991)
S. cerevisiae Rice 6 Yang and Tung (1996)
Fed batch S. cerevisiae Rice suppl. 20 Yang and Tung (1996)
C. utilis CMS suppl. 15 41 Cabib et al. (1983)
Continuous C. utilis CMS suppl. 4 – 24 40 – 52 30 – 40 Several references
Hansenula sp. BMS 5.7 39.6 31 Shojaosadati et al. (1999)
Hansenula sp. BMS suppl. 8.5 50.6 35.7 Shojaosadati et al. (1999)
K. marxianus BMS 10 – 11 60 – 70 Braun and Meyrath (1981)
S. cerevisiae Rice suppl. 70 Yang and Tung (1996)
CMS, cane molasses stillage; suppl., supplemented; BMS, beet molasses stillage; WV, wine vinasse; HHS, hemicellulose hydrolyzate stillage; MS, molasses stillage of unknown origin.
Friedrich
Bioconversion of Distillery Waste 435

phosphorus may be necessary. Sometimes enrichment of supplementation a concentration of biomass of up to 25 g/l

stillage with an easily metabolizable carbon source can could be obtained with C. utilis in cane molasses stillage
improve growth and substrate utilization (Cabib et al. 1983; (Cabib et al. 1983). The high quantity of C. utilis biomass of
Wang et al. 1977). The proportions of elements C:N:P of 22 g/l obtained by Wang et al. (1980) in continuous
50:5:1 were used in growing filamentous fungi in sugarcane fermentation was a result of cell recycling. Crude protein
stillage (Araújo et al. 1977; De Lamo and De Menezes 1978), content in the biomass of Candida utilis accounted for 28 –
while 40:10:1 was optimal for A. fusidioides in the same 58% of the cell mass. With the same species the maximal
substrate (Rosalem et al. 1985). Cells from agar slants can be COD reduction in the effluent was 52% (Nudel et al. 1987)
used directly as inocula for stillage fermentation, or the and the highest BOD reduction was 55% (Matsuo et al. 1965).
relevant microbes can be precultured under submerged Among yeasts shown in Table 2, other than C. utilis,
conditions. The usual quantity of inoculum for inoculation is outstanding biomass concentration of 70 g/l was produced by
10% of the broth volume. Previous adaptation of the S. cerevisiae during continuous cultivation in enriched rice
microbial strain to the stillage may have a positive effect on spirit stillage (Yang and Tung 1996). In general, concen-
bioconversion. Submerged aerobic processes are used with trations of about 20 g/l were relatively high. The biomass of
laboratory experiments carried out in shake flasks and Candida brumptii showed the highest protein content of 53%.
fermentors. In large-scale experiments culture volumes of Kluyveromyces marxianus seemed to be outstandingly
9 m3 (Cabib et al. 1983) and even 80 m3 (Wang et al. 1980) efficient, considering the COD reduction of 60 –70%, while
have been reported. Batch, fed batch, or continuous operation its biomass concentration of 10 g/l was low in comparison
modes are employed in trial fermentation. In addition to with C. utilis. The results of reduced COD and BOD are given
single fungus inocula, mixed cultures of microorganisms or a in terms of relative values and the absolute reduction is
two-stage cultivation with successive cultivation of two dependent on the initial value.
different microorganisms have been used. Cell recycling Filamentous fungi are cultivated mostly in batch
proved to be beneficial for higher yields of biomass as well as processes. Results of bioconversion of distillery waste by
for better consumption of organic substances (Staheeu et al. filamentous fungi are given in Table 3.
1985; Wang et al. 1980). Cultivation temperatures were kept When compared with yeasts, filamentous fungi are more
between 22 and 388C, suitable for mesophilic microorgan- effective in consuming polluting substances, since COD and
isms; pH control at the predetermined value often proved to BOD reductions of over 80 and 90%, respectively, have been
be beneficial to the microorganism. The fermentation broth recorded. A. oryzae (Araújo et al. 1977) and G. deliquescens
required vigorous aeration to ensure good growth. Aeration (Rolz et al. 1975) in sugarcane stillage, Penicillium strains in
values of 0.5– 1.0 vvm generally were employed; exceptions wine vinasse (Magny et al. 1977), and G. candidum in malt
being the very high aeration rates in small bioreactors or whisky stillage (Quinn and Marchant 1980) proved to be the
lower aeration rates of 1 vvh (Wang et al. 1980) in large scale most promising in this respect. The highest amount of
experiments. Agitation rates range from 200 to 1000 rpm. In biomass was reached with A. awamori var. kawachi in rice-
continuous cultivation, dilution rates of 0.016 h21 (Yang and shochu distillery wastewater (Morimura et al. 1994a). It was
Tung 1996) to 0.383 h21 (Wang et al. 1980) were employed. observed that on average the protein content in the biomass of
The dilution rate could be increased by the use of a cell- filamentous fungi was lower than that of yeasts. G. candidum,
recycling technique (Wang et al. 1980). In batch processes the however, was found to produce the highest protein content;
fermentation time is dependent on the microorganism and there was 45.5% “true” protein in the biomass as determined
ranged from several hours to 2 days with yeasts, to several by the biuret method (Quinn and Marchant 1980).
days with molds, and up to 12 days with mushrooms. After An overview of the bioconversion products and effects
fermentation the yeast biomass is generally harvested by using mixed cultures of different microorganisms is given in
centrifugation, whereas with filamentous fungi simple Table 4.
filtration is possible. In general, mixed cultures were the most effective in
substrate utilization, based on COD values. A co-culture of 15
yeast strains in a high-loaded beet molasses stillage of initial
5 PRODUCTS AND EFFECTS 74.5 g/l COD, produced the highest amount of biomass,
28.9 g/l, in batch culture. When diluted medium was applied,
Bioconversion of distillery waste by means of fungi brings a the biomass yield was much lower (Huyard et al. 1986). A
double benefit: the effluent is substantially purified and, in successful approach seems to be a two-step separated
addition, it is possible to obtain useful products, such as cultivation of C. utilis and P. varioti by which a total amount
protein-rich fungal biomass, ethanol, enzymes, etc. Results of of 22 g/l cell material and a COD reduction of as much as 90%
bioconversion by yeasts are given in Table 2. were obtained. During the first step, C. utilis was used mainly
In most experiments C. utilis was used as the chosen for SCP production, whereas P. varioti in the second step
microorganism, especially for cane molasses stillage consumed the reducing organic substances in the liquid phase
(Friedrich et al. 1992). The quantity of cells produced varied (Azzam and Heikel 1989; Bottaro-Castilla et al. 1984).
significantly with the concentration of the substrate and with Similar results were observed in a two-step continuous
the addition of N sources. With increasing nitrogen cultivation of C. utilis and A. niger grown separately (Nudel
 436

Table 3 Products and effects of bioconversion of distillery waste by filamentous fungi

Biomass Protein COD reduction BOD reduction

Fungus Stillage (g/L) (%) (%) (%) References
A. campestris CMS 13 45 Falanghe (1962)
A. fusidioides CMS 11 40 Rosalem et al. (1985)
A. niger CMS 8 – 13 30 – 40 46 – 78 De Lamo and De Menezes (1978) and Rosalem et al. (1985)
A. awamori var. Rice 40 40 76 (TOC) Morimura et al. (1994b)
kawachi
A. niger Fruit 4 – 20 12 – 36 50 – 70 Friedrich et al. (1983, 1986) and Gunde-Cimerman et al. (1986)
A. oryzae CMS 14 – 17 35 – 50 61 – 88 79 – 83 Araújo et al. (1977)
A. oryzae CMS 12 – 15 39 48 – 72 78 De Lamo and De Menezes (1978)
A. phoenicis CMS 20 21 58 De Gonzáles and De Murphy (1979)
(rum)
G. candidum Whiskya 3.5 80.6 92 Quinn and Marchant (1980)
G. candidum Whisky 11 – 27 32 – 78 63 – 91 Quinn and Marchant (1980)
G. candidum Whiskyb 34 46 87 Quinn and Marchant (1980)
G. deliquescens CMS 11 – 19 60 – 85 Rolz et al. (1975)
(rum)
M. verrucaria CMS 11 – 12 79 – 82 Rolz et al. (1975)
(rum)
P. elegans CMS 11 – 14 62 – 66 Rolz et al. (1975)
(rum)
P. varioti CMS 13 – 25 40 43 – 70 Bottaro Castilla et al. (1984), Cabib et al. (1983), and Nudel et al.
(1987)
P. varioti CMS 5 70 Azzam and Heikel (1989)
P. oxalicum Raisins 12 34 Aran (1977) and Yazicioğlu et al. (1980)
Penicillium sp. CMS 12 – 16 65 Rolz et al. (1975)
(rum)
Penicillium spp. WV 13 41 91 Magny et al. (1977)
T. viride CMS 12 – 28 59 – 79 Nudel et al. (1987) and Rolz et al. (1975)

CMS, cane molasses stillage; WV, wine vinasse.

a
Diluted five times.
b
Continuous, two stage.
Friedrich
 Bioconversion of Distillery Waste

Table 4 Bioconversion of distillery waste by mixed microbial cultures

Microorganisms Stillage Biomass (g/L) COD reduction (%) Cultivation mode References
P. varioti þ T. viride CMS 12 – 17 50 – 64 Batch Nudel et al. (1987)
T. viride þ A. oryzae CMS 10 – 13 50 – 54 Batch Nudel et al. (1987)
C. utilis þ P. varioti CMS 8 – 12 45 – 50 Batch Nudel et al. (1987)
C. utilis þ C. acetoacidophilum CMS 16 – 17 65 Batch Nudel et al. (1987)
C. utilis þ B. flavum CMS 16 65 Batch Nudel et al. (1987)
Azotobacter þ C. utilis CMS 16 Batch Nudel et al. (1987)
C. utilis þ A. niger CMS 16 – 17 89 Cont. serial Nudel et al. (1987)
C. utilis þ P. varioti CMS 22 92 Batch Bottaro Castilla et al. (1984)
C. utilis þ P. varioti MS 22 90 Batch, two step Azzam and Heikel (1989)
C. utilis þ P. varioti CMS 14 – 16 85 Cont. serial Bottaro Castilla et al. (1984)
15 yeasts BMS dil. 7 79 Batch Huyard et al. (1986)
15 yeasts BMS dil. 9 72 Cont. Huyard et al. (1986)
15 yeasts BMS 29 74 Batch Huyard et al. (1986)
13 yeasts BMS dil. 7 – 12 68 – 75 Cont. Malnou et al. (1987)
G. candidum þ C. crusei þ H. anomala MWS 13 55 Batch Barker et al. (1982)
G. candidum þ C. crusei þ H. anomala MWS 5 32 Cont. Barker et al. (1982)
A. awamori þ T. reesei Apple dil. 5 31 Batch Friedrich et al. (1987)
CMS, cane molasses stillage; BMS, beet molasses stillage; MS, molasses stillage (unknown origin); dil, diluted; MWS, malt whisky stillage.
437
 438

Table 5 Bioconversion of stillage by continuous cultivation of fungi

Dilution rate Productivity Biomass COD reduction BOD reduction

Fungus (h21) (g/L/h) (g/L) (%) (%) References
C. utilis 0.383 4.24 17.9 34.6 Wang et al. (1980)
C. utilis 0.365 4.06 21.9 37.1 Wang et al. (1980)
C. utilis 0.22 2.64 12 Cabib et al. (1983)
C. utilis 0.27 3.24 12 Cabib et al. (1983)
C. utilis 0.2 1.8– 2.0 9 –10 35 Bottaro Castilla et al. (1984) and Nudel
et al. (1987)
C. utilis 0.2
P. variotia 0.1 2.3– 2.6 19 –21 85 Bottaro Castilla et al. (1984)
C. utilis 0.2
A. nigera 0.1 2.7– 3.2 15 –17 89 Nudel et al. (1987)
K. marxianus 0.3 3 10 –11 60 – 70 Braun and Meyrath (1981)
G. candidum 0.125 2.24 30 50 Quinn and Marchant (1980)
G. candidum 1st step 0.125
2nd step 0.10 31 50 Quinn and Marchant (1980)
G. candidum 1st step 0.125
2nd step 0.085 1.72 34 87 Quinn and Marchant (1980)
G. candidum þ C. crusei þ 0.10 0.48 4.8 31.5 Barker et al. (1982)
H. anomala
G. candidum þ C. crusei þ 0.20 0.36 15.9 Barker et al. (1982)
H. anomala
G. candidum þ C. crusei þ 0.35 0.42 2.0 Barker et al. (1982)
H. anomala
Hansenula sp. 0.12 ,1 8.5 35.7 Shojaosadati et al. (1999)
G. deliquescens 0.03 17 –22 61 – 50 Rolz et al. (1975)
S. cerevisiae 0.016 70b Yang and Tung (1996)
Association of 15 yeasts 0.133– 0.134 0.5– 1.6 11 68 Huyard et al. (1986) and Malnou et al.
(1987)
a
Two step process.
b
Enriched stillage.
Friedrich
Bioconversion of Distillery Waste 439

et al. 1987). Summarizing the results of conversion of both acceptability, good weight gain, and no toxicity; the biomass
steps, 17 g/l biomass and 89% COD reduction were obtained. had a very good protein efficiency ratio, being comparable to
In general, it was observed that the combination of yeast with meat and soya meal (Araújo et al. 1977). A 1% methionine
filamentous fungi resulted in improvement of COD reduction, addition to Candida yeast biomass increased the biological
and improved wastewater purification. value, which was not much lower than that of casein (Cabib
While the batch cultivation mode is most frequently used, et al. 1983).
continuous cultivation of fungi in stillages can have some With the use of fungi for bioconversion of distillery
advantages regarding the product yield (Table 5). wastewaters, not only fungal biomass but also other products
As shown in Table 5, an outstanding biomass yield of can be obtained. Secondary ethanol can be produced by the
70 g/l was produced by S. cerevisiae in enriched stillage from original yeast used in a primary alcohol fermentation, if the
rice spirit distillation when the dilution rate was low (Yang distillation is performed at low temperatures in vacuum
and Tung 1996). On the other hand, dilution rates of over (Teramoto et al. 1993; Ueda and Teramoto 1995; Ueda et al.
0.36 h21 with cell recycling resulted in a productivity of over 1991). Microbial polysaccharides can be produced, such as
4 g/l/h Candida biomass and a steady-state biomass pullulan with Aureobasidium sp. (Leathers and Gupta 1994)
concentration of 18–22 g/l (Wang et al. 1980). In continuous or chitosan with A. atrospora or G. butleri strains (Yokoi et al.
culture, COD was reduced most effectively by using 1998). Hydrolytic enzymes can be produced by filamentous
K. marxianus (Braun and Meyrath 1981). A serial culture of fungi. Cellulases and other glucosidases can be products of
a yeast and a filamentous fungus in two steps gave the most bioconversion when cellulolytic fungi are grown in fruit
promising results with respect to both biomass yield and distillery slops (Friedrich et al. 1986; 1987; Gunde-Cimerman
efficient consumption of organic matter (Bottaro Castilla et al. 1986). Some ascomycetes, especially A. awamori var.
et al. 1984; Nudel et al. 1987). Two-step cultivation of kawachi, are able to produce starch saccharifying enzymes
G. candidum also seemed to be very suitable, with the from shochu distillery wastewater (Morimura et al. 1991;
steady-state biomass of 31 –34 g/l (Quinn and Marchant 1992). Acid proteases are produced mainly with Aspergillus
1980). The continuous cultivation process could lead to a species in rice (Morimura et al. 1994a; Yang and Lin 1998)
higher protein content in the biomass and enhance the and barley (Morimura et al. 1994a) shochu stillages. Possible
stability of the culture as well as its resistance to products are a yeast pigment, astaxanthin, from P. rhodozyma
contamination (Barker et al. 1982). (Fontana et al. 1997) and plant growth hormones, obtained by
Microbial biomass rich in protein is the main product of growing white rot fungi, such as F. trogii or T. versicolor
fungal bioconversion of stillages. Some information is (Yürekli et al. 1999).
available about cell material composition. It appears that
C. utilis biomass composition is similar to that of other
yeasts grown in carbohydrate media. Microbial protein has a 6 ECONOMIC CONSIDERATIONS
good balance of amino acids with the exception of those
containing sulfur, such as cysteine and methionine; the Little information is available concerning the economics of
content of the latter amino acids is generally low in the bioconversion process. Only rough estimates of costs were
microbial biomass (Cabib et al. 1983; Quinn and Marchant given by Maiorella et al. (1983) and Tomlinson (1976b). The
1979b; Yazicioğlu et al. 1980). However, lysine is in excess main costs are high capital investment (Tomlinson 1976b),
when compared to the recommended level of the FAO energy needed for aeration, agitation and cooling (Maiorella
standards (Quinn and Marchant 1979b). Yeast biomass from et al. 1983), biomass separation (Tomlinson 1976b), nutrient
vinasse is rich in nitrogen, vitamins, and other biologically supplementation, sterility problems, limited production, and
active substances. The vitamin B complex of yeast is very irregularities in raw material composition. However, capital
efficient and could not be adequately substituted by a cost may not be too high, as was shown in the cost evaluation
mixture of analytically pure vitamins (Cabib et al. 1983; for molasses stillage treatment methods, where aerobic yeast
Yazicioğlu et al. 1980). growth was estimated to require the lowest capital investment
Nutritional evaluation of yeast biomass grown in malt (Maiorella et al. 1983). Separation of cell materials from the
whisky distillery slop showed that it was suitable for broth would be easy with the application of filamentous fungi.
nonruminants with a net protein utilization value of 0.40, and The addition of nutrients is not always required. Sterility
a digestibility of 0.67. The biomass was not toxic, as problems could be managed or at least reduced by
confirmed by toxicological tests (Barker et al. 1982). When maintaining a low pH (Cabib et al. 1983; Huyard et al.
molasses stillages were used, K and Mg contents could have 1986; Matsuo et al. 1966), and the use of mixed cultures
laxative effects (Araújo et al. 1977). The nucleic acid content (Barker et al. 1982). The problem with large-scale plants is
in cell material should be as low as possible; filamentous the need for a continuous and relatively homogeneous supply
fungi have an advantage over yeasts in this respect (Araújo of stillage. Comparison of cost with that for conventional
et al. 1977). It has been observed that levels of DNA and RNA biological treatment has shown that annual savings could be
were lower in batch than in continuous cultures (Quinn and increased by applying fungal bioconversion of stillages
Marchant 1979b). Feeding experiments of Aspergillus (Tomlinson 1976b). Similarly, rough estimates of capital and
biomass in a diet for chicks, demonstrated excellent operating costs for molasses stillage treatment by fungi show
440 Friedrich

that savings could be gained, even though wastewater would Bewick MWM (1980). Handbook of Organic Waste Conversion.
still have to be purified. Comparison of different treatment New York: Van Nostrand Reinhold.
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Paecilomyces varioti in two step continuous cultures.
was proposed for sulfite waste liquor and wood hydrolysis
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37
Degradation of Hydrocarbons by Yeasts and Filamentous Fungi

John B. Sutherland National Center for Toxicological Research, U.S. Food and Drug Administration,
Jefferson, Arkansas, USA

1 INTRODUCTION Napolitano and Juárez 1997). Many filamentous fungi and

yeasts grow abundantly in soils contaminated by petroleum
Hydrocarbons are ubiquitous in the environment, but they are residues (April et al. 1998; 2000; Ekundayo and Obuekwe
most abundant where coal and petroleum fuels are stored, 2000). Fungi also degrade hydrocarbons in streams and lakes
processed, or burned (Tuháčková et al. 2001; Warshawsky (Griffin and Cooney 1979; Romero et al. 2001) and in oil-
1999). Because many hydrocarbons are toxic, their biological polluted seawater (Cerniglia 1997; Zinjarde et al. 1998).
effects have been studied extensively. Occupational exposure Filamentous fungi, especially Hormoconis resinae [the
to polycyclic aromatic hydrocarbons (PAHs) in the anamorph of Amorphotheca resinae], may contaminate
aluminum, coke, and steel industries has been linked to aviation fuels when water is present (Parbery 1971). Yeasts
lung and bladder cancer (Mastrangelo et al. 1996) and are responsible for biodeterioration of liquid fuels at the
exposure to hydrocarbon fuels may produce neurotoxic interface with water and mycelial fungi may physically block
effects (Ritchie et al. 2001). Mammalian metabolites of the fuel lines of ships and aircraft (Lindley 1992). The ability
benzo[a]pyrene, the most important carcinogen in natural of fungi to metabolize hydrocarbons is now being exploited to
PAH mixtures, form adducts with macromolecules and clean up the environment. Several fungi are used to remove
interfere with cellular signaling pathways (Miller and Ramos PAHs from wastewater (Giraud et al. 2001; Liao et al. 1997)
2001). Some PAHs are either weakly estrogenic or and yeasts are used to decompose petroleum residues in
antiestrogenic (Santodonato 1997). The widespread ability estuaries (Nwachukwu 2000). Fungi have been used to
of yeasts and filamentous fungi to transform hydrocarbons inoculate contaminated soils to degrade hydrocarbons (May
suggests that they may be involved in the recycling of et al. 1997; Novotny et al. 1999; Rama et al. 2000). Cultures
naturally occurring hydrocarbons in the environment as well that grow on toluene, ethylbenzene, and styrene can be used in
as in the biodeterioration of liquid fuels (Lindley 1992). biofilters to remove these compounds from industrial waste
Their versatility in degrading hydrocarbons is due to the gases (Cox et al. 1996; Garcı́a-Peña et al. 2001; Prenafeta-
broad substrate specificity of their enzymes (Cerniglia and Boldú et al. 2001).
Sutherland 2001). Selected fungi are now being exploited Many purified hydrocarbons can be transformed stereo-
for the bioremediation of soils contaminated with hydrocarbon specifically by fungi to produce higher-value products. The
wastes (Atlas and Cerniglia 1995; Cerniglia and Sutherland monoterpenes a- and b-pinene (Prema and Bhattacharyya
2001) and other species are used for the biotransformation of 1962), b-myrcene (Yamazaki et al. 1988), and limonene
hydrocarbons to higher-value compounds (Trudgill 1994). (Noma et al. 1992; de Oliveira and Strapasson 2000) as well
The recycling of hydrocarbons by fungi is probably as several sesquiterpenes (Abraham et al. 1992; Miyazawa
common, though usually unnoticed, in the environment. For et al. 1995; 1997; 1998) are transformed to chiral metabolites.
instance, certain yeasts can utilize the methane produced in (þ)-Limonene can be transformed to perillyl alcohol, an
lakes (Wolf and Hanson 1979; 1980). Fungi that are anticancer drug (de Oliveira and Strapasson 2000). Ethyl-
pathogenic to insects can degrade the hydrocarbons found benzene and propylbenzene are transformed stereospecifi-
in the epicuticular waxes of their hosts (Crespo et al. 2000; cally to (þ)-1-phenylethanol and (þ)-1-phenylpropanol,

443
444 Sutherland

which may be used as chiral building blocks for chemical conjugated later with sulfate or glucoside (Casillas et al.
synthesis (Uzura et al. 2001). Some species of fungi are more 1996; Pothuluri et al. 1990; 1996). Another species,
abundant in sites with hydrocarbon deposition (Tuháčková C. blakesleeana, oxidizes cyclohexylcyclohexane (Davies
et al. 2001). The fungal genera that have been studied most et al. 1986).
extensively for use in the biotransformation of hydrocarbons Aspergillus niger and A. fumigatus both metabolize
are the white-rot basidiomycetes Phanerochaete, Pleurotus, terpenes and PAHs. A. niger converts the terpene b-myrcene
and Trametes, the zygomycete Cunninghamella, the hypho- to dihydroxylated derivatives (Yamazaki et al. 1988). Another
mycetes Aspergillus and Penicillium, and the yeast Candida. strain of this species oxidizes the PAHs phenanthrene and
Several of these fungi have been used in the experimental pyrene to phenols and methyl ethers (Sack et al. 1997a), and
bioremediation of soils contaminated with oil (Atlas and there is even a report of the ability of A. niger to cleave
Cerniglia 1995; Colombo et al. 1996). Phanerochaete the rings of naphthalene, anthracene, and phenanthrene
chrysosporium causes a white rot of wood by degrading (Yogambal and Karegoudar 1997). A. cellulosae
lignin (Hammel 1995). It oxidizes PAHs to trans-dihydro- [A. fumigatus] transforms (þ )- and (2 )-limonene by
diols, phenols, quinones, glucoside conjugates, aromatic hydroxylation, double-bond reduction, and ketone formation
acids, and eventually CO2 (Bumpus 1989; Cerniglia and (Noma et al. 1992). A. fumigatus also produces a cytochrome
Sutherland 2001; Sutherland et al. 1991). Several enzymes are P450 that hydroxylates benzo[a]pyrene (Venkateswarlu et al.
responsible, including lignin peroxidase, manganese per- 1996). Penicillium glabrum oxidizes pyrene to mono- and
oxidase, and cytochrome P450 (Bogan et al. 1996; Cerniglia dihydroxylated derivatives, quinones, methyl ethers, and a
and Sutherland 2001; Hammel et al. 1991; 1992). sulfate (Wunder et al. 1997). P. digitatum hydrates one of the
P. chrysosporium also cometabolizes single-ring aromatic double bonds in (þ)-limonene to form (þ)-a-terpineol
compounds, such as benzene and toluene, to CO2 (Yadav and (Demyttenaere et al. 2001; Tan et al. 1998) and a different
Reddy 1993). P. laevis, another species which oxidizes PAHs Penicillium sp. strain transforms a-pinene to verbenone
to polar metabolites (Bogan and Lamar 1996), produces (Agrawal and Joseph 2000). P. janthinellum oxidizes high-
manganese peroxidase and a small amount of laccase but not molecular-weight PAHs to phenols and quinones (Boonchan
lignin peroxidase. et al. 2000; Launen et al. 2000). Candida maltosa, a yeast that
Pleurotus ostreatus, the cultivated oyster mushroom, is grows on n-alkanes, and some other Candida spp. produce
another white-rot fungus with the ability to mineralize PAHs cytochrome P450 monooxygenases that hydroxylate
to CO2 (Bezalel et al. 1996a; Wolter et al. 1997). Other n-hexadecane and n-octadecane (Scheller et al. 1996).
metabolites include trans-dihydrodiols, quinones, and aro- Alkane-utilizing Candida sp. strains from oil-polluted soils
matic acids (Bezalel et al. 1996b). Evidence supports the may produce biosurfactants as well as oxidative enzymes
involvement of a cytochrome P450 and an epoxide hydrolase (Ekundayo and Obuekwe 2000). A strain of C. utilis that
in the early steps of PAH metabolism; manganese peroxidase utilizes n-alkanes has been used for the bioremediation of
and laccase may be involved in the later steps (Bezalel et al. mangrove sites (Nwachukwu 2000). Among the hydrocarbons
1996b; Schützendübel et al. 1999). P. pulmonarius, a related that can be transformed by fungi are alkanes (Lindley
species, produces a cytochrome P450 that hydroxylates 1992; Morgan and Watkinson 1994), terpenes (Trudgill
benzo[a]pyrene (Masaphy et al. 1999). Trametes versicolor, 1994), monocyclic aromatic compounds (Prenafeta-Boldú
another basidiomycete that causes a white rot of wood, also et al. 2001; Yadav and Reddy 1993), and PAHs (Cerniglia
degrades PAHs in soil (Rama et al. 2000). It produces a 1992; 1993; 1997; Cerniglia and Sutherland 2001; Cerniglia
manganese peroxidase that oxidizes fluorene and phenan- et al. 1992; Juhasz and Naidu 2000; Hammel 1995;
threne (Collins and Dobson 1996). T. versicolor also produces Müncnerová and Augustin 1994; Pothuluri and Cerniglia
laccases, which can oxidize anthracene and benzo[a]pyrene in 1994; Sutherland 1992; Sutherland et al. 1995). This
the presence of chemical mediators (Collins et al. 1996; chapter will emphasize the recent research on hydrocarbon
Johannes and Majcherczyk 2000). A second species, transformation by fungi.
T. hirsuta, produces a laccase that has been shown to oxidize
phenanthrene in the presence of linoleic acid and the mediator
1-hydroxybenzotriazole (Böhmer et al. 1998). Cunning- 2 ALKANES AND ALKENES
hamella elegans, a zygomycete, oxidizes biphenyl and a large
variety of PAHs to metabolites with higher water solubility Many soil fungi, such as Aspergillus terreus, Pseudal-
(Cerniglia 1992; 1993; 1997; Dodge et al. 1979; Cerniglia lescheria boydii, and the yeast Candida tropicalis, degrade
et al. 1992; Sutherland 1992; Pothuluri and Cerniglia 1994; the mixtures of n-alkanes found in crude oil (April et al. 1998;
Cerniglia and Sutherland 2001). C. elegans first oxidizes a 2000; Colombo et al. 1996; Nwachukwu 2000), often with the
PAH to an arene oxide by a cytochrome P450 monooxygen- help of biosurfactants (Lindley 1992). Candida utilis has also
ase reaction (Cerniglia 1992; 1997). It then quickly hydrates been used for the bioremediation of n-alkanes in aquatic
the arene oxide, forming a trans-dihydrodiol, by an epoxide ecosystems (Nwachukwu 2000).
hydrolase reaction (Sutherland et al. 1995). Alternatively, the Methane, the major component of natural gas, is produced
arene oxide may rearrange nonenzymatically to form a by a variety of methanogenic Archaea. It is utilized for growth
phenol. The trans-dihydrodiols and phenols may be by methanotrophic yeasts (Wolf and Hanson 1979; 1980) that
Hydrocarbon Degradation by Fungi 445

lack the ability to grow on methanol, formaldehyde, or found to make limited growth on cyclohexane (Prenafeta-
formate. Ethane, propane, and n-butane, which are also found Boldú et al. 2001). The synthetic compound cyclohexyl-
in natural gas, are oxidized by Acremonium sp. and some cyclohexane is cometabolized to a diol by B. bassiana,
other fungi to alcohols that can be utilized for growth (Davies Cunninghamella blakesleeana, and Gongronella lacrispora
et al. 1976). (Davies et al. 1986).
Straight-chain alkanes, from n-decane to n-octadecane, are Alkenes, produced during the cracking of petroleum and
utilized for growth by several yeasts (Ekundayo and Obuekwe natural gas, have seldom been investigated as possible
2000; Iida et al. 2000). They are hydroxylated by inducible substrates for fungal degradation. Hexadec-1-ene and
cytochrome P450 monooxygenases in C. tropicalis, heptadec-1-ene are oxidized by Candida lipolytica
C. maltosa, and C. apicola (Scheller et al. 1996) and [Y. lipolytica] via three different pathways (Figure 1) to
Yarrowia lipolytica (Iida et al. 2000). Several genes encoding epoxides, alcohols, diols, and carboxylic acids (Klug and
cytochromes P450 have been identified in the alkane- Markovetz 1968). 1,13-Tetradecadiene is utilized for growth
metabolizing yeasts (van den Brink et al. 1998; Iida et al. by unidentified fungi (Griffin and Cooney 1979). Cyclo-
2000). n-Hexadecane induces conversion of the mycelial hexene is transformed via 2-cyclohexenol to 2-cyclohexenone
form of Y. lipolytica to the yeast form (Zinjarde et al. 1998). and 1-methylcyclohexene is transformed via 3-methyl-2-
Cladosporium resinae [H. resinae], a filamentous fungus cyclohexenol to 3-methyl-2-cyclohexenone by Aspergillus
which grows on n-alkanes, produces an NADH-dependent cellulosae [A. fumigatus] (Noma et al. 1992).
alkane monooxygenase located in the cytosol (Goswami and
Cooney 1999). The insect pathogens Metarhizium anisopliae
and Beauveria bassiana grow on n-hexadecane, n-tetracosane, 3 TERPENES
n-pentacosane, and n-octacosane, apparently by b-oxidation
(Napolitano and Juárez 1997; Crespo et al. 2000). Terpene hydrocarbons (Figure 2) are plant products,
The branched alkanes pristane (2,6,10,14-tetramethyl- synthesized from isoprene units, that are found in the
pentadecane) and phytane (3,7,11,15-tetramethylhexadecane) essential oils of plants (Trudgill 1994). Several mono-
are usually considered recalcitrant to microbial attack but can terpenes, sesquiterpenes, and triterpenes are transformed
be degraded by several fungi, including P. boydii (April et al. stereoselectively by fungi.
1998; Griffin and Cooney 1979). 3,11-Dimethylnonacosane, (þ)-a-Pinene from oil of turpentine is transformed by
another branched alkane, is degraded to shorter-chain A. niger to (þ)-verbenone, (þ )-cis-verbenol, and (þ)-trans-
hydrocarbons by M. anisopliae (Napolitano and Juárez sobrerol (Prema and Bhattacharyya 1962); by Armillariella
1997). mellea to (þ )-trans-sobrerol, (þ )-trans-verbenol, and
Cyclohexane, which occurs in petroleum and is used as a (þ)-verbenone plus seven minor metabolites (Draczyńska
solvent, is transformed cometabolically by several fungi et al. 1985); and by Penicillium sp. to verbenone (Agrawal
(Griffin and Cooney 1979). A Cladophialophora sp. has been and Joseph 2000). (2)-a-Pinene is converted by A. mellea

Figure 1 Pathways for the oxidation of 1-alkenes by the yeast Yarrowia lipolytica (adapted from Klug and Markovetz 1968).
446 Sutherland

Figure 2 Structures of several terpene hydrocarbons and the steroid hydrocarbon stigmasta-3,5-diene.

to (2 )-trans-sobrerol, (2 )-verbenone, (2 )-7-hydroxy-a- The triterpene squalene (Figure 2) and steroid hydro-
terpineol, and five other compounds (Draczyńska et al. carbons, such as stigmasta-3,5-diene, are among the lipophilic
1985) and by the yeast Hormonema sp. to trans-verbenol and extractives in eucalyptus wood that have been found to be
verbenone (van Dyk et al. 1998). (2 )-b-Pinene is partially degraded by Phlebia radiata and other white-rot
transformed by A. mellea to trans-pinocarveol, isopinocam- fungi (Martı́nez-Íñigo et al. 2000).
phone, pinocarvone, and six minor metabolites (Draczyńska
et al. 1985) and by Hormonema sp. to pinocamphone and
3-hydroxypinocamphone (van Dyk et al. 1998). 4 MONOCYCLIC AROMATIC
b-Myrcene, from oil of bay, is transformed by A. niger, HYDROCARBONS
which adds two hydroxyl groups at any of the double bonds to
form three isomeric diols (Yamazaki et al. 1988). The BTEX compounds (benzene, toluene, ethylbenzene, and
(þ)-Limonene, found in orange-peel oil, is transformed by the xylene isomers) are toxic components of liquid fuels
yeasts and filamentous fungi to several products, including (Ritchie et al. 2001). They are also commonly used in industry
(þ)-isopiperitenone, (þ)-limonene trans-1,2-diol, (þ)-cis- as solvents and chemical feedstocks. Toluene and ethyl-
carveol, (þ )-perillyl alcohol, (þ )-perillic acid, trans- benzene support the growth of a few fungi, which have
isopiperitenol, and a-terpineol (de Oliveira and Strapasson consequently been used for the bioremediation of the volatile
2000; Noma et al. 1992; van Rensburg et al. 1997; van Dyk components of toxic wastes (Prenafeta-Boldú et al. 2001).
et al. 1998). Penicillium digitatum converts it mainly to Benzene and xylenes do not appear to serve as carbon or
(þ )-a-terpineol (Tan et al. 1998). (2 )-Limonene is energy sources for fungi, although there are reports of the
transformed by A. fumigatus to (2 )-perillyl alcohol, fungal cometabolism of benzene to phenol (Griffin and
(2 )-limonene trans-1,2-diol, and (þ )-neodihydrocarveol Cooney 1979) and even of its mineralization to CO2 (Yadav
(Noma et al. 1992). Corynespora cassiicola converts the and Reddy 1993).
(þ) and (2) isomers of limonene mainly to (þ)- and Toluene is utilized as a carbon and energy source by
(2 )-limonene trans-1,2-diol (Demyttenaere et al. 2001). Cladosporium sphaerospermum, Scedosporium apiospermum
Several sesquiterpenes (Figure 2) are transformed by [the anamorph of P. boydii ], Cladophialophora sp.,
fungi. Calarene is oxidized by Diplodia gossypina at two of Exophiala sp., and Pseudeurotium zonatum; these fungi
the ring carbons and one of the methyl groups of the may be used in biofilters to clean up toluene in waste gases
cyclopropane ring (Abraham et al. 1992). b-Selinene, (Garcia-Peña et al. 2001; Prenafeta-Boldú et al. 2001; Weber
(þ)-aromadendrene, and its isomer (2 )-alloaromadendrene et al. 1995). In high-nitrogen media, P. chrysosporium
are transformed by Glomerella cingulata to triols (Miyazawa cometabolizes toluene to CO2 (Yadav and Reddy 1993).
et al. 1995; 1997) and (þ)-g-gurjunene is transformed by Ethylbenzene is used as a growth substrate by
G. cingulata to a diol and a triol (Miyazawa et al. 1998). C. sphaerospermum, Cladophialophora sp., and Exophiala
Hydrocarbon Degradation by Fungi 447

sp. (Prenafeta-Boldú et al. 2001) and is cometabolized by groups by Sporotrichum sulfurescens [B. bassiana] (Johnson
P. chrysosporium (Yadav and Reddy 1993). Fusarium et al. 1973).
moniliforme [F. verticillioides] oxidizes ethylbenzene and Styrene (C6H5CHvCH2) serves as a growth substrate
propylbenzene to (þ)-1-phenylethanol and (þ)-1-phenyl- for Exophiala jeanselmei, C. sphaerospermum, and
propanol, respectively (Uzura et al. 2001). Cladophialophora sp. (Cox et al. 1996; Prenafeta-Boldú
The o-, m-, and p-isomers of xylene are all cometabolized et al. 2001). In E. jeanselmei, it is oxidized by a
by P. chrysosporium but the products are unknown (Yadav cytochrome P450 monooxygenase to styrene oxide, which
and Reddy 1993). Several other dialkylbenzenes, such as is converted to phenylacetaldehyde and phenylacetic acid
1,4-di-tert-butylbenzene, are hydroxylated on the methyl (Cox et al. 1996). P. ostreatus oxidizes styrene to

Figure 3 Structures of several polycyclic aromatic hydrocarbons.

448 Sutherland

phenyl-1,2-ethanediol and other products (Braun-Lüllemann Fluorene is oxidized by C. elegans to 9-hydroxyfluorene,

et al. 1997). 9-fluorenone, and 2-hydroxy-9-fluorenone (Pothuluri et al.
1993). The first two are also produced by P. chrysosporium,
P. ostreatus, and other fungi (Bezalel et al. 1996c; Bogan et al.
5 BIPHENYL 1996; Bumpus 1989; Garon et al. 2000; Schützendübel et al.
1999). The oxidation of fluorene to 9-fluorenone requires
Biphenyl (C6H5C6H5), found in coal tar, oil, and natural gas, manganese peroxidase activity in P. chrysosporium and
is hydroxylated at various positions by C. elegans (Dodge T. versicolor (Bogan et al. 1996; Collins and Dobson 1996)
et al. 1979). Several yeasts cometabolize biphenyl to but not in P. ostreatus or Bjerkandera adusta (Schützendübel
4-hydroxybiphenyl, which Debaryomyces vanrijiae, Rhodo- et al. 1999). The laccases of C. gallica and T. versicolor are
torula glutinis, and Y. lipolytica convert further to 4-phenyl-2- also able to oxidize fluorene (Bressler et al. 2000; Johannes
pyrone-6-carboxylic acid (Lange et al. 1998; Romero et al. and Majcherczyk 2000).
2001). The yeast Trichosporon mucoides cometabolizes Anthracene is transformed by C. elegans to a trans-1,2-
biphenyl to 2-, 3-, and 4-hydroxybiphenyl, eight dihydroxy- dihydrodiol and 1-anthryl sulfate (Cerniglia and Yang 1984);
lated biphenyls, three trihydroxylated biphenyls, and a 9,10-anthraquinone has also been reported (Lisowska and
quinone (Sietmann et al. 2000; Sietmann et al. 2001). It Dlugonski 1999). Rhizoctonia solani produces the (þ) and
further transforms the di- and trihydroxylated biphenyls to (2 ) trans-1,2-dihydrodiols and three xyloside conjugates
eight different ring-cleavage products (Sietmann et al. 2001). (Sutherland et al. 1992). Absidia cylindrospora, Rhizopus
arrhizus, Ulocladium chartarum, A. niger, Cryphonectria
parasitica, and several white-rot fungi also transform
6 POLYCYCLIC AROMATIC anthracene (Field et al. 1996; Giraud et al. 2001; Gramss
HYDROCARBONS et al. 1999; Krivobok et al. 1998; Schützendübel et al. 1999;
Yogambal and Karegoudar 1997). P. chrysosporium degrades
The first step in the biotransformation of PAHs (Figure 3) by anthracene via anthraquinone and phthalate to CO2,
most fungi is initiated by a cytochrome P450 mono- presumably with the involvement of lignin peroxidase
oxygenase; white-rot basidiomycetes may also employ lignin (Hammel et al. 1991). P. ostreatus produces anthraquinone
peroxidase, manganese peroxidase, or laccase (Hammel and anthracene trans-1,2-dihydrodiol (Andersson and
1995; Cerniglia 1997; Cerniglia and Sutherland 2001; Henrysson 1996; Bezalel et al. 1996c; Novotny et al. 1999;
Sutherland et al. 1995). Later steps may involve conjugation Schützendübel et al. 1999); in soil, it incorporates the
to form sulfates, glucosides, glucuronides, or xylosides products into humus (Bogan et al. 1999) or degrades them to
(Casillas et al. 1996). Although PAHs do not generally serve CO2 (Márquez-Rocha et al. 2000). Pleurotus sajor-caju
as growth substrates for fungi, the yeast R. glutinis is reported [P. pulmonarius], T. versicolor, and other fungi also produce
to utilize phenanthrene for growth (Romero et al. 1998). The anthraquinone (Andersson and Henrysson 1996). Phanero-
ability of fungi to transform PAHs has been exploited for the chaete laevis produces manganese peroxidase and degrades
bioremediation of contaminated soil (Juhasz and Naidu 2000; anthracene without accumulating anthraquinone (Bogan and
Cerniglia and Sutherland 2001). Lamar 1996). The laccases of C. gallica and T. versicolor
Naphthalene is oxidized by C. elegans to 1- and oxidize anthracene to anthraquinone (Collins et al. 1996;
2-naphthol, naphthalene trans-1,2-dihydrodiol, 4-hydroxy-1- Johannes and Majcherczyk 2000; Pickard et al. 1999); that of
tetralone, the 1,2- and 1,4-naphthoquinones, and sulfate and Nematoloma frowardii, in the presence of glutathione, can
glucuronide conjugates (Cerniglia and Gibson 1977; mineralize it (Sack et al. 1997b).
Cerniglia 1992). Both constitutive and inducible hydroxylases Phenanthrene is oxidized by C. elegans, Syncephalastrum
appear to be involved in the pathway (Faber et al. 2001). racemosum, and A. niger to trans-dihydrodiols, phenanthrols,
A. niger has been reported (Yogambal and Karegoudar 1997) and sulfate conjugates (Cerniglia and Yang 1984; Casillas
to metabolize naphthalene via gentisate, a naphthalene et al. 1996; Sutherland et al. 1993). Glucoside and
metabolite produced by some bacteria. Naphthalene is glucuronide conjugates may also be produced (Casillas et al.
removed from wastewater by the immobilized mycelia of 1996). A. niger is reported to produce 1-methoxy-
P. chrysosporium (Liao et al. 1997). phenanthrene (Sack et al. 1997a) as well as a ring-cleavage
Acenaphthene is oxidized by C. elegans to 6-hydro- product, protocatechuate (Yogambal and Karegoudar 1997).
xyacenaphthenone, 1,2-acenaphthenedione, acenaphthene P. chrysosporium degrades phenanthrene (Bumpus 1989),
trans-1,2-dihydrodiol, and four minor metabolites (Pothuluri producing trans-9,10- and 3,4-dihydrodiols, 9-, 3-, and
et al. 1992). The laccase of Coriolopsis gallica oxidizes 4-phenanthrols, and a glucoside in high-nitrogen media
acenaphthene and biphenylene in the presence of an artificial (Sutherland et al. 1991; Sutherland et al. 1993) and
mediator compound, 2,20 -azinobis-(3-ethylbenzthiazoline-6- phenanthrene-9,10-quinone and 2,20 -diphenic acid in low-
sulfonate) (ABTS) (Pickard et al. 1999), and the laccase of nitrogen media (Hammel et al. 1992). P. ostreatus also
T. versicolor oxidizes acenaphthene and acenaphthylene in degrades phenanthrene (Bezalel et al. 1996a; Márquez-Rocha
the presence of 4-hydroxybenzoic acid (Johannes and et al. 2000; Schützendübel et al. 1999), producing the
Majcherczyk 2000). trans-9,10-dihydrodiol and 2,20 -diphenic acid (Bezalel et al.
Hydrocarbon Degradation by Fungi 449

1996b) and phenanthrenequinone (Novotny et al. 1999). The manganese peroxidase of N. frowardii oxidizes
Several other fungi, including white-rot and ectomycorrhizal benz[a]anthracene in the presence of glutathione (Sack et al.
fungi, degrade phenanthrene (Colombo et al. 1996; Braun- 1997b).
Lüllemann et al. 1999; Gramss et al. 1999; Field et al. 1996; Chrysene, a weak carcinogen, is metabolized by C. elegans
Lisowska and Długoński 1999; Schützendübel et al. 1999). to chrysene 2-sulfate and chrysene 2-hydroxy-8-sulfate
The manganese peroxidases of P. laevis (Bogan and Lamar (Pothuluri et al. 1995) and by P. janthinellum to the trans-
1996), T. versicolor (Collins and Dobson 1996; Rama et al. 1,2-dihydrodiol (Boonchan et al. 2000; Kiehlmann et al.
2000), and N. frowardii (Sack et al. 1997b) all oxidize 1996). S. racemosum, P. ostreatus, and the ectomycorrhizal
phenanthrene. The laccase of Trametes hirsuta, in the fungi Boletus edulis and Amanita muscaria also degrade
presence of the mediator 1-hydroxybenzotriazole and linoleic chrysene (Braun-Lüllemann et al. 1999; Kiehlmann et al.
acid, oxidizes phenanthrene via the quinone to 2,20 -diphenic 1996; Wolter et al. 1997).
acid (Böhmer et al. 1998); that of C. gallica, in the presence of Benzo[b]fluoranthene and benzo[k]fluoranthene, which
ABTS, also oxidizes it (Pickard et al. 1999). Unlike other are both carcinogenic, are degraded in soil extracts by
fungi, the yeast R. glutinis has been reported to utilize Bjerkandera sp. (Field et al. 1996) and in wheat straw by
phenanthrene as a sole carbon and energy source (Romero P. ostreatus (Wolter et al. 1997).
et al. 1998). Benzo[a]pyrene, a potent carcinogen, is oxidized by a
Fluoranthene is metabolized by C. elegans to a trans-2,3- cytochrome P450 monooxygenase in C. elegans, which
dihydrodiol, the 8- and 9-hydroxyfluoranthene trans-2,3- transforms it to at least 13 compounds (Figure 4) (Cerniglia
dihydrodiols, and two glucoside conjugates (Pothuluri and Gibson 1979; Cerniglia 1997). The cytochromes P450 of
et al. 1990). A. cylindrospora, A. niger, U. chartarum, the Aspergillus fumigatus and other fungi also hydroxylate it
yeast Cryptococcus albidus, and the white-rot fungi (Juhasz and Naidu 2000; Venkateswarlu et al. 1996). Several
P. chrysosporium, P. ostreatus, and B. adusta also degrade white-rot and ectomycorrhizal fungi degrade benzo[a]pyrene
fluoranthene (Liao et al. 1997; Giraud et al. 2001; Gramss (Bezalel et al. 1996a; Bumpus 1989; Braun-Lüllemann
et al. 1999; Salicis et al. 1999; Schützendübel et al. 1999). et al. 1999; Kotterman et al. 1998; Márquez-Rocha et al.
P. ostreatus binds fluoranthene metabolites to humus in soil 2000; Rama et al. 2000; Wolter et al. 1997; Wunch et al.
(Bogan et al. 1999). 1999). P. chrysosporium has been used to remove it from
Pyrene is metabolized to 1-hydroxypyrene, 1,6- and water (Liao et al. 1997), and both P. chrysosporium and
1,8-pyrenequinones, and three glucoside conjugates by P. ostreatus form quinones that can be polymerized in soil
C. elegans (Cerniglia et al. 1986). A. niger produces (Bogan et al. 1999; May et al. 1997). P. laevis produces
1-methoxypyrene and 1-hydroxypyrene (Sack et al. 1997a) manganese peroxidase and transforms benzo[a]pyrene to
and P. glabrum produces these two products plus 1-pyrenyl polar products (Bogan and Lamar 1996); the manganese
sulfate, 1,6-dimethoxypyrene, 1,6- and 1,8-dihydroxypyrene, peroxidase of N. frowardii can mineralize it in the presence of
and both quinones (Wunder et al. 1997). Penicillium glutathione (Sack et al. 1997b). The laccases of T. versicolor,
janthinellum and other fungi found in sediments also degrade Pycnoporus cinnabarinus, and C. gallica oxidize benzo[a]-
pyrene (Launen et al. 2000; Ravelet et al. 2000). P. ostreatus pyrene to quinones in the presence of ABTS (Collins et al.
hydroxylates pyrene to the trans-4,5-dihydrodiol and partially 1996; Pickard et al. 1999; Rama et al. 1998).
mineralizes it (Bezalel et al. 1996a; Bezalel et al. 1996c; Benzo[e]pyrene, a noncarcinogenic isomer of benzo-
Wolter et al. 1997), even in soil (Novotny et al. 1999; pyrene, is metabolized by C. elegans to sulfate and glucoside
Márquez-Rocha et al. 2000). Crinipellis sp., Marasmius conjugates (Pothuluri et al. 1996). Perylene is metabolized by
sp., and Marasmiellus sp. metabolize it to 1-hydroxy- several wood- and straw-degrading fungi (Gramss et al. 1999)
pyrene, 1,6- and 1,8-dihydroxypyrene, both quinones, and the carcinogenic benzo[ghi]perylene is degraded by
pyrene trans-4,5-dihydrodiol, and three sulfate conjugates P. ostreatus (Wolter et al. 1997). Dibenz[a,h]anthracene, a
(Lange et al. 1996). Several other wood-decay and suspected carcinogen, is degraded by the white-rot fungi
mycorrhizal fungi mineralize pyrene (Braun-Lüllemann P. ostreatus and P. sajor-caju [P. pulmonarius] (Andersson
et al. 1999; Field et al. 1996; Gramss et al. 1999; in der and Henrysson 1996; Wolter et al. 1997) and by
Wiesche et al. 1996; Schützendübel et al. 1999; Song P. janthinellum (Boonchan et al. 2000). Indeno[1,2,3-cd ]
1999), as does the manganese peroxidase of N. frowardii pyrene, which is also considered carcinogenic, is metabolized
in the presence of glutathione (Sack et al. 1997b). by Bjerkandera sp. in solvent extracts of contaminated soil
Benz[a]anthracene, a carcinogen, is hydroxylated by (Field et al. 1996). The methylated PAHs, many of which are
C. elegans to the trans-8,9-,10,11-, and 3,4-dihydrodiols and carcinogenic, can be oxidized to a variety of metabolites.
the 8,9,10,11-tetraol (Cerniglia et al. 1994). P. janthinellum Methylnaphthalenes, methylanthracenes, methyl- and
and the white-rot fungi P. chrysosporium, P. ostreatus, dimethylbenz[a]anthracenes, and 3-methylcholanthrene are
P. sajor-caju [P. pulmonarius], and T. versicolor also degrade transformed by C. elegans (Cerniglia et al. 1982a; 1982b;
it (Andersson and Henrysson 1996; Wolter et al. 1997; 1983; 1984; 1990; Wong et al. 1983). In addition,
Boonchan et al. 2000). The manganese peroxidase of P. laevis methylanthracenes are transformed by C. gallica (Pickard
degrades it during lipid peroxidation; the 7,12-dione is et al. 1999) and methylphenanthrenes by Fusarium solani
produced but does not accumulate (Bogan and Lamar 1996). (Colombo et al. 1996).
450 Sutherland

7 HYDROCARBON DEGRADATION BY enhanced by soil microorganisms (in der Wiesche et al.

MIXED CULTURES 1996). The mineralization of benzo[a]pyrene by Bjerkandera
sp. more than doubled after the addition of activated sludge
Several experiments have shown that the mineralization of (Kotterman et al. 1998). P. janthinellum, when mixed with the
hydrocarbons in nature may require the combined efforts of bacterium Stenotrophomonas maltophilia, mineralized
both fungi and bacteria (Juhasz and Naidu 2000). Pyrene pyrene, chrysene, benz[a]anthracene, benzo[a]pyrene, and
mineralization by Dichomitus squalens and Pleurotus sp. was dibenz[a,h]anthracene (Boonchan et al. 2000).

Figure 4 Benzo[a]pyrene and the metabolites that have been identified in cultures of Cunninghamella elegans (adapted from Cerniglia
and Gibson 1979; Cerniglia et al. 1992; Cerniglia 1997). The absolute stereochemistry of some of the compounds and the structures of the
glucuronide and sulfate conjugates are not known.
Hydrocarbon Degradation by Fungi 451

8 BIOTECHNOLOGICAL IMPORTANCE and biochemicals (Johnson et al. 1973; Uzura et al.

2001).
At one time, most of the industrial research on hydrocarbon
metabolism consisted of attempts to produce yeast single-cell
protein from petroleum, which was cheap and widely 9 CONCLUSIONS
considered to be surplus (Lindley 1992). Little of that type
of research is being done now, but there has been extensive Fungi are capable of cometabolizing most hydrocarbons.
progress on the biotransformation of terpenes for the However, only a few fungi are known to grow on
production of flavors, fragrances, and drug intermediates hydrocarbons, such as methane, n-hexadecane, toluene,
(Trudgill 1994) and on the degradation of PAHs and other styrene, or phenanthrene, as sole carbon sources. Since
toxic hydrocarbons during the bioremediation of soils (Atlas various fungi oxidize hydrocarbons, often in a stereoselective
and Cerniglia 1995). manner, some strains have been selected to carry out specific
Many of the hydrocarbons contaminating soils and biocatalytic transformations to produce higher-value pro-
groundwater near leaking fuel tanks, oil spills, and chemical ducts. Many fungi transform toxic hydrocarbons to oxidized
waste dumps can be degraded by fungi (Atlas and Cerniglia derivatives and a few strains are able to cleave aromatic rings;
1995; Cerniglia and Sutherland 2001; Colombo et al. 1996). thus these fungi have the potential for use, preferably together
Most of the recent work in this field has been done on PAHs, with bacteria, for the bioremediation of toxic wastes in the
but some of it has been on BTEX compounds (Yadav and environment. Several lines of research on the fungal
Reddy 1993) and on alkanes (Nwachukwu 2000). Although degradation of hydrocarbons are likely to be productive in
gigantic oil spills from marine supertankers and offshore oil the future. These include: (a) Investigating the roles of yeasts
wells attract immediate public concern, smaller spills from and filamentous fungi in transforming naturally occurring
fuel storage tanks are numerous and usually more amenable to hydrocarbons at the concentrations usually found in the
bioremediation (Atlas and Cerniglia 1995). The fungal environment. (b) Finding additional ways to transform
bioremediation of contaminated soils may be accomplished abundant renewable resources, including by-products from
either by removing soil for off-site treatment (May et al. agriculture and forestry, into chiral drug precursors and other
1997) or by using organic solvents to extract hydrocarbons high-value products. (c) Developing practical combinations
from the soil for later degradation (Field et al. 1996). On-site of fungi and bacteria that can be used for the large-scale
bioremediation may be done with fungi that can grow through bioremediation of soils contaminated with mixtures of
the soil mass to reach the PAHs (Cerniglia 1997; Novotny hydrocarbons.
et al. 1999). Since few soil microorganisms have the ability to
degrade high-molecular-weight PAHs alone (Juhasz and
Naidu 2000), fungi and bacteria are now being added together ACKNOWLEDGEMENTS
to achieve bioremediation (Boonchan et al. 2000). Fungi have
also been used to remove PAHs from wastewater (Liao et al.
I am grateful to Carl E. Cerniglia for his encouragement and
1997) and biofilters containing selected fungi have been used
helpful comments on this manuscript.
to remove toluene and other hydrocarbons from waste gases
(Garcı́a-Peña et al. 2001).
Another application of fungal biotechnology is the
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38
Biodegradation of Azo Dyes by Fungi

John A. Bumpus University of Northern Iowa, Cedar Falls, Iowa, USA

1 INTRODUCTION selective pressures on micro-organisms would have favored

survival of those species that evolved enzymes capable of
The manufacture and use of dyes and pigments is a specifically destroying azo dyes. The toxicity of azo dyes
multibillion-dollar industry. The use of these substances is makes it important that ways be found to treat wastewaters
an integral part of almost all manufacturing processes. that contain these substances.
Interestingly, the widespread use of synthetic colorants and This review focuses on the ability of fungi to degrade azo
the modern dye industry dates only to 1856 with the synthesis dyes. Before, 1990, there were few reports of the
of mauveine by W.H. Perkin (Kirk-Othmer Encyclopedia of biodegradation of azo dyes by aerobic micro-organisms.
Chemical Technology 1992). Of all the different types of Cripps et al. (1990) showed that three azo dyes were
dyes, azo dyes are the most useful and widely used colorants. extensively, degraded by the white rot fungus Phanerochaete
chrysosporium under aerobic conditions. Since that time,
In 1858, J. P. Griess synthesized a yellow azo dye that was
many manuscripts have been published documenting
commercialized briefly (Kirk-Othmer Encyclopedia of
degradation of azo dyes by P. chrysosporium and other
Chemical Technology 1992). Other azo dyes that saw early
fungi. Like P. chrysosporium, most of the other fungi studied
commercialization included Chrysodine (in 1875), Congo
and shown to degrade azo dyes are white rot fungi.
Red (in 1884), and Bismark Brown. Following these early
Interestingly, however, a few fungi that are not white rot
syntheses, numerous azo dyes have been made and it is
fungi have been reported to have some ability to degrade this
estimated that over 2,000 are in use (Colour Index, 3rd Ed.).
important class of compounds. The ability of white rot fungi
Wastewaters are produced during the synthesis and use of and other micro-organisms to degrade azo dyes was reviewed
dyes. Such wastewaters must be treated. The most important by this author several years ago (Bumpus 1995). A number of
criterion for treatment of wastewater is that associated reviews addressing, at least in part, the biodegradation and/or
toxicity is reduced to acceptable levels. Similarly, odors must biodecolorization of azo dyes in water by white rot fungi have
be reduced and the amount of dissolved material must be appeared recently (Stolz 2001; Fu and Viraraghavan 2001;
reduced. Decolorization of water is also a primary goal of Knapp et al. 2001; Robinson et al. 2001; McMullan et al.
water purification processes and one that is critical to 2001). This review summarizes this area of research and
dye-containing wastewaters. important recent observations while attempting to keep
There are several reasons for the popularity and use of azo overlap with other reviews at a minimum.
dyes. Azo dyes are available in almost every color across the
spectrum, as a group they are colorfast and many can be
structurally modified to bind to a variety of natural and
synthetic fabrics (Kirk-Othmer Encyclopedia of Chemical 2 DECOLORIZATION AND
Technology 1992). Unfortunately, azo dyes are resistant to BIODEGRADATION OF AZO DYES
biological degradation. This is likely due to the fact that azo BY WHITE ROT FUNGI
linkages are rarely found in nature. Thus micro-organisms
have not often been exposed to compounds containing this The remarkable biodegradative abilities of the white rot
functional group and there have been few instances in which fungus Phanerochaete chrysosporium (Bumpus et al. 1985)

457
458 Bumpus

led Cripps et al. (1990) to study the ability of this fungus to limited conditions were selected because, in this fungus, such
degrade azo dyes. It was shown that Orange II, Tropaeolin O, conditions promote expression of the lignin degrading system
and Congo Red were all extensively decolorized in nutrient and it is this system that is thought to be instrumental in
nitrogen limited cultures of this fungus. Nutrient nitrogen biodegradation of xenobiotics. Involvement of the lignin

Figure 1 Representative azo dyes degraded by white rot fungi.

Biodegradation of Azo Dyes by Fungi 459

degrading system was confirmed by the fact that these dyes Decolorization as measured by visible spectroscopy is
were all found to be oxidized by lignin peroxidases. Although an exceptionally easy and inexpensive way to monitor
initial studies (Cripps et al. 1990) suggested that lignin degradation of dyes by micro-organisms. However, other
peroxidases did not oxidize Congo Red, subsequent techniques are required in order to obtain a true picture of the
investigations (Tatarko and Bumpus 1998) showed that this extent of degradation that occurs. Degradation of azo dyes to
dye is indeed, a substrate for these enzymes. carbon dioxide by P. chrysosporium was demonstrated by

Figure 1 (continued)
460 Bumpus

Spadaro et al. (1992) who showed that 23.1 –48.1% of the 4-(40 -nitrophenylazo)-aniline (Disperse-Orange 3), and
aromatic ring labeled carbon atoms in 7 azo dyes 1-phenylazo-2-naphthol (Solvent Yellow 14) were oxidized
(4-phenylazophenol, 4-phenylazo-2-methoxyphenol, 2-(40 - to carbon dioxide by P. chrysosporium. Paszczynski et al.
acetamidophenylazo)-4-phenylazophenol (Disperse Yellow (1992) synthesized several ring-labeled sulfonated azo dyes
3), 4-phenylazoaniline, N,N-dimethyl-4-phenylazoaniline, and showed that substantial quantities (17.2 –34.8%) of

Figure 1 (continued)
Biodegradation of Azo Dyes by Fungi 461

the labeled carbon atoms in the dyes were degraded to carbon The effect of carbon source on the decolorization of water
dioxide by P. chrysosporium. Among the dyes degraded to containing Amaranth and Orange G was studied by Chao and
carbon dioxide were Acid Yellow, Orange I, Orange II, 4-(3- Lee (1994). Both dyes as well as the heterocyclic dye Azure B
methoxy-4-hydroxyphenylazo)benzenesulfonic acid, and were decolorized by two strains of P. chrysosporium and
4-(2-sulfa-30 -methoxy-4-hydroxyazobenzenesulfonic acid. another unidentified white rot fungus. Interestingly, decolor-
P. chrysosporium has served well as a model micro- ization was reported to occur if fungal strains were pre-
organism for the study of the degradation of azo dyes and cultured in a high nitrogen medium in the presence of glucose.
other environmentally persistent organic pollutants. However, This is of interest as high nitrogen conditions might be
it has been shown that a number of other white rot fungi are expected to repress expression of lignin peroxidases and
able to degrade hard-to-degrade compounds, including azo manganese peroxidases, enzymes that would be expected to
dyes. Representative azo dyes degraded by white rot fungi are mediate decolorization of these dyes.
illustrated in Figure 1. Knapp et al. (1995) compared the ability of 7 wood-rotting
As discussed below, some of these fungi have degradative fungi to degrade a variety of dyes, including the azo
abilities and other properties that will likely make them dyes, Mordant Yellow 10, Cibacron Brilliant Yellow 3G-P,
superior to P. chrysosporium for use in bioremediation Cibacron Brilliant Red 3B-A, Orange II, Brilliant Yellow,
systems. White rot fungi that have been reported to Chrysophenine, Chlorazol Yellow, and Acid Red 106. Results
degrade/decolorize azo dyes are listed in Table 1. were variable in that all of the fungi were able to degrade
Several Myrothecium spp. and Ganoderma spp. were extensively some of the dyes. No strain was able to degrade
shown by Mou et al. (1991) to be able to decolorize Orange II extensively all of the dyes. It is interesting to note that
as well as two other diazo sulfonated dyes designated as P. chrysosporium, was able to mediate 99–100% decoloriza-
RS(WC) and lOB(H/C). Several experiments were performed tion of 4 of the 14 dyes and 27 –69% decolorization of the
using M. verrucaria. Results showed that this fungus was other 10. Nevertheless, P. chrysosporium was among the least
more effective than activated sludge in decolorizing effective of the fungal strains investigated. This study shows
wastewater from a dye-manufacturing plant. Decolorization clearly that the biodegradative abilities of P. chrysosporium
of wastewater from a textile-dyeing factory was also studied. are substantial, but it is necessary to assess the biodegradative
Five of six highly colored wastewaters were substantially abilities of other white rot fungi as many will have even
decolorized. It is interesting to note that some experiments greater abilities. These investigations also showed that no one
were performed at high pH values, one as high as pH 11. In species of fungus was best for all of the dyes studied.
some wastewaters, the concentration of sodium chloride was Dye decolorization may be achieved using intact fungal
as high as 15% (w/v). Adsorption to mycelium was shown to cultures or by using selected fungal enzymes. This may have
be an important mechanism for wastewater decolorization. important implications in bioreactor development. Young and
In some experiments, adsorption was followed by slow Yu (1997) showed that a variety of dyes, including four azo
decolorization of the mycelium. However, it should be noted dyes (Acid Violet 7, Reactive Black 5, Acid Orange 74, and
that decolorization of mycelium was not observed in all Acid Black 24) were decolorized in culture by
experiments even after extended (1 month) incubation. P. chrysosporium and Trametes versicolor. Of interest,

Table 1 Representative manuscripts documenting biodegradation of azo dyes by white rot fungi

Fungus Dye Reference

P. chrysosporium Congo Red, Orange II, Tropaeolin O Cripps et al. (1990)
Myrothecium spp. Orange II, RS(H/S), 10B(H/C) Mou et al. (1991)
T. versicolor Acid Violet 7, Reactive Black 5, Young and Yu (1997)
Acid Orange 74, Acid Black 24
Strain F29 Orange II Knapp et al. (1997)
P. ostreatus Congo Red, Methyl Orange Shin and Kim (1998)
B. adusta Reactive Violet 5, Reactive Black 5, Heinfling et al. (1998a,b)
Reactive Orange 96, Reactive Red 198
Bjerkandera BOS55 Amaranth, Remazol Black B, Reactive Blue Swamy and Ramsay (1999a,b)
15, Remazol Brilliant Orange 3R, Tropaeolin O
T. hirsuta Amaranth, Remazol Black B Swamy and Ramsay (1999a,b)
P. sanguineus Orange G, Amaranth Pointing and Vrijmoed (2000)
I. lacteus Congo Red, Methyl Orange, Reactive Orange Novotny et al. (2001)
16, Reactive Black 5
P. sajorcaju Amaranth, New Coccine, Orange G Chagas and Durrant (2001)
F. trogii Astrazon Red FBL Yesilada et al. (2002)
462 Bumpus

however, was the observation that some dyes were more of these fungal strains substantially degraded representative
extensively decolorized by a lignin peroxidase preparation azo and diazo dyes as well as anthraquinone, heterocyclic,
from P. chrysosporium. For example, 6.6% decolorization of triphenylmethane, and phthalocyanine dyes. It is worth
the azo dye Acid Orange 74 was observed in solid agar mentioning, however, that the diazo dye, Congo Red appeared
cultures of P. chrysosporium. However, when incubated in to be one of the dyes that was comparatively more resistant to
the presence of hydrogen peroxide and lignin peroxidase from degradation. I. lucteus caused only 58% decolorization of this
this fungus, 79.1% decolorization was observed. In some dye during 14 days of incubation.
cases, the opposite was observed. In solid agar culture, 98% Recently, several other white rot fungi have been shown to
decolorization of Acid Black 24 occurred whereas when this be able to degrade azo dyes. Heinfling et al. (1998a,b) showed
dye was incubated with lignin peroxidase in the presence of that Bjerkundera adusta is able to degrade azo dyes. These
hydrogen peroxide, only 25.4% decolorization occurred. authors have also purified and characterized manganese
Shin and Kim (1998) showed that a peroxidase isolated peroxidases and lignin peroxidases from this fungus and have
from Pleurotus ostreatus decolorized several types of dyes documented their involvement in azo dye degradation. Azo
including the azo dyes Congo Red and Methyl Orange. After dyes degraded by B. adusta include Reactive Violet 5,
4 min, 77 and 26% decolorization of these dyes was noted. Of Reactive Black 5, Reactive Orange 96, and Reactive Red 198.
interest was the observation that addition of veratryl alcohol It is worth noting that B. adusta is the most common
did not enhance the rate and extent of decolorization. This is Bjerkandera species in Europe (Heinfling et al. 1998a).
in contrast to the observations by Ollikka et al. (1993) for Swamy and Ramsay (1999a,b) studied the ability of
lignin peroxidase from P. chlysosporium. P. chrysosporium, Bjerkandera sp. BOS55, P. ostreatus,
Martins et al. (2001) synthesized several azo dyes having Trametes hirsuta and T. versicolor to decolorize 6 dyes,
substituent groups [2-methoxyphenol (guaiacol) and including 4 azo dyes, in solid plate cultures. P. ostreatus was
2,6-dimethoxyphenol (syringol)] that would be expected to not able to decolorize any of the dyes (the azo dyes Amaranth,
be found in lignin. Such groups were designated as Remazol Black B, Tropaeolin O, and Remazol Brilliant
bioaccessible as they would be expected to be attacked by Orange 3R and the phthalocyanine dye Reactive Blue 15 and
enzymes from lignin degrading fungi. Not unexpectedly, the the anthroquinone dye Remazol Brilliant Blue R) under
extent of dye degradation depended on the concentration of investigation. Bjerkandera sp. BOS55 and Trametes versi-
sucrose used as a growth substrate and on the structure of the color were most effective in their ability to decolorize these
dye. Interestingly, degradation also depended on the structure dyes. Both were able to decolorize all of the dyes to some
of the dye present in cultures of P. chrysosporium that were extent. P. chrysosporium and T. hirsuta possessed inter-
allowed to grow in the presence of the dye and, presumably, mediate decolorization ability. P. chrysosporium did not
the fungus became acclimated to the dye. This is similar to the decolorize Remazol Brilliant Orange 3R and only marginally
approach taken by Paszczynski et al. (1991) who synthesized decolorized Tropaeolin O. This fungus did cause substantial
from Acid Yellow 9 and from sulfanilic acid two azo dyes decolorization of Amaranth, Remazol Black B, and Reactive
having guaiacol groups. In this study, P. chrysosporium Blue 15. T. hirsuta decolorized Amaranth and Remazol Black
degraded these dyes more readily than the parent compounds B. T. hirsuta did not decolorize Remazol Brilliant Orange 3R
that lacked the guaiacol group. Interestingly, none of the and only marginally decolorized Reactive Blue 15 and
Streptomyces spp. degraded the parent compounds and only 5 Tropaeolin O. It appears that the ability of a given fungus to
of the 9 Streptomyces spp. degraded the new guaiacol-linked decolorize a dye depends on certain culture conditions. For
azo dyes. example, P. chrysosporium did decolorize Remazol Brilliant
Pasti-Grigsby et al. (1992) synthesized 16 azo dyes and Orange 3R in solid culture. However, in agitated (200 rpm)
assessed there ability, along with 6 commercially available pelleted cultures substantial (85% in 18 days) decolorization
azo dyes (Methyl Orange 52, Ethyl Orange, Acid Yellow 9, occurred. Similarly, it was shown that decolorization occurred
Acid Orange 12, Orange 1, and Orange 2), to be degraded by more rapidly in agitated liquid cultures, in which fungi
P. chrysosporium and by Streptomyces spp. P. chrysosporium formed pellets, than in stationary cultures in which the fungi
decolorized all of the newly synthesized dyes and the were present as mycelial mats. Although Bjerkundera sp.
commercially available azo dyes. Most of the newly BOS55 was superior in its ability to cause decolorization in
synthesized dyes underwent substantial (i.e., # 85%) solid agar cultures, T. versicolor was superior in agitated
decolorization when the initial concentration of dye was pelleted liquid cultures. This will be problematic for reactor
150 mg/L. However decolorization ranged from 27 to 99% at development because selection of micro-organisms to be used
this concentration and from 15 to 97% when the initial cannot necessarily be based on their performance in solid agar
concentration was 300 mg/L. culture. Thus the effectiveness of a micro-organism for dye
Novotny et al. (2001) studied the biodegradative ability of decolorization will have to be judged on the basis of
103 strains of wood-rotting fungi. A strain of Irpex lacteus conditions similar to those under which it will be used in
and a strain of P. ostreatus were selected for further actual wastewater treatment systems. Swamy and Ramsay
investigation to decolorize several different types of dyes (1999a,b) also assessed the ability of Bjerkandera sp. BOS55,
including the azo dyes Congo Red, Methyl Orange, Reactive P. chrysosporium and T. versicolor to decolorize the 5 dyes
Orange 16, Reactive Black 5, and Naphthol Blue Black. Both added sequentially, each after the dye last added had been
Biodegradation of Azo Dyes by Fungi 463

completely decolorized. In the last step of this experiment all fungal pellets could be reused up to five times with great
of the dyes were added as a mixture. In this experiment, only effectiveness (i.e., 92 –98% color removal occurred). At
T. versicolor was able to degrade all of the dyes. Indeed, higher concentrations (264 mg/L) similar color removal was
P. chrysosporium and Bjerkandera sp. BOS55 lost their observed only when an increased amount of fungal mass was
decolorization ability following decolorization of Amaranth used. However, in these experiments, repeated use of the
and Remazol Black. This investigation demonstrates that the fungal pellets resulted in lower amounts (0 – 73%) of
most effective fungal system for decolorization will depend decolorization. It appears that the dye is toxic to the fungus
on several things including the choice of fungus, the buffer, at high concentrations. Nevertheless, it is clear that fungal
the dyes to be degraded, and culture agitation. pellets can be reused several times to remediate water
Knapp et al. (1997) showed that strain F29, obtained from containing dyes at concentrations that might be expected to be
an unidentified fruiting body from a white rot fungus found present in wastewater. Also of importance was the
growing on rotting willow wood was able to cause substantial observation that dye degradation occurred over the pH
(98%) decolorization of water containing up to 1000 mg/L of range 6–11. It should also be noted that, like several other
Orange II in only two days. This is remarkable as Pasti- white rot fungi, dye adsorption by fungal mycelium was
Grigsby et al. (1992) showed that it took P. chrysosporium followed by decolorization of the mycelium.
15 days to achieve 53 and 99% decolorization of water Chagas and Durrant (2001) compared the ability of
containing 300 and 150 mg/L Orange II, respectively. Knapp P. chrysosporium and Pleurotus sajorcaju to decolorize four
et al. (1997) reported that a carbon source other than Orange II azo dyes. P. chrysosporium in solid culture medium partially
was required for decolorization and that a small amount of decolorized Amaranth, New Coccine, Orange G, and
nutrient nitrogen (0.25 g dm21) seemed optimal and necessary Tartrazine. P. sarorcuju decolorized Amaranth, New Coccine,
to maintain decolorization ability in long-term experiments. A and Orange G. It did not decolorize Tartrazine. In liquid cultures
pH of 5.3–6.3 appeared to be optimal. Addition of veratryl of P. chrysosporium total decolorization of Amaranth, New
alcohol provided a slight improvement in the rate of Coccine, and Orange G and 60% decolorization of Tartrazine
decolorization. However, addition of Mn(II) resulted in a was observed. P. sajorcaju mediated total decolorization of
decolorization rate 70% greater than that of controls to which Amaranth and Coccine, 50% of Orange G and 20% of
no additional Mn(II) was added. These results led this Tartrazine. In these investigations, lignin peroxidase activity
research group to use this fungus in the development of was not observed. However, manganese peroxidase activity
bioreactors (Zhang et al. 1998) as discussed elsewhere in this was observed in cultures of P. chrysosporium and laccase
review. activity was found in cultures of P. sajorcaju suggesting that
Pointing and Vrijmoed (2000) studied the ability of these enzymes are important in dye degradation.
Pycnoporus sanguineus to decolorize two triphenylmethane
dyes (Bromophenol Blue and Malachite Green) and two azo
dyes (Orange G and Amaranth). These investigators also
found that the azo dyes were more resistant to degradation
3 DECOLORIZATION AND
than the other dyes studied. Complete or nearly complete BIODEGRADATION OF AZO DYES BY
decolorization of the triphenylmethane dyes occurred while FUNGI OTHER THAN WHITE ROT FUNGI
only partial (up to 60%) decolorization of the azo dyes was
observed. Adsorption of dye to mycelium was not a major In recent years there have been many reports regarding
factor in decolorization by this fungus. biodegradation of azo dyes by white rot fungi (Bumpus 1995;
Yesilada et al. (2002) showed that Funalia trogii in Stolz 2001; Yuzhu and Viraraghavan 2001; Knapp et al.
pelleted form could decolorize relatively high concentrations 2001; Robinson et al. 2001; McMullan et al. 2001). In
(264 mg/L) of Astrazon Red FBL. Astrazon dyes are widely contrast, there have been only a few reports focusing on
used basic dyes. In these investigations, which were the first biodegradation and/or decolorization of azo dyes by fungi that
to study biodegradation of Astrazon dyes by a white rot are not white rot fungi. Some of these fungi are listed in
fungus, it was shown that at a dye concentration of 13 mg/L, Table 2.

Table 2 Manuscripts describing adsorption of or biodegradation of azo dyes by fungi other than
white rot fungi

Fungus Dye Reference

N. crassa Vermelho Reanil P8B Corso et al. (1981)
A. sojae B-10 Amaranth, Sudan II, Congo Red Behung-Ho and Weon (1992)
A. foetidus Drimarene Red, Drimarene Blue Sumanthi and Phatak (1999)
Drimarene Black Sumanthi and Manju (2000)
C. inaequalis Chicago Sky Blue 6B ten Brink et al. (2000)
464 Bumpus

As noted by Chung and Stevens (1993) an early report by nature substantial amounts of cellulose often remain giving
Riedel (1942) showed that azo dyes were reduced by yeasts the decayed wood a white appearance. Conversely when
but that pure culture isolations were not reported in this study. brown rot fungi attack wood residual lignin, which is brown in
In a study focusing on the use of fungi to treat industrial color, remains. Thus one of the consequences of biodegrada-
effluents containing an azo dye it was shown by Corso et al. tion by white rot fungi is decolorization of naturally occurring
(1981) that the Ascomycete Neurospora crassa was able to substances. The enzymes responsible for the initial stages of
decolorize water containing Vermelho Reanil P8B. In these biodegradation and decolorization are oxidative enzymes,
studies 91.3 –89.1% decolorization was achieved during a primarily lignin peroxidases (ligninases), manganese peroxi-
24-hr incubation period in water in which the dye dases, and laccases (Kirk and Farrell 1987). Lignin
concentration was 16–32 mg/L. These dye concentrations peroxidases and manganese peroxidases have reaction
are typical of those that would be found in wastewater. mechanisms similar to those of other peroxidases (e.g.,
Behung-ho and Weon (1992) reported that the Ascomycete horseradish peroxidases and lactoperoxidase) (Dunford
Aspergillus sojae B-10 was able to mediate substantial 1999). These peroxidases contain a heme prosthetic group
decolorization of Amaranth, Sudan III, and Congo Red. The and require hydrogen peroxide as an oxidizing cosubstrate. In
effect of several nutrient nitrogen sources on decolorization the first step of the peroxidase reaction mechanism the
was assessed. Nitrogen containing salts (ammonium tartrate, enzyme undergoes a two-electron oxidation in which it is
sodium nitrate, ammonium nitrate, and ammonium sulfate) converted to compound I. Compound I is an activated form of
appeared to promote decolorization relative to biologically the enzyme in which the heme iron exists as an oxyferryl
derived nitrogen sources (peptone, malt extract, and yeast species in the 4 þ oxidation state. The other equivalent exists
extract). It was also shown that decolorization decreased as a as a porphyrin cation radical or, in some cases, as another
function of sodium nitrate concentration for all three dyes that radical species. The oxygen atom in the oxyferryl species is
were studied. Adsorption contributed to the decolorization that donated by hydrogen peroxide. The other oxygen atom
was observed. However, the authors assert that biodegradation originally present in hydrogen peroxide is reduced to water.
was also important in this process. If this decolorization is, Compound I is a good oxidant and is able to mediate the one-
indeed, due to biodegradation and not simple adsorption it will electron oxidation of a variety of organic pollutants, including
be interesting to determine the mechanisms by which several azo dyes. During the one-electron oxidations
biodegradation occurs as they will almost certainly be mediated by compound I, this activated peroxidase inter-
different than those which occur in white rot fungi. mediate is reduced to compound II, which is also an activated
Sumanthi and Phatak (1999) and Sumanthi and Manju oxyferryl peroxidase intermediate. Unlike compound I, which
(2000) showed that Aspergillus foetidus was able to has an oxidation state that is two electron equivalents greater
decolorize in culture several azo dyes (Remazol Red, than the ferric resting state, the oxidation state of compound II
Remazol Dark Blue HR, Remazol Brown GK, Drimarene is one electron equivalent greater than the ferric resting
Red, Drimarene Blue, Drimarene Black, Procion Green, and stating. Thus another one-electron oxidation of an azo dye or
Procion Turquoise). However, these investigators noted that other reducing substrate converts compound II back to the
decolorization was due primarily to biosorption and that little resting state thereby completing the reaction cycle.
biotransformation occurred. Lignin peroxidases and manganese peroxidases are
Curvularia spp. are members of the Ascomyctes and relatively nonspecific in that they are able to oxidize a
include animal and plant pathogens. Unlike the other fungi variety of substrates. On the other hand, these enzymes also
discussed in this review, Curvularia inaequalis produces a exhibit some specificity. This has been demonstrated nicely
peroxidase that contains vanadate rather than heme (ten Brink by Pasti-Grigsby et al. (1992) who showed that some azo dyes
et al. 2000). This vanadium containing peroxidase requires are preferentially oxidized by lignin peroxidases whereas
hydrogen peroxide as a cosubstrate and mediates oxidation of others are preferentially oxidized by manganese peroxidases.
halides to hypohalous acid. It also mediates the sulfoxidation Veratryl alcohol is an endogenous substrate for lignin
of organic sulfides to sulfoxides and the one-electron peroxidases produced by Phanerochaete chrysosporium.
oxidation of 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulfonic When this compound serves as substrate it undergoes two
acid) to its free radical product (ten Brink et al. 2000). It has successive one-electron oxidations to form veratraldehyde. Of
been shown that the vanadate-containing peroxidase from this interest to this review is the fact that veratryl alcohol enhances
fungus mediates oxidation of the azo dye Chicago Sky Blue oxidation of some substrates, including some azo dyes. It
6B (ten Brink et al. 2000). appears that some azo dyes (e.g., Biebrich Scarlet and
Tartrazine) can undergo a one-electron oxidation by lignin
peroxidase compound I forming the one-electron oxidation
4 SELECTED ASPECTS OF THE product of the dye and lignin peroxidase compound II
ENZYMOLOGY OF AZO DYE (Paszyzynski and Crawford 1991). However, compound II
DEGRADATION BY WHITE ROT FUNGI does not appear to be able to mediate oxidation of the azo dye.
This results in accumulation of lignin peroxidase compound II
White rot fungi are so named because they are able to and the reaction ceases. When veratryl alcohol is added,
preferentially degrade lignin in wood. When this occurs in compound II oxidizes it and returns to the ferric resting state
Biodegradation of Azo Dyes by Fungi 465

of the enzyme which may then participate in another round of produce two molecules of water. During this reaction Cu(I) is
catalysis. By preventing accumulation of compound II oxidized back to Cu(II) thus completing the reaction cycle.
veratryl alcohol enhances oxidation of the azo dye. Laccases can mediate other reactions. Wong and Yu (1999)
Wu et al. (1996) showed that nutrient carbon or nutrient showed that Trametes versicolor decolorized Acid Violet 7.
nitrogen limited cultures of P. chrysosporium are able to Of interest was the observation that laccase did not directly
decolorize Reactive Red 22. It was also reported that a crude oxidize this azo dye. However, in the presence of a low
preparation of lignin peroxidases mediated decolorization of molecular weight compound substantial oxidation (i.e.,
Reactive Red 22 and another azo dye, Evan’s Blue. Similar to decolorization) of this dye occurred. It was proposed that
the observations of Paszyzynski and Crawford (1991) it was the low molecular weight compound was oxidized by laccase
found that the presence of veratryl alcohol enhanced producing a reactive radical species, which then oxidized the
decolorization of Reactive Red 22. dye molecule resulting in a colorless product.
Manganese peroxidases are so named because they Pointing and Vrijmoed (2000) have presented evidence
mediate the one-electron oxidation of Mn(II) to Mn(III) implicating laccases produced by Pycnoporus sanguineus in
(Kuwahara et al. 1984; Glenn et al. 1986; Wariishi et al. the oxidation of the azo dyes Orange G and Amaranth and the
1988). In vitro, this reaction is often performed in the triphenylmethane dyes Bromophenol Blue and Malachite
presence of a metal chelator such as lactate or tartrate, the Green. Evidence for this, however, was indirect as oxidation
purpose of which is to facilitate dissociation of Mn(III) from of these dyes was only correlated with laccase activity in
the enzyme in the form of a Mn(III)-lactate or Mn(III)-tartrate cultures of this fungus. It should be noted, however, that
complex (Wariishi et al. 1992). P. sanguineus did not produce lignin peroxidase or
In vivo, oxalate or malonate likely performs this function. manganese peroxidase.
This is of interest because Mn(III)-complexes are relatively Yesilada and Ozcan (1998) studied the ability of crude
good oxidants. They are also relatively stable. It is thought culture filtrates to decolorize Orange II. It was shown that
that Mn(III)-complexes are stable enough to diffuse away filtrates from Coriolus versicolor but not P. chrysosporium,
from the active site and mediate oxidation of lignin and other were able to mediate substantial decolorization of water
materials that are inaccessible or otherwise not amenable to contaminated with this dye. Decolorization was dependent on
direct oxidation by laccases, lignin peroxidases, or manganese the age of the culture and was not dependent on the presence
peroxidases. Recent studies have shown that the specificity of of hydrogen peroxide. Decolorization activity was inactivated
manganese peroxidases varies considerably between fungal by heat. Given the fact that C. versicolor produces laccase in
species. For example, Heinfling et al. (1998a,b) showed that abundance it is reasonable to suggest that this enzyme is
Reactive Orange 96 and Reactive Red 198 were not oxidized responsible for the decolorization observed.
by manganese peroxidase P1 from P. chrysosporium and that Production of veratryl alcohol radical and Mn(III)
oxidation of Reactive Blue 5 by this enzyme was negligible. complexes may be of importance in lignin degradation as it
This enzyme slowly oxidized Reactive Violet 5 and activity has been suggested that these low molecular weight reactive
was increased by.approximately five-fold when 0.3 mM species may be stable enough to diffuse into the 3-D lignin
MnSO4 was present in the reaction mixture. In contrast, the structure and mediate oxidations that are not accessible by
presence of 0.3 mM MnSO4 did not enhance oxidation of the direct enzymatic oxidation by lignin peroxidases, manganese
other azo dyes studied. The ability of manganese peroxidases peroxidases, or laccases (Barr and Aust 1994; Harvey et al.
from other white rot fungi to oxidize these azo dyes was also 1986; Glenn et al. 1986). Veratryl alcohol radical may be too
studied. Of importance is the observation that substantial rates unstable and short-lived to accomplish this. However, Mn(III)
of dye oxidation occurred in reactions that were independent complexes do appear to be sufficiently stable for this purpose.
of manganese. Indeed, Mn(II) was shown to function as a Mn(III) complexes appear to be able to oxidize phenolic
noncompetitive inhibitor of Reactive Black 5 oxidation by units in lignin. They do not appear to be able to oxidize
MnP1 from B. adusta. Manganese peroxidases from these nonphenolic units (Popp and Kirk 1992; Wariishi et al. 1992).
fungi allow a strategy of direct oxidation (i.e., by the enzyme) However, when unsaturated fatty acids are added to reaction
or indirect oxidation (by Mn(III)-complexes) to be pursued. mixtures containing manganese peroxidase, Mn(II) com-
This may become important in bioreactor design and strategy plexes, and hydrogen peroxide, lipid peroxidation occurs
development. (Kapich et al. 1999). Of interest is the observation that
In addition to lignin peroxidases and manganese oxidants are produced, in such reaction mixtures, which are
peroxidases, white rot fungi often produce laccases. These capable of oxidizing nonphenolic subunits in lignin as well as
oxidases are also important in lignin degradation but unlike selected organic pollutants such as phenanthrene. Kapich et al.
peroxidases, they do not contain heme. Laccases are more (1999) suggest that peroxyl radicals are produced that are
formally known as benzenediol:O2 oxidoreductases, EC responsible for the oxidations that occur.
1.10.3.2). Instead of heme, laccases require active site Cu(II) Lignin peroxidases, manganese peroxidases, and laccases
ions for activity. During laccase-mediated reactions, diphe- are by far the most studied oxidases found in white rot fungi.
nolic compounds undergo a four-electron oxidation. During Because of their ability to degrade a wide range of organic
this reaction, Cu(II) is reduced to Cu(I). During the next step compounds, it was reasonable to suspect that cytochrome
in the reaction, Cu(I) reduces molecular oxygen (O2) to P-450 monooxygenases may be responsible for some of
466 Bumpus

the oxidations that have been observed. Indeed, some of the Blue. This fungus did not decolorize Amaranth, Congo Red,
documented oxidations that are mediated by P. chrysosporium and Ponceau 2R. These investigators also studied the ability
have not been attributed to lignin peroxidases, manganese of these fungi to decolorize several triphenylmethane,
peroxidases, or laccases. As a case in point, during DDT heterocyclic, and polymeric dyes. Several dyes were
degradation, the parent compound undergoes hydroxylation, decolorized. However, in general, the azo dyes appeared to
forming dicofol (Bumpus et al. 1985). To date, the enzyme be more resistant to degradation. Also studied were the
responsible for this oxidation has not been identified and may inhibitory effects of three metal ions [Cd(II), Cu(II), and
well, indeed, be a cytochrome P-450 monooxygenase. This Zn(II)] on dye degradation. These studies were performed
family of enzymes has not been extensively investigated in using Poly R 478. It was shown that T. versicolor (HKUCC)
white rot fungi. Studies by Knapp et al. (1997) and by Yadav and the unidentified strain (HKUCC 4062) decolorized this
and Loper (2000) showed that P-450 monooxygenase genes dye in the presence of 0.1 mM Cu(II), and Zn(II). Cd(II)
exist in this fungus. Substantial evidence for P-450 mediated appeared inhibitory to T. versicolor at this concentration.
hydroxylation of benzo(a)pyrene was presented by Masaphy Interestingly, the unidentified strain was able to mediate
et al. (1996) who showed that microsomal and soluble fractions decolorization of Poly R 478 at a Cd(II) concentration of
from P. chrysosporium exhibited characteristic reduced 0.25 mM. In contrast, when P. chrysosporium was studied,
carbon monoxide difference spectra. Benzo(a)pyrene decolorization was inhibited by metal ion concentrations of
hydroxylation was shown to be dependent on NADPH and 0.1 mM. Although Poly R 478 is not an azo dye, this study
was inhibited by carbon monoxide. Furthermore benzo(a)pyr- suggests that the presence of metal ions in dye containing
ene caused a type I spectral shift (indicative of substrate wastewater is one of the factors that must be taken into
binding) when added to soluble and microsomal preparations. consideration in developing bioreactors based on the use of
A cytochrome P-450 monooxygenase is also thought to be basidiomycetaceous fungi.
responsible for phenanthrene oxidation during biodegradation Fungi often grow in conditions where iron concentrations
of this compound by Pleurotus ostreatus (Bezalele et al. 1996). are vanishingly low. Iron is essential for metabolism (i.e.,
Most recently, the genome of P. chrysosporium has been electron transport system). Thus fungi have evolved ways of
sequenced at the U.S. Department of Energy’s facility at sequestering this essential element. To do this, fungi secrete
Walnut Hill, California. This fungus is the first basidiomycete siderophores having very high affinities for iron and certain
whose genome has been sequenced. The genome is about other polyvalent cations and it is in the form of these metal
30 Mb and is comprised of 10 chromosomes. Of interest is the siderophore complexes that metals are absobed by fungi
observation that this fungus may have the genetic potential to (Fekete et al. 1989). In addition to their role in sequestering
produce over one-hundred P-450 monooxygenases (Nelson metals, siderophores may have another important role as
2001). Clearly, elucidation of the contribution of P-450 some siderophore – metal complexes appear to exhibit
monooxygenases to azo dye degradation and xenobiotic phenoloxidase-like activity. Some siderophore iron com-
metabolism, in general, will be an area of considerable plexes are reduced from the ferric (III) to the ferrous(II)
research interest. oxidation state. When lignin is the electron donor, measurable
lignin degradation takes place. Minussi et al. (2001) have
studied this phenomenon using several dyes and have
presented circumstantial evidence that siderophore –metal
5 METAL IONS AND THE POSSIBLE ROLE complexes might be involved in the decolorization of
OF SIDEROPHORES IN Reactive Blue 19, Reactive Red 195, Reactive Yellow 145,
BIODEGRADATION OF DYES and Reactive Black 5 by wood rotting fungi. Although these
studies are inconclusive, the involvment of metal – side-
Kirk et al. (1986) developed culture media for Phanerochaete rophore complexes in the decolorization of azo dyes is an area
chrysosporium containing certain supplemental nutrient that deserves further scrutiny.
mixtures of metal salts that enhance the level of lignin
peroxidase activity produced in cultures of this fungus.
Pointing et al. (2000) studied the ability of P. chrysosporium
(IMI 284010), Pycnoporus sanguineus (HKUCC 4065), 6 DEVELOPMENT OF BIOREACTORS USED
Trametes versicolor (HKUCC 4063), and an unidentified FOR REMEDIATION OF AZO DYE
fungal strain (HKUCC 4062) to decolorize several azo, CONTAINING WASTEWATER
triphenylmethane, heterocyclic, and polymeric dyes. All four
strains were described as sub-tropical basidiomyceteous White rot fungi have been proposed for remediation of soils
fungi. P. chrysosporium, T. versicolor, and the unidentified contaminated with toxic organic pollutants such as DDT,
strain completely decolorized in liquid cultures the azo dyes polychlorinated phenols, and polycyclic aromatic hydro-
Amaranth, Amido Black, Aniline Blue, Congo Red, Methyl carbons (Bumpus 1993). However, decolorization and
Orange, Orange G, Ponceau 2R, and Trypan Blue during a remediation of wastewater from a variety of industries that
14-day incubation period. P. sanguineus decolorized Amido produce colored wastewater may be the most promising place
Black, Aniline Blue, Methyl Orange, Orange G, and Trypan for use of these fungi in bioremediation systems. Processes
Biodegradation of Azo Dyes by Fungi 467

developed to remediate colored wastewaters have been the contamination, which appeared to interfere with peroxidase
subject of considerable research that has been reviewed activity, resulting in decreased decolorization. Biodegrada-
recently by Knapp et al. (2001). Although many investi- tion of Acid Violet 7 was also studied in liquid batch cultures
gations have focused on the ability of white rot fungi to and in a fluidized bed reactor (Zhang and Yu 2000). In both
decolorize/degrade azo dyes, only a few investigations have cases, the ability of mycelial pellets to degrade this dye was
specifically focused on development of bioreactors that could studied. An interesting aspect of this research was that added
be scaled up and used for this purpose. Zhang et al. (1998) activated charcoal had a positive effect on biodegradation. It
showed that an unidentified basidiomycete designated F29 was shown that fungal mycelium surrounded a core of
was able to extensively and rapidly decolorize high activated charcoal to form complex pellets. These complex
concentrations of the azo dye Orange II in three different pellets appeared to enhance biodegradation by adsorbing both
bioreactor configurations. Continuous packed-bed, fedbatch the dye and fungal enzymes responsible for biodegradation.
fluidized-bed, and continuous fluidized-bed bioreactors were Furthermore, decolorization by the complex pellets was
studied. All of the bioreactors studied proved suitable for superior to that observed for fungal pellets without activated
bioremediation of water-containing Orange II. Concen- charcoal, for fungal pellets to which activated charcoal was
trations of dye up to 1000 mg/L were removed relatively added (after pellet formation) and to activated charcoal alone.
rapidly (1– 3.5 days) and the mycelium could be used It was also shown that reactors operated in repeated-batch-fed
repeatedly. Particular success was achieved with the fedbatch mode resulted in greater and more efficient decolorization
fluidized bed-immobilized reactor. In this system average than reactors operated in a continuous flow mode. In one
decolorization rates of 40– 50 mg l21 hr21 were achieved experiment a repeated-batch-fed reactor was able to
resulting in 97% color removal in 24 hr. This study is decolorize 9 additions of dye over a period of 130 h.
significant as it demonstrates that rapid degradation occurs Batch-fed additions of dye ranged from 100 to 500 mg/L.
and the fungal mycelium are robust and unaffected by Mielgo et al. (2001) studied the use of immobilized
exposure to high concentrations of Orange II during repeated P. chrysosporium in continuous flow packed bed bioreactors.
exposure over 1 –2 months. At dye loading rates of 0.2 g l21 d21 greater than 95%
Swamy and Ramsay (1999a,b) studied conditions, which decolorization was achieved at hydraulic retention times of
promote azo dye degradation by Trametes versicolor, 24 h. In these experiments, the temperature was maintained at
Bjerkundera adusta and Phanerochaete chrysosporium 378C and oxygen was supplied in a pulsed flow.
sequentially in batch reactors. Only T. versicolor proved to Trametes hirsuta was shown to effectively decolorize the
be able to maintain its ability to cause decolorization of water azo dyes Reactive Black 5 and Direct Blue 71 as well as
following repeated addition of the several dyes and dye representative triphenylmethane, indigoid, and anthraquinone
mixtures. dyes (Abadulla et al. 2000). All of the dyes were decolorized
Wang and Yu (1998) studied the ability of T. versicolor to by the laccase isolated from T. hirsuta. This research is
decolorize water containing an anthroquinone dye, an indigo important as it addressed the issue of recycling dye-
dye, and the azo dye Acid Violet 7. All of the dyes were contaminated water. Water decolorized using the intact
degraded. These investigators also showed that adsorption to fungus or laccase in solution was not suitable for reuse in
mycelium followed by dye degradation occurred. Adsorption dyeing operations due, presumably, to interference by soluble
was rapid and occurred with living mycelium and with heat protein. However, when a reactor containing immobilized
killed controls. With live fungi, mycelium was regenerated by laccase was used, the recycled decolorized wastewater was
physical desorption and enzymatic degradation. The authors suitable for dyeing operations as the laccase was retained in
suggest that a sequential treatment system could be developed the bioreactor.
in which dyes in wastewater could be first adsorbed on fungal
mycelium followed by decolorization/degradation. It is
reasonable to suggest that sequencing batch reactor 7 CONCLUSIONS
technology might function well in this situation. Indeed,
Borchert and Libra (2001) have used T. versicolor in 4-L It is clear that white rot fungi are able to mediate extensive
sequencing batch stirred tank reactors to decolorize water and often rapid degradation of azo dyes. Although several
containing the azo dyes Reactive Black 5 and Reactive Red micro-organisms (other fungi and some bacteria) have been
198 and the anthroquinone dye Brilliant Blue R. Reactors reported to be able to degrade this class of pollutant, it appears
were cycled repeatedly over the course of the experiments. that white rot fungi have superior biodegradative abilities in
During long-term (200 days) experiments under sterile this regard. The enzymes (lignin peroxidases, manganese
conditions, 18 dye additions occurred. In each case substantial peroxidases, laccases, and other enzymes) traditionally
color removal occurred. When the initial concentration of dye associated with the lignin degrading system of this fungus
was 100 mg/L, 97– 99.5% decolorization occurred. When the are important. However, it is likely that other oxidative
initial concentration was 500 mg/L, 91– 99% decolorization enzymes, especially cytochrome P-450 monooxygenases may
occurred. When similar experiments were performed under also be important and they require further scrutiny for a better
nonsterile conditions, only 5 decolorization cycles (during a understanding of how azo dyes (and other organic pollutants)
55-day experiment) were possible due to bacterial are degraded by these fungi. There is substantial interest in
468 Bumpus

harnessing the biodegradative abilities of white rot fungi to Neurospora crassa Strain 74A. Eur J Appl Microbiol
treat contaminated soil and water and considerable progress is Biotechnol 13:64 – 66.
being made in the development of bioreactors that are able to Cripps C, Bumpus JA, and Aust SD (1990). Biodegradation of azo
effect remediation of water contaminated with a variety of and heterocyclic dyes by Phanerochaete chrysosporium. Appl
Environ Microbiol 56:1114 –1118.
colored substances, including azo dyes.
Dunford HB (1999). Heme Peroxidases. New York: Wiley-VCH.
p 507.
Fekete FA, Chandhoke V, and Jellison J (1989). Iron-binding
ACKNOWLEDGEMENT compounds produced by wood decaying basidiomycetes. Appl
Environ Microbiol 55:2720 –2722.
Research in the author’s laboratory has been supported by the Fu Y and Viraraghavan T (2001). Fungal decolorization of dye
National Institutes of Health, the U.S. Department of the wastewaters: a review. Bioresour Technol 79:251 –262.
Interior, the U.S. Department of Energy, the Air Force Office Glenn JK, Aikeswan L, and Gold MH (1986). Mn(II) oxidation is the
for Scientific Research and the University of Northern Iowa. principal function of the extracellular manganese-peroxidase
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Harvey PJ, Shoemaker HE, and Palmer JM (1986). Veratryl alcohol
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biodegradation by Phanerochaete chrysosporium. FEBS Lett
195:242 – 246.
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39
Fungal Degradation of Explosives

J.L. Faull / S.C. Baker School of Biological and Chemical Sciences, Birkbeck College,
London, United Kingdom

S. Wilkinson / S. Nicklin DSTL, Sevenoaks, Kent, United Kingdom

1 INTRODUCTION 2 EXPLOSIVES AS SUBSTRATES FOR

MICROBIAL DEGRADATION
Explosive compounds are very significant environmental
2.1 2,4,6-Trinitrotoluene (TNT)
contaminants. Their production, testing, use in conflict, and
disposal has led to contaminated soils, sediments, and water
around the world (Spain et al. 2000). These materials are The nitro-aromatic compound 2,4,6-trinitrotoluene (TNT) is
intrinsically toxic to microbes, plants, animals, and man. In of greatest concern as an environmental contaminant
the United States, it is now a requirement that sites (Figure 1a). It is intrinsically difficult for microorganisms to
contaminated by these compounds are risk assessed and mineralize as the three symmetrically placed nitro- groups on
remediated to acceptable standards determined by site- the aromatic ring reduce its electron density and limits attack
specific clean-up goals (Jerger and Woodhull 2000). Various by electrophilic di-oxygenase enzymes (Nishino et al. 2000).
remediation strategies have been adopted to date, and these Moreover, TNT is poorly soluble in water (140 mg/l at 258C),
have involved physical, chemical, and biological approaches relatively stable and persistent in the environment (Gorontzy
(Newcombe and Crawford 2002). Physical techniques such as et al. 1994). Its partial breakdown products are more
incineration, activated carbon absorption, and filtration are hazardous, recalcitrant, carcinogenic, and mutagenic than the
effective, but generate unwanted residues that still have to be parent compound (Bennett 1994; Whong and Edwards 1984;
treated. Chemical treatments have been tried, for example Won et al. 1976).
precipitation using surfactants and solvent extraction (Kaplan Natural decomposition is often the result of co-metabolic
1990) but again these approaches generate further residues bio-transformation but not mineralization and breakdown
that have to be disposed of. Biological treatments have products include mutagenic reduced amines (Yinon 1990).
included biostimulation of existing indigenous microflora and Nitroaromatic compounds such as TNT are very rarely
bioaugmentation where an explosive degrading microbial produced in nature. A few antibiotics containing nitro-
inoculum is added to the contaminated environment (Kaplan aromatic groups are produced by Pseudomonas sp., for
1990). Biological approaches appear to be cost effective example nitropyrrolluteorin (Ohmori et al. 1978) and
alternatives to other methods, but they have had limited oxypyrrolenitrin (Hattari et al. 1970). Streptomyces
success because of microbial sensitivity to toxic levels of venezuelae has also been reported to produce chloramphe-
explosive, toxic by-products of incomplete degradation and nicol, and bryozoans have been reported to produce the
the length of time biological clean-up of a contaminated antibiotic phidolophin (Tischler et al. 1986). Nitrophenolic
environment can take (Reddy 1995). This review seeks to compounds such as aristolochic acid can be produced by
summarize available information about the metabolic path- plants (Williams and Barnaby 1977) and this compound
ways involved in fungal biodegradation of explosives and to has been identified as a potent carcinogen, intercalating or
evaluate the distribution of these pathways across the forming adducts with DNA leading to oncogensis (Arlt
taxonomic groups. et al. 2000).

471
472 Faull et al.

RDX production with a higher melting point than RDX. This

explosive has a very low aqueous solubility of 5 mg/l at 258C.
These compounds are truly xenobiotic, and do not occur
naturally. Nitramines are acutely toxic and are class C
carcinogens (Hawari et al. 2000a).

2.3 Origins and Evolution of Degradation

Pathways

The xenobiotic nature of these compounds and their poor

aqueous solubility may help explain why, to date, they have
accumulated in the environment. However, within the last
decade, after considerable exposure, bacteria are now being
isolated from contaminated environments that are capable of
mineralizing some of these explosives. For example, a TNT
degrading Pseudomonas sp. has been isolated from
contaminated water samples from a nitrobenzene production
site (Parales 2000). Examination of the gene clusters
associated with the nitroarene degradation pathway of this
bacterium indicates that the evolution of the pathways is a
recent event. This is suggested by the fact that the pathway is
not optimized, there are vestigial genes, a lack of co-ordinated
regulation, and long acclimatization times (Parales 2000).

3 BIODEGRADATION OF TNT BY FUNGI

In contrast to the newly evolved pathways we see in the

bacteria, fungi have the capability to biodegrade explosives
Figure 1 Chemical structures of the major classes of explosives. using existing, initially co-metabolic, pathways that lead to
(a) TNT. (b) PETN. (c) Nitrocellulose. (d) RDX. (e) HMX. mineralization. Parrish (1977) published the results of a
screen of the explosive degrading capability of 190 fungi.
This study showed that fungi were capable of TNT
2.2 Nitrate Esters and Methylene Nitramine degradation, but the sensitivity of the fungi to levels
Cyclic Esters exceeding 20 ppm was such that Parrish discounted their
use in bioremediation. Bennett (1994) comments that the
interpretation of results from this work appears to have
Nitrate esters, including glycerol trinitrate (nitroglycerin) delayed more serious consideration of fungi as explosive
and pentaerythritol tetra nitrate (PETN) (Figure 1b) are degraders for some years. However, more recent screens have
pharmacologically active at low concentrations and at high shown that the ability to degrade TNT to some degree was
concentrations they are acutely toxic (Gorontzy et al. 1994). distributed across many genera within the Zygomycota,
Other nitrate esters, like cellulose nitrate (Figure 1c), are Ascomycota, and Basidiomycota (Scheibner et al. 1997b;
nontoxic and relatively stable. Naturally occurring, biologi- Weber et al. 2002). Recent data suggests that under the right
cally generated, organic nitrate esters appear very rarely. conditions fungi are capable of achieving mineralization of
There is a single report of an insect sex pheromone that TNT at rates far higher than bacteria (Hawari et al. 2000a).
contains a nitrate ester (Hall et al. 1992). The best studied fungal TNT decomposer is the lignin
Another group of energetic compounds that are significant decomposing Basidiomycete Phanaerochaeta chryso-
environmental contaminants are cyclic trimers of methylene sporium. This fungus was first noted for its lignolytic
nitramine (Gorontzy et al. 1994) (Figure 1d and e). This group capabilities when it was found to cause the overheating of
includes the most powerful military explosives in use today, woodchip piles (Burdsall 1981) but to date its natural niche is
RDX (cyclotrimethlenetrinitramine) and HMX (octahydro- unknown. It has been demonstrated in numerous studies to be
1,3,5,7-tetranitro-1,3,5,7-tetrazocine). RDX is often mixed capable of TNT degradation (Esteve-Nunez et al. 2001;
with TNT and is therefore a common environmental Fernando et al. 1990). The initial reduction of TNT is
co-contaminant. It also has a limited aqueous solubility of independent of the ligninase enzymes associated with its
42.3 mg/l at 208C (Gorontzy et al. 1994). HMX is used in mineralization, and is mediated by nonspecific nitro-
shaped charges or as a rocket propellant and is a by-product of reductases that catalyze the conversion of highly oxidized
Fungal Degradation of Explosives 473

nitro-functional groups to mono- and di-amino toluenes including nitroso-toluene (NST), ortho-hydroxyl amino 2,4-
(ADNT and DANT), and nitroso- and hydroxylamine dinitrotoluene (HADNT), ADNT, and DANT are illustrated
containing intermediates (Esteve-Nunez et al. 2001). Stahl in a putative pathway constructed by Hawari et al. (2000a)
and Aust (1993) demonstrated that this reaction is (Figure 2) where the initial products are azo, azoxy, phenolic,
dependent on the presence of a living mycelium and and acylated derivatives. These compounds are already
appears to be associated with a membrane bound redox known to be mineralized by white rot fungi (Esteve-Nunez
system. This work was recently confirmed by Van Aken et al. 2001). However in P. chrysosporum, HADNT is known
et al. (1999). However, Michels and Gottschalk (1995) have to inhibit veratryl alcohol oxidation by LiP (Bumpus and
reported intracellular NADPHþ Hþ dependent TNT Tartako 1994). The veratryl alcohol to veratryl aldehyde
reductase activity. A further mechanism was reported by conversion is essential for the production of free radicals that
Eiler et al. (1999). Using Bjerkandera adusta, they observed are involved in the oxidation of primary substrates of LiP.
TNT breakdown was associated with a microsomal Thus biodegradation of TNT will be inhibited if levels of
cytochrome P 450. HADNT are allowed to accumulate. Bumpus and Tatarko
The initial co-metabolic reactions increase the electron (1994) reported that levels as low as 30 mM will inhibit
density of the aromatic ring and facilitate electrophilic attack enzyme activity. This sensitivity is regarded as a key
by lignin-degrading enzymes (Field et al. 1993). Lignin and limitation to the use of this fungus in biodegradation in the
TNT degradation occurs by a series of co-metabolic, field (Michels and Gottschalk 1995).
synergistic reactions that involve three enzymes, manganese For several years, research concentrated on P. chryso-
peroxide (MnP), lignin peroxidase (LiP), and laccase. The sporium as a prime candidate for explosive degradation.
peroxidase enzymes are haem-containing glycoproteins that However, other fungi have been screened and Table 1
require hydrogen peroxide to function and they catalyze summarizes published work to date. In some of the other
single electron oxidations that generate free radicals. fungal species screened, tolerance to TNT and its breakdown
Hydrogen peroxide is generated by oxidase enzymes products can be much higher than that of P. chrysosporium.
including glyoxyl- and aryl-oxidase. Laccase is a copper For example, Rhizopus nigricans was reported as being able to
containing phenol oxidase, which uses molecular oxygen as remove TNT from a medium containing 100 mg/ml
its terminal electron acceptor (Fritsche et al. 2000). Laccase TNT (Klausmeier et al. 1974) and Irpex lacteus tolerated up
can catalyze both polymerization and depolymerization to 50 mg/ml of TNT, and was able to degrade TNT by
reactions via oxidation (Harvey and Thurston 2001). more than one route, forming transient hydride-Meisen-
Subsequent mineralization steps of TNT reduction products, heimer complexes (Kim and Song 2000).

Figure 2 One of the putative pathways of TNT degradation by fungi.

Table 1 Species of fungi reported as capable of TNT degradation
474
Division Family Genus/species Type Habitat Comments

Basidiomycetes/ Gloeophyllaceae G. separium Brown rot Wood Hess et al. 1998

Polyporales Phanaerochaetaceae P. chrysosporium White rot Wood ATCC 24725
P. sordida White rot Wood HHB 8922, Donelly et al. (1997)
Meruliaceae P. radiata White rot Wood ATCC 64658, Van Aken et al. (1999)
P. brevispora White rot Wood HHB7030, Donelly et al. (1997)
P. ochracheofulva White rot Wood Scheibner et al. (1997a, b)
Polyporaceae T. versicolor White rot Wood DSM 11269, Scheibner et al. (1997a, b)
T. suevolens White rot Wood MWT 03-2, Scheibner et al. (1997a, b)
L. sulphureas White rot Wood Scheibner et al. (1997a, b)
F. fomentarius White rot Wood MWF 01-4, Scheibner et al. (1997a, b)
S. commune White rot Wood Kim and Song (2000)
C. versicolour White rot Wood KR11W, KR 65W, Kim and Song (2000)
P. coccineus White rot Wood Kim and Song (2000)
L. lacteus White rot Wood Kim and Song (2000)
Atheliaceae T. fibrillosa Ectomycorrhizal Meharg et al. 1997
Hapalopilaceae B. adusta White rot Wood Field et al. 1993
Basidiomycete/ Bondarzeiaceae H. annosum White rot Wood TM5P2. Scheibner et al. (1997a, b)
Agaricales Strophariaceae H. fasciculare White rot Wood TM 52, Scheibner et al. (1997a, b)
K. mutablis White rot Wood TME, TM 1/68, Scheibner et al. (1997a, b)
N. frowardii White rot Wood ATCC 201144, DSM 1239, Scheibner et al. (1997a, b)
S. ruguloso-annulata Litter DSM 11373, TME, Scheibner et al. (1997a, b)
Tricholomatacea C. dusenii White rot Wood DSM 11238, TMb 12, Scheibner et al. (1997a, b)
Nidulariaceae C. stercorus White rot Wood 36910 Donelly et al. (1997)
Tricholomatacea C. (Lepista) nebularis Litter TM, Scheibner et al. (1997a, b)
C. odora Litter TM3, Scheibner et al. (1997a, b)
Plutaceae A. muscaria Ectomycorrhizal Scheibner et al. (1997a, b)
H. fasciculare White rot Wood TM5.2
Agaricaceae A. aestivalis Litter TMAEST1, Scheibner et al. (1997a, b)
A. bisporus Litter MWA 80-7, Scheibner et al. (1997a, b)
A. praecox Litter TM 70 84, TM70.3.1 Scheibner et al. (1997a, b)
Basidiomycetes/ Suillaceae S. granulatus Ectomycorrhizal Scheibner et al. (1997a, b)
Boletales S. variegates Ectomycorrihazal Meharg et al. 1997
Sclerodermataceae P. tinctorus Ectomycorrihizal Meharg et al. 1997
Ascomycete Moniliales P. frequentens Ubiquitous ATCC 96048, Scheibner et al. (1997a, b)
anamorphs Penicillium sp. Ubiquitous DSM 11168, Scheibner et al. (1997a, b)
P. chrysogenum Ubiquitous IFO 31249, Kim and Song 2000
A. terreus Ubiquitous MW 458, Scheibner et al. (1997a, b)
Faull et al.
Fungal Degradation of Explosives 475

4 BIODEGRADATION OF NITRATE ESTERS

TM ¼ Culture collection of the Institute of Microbiology, Jena, Germany; MW ¼ Stock culture collection, Weimar, Germany; DSM ¼ German Culture Collection, Braunschweig, Germany;
BY FUNGI

v. elegans DSM (1980), Scheibner et al. (1997a, b) Substituted nitrate esters have a fairly low aqueous solubility.
Glycerol tri-nitrate saturates at 1.5 g/l, whilst fully substituted
nitro-cellulose is completely insoluble in water (Williams and
DSM 810, Scheibner et al. (1997a, b)
TM 821, Scheibner et al. (1997a, b)

Bruce 2000). Although these compounds are relatively stable,

TMR 2, Scheibner et al. (1997a, b)

there is some evidence that in the natural environment they

TM, Scheibner et al. (1997a, b)

can be reductively transformed, forming alcohols and nitrates

(Williams and Bruce 2000). There is a dearth of published
Scheibner et al. (1997a, b)

literature on fungal biodegradation of nitrate esters and the

Klausmeier et al. 1974

possible pathways by which such degradation could occur.

The few fungi reported as having some degradative effects on
nitrate esters are distributed across the mitosporic fungi and
wood rotting Basidiomycete genera. Geotrichium candidum
was shown to have denitration capability, generating glycerol
dinitrate and glycerol mono-nitrate with glycerol-2 mono-
nitrate the predominant product (Ducroq et al. 1990). This
work demonstrated a regio-specificity in the de-nitration, and
they suggested that different enzymes were involved in the
de-nitration reactions. P. chrysogenum appears also to have
the ability to denitrate nitrate esters and in this Basidio-
mycete, the enzymes responsible for these reactions appear to
Ubiquitous
Ubiquitous
Ubiquitous
Ubiquitous
Ubiquitous
Ubiquitous
Ubiquitous
Ubiquitous

be glutathione-S-transferases (GST) (Servent et al. 1991;

1992). Glutathione-S-transferase enzyme activity has been
located in the cytosol. Furthermore, these workers also
identified a cytosolic and microsomal P450-like enzyme with
similar activity. There are therefore at least two different
classes of enzymes involved in the de-nitration of GTN, an
oxygen insensitive glutathione-dependent enzyme that
liberates nitrate, and an oxygen-sensitive NADPred dependent
P450-like activity that liberates nitrous oxide. In all cases of
fungal degradation of nitrate esters, additional carbon sources
have had to be supplied, and degradation is only partial. Even
when a cellulolytic species is combined in co-culture with a
de-nitrating species (Sclerotium rolfsii plus Fusarium solani)
Alternaria sp.

C. echinulata
Fusarium sp.

decomposition is incomplete (Sharma et al. 1995; Sundaram

R. nigricans
M. mucedo
A. resinaea

ATCC ¼ American Type Culture Collection, http://www.atcc.org

et al. 1995).
N. crassa
F. solani

5 BIODEGRADATION OF RDX/HMX BY
FUNGI

In contrast to TNT, RDX and HMX are intrinsically less

Also known as Cladosporium resinae.

recalcitrant. Once there is a change in the ring structure, for

example by cleavage of a N –NO2 bond or C– C bond, the ring
structure undergoes spontaneous decomposition, producing
small nitrate and carbon containing compounds that are easily
mineralized by many species of microorganisms (Hawari
2000). However, there is only a limited literature on
biodegradation of cyclic triamines. Bayman et al. (1995);
Bayman and Redkar (1997) report that mycelium of
Zygomycetes

P. chrysogenum, Cladosporium resinae, Cyathus pallides,

and Cunninghamella echinulata var elegans were not
inhibited by 50 – 100 mg/ml RDX, and that the RDX
“disappeared” when incubated with these fungal species.
a
476 Faull et al.

However, it was not possible to trace the breakdown products 6.2 Landfarming and Soil Slurry Reactors
or to determine if the disappearance was due to mineralization
reactions or bio-transformations. Fernando and Aust (1991) Land farming, where inoculants and moisture are ploughed
reported that P. chrysogenum could mineralize RDX, and into soil on a large scale, has had little success using fungal
they provided some evidence that a free-radical chain inoculants and soil slurry reactors have also proven to work
mechanism was involved in the degradation, but again were better with bacterial inoculants than fungal (Hawari et al.
unable to identify the initial products of decomposition. 2000b). However, Fritsche et al. (2000) remain optimistic
about the potential of the land farming approach using fungal
inoculants.
6 USE OF FUNGI IN BIOREMEDIATION IN
THE FIELD
6.3 Bioreactors
A number of different strategies have been adopted where
attempts have been made to exploit fungi in bioremediation. Fungal inoculants have also been used in aqueous-based
Early findings demonstrated that attempts to establish wood systems using fixed fungal films. They may be attached to
rotating discs in a bioreactor (Sublette et al. 1992)
rotting species in soil without amendments or soil
(P. chrysosporium to degrade TNT and RDX) or in
sterilization failed. Like the early failures in establishing
continuous culture air lift fermenters (Rho et al. 2001)
biological control agents in soil (Faull 1986), the indigenous
(P. chrysosporium with TNT).
micro flora out-competed the inoculants unless there were
large additions of substrates such as wood chips and other
ligno-cellulosic materials to the soil (Bennett et al. 2002;
Cerniglia and Sutherland 2002; Radtke et al. 1994). 7 FUTURE PROGRESS
Currently, a number of approaches are being tried, including
soil piles and windrows (where “composting” may occur), It is possible to identify some key features that limit
soil farming, soil slurry reactors, and fixed film reactors successful use of fungi in bioremediation processes and
(Rogers and Bunce 2001). identify possible strategies that might be employed to
overcome them.

6.1 Soil Piles and Windrows

7.1 Use of Photocatalysis to Reduce Toxic Effects
of Breakdown Products
Soil piles and windrows are created by mixing soil with
wood chips, corn cobs, or other ligno-cellulosic materials
and adding fungal inoculum on a lignocellulosic base. The There is a sensitivity of key fungal enzymes to explosive
pile is then left for an extended period of time with regular degradation products that currently limits the use of fungi in
turning and wetting for composting, or no turning for the bioremediation of contaminated materials. This is exempli-
static pile. This approach can lead to a rapid disappearance fied by the sensitivity of veratryl alcohol oxidase activity of
of explosive contaminants. A number of different fungal P. chrysosporim LiP to HADNT. A solution to this problem
inoculants have been tried. For example, Jerger and Woodull may be in the use of combined photo-catalytic and biological
(2000) used Trametes versicolour and P. chrysosporium as processes. The photo catalytic process degrades the toxic
soil pile inoculants, Fritsche et al. (2000) used Stropharia intermediates of breakdown and allows the fungal enzymes
rugosoannulata and Spreinart et al. (1998) used B. adusta. to continue mineralizing the products of photo-catalytic
breakdown (Hess et al. 1998).
This approach is being widely used in the United States
for the clean up of military sites (USAEC 1999). However,
criticisms of this technique include the long incubation
times needed for complete disappearance of the target 7.2 Use of Anti-Oxidants to Control Quenching
substrate, and the high costs of set-up and maintenance. The
process is further criticised for being based on unknown There is evidence that one of the key elements of the
biological processes that may produce toxic breakdown degradation process, the production of free radicals via the
products that bind to soil organic matter and undergo activity of Lip and MnP, is quenched by the presence of
no further mineralization (Hawari 2000). Furthermore, in humic compounds (Hawari 2000). The use of fungi in
many cases it has been shown that added microbial controlled environments (particularly in fixed film reactors),
inoculants did not persist in the environment and it was where reduced glutathione and other thiols can be added
the indigenous micro flora that achieved much of the to reverse the quenching effects, has produced promising
degradation (Gerth et al. 2001). results (Scheibner and Hofrichter 1998; Fritsche et al. 2000).
Fungal Degradation of Explosives 477

7.3 Screening for New Fungal Species Strains Hedger 2001). Species from the Sphaerales are slow lignin
degraders with great tolerance to water stress. It is essential to
Strain selection for effective explosive-degrading fungi is far understand such ecological effects and to match these
from complete. The rather random screening approach used to biodegradative abilities with the desired end result before
date has shown that the ability to degrade substrates like TNT is selecting a species of fungus to use in bioremediation of a
widespread across fungal genera (Scheibner et al. 1997a,b). particular substrate.
Most of the species studied have been from temperate regions.
However, the tropical species may well have greater lignolytic
capabilities as they are able to decompose far more woody 7.5 Use of Sorption Strategies
litter per year than the temperate species can (Swift et al.
1976). Furthermore, many species of fungi with no lignin- Bioattenuation/humification strategies are currently being
degrading capability seem able to partially biodegrade seriously considered as alternatives to mineralization (Hawari
TNT and other explosives by unknown metabolic routes. et al. 2000a,b; Isbister et al. 1984; Weber et al. 2002). In many
Understanding of the enzymology associated with these experiments, mass balance equations prove that mineraliz-
reactions would seem to be a priority. This would lead to a ation does not occur, but TNT breakdown products
more rational screening approach. “disappear.” The process appears to be due to irreversible
binding (sorption) of TNT breakdown products with the soil
humic and clay fraction (Achtnich et al. 1999; Elovitz and
7.4 Matching Colonization Strategies with Weber 1999; Weber et al. 2002). Sorption mechanisms
Bioremediation Requirements include electrostatic interactions and co-valent bonding (Head
1998) (Figure 3). Sorption interactions will also depend on the
It is essential to understand the way in which different fungi chemistry of the pollutant, and the amount of clay and organic
colonize and function in different niches. The initial phase of content of the soil. Binding can also be mediated via oxido-
substrate colonization has little to do with ligninases (Evans reductive enzymes including laccase (Bollag et al. 1992). The
and Hedger 2001). Lignin has such a low calorific value that it driving force behind accepting that “disappearence” is a
cannot act as a sole carbon source, therefore white rot fungi satisfactory end point in bioremediation is the poor results so
only degrade lignin when they cannot utilize cellulose. For far obtained from in situ remediation of soil using fungal
example T. versicolour enters wood through cuts in vessels or inoculants. Such an approach needs careful evaluation as
tracheids and it colonizes rapidly through these structures, Palmer et al. (1997) found that apparently irreversibly bound
utilizing soluble materials whilst sequestering and retaining products could release sufficient residues under certain
substrate. Only after colonization and substrate possession is conditions to be detectable in mammalian bioassays. The
complete does cellulose availability become limited, and as presence of these pollutants was not detectable by
nitrogen levels reduce to below 200:1 C:N lignin degradation conventional analytical chemical techniques. Sorption is
begins. Other members of the Aphyllophorales, including influenced by pH and high will release sorbed molecules
Ganoderma sp., Fomes sp., and lnonotus sp., are slow to (Head 1998). Natural surfactants produced by indigenous soil
colonize and the onset of lignin degradation is even slower, microbes can also release sorbed products. There is an
but very recalcitrant molecules can be degraded (Evans and increasing awareness that irreversible binding may not

Figure 3 Interaction of TNT degradation products with materials in soil.

478 Faull et al.

actually occur, and there are many on-going studies on the use Cerniglia CE and Sutherland JB (2002). Bioremediation of
of toxicity assays to monitor composts produced by polycyclic aromatic hydrocarbons by lignolytic and non-
bioremediation (Gundersen et al. 1997; Rocheleau et al. lignolytic fungi. In: Gadd G ed. Fungi in Bioremediation.
1999). Cambridge, UK: CUP. pp 136 – 187.
Donelly KC, Chen JC, Huebner HJ, Brown KW, Autenreith RL, and
Bonner JS (1997). Utility of four strains of white rot fungi for
the detoxification of 2,4,6-trinitrotoluene in liquid culture.
8 CONCLUSIONS Environ Toxicol Chem 16:1105 – 1110.
Ducroq C, Servy C, and Lenfant M (1990). (Formation of glyceryl-2-
There is clearly a pressing need for new approaches to mononitrate by regioselective bioconversion of glyceryl
bioremediation of explosives-contaminated soils and sedi- trinitrate-efficiency in the filamentous fungus Phanaerochaeta
chrysosporium. Biotechnol Appl Biochem 12:325– 330.
ments. There is increasing evidence that co-metabolically
Eiler A, Rungeling E, Stundl UM, and Gottschalk G (1999).
produced products of the incomplete degradation of explosive Metabolism of 2,4,6-trinitrotoluene by the white rot fungus
compounds such as TNT are not as tightly bound to clays and Bjerkandera adusta DSM 3375 depends on Cytochrome p450.
organic matter as was first thought. Their presence in an Appl Microbial Biotechnol 53:75 – 78.
ecosystem can be detected by bioassay even though, in terms Elovitz MS and Weber EJ (1999). Sediment mediated reduction of
of chemical analysis, they have disappeared. Current 2,4,6-trinitrotoluene and fate of resulting aromatic (poly)
techniques have failed to fully exploit the capacity of fungi amines. Environ Sci Technol 33:2617 –2625.
to mineralize these compounds via naturally occurring Esteve-Nunez A, Caballero A, and Ramos JC (2001). Biological
pathways. This appears to be because a limited selection of degradation of 2,4,6-trinitrotoluene. Micro Mol Biol Revs
strains and species of fungi have been screened for their 65:335 –352.
explosive degrading capability. We have an incomplete Evans CS and Hedger JN (2001). Degradation of plant cell wall
polymers. In: Gadd G ed. Fungi in Bioremediation. Cambridge,
understanding of metabolic pathways involved in the strains
UK: CUP. pp 1– 26.
that have been selected. We do not fully understand the
Faull JL (1986). Fungi and their role in crop protection. In: Day
microbial ecology of these fungi when used in bioremedia- PR ed. BCPC monograph 34: Biotechnology and Crop
tion. Further work is needed within all three of these areas Improvement and Protection. UK: BCPC publications.
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create as many challenges as they overcome. Fernando T and Aust SD (1991). Biodegradation of munitions waste
TNT (2,4,6-trinitrotoluene) and RDX (hexahydro-1,3-5 trinitro
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40
Restoration of Mycorrhizae in Disturbed Arid Ecosystems

Season R. Snyder Sapphos Environmental, Inc., Pasadena, California, USA

Michael F. Allen Center for Conservation Biology, University of California, Riverside, California, USA

1 INTRODUCTION can survive and contact each other (Allen et al. 1992).
Alternatively, inoculations of a disturbed site with native or
Mycorrhizae are mutualistic symbioses between plants and exotic fungi facilitate formation of mycorrhizae in a shorter
fungi. As such, mycorrhizal fungi play an important role in period, given certain circumstances. The simple application
structuring plant communities by improving the performance of inoculum, however, does not guarantee the formation of
of individual plants. Improved growth and survival of host a functional mycorrhiza. The potential benefits of
plants is mostly attributed to the ability of fungal partners to inoculation could be outweighed by cost and efficiency
improve plant nutrition and rooting structure (Allen and Allen limitations, if the probability of plants benefiting from
1986; Allen et al. 1989; Lewis 1973) and enhance resistance mycorrhizae is low (Findlay and Kendle 2001). Under-
to environmental stresses and soil pathogens (Sylvia and standing mycorrhizal function and limits of mycorrhizal
Williams 1992). However, a variety of mycorrhizal types function are critical in studying ecosystem response to
differentially affects their host plants. If the appropriate group disturbance and recovery from disturbance (Allen et al.
of fungi is unavailable to form the type of mycorrhizal 1999).
association to which a plant belongs, that group of plants may In addition to examining the integral role of mycorrhizal
not remain or recover from a disturbance (Allen et al. 1993). fungi in ecosystem restoration, there has been considerable
Disturbance alters fungal communities in different ways and interest in determining the influence of mycorrhizal fungi
fungal recovery typically occurs after initial plant establish- on revegetation of severely polluted soils. The use of
ment. As a consequence, the formation of functional mycorrhizal fungi in bioremediation of metal-polluted soils
mycorrhizae may require decades or may never occur. has received increasing attention. Recent studies suggest
The importance of mycorrhizae to the restoration of arid that mycorrhizal fungi may exhibit some degree of heavy
plant communities has received considerable attention, most metal tolerance and as a result, confer heavy-metal
notably because successful establishment of mycorrhizae has tolerance in host plants. However, under natural conditions,
implications to rates and patterns of plant succession (Allen the extent to which mycorrhizal associations benefit plants,
and Allen 1990). It is critical, therefore, to understand the key in terms of alleviating metal toxicity, remains largely
factors that regulate the natural reestablishment of mycor- uncertain (Leyval et al. 1997). In this chapter, we will
rhizae (Allen et al. 1992), and determine whether mycorrhizal address in more detail the response of mycorrhizal fungi to
inoculation is a viable alternative to managing existing fungal disturbance and the factors that inhibit or facilitate their
populations or manipulating their natural reinvasion. natural recovery. We will also discuss management options
Although the specific factors that regulate natural reestablish- for enhancing native fungal reestablishment and the
ment of mycorrhizae are not entirely known, they involve potential benefits and drawbacks of using mycorrhizal
survival of residual fungal propagules, dispersal of propagules inoculum. Lastly, we will briefly discuss the potential role
from adjacent undisturbed areas or from local sites of residual of mycorrhizal fungi as bioindicators and their application
survival, and available microsites where plants and fungi to bioremediation.

481
482 Snyder and Allen

2 TYPES OF MYCORRHIZAE only 48 species of AM fungi, with no one site containing more
than 12 species. In a seasonal tropical forest, Allen et al.
The term mycorrhiza describes the symbiotic relationship (1998) found approximately 25 –30 species of AM fungi in a
between diverse assemblages of fungi and plant roots. forest with plant species richness exceeding 1,000.
Mycorrhizal associations are considered mutualistic because Unlike AM fungi, the formation of EM involves a greater
they are both the normal state (Smith and Read 1997) and a diversity of fungal species (. 5400 species), exhibiting
constant feature (Smith 1995) of most plants under most varying degrees of host specificity (Molina et al. 1992). For
ecological conditions. There are several types of mycorrhizae example, the EM fungal genera, Hydnangium, is found only
characterized with respect to the organisms involved in the on Eucalyptus and Suillis and Rhizopogon are restricted to
symbiosis and their anatomy and function. The most common Pinaceae, while Amanita and Laccaria associate with most
is called an arbuscular mycorrhiza (AM). The AM fungi EM hosts (Molina et al. 1992). Further, EM fungal diversity
(Glomales, Zygomycetes) occur in most terrestrial environ- can be high in areas where plant community diversity is low.
ments (Allen et al. 1995; Janos 1980) and are the dominant Early studies by Trappe (1977) estimated 2000 species of EM
association in grassland, shrubland, and agricultural eco- associated with Douglas Fir alone. In the Jarrah forest of
systems. These fungi are characterized by the formation of southwestern Australia dominated by Eucalyptus marginate
arbuscules, thinly branched hyphal structures within root and E. calophylla, 90 species of EM fungi were found
cortical cells responsible for transfer of immobile nutrients (Hilton et al. 1989). Over 50 species of EM fungi were
such as phosphorous (P) from fungi to plant. Arbuscular identified in a Quercus agrifolia stand near Temecula in
mycorrhizae fungi are obligate biotrophs, obtaining all of southern California. These included truffle fungi in the genera
their carbon from a host plant, which makes culturing and Hydnotryposis, Hydnotrya, and Tuber, as well as epigeous
mass-producing fungal isolates for large-scale inoculation mushrooms such as Amanita, Boletus, Cortinarius, Laccaria,
efforts extremely difficult. The second most common and Russula. Still, most studies underestimate EM diversity
mycorrhizal type is ectomycorrhiza (EM). The majority of because many of these fungi fruit irregularly, and fungi with
EM fungi are basidiomycetes and ascomycetes, with few abundant sporocarps may not form functional EM (Gardes
species of zygomycetus fungi in the genus Endogone (Smith and Bruns 1996; Gehring et al. 1998).
and Read 1997). These fungi associate almost exclusively
with woody plants in the Pinaceae and Fagaceae (Allen et al.
1995). Ectomycorrhizae fungi do not penetrate the plant cell 4 MYCORRHIZAL FUNCTION
walls but surround them forming a Hartig net and encase 4.1 Nutrient Uptake
individual roots with a mantle of fungal tissue. These fungi
are used extensively in forest reclamation worldwide, the
most common being Pisolithus tinctorius (Marx and Cordell Despite the differences in associations among mycorrhizal
1989; Marx et al. 1976), Rhizopogon spp. (Trappe 1977), and types, the function of all mycorrhizal systems depends on the
Suillus spp. (Chapela et al. 2001). Lastly, other less dominant ability of the fungal mycelium to absorb available inorganic
mycorrhizal types that form important species-specific or organic nutrients from the soil and translocate them to the
associations with ericaceous and orchidaceous plants should host roots (Smith and Read 1997). Hyphae outside the root
also be included in their restoration efforts. may form an extensive network extending several centimeters
from the root surface (Friese and Allen 1991), enabling plants
to obtain resources well beyond depletion zones (Nye and
3 MYCORRHIZAL DIVERSITY Tinker 1977) and to forage effectively for patchily distributed
resources (Cui and Caldwell 1996). Fine, highly branched
3.1 Fungal and Plant Species Richness hyphae increase the absorptive surface area of the root
(Harley and Smith 1983; Rousseau et al. 1994), produce
While plants vary in their dependence on mycorrhizal fungi, enzymes for mineralization of nutrient sources that are
the fungi are generally obligate mutualists (Lewis 1973). normally unavailable to plants (Abuzinadah and Read 1986;
Interestingly, patterns of mycorrhizal diversity do not always Bauer et al. 2000), and because of their extremely fine
follow those of plant diversity (Allen et al. 1995). There are diameter they are considerably less expensive than roots. As a
approximately 150 species of AM fungi (Morton et al. 1995; result, mycorrhizal plants undergo physiological changes
Schenck and Perez 1990) forming associations with about which include increased rates of growth and seed production,
70% of plants worldwide. Virtually any AM fungus can increased nutrient status, enhanced water uptake and drought
associate with any vascular plant capable of forming an AM tolerance, and improved resistance to plant pathogens.
(Allen et al. 1995). Despite low fungal species richness, many
AM communities can exhibit high plant species diversity. In
Wyoming sagebrush-steppe, 11 species of AM fungi were 4.2 Carbon Allocation
found where plant species richness exceeded 150 species
(Allen et al. 1993). In a survey of 80 sites across the Great Arbuscular mycorrhiza fungi are obligate symbionts and must
Basin in the western United States, Allen et al. (1993) found obtain carbon (C) from the host plant, whereas EM fungi
Restoration of Mycorrhizae in Disturbed Arid Ecosystems 483

primarily uses host plant C. Yet, some taxa are capable of the extraradical hyphae of AM fungi, together with fine roots,
supplementing host C with supplies from the soil (Durall et al. physically bind soil particles into larger macroaggregate units
1994; Smith and Read 1997). Studies have estimated that (Miller and Jastrow 1990). Second, AM fungi channel a
10– 85% of net photosynthate is allocated to mycorrhizal significant amount of carbon into the soil by which soil
fungi depending on the ecosystem (Allen 1991). Mycorrhizal particles and organic material become bound, contributing to
plants consistently transfer more C belowground than soil aggregation and stability (Rillig et al. 1999). Typically,
nonmycorrhizal plants. Under low light or high nutrient there are between 1–20 m of AM hyphae/g of soil (Sylvia
conditions, mycorrhizae may suppress plant growth and the 1990). In cold deserts, Allen and MacMahon (1985) reported
fungal partner has sometimes been considered a drain on the hyphal length of 2 –6 m/g of soil. Treseder and Allen
host (Johnson et al. 1997). However, a number of studies have (2001) reported similar values (2– 4 m of live AM hyphae/g
shown that photosynthetic rates are enhanced in mycorrhizal of soil) in a chaparral ecosystem. In contrast, Miller et al.
plants to compensate for the cost of mycorrhiza development, (1995) found that soils associated with mycotrophic plants in
regardless of nutrient availability (Allen 1991). tallgrass prairie had 111 m/cm3 and Allen and Allen (1988)
Mycorrhizal fungi are an important link in the chain of C reported that in Wyoming sagebrush steppe, AM fungi
transfer from plant to soil. We now realize the important role produced 28 –54 m total hyphae/g of soil. The significant
of mycorrhizae in sequestration of C in soil (Bonkowski et al. contribution of fungal hyphae to the formation of stable soil
2000; Rillig et al. 2001; Treseder and Allen 2000) and the aggregates that prevent wind and water erosion which may be
potential for mycorrhizae to influence C cycling rates. crucial to restoration success (Miller and Jastrow 1991) and
Estimates of AM fungal biomass can account for 1 –17% of carbon sequestration (Jastrow et al. 1996).
the root weight (Fujiyoshi et al. 2000) and EM can comprise
20– 30% of the root volume (Harley and McCready 1952).
Processes involved in the cycling of fungal C include 5 MYCORRHIZAL RESPONSE TO
production, survivorship, and decomposition rates of fungal DISTURBANCE
tissue (Treseder and Allen 2000). The production and
turnover of fine hyphal networks exceeds the turnover rates The fundamental role of mycorrhizal fungal hyphae is to
for both root and shoot material (Fitter et al. 2000). However, provide a direct physical link between the plant community
a substantial amount of the C allocated to mycorrhizal tissue and surrounding soil resources (Miller and Jastrow 1992).
could remain in the soil for a longer period. Chitin, a Disturbance to soil systems always alter in some way the
recalcitrant polysaccharide, can constitute 60% of fungal cell spatial heterogeneity between soil fungi and plants, organic
walls and may persist years to decades in the soil. The matter, and nutrient pools (Allen and MacMahon 1985; Allen
functioning hyphal mycelium also secretes an insoluble et al. 1999). Major disturbances usually reduce mycorrhizal
glycoprotein called glomalin (Wright and Upadhyaya 1996) inoculum densities. Disturbance to hyphae can occur through
that sloughs from hyphae during the life span of root compaction of soil pores or physical destruction of the hyphae
colonization (Wright and Upadhyaya 1999; Wright et al. (Allen and MacMahon 1985) and through vegetation removal
1996). Glomalin is important in forming water-stable or loss of plant cover. The impact of disturbance on
aggregates and in soil fertility; measurement of this protein mycorrhizal infectivity has been the topic of numerous studies
concentration in soils will help with the comparison of soils of (Jasper et al. 1989; Jasper et al. 1991; McGonigle et al. 1990).
different compositions and/or tillage or disruption practices
(Wright and Upadhyaya 1996). Because hyphal productivity
varies among AM fungi, glomalin production also varies 6 RECOVERY OF MYCORRHIZAL FUNGI
(17 mg/mg hyphae –63 mg/mg hyphae) (Wright et al. 1996).
Glomalin levels can be present in soil as high as 1.5% of the The reestablishment of mycorrhizal fungi following disturb-
soil dry weight (Wright and Upadhyaya 1996) and constitute ance has been extensively studied (Allen et al. 1999). Factors
30– 60% of the recalcitrant carbon in undisturbed soils that are most influential to the recovery of mycorrhizae
(Treseder and Allen 2000). Carbon dating of glomalin include the dispersal of plant and fungal propagules, the
indicates that turnover occurs at time scales of several years to physicochemical soil environment, and the survival of fungal
decades, much longer than the turnover estimates for AM residuals. Plant and fungal propagules typically disperse
hyphae (Rillig et al. 2001). independently of one another and then must encounter each
other in a suitable microsite favorable to their survival. Plant
and fungal propagules arriving at that microsite must also be
4.3 Soil Stabilization viable and germinable (Cooke and Whipps 1993). Soil
fertility, especially artificially high levels of available nitrogen
In addition to direct benefits of plant growth and health, the (N) and P, could inhibit formation of mycorrhizae and slow the
contribution of fungal mycelium to soil aggregation and soil recovery of fungal propagules densities in soil. Therefore,
organic matter development may be a critical component to survival of infective residual fungal propagules would greatly
most restoration programs (Miller and Jastrow 1991). The enhance mycorrhizal reestablishment (Allen and MacMahon
role of fungi in enhancing soil stabilization is two-fold. First, 1985; Allen et al. 1992).
484 Snyder and Allen

7 MANAGING EXISTING INOCULUM 7.3 Maintaining Source Areas

7.1 Protecting Topsoil
The ability of mycorrhizal propagules to colonize disturbed
sites from across relatively long distances supports the need to
If the disturbance event can be anticipated, as in the case of maintain source areas with diverse and abundant mycorrhizal
mining or logging, it may be possible to minimize the adverse populations. The natural recovery of mycorrhizae was first
effects to mycorrhizal communities through topsoil protec- presumed to be a limited process, relying heavily on the slow
tion. In work undertaken by Miller et al. (1998), EM fungi
recolonization of plants and animals from disturbance edges.
persisted on dying roots following fires for over a year after
However, Allen et al. (1989); Warner et al. (1987) have
the trees were dead: new seedling roots could tap this resource
demonstrated that both wind and animals can disperse AM
if it remains intact. Since most mycorrhizal fungi are present
spores quite rapidly and as far as 2 km across disturbed arid
in the upper soil profile, topsoil serves as a reservoir of fungal
landscapes. On Mount St. Helens, wind rapidly dispersed EM
inoculum, as well as a hospitable environment for invading
inoculum, but animals were necessary for dispersal of AM
propagules. Therefore, the careful management of topsoil
(Allen et al. 1992).
should alleviate the need for intense inoculation efforts after
disturbance. For example, when possible, disturbance to soil
should be minimized by limiting or eliminating activities that
exacerbate the level of impact (Allen et al. 1999). This allows
mycorrhizae associated with plants and organic matter to
7.4 Managing Natural Reinvasion
stabilize soil and reduce erosion. It also protects intact hyphal
networks that can rapidly form mycorrhizae on uncolonized
roots of neighboring plants (Francis et al. 1986; Smith and Of the diverse array of AM and EM fungi that could
Read 1997). In situations where land is to be eventually potentially inhabit a site, relatively few are used to replace
disturbed, the most effective means to preserve mycorrhizae the hundreds that are lost with disturbance. Managing a site
may include salvaging the topsoil before disturbance and to facilitate natural reinvasion may therefore be critical to
respreading it afterwards or transferring it away from the site the restoration process. In arid habitats, mycorrhizal
to another disturbed area (Allen et al. 1999). Top-soiling can propagules are dispersed across sites by both wind and
be a significant source of microbes, plant propagules, and animals (Warner et al. 1987). Evaluating and manipulating
organic matter (Allen and Allen 1980); however, critical to these two factors could potentially enhance the rate of
the benefits of top-soiling is the length of time, the soil spends mycorrhizal recovery. For example, Allen et al. (1989) found
without plant cover necessary to sustain symbiotic relation- that wind dispersal and deposition patterns of fungal spores
ships. Soil may be stockpiled for several years, during which were predictable given an understanding of the physical and
time organic matter (Schwenke et al. 2000), chemical biological characteristics of a site. Knowing potential source
composition (Kundu and Ghose 1997), and infectivity of and sink areas, it is possible to enhance the trapping of wind-
mycorrhizal propagules can change greatly (Miller et al. blown propagules using artificial barriers such as snow
1985). Even after soil reapplication, slow or poor seedling fences or by manipulating the distances between individual
recruitment may further hinder mycorrhizal establishment. plants thereby creating islands of fungal inoculum (Allen
Planting a dense cover crop of the appropriate plants on soil et al. 1997).
stockpiles could enhance the survival and subsequent Animal activity on disturbed sites is also important to
establishment of mycorrhizal fungi on restoration sites. mycorrhizal recovery. Animals disperse fungal propagules,
either by directly feeding on them (Allen 1988; Allen et al.
1997; Blaschke and Bäumler 1989; Rabatin and Skinner
7.2 Soil Amendments 1985) or by moving soil and root material containing
propagules (Allen and MacMahon 1988; Friese and Allen
Improving the structure of soil organic matter is an important 1993). Numerous animals consume the hypogeous fruiting
management option for enhancing mycorrhizal development bodies (truffles) of some EM fungi and deposit these in new
(Allen et al. 1999). Compost, bark, or some other recalcitrant locations, a critical element to the reforestation of many
carbon source provides a slow release of nutrients while habitats (Allen et al. 1997). Pocket gopher (Thomomys
increasing soil moisture, facilitating infiltration, and reducing talpoides) and harvester ant (Pogonomrymex occidentalis)
soil compaction. These conditions are not only beneficial to move substantial amounts of soil, bringing spores, root
mycorrhizal fungi, but also many other microorganisms that fragments, and plant propagules to the surface (Allen et al.
improve nutrient availability and plant performance. For EM 1984). Their mounds provide refuge and favorable microsite
particularly, the absence of organic matter following severe conditions for the establishment of late-seral obligately
disturbance limits the establishment of mycorrhizae despite mycorrhizal plant species (Allen 1987). Interestingly, the
adequate dispersal onto the site (Allen et al. 1992; Read dispersal of mycorrhizal propagules via animal activity could
1984). The use of soil amendments could therefore alleviate play an influential role in determining rates and patterns of
the need for further mycorrhizal inoculation. plant reestablishment success.
Restoration of Mycorrhizae in Disturbed Arid Ecosystems 485

7.5 Managing Plants to Enhance Recovery Thomson 1994). Large-scale disturbances always change soil
characteristics, alter plant communities, and reduce mycor-
The establishment of mycorrhizal associations depends rhizal abundance and diversity. Where native fungi have low
largely on two facets: the characteristics of the host plant colonization capacity, but provide benefits to host plants,
and the spatial pattern of planting. Plants exhibit different managing to increase the abundance of fungal populations
degrees of mycotrophy, primarily based on their capacity for may be more appropriate than augmenting with nonnative
nutrient uptake and their growth response to fungal fungi (Dodd and Thomson 1994). Most areas to be restored
colonization. In early work by Stahl (1900), plants were vary greatly from their predisturbance state. Native ecotypes
divided into nonmycotrophic, facultatively mycotrophic, and may or may not be better adapted to the prevailing site
obligately mycotrophic categories, forming a continuum from conditions (Azcón-Aguilar and Barea 1997). Consequently,
the least to most responsive mycorrhizal fungi (Allen and using nonnative mycorrhizae better adapted to the current
Allen 1990). Studies conducted along successional sequences environmental conditions is an important consideration. For
have demonstrated that early-seral plant species are often instance, management for native populations of mycorrhizal
nonmycorrhizal, followed by facultative and late-seral fungi may not be appropriate where exotic trees are planted on
obligate species (Janos 1980). Interestingly, many pioneer disturbed sites. Dunstan et al. (1998) noted that the first
species in the Chenopodiaceae, Brassicaceae, and attempts to establish pine plantations, especially Pinus
Amaranthaceae (Gerdemann 1968) are nonmycotrophic and radiata, in Western Australia were large-scale failures, and
persist on disturbed soils where mycorrhizal inoculum it was not until inoculation with compatible EM fungi that
densities are low (Allen and Allen 1980). For most restoration pines were successfully introduced. Nevertheless, after
projects, the goal is to establish late-seral plant species in 100 years of successive inoculation with exotic EM, the
early-seral soils. The appropriate mycorrhizal association diversity of fungi colonizing roots of pines in plantations
must be considered. Because mycorrhizae are generally remains low, attributed to the host-specific nature of some EM
adapted to local plant populations (Weinbaum et al. 1996), fungi. Another important consideration for inoculating with
using locally adapted seeds for the revegetation of a particular mycorrhizal fungi is the ability of the symbiosis to reduce the
species could facilitate mycorrhiza formation. Transplanting use of fertilizers and pesticides. The use of fungi to reduce
mycorrhizal seedlings onto disturbed sites can quickly fertilizer and pesticide application has been achieved in plant
increase AM infectivity because fungal hyphae expand into production systems for agriculture, horticulture, and recently,
open habitats and along roots, slowly spreading the ecosystem restoration (Azcón-Aguilar and Barea 1997).
association to adjacent plants (Warner and Mosse 1980). Maximum benefits will only be obtained from careful
Manipulating the spatial arrangement of transplants to selection and inoculation of compatible host-fungus –soil
concentrate resources and create resource islands may provide combinations (Azcón-Aguilar and Barea 1997).
greater benefits than less intensive treatments over a large
area (Allen 1988; Allen and MacMahon 1985). These
resource islands can provide seed and inoculum for
surrounding areas. Replanting multiple shrubs in a clumped
8.2 Isolating and Culturing Fungi
pattern enhances mycorrhizal recovery by trapping more
wind-carried propagules between plants than would be Despite the benefits of mycorrhizal associations, application
deposited around individually spaced plants (Allen et al. has not been widely used on a commercial scale. For
1997). commercial development, large quantities of inoculum must
be produced. Since AM are obligate biotrophs, they must be
grown and maintained on living plant roots. This is often
8 USE OF MYCORRHIZAL INOCULUM problematic because of the high risk of introducing plant
pathogens and other contaminants into the culture system
8.1 When is it Appropriate? (Jarstfer and Sylvia 1997). In contrast, EM fungi may grow on
agar media in the absence of a host plant root or from the
Over the past three decades, there has been increasing interest vegetative mycelium of fruiting bodies collected directly
in using mycorrhizal inoculation in large-scale plant from the field (Molina and Palmer 1982).
production situations, including manipulating and managing The first step in culturing mycorrhizal fungi is producing
the effectiveness of plant –fungus relationships (Miller et al. stock of the individual fungal isolate on host plant roots. For
1994). Before the selection and culture of fungi begins, it AM fungi, spores or colonized root fragments from the stock
should be determined that inoculation is more appropriate as a are used to produce larger quantities of inoculum for growth
management option than manipulation of the native on soil-based or soil-free substrates (Schenck and Perez
mycorrhizal population. Identifying whether a disturbed site 1990). Although large amounts of EM fungal spores can be
would respond favorably to AM or EM inoculation is the first easily collected in the field, spores are rarely used to isolate
step. This involves knowing the limitations to plant growth or EM fungi. Instead, many inoculation programs use the EM
establishment in a particular soil, and determining whether vegetative mycelium for its effective growth and storage on
mycorrhizal fungi can alleviate those restrictions (Dodd and agar (Molina and Palmer 1982).
486 Snyder and Allen

There are several benefits to using a soil-based culture fungi in the field after transplantation if several isolates were
system. Soil-based inocula are easy to produce, highly better adapted to conditions in the nursery, rather than
infective, and can be stored for several months or years. extremes in the environment (Dodd and Thomson 1994).
Jarstfer and Sylvia (1997) outline the most basic procedure for Under most circumstances, attempts to increase mycorrhizal
isolating and culturing spores on plants in sterile soil. Host fungi in soil have involved inoculation with exotic fungal
plants propagated from seed are preferred over cuttings species (Miller et al. 1994). Exotic fungi must outcompete
because they are easily disinfected. Disinfecting fungal native mycorrhizal populations, persist on roots, and colonize
propagules prior to inoculation is also critical because other the root systems of neighboring hosts to be effective.
microorganisms may be propagated with or instead of the AM Consequently, most exotic AM and EM fungi are eventually
fungi (Jarstfer and Sylvia 1997). To avoid contamination, replaced with native mycorrhizae over time (Marx and
cultures should be isolated from nonsterile environments. Cordell 1987). However, their importance to the initial stages
Cultures are typically grown for 4 –6 months, ensuring of plant establishment is often critical.
sporulation of all genera (Sieverding 1991), and are then
stored as air-dried soil at room temperature (Dodd and
Thomson 1994). Fungal inoculum can be stored for long 8.4 Methods for Applying Inoculum
periods (.5 years) as air-dried soil; however, the viability of
individual isolates during storage remains uncertain (Jarstfer Most commercially available AM bulk inoculum is a mixture
and Sylvia 1997). The most convenient use of soil-based of spores, colonized roots, hyphae, and the substrate on which
systems has been for inoculating nursery grown plants that are pot cultures were grown. For EM fungi, many inoculation
later transplanted in the field. This type of culture system may programs have successfully used the EM vegetative
be otherwise too cumbersome for extensive use on a mycelium. The production of mycelial inoculum for large-
landscape-scale (Jarstfer and Sylvia 1997). scale inoculation programs is often costly and pure culture
Soil-free systems, like hydroponics and aeroponics, were isolates can be difficult to maintain (Marx and Kenney 1982).
developed to overcome the limitations and drawbacks In contrast, EM sporocarps contain a significant amount of
associated with soil-based systems. Culturing fungi in soil- spores that can be collected from the fruiting populations and
less media provides greater control over the physical and easily dried and stored until application. Spores from EM
chemical characteristics of the growth medium and minimizes fruiting bodies of Pisolithus, Scleroderma, and Rhizopogon
the detrimental impacts of contamination with other are most frequently used as inoculum. Spores of Laccaria,
organisms (Jarstfer and Sylvia 1995). As such, the ideal Descolea, Scleroderma and Pisolithus spp. have recently been
conditions conducive to AM development are capable of proposed as candidates for nursery inoculation programs for
being achieved. Better control of nutrients in soil-less systems eucalypti (Lu et al. 1998) and Rhizopogon spp. are commonly
can result in greater root proliferation and higher numbers of used to inoculate Doulgas-fir seedlings in commercial
spores per centimeter of colonized root length (Sharma et al. nurseries (Castellano 1994).
2000). Colonized roots and spores free of substrate allow for The amount, timing, and method of inoculation are
more efficient production and distribution of inocula (Jarstfer important factors in properly managing the establishment of
and Sylvia 1997). Consequently, soil-free systems produce mycorrhizae. Little is known about appropriate application
greater propagule densities than soil-based pot cultures of rates. Estimates of 1 – 2 kg of bulk soil inoculum
the same age (Jarstfer and Sylvia 1995). Aeroponic culture in (5000–10,000 propagules) have been used for AM inocu-
particular allows for easy extraction of AM fungal lations (Lovato et al. 1995) and spore suspensions at densities
propagules, and mycorrhizal roots can be sheared to produce of 106 –107 spores per ml have been used for EM inoculations
high-density inoculum that is both efficient and easy to handle (Lu et al. 1998). The earlier plants are inoculated with fungi,
(Jarstfer and Sylvia 1995). Commercial nurseries currently the greater the benefits are to those plants. Inoculation is
use aeroponic culture systems for revegetation programs typically applied in either by broadcasting at the soil surface
where on-site production of inocula allows for the use of fresh or by banding inoculum at the root zone. Broadcasting
mycorrhizal propagules at optimal times (Jarstfer and Sylvia requires more inoculum whereas banding concentrates
1995). inoculum near the area of developing roots. Transplanting
mycorrhizal seedlings can also be used to inoculate other
plant roots. Fungal hyphae expand into open habitats and
8.3 Inoculum Diversity along roots, slowly spreading the association to adjacent
plants (Warner and Mosse 1980).
Inoculating with several AM or EM fungal isolates may be
appropriate for reestablishing a range of mycotrophic plant
species on a disturbed site, especially if plant diversity was 9 MYCORRHIZAE AND BIOREMEDIATION
high, prior to disturbance (Dodd and Thomson 1994).
Whether fungal inocula are native, exotic, or both depends In the mining process, not only plants are removed, but also
largely on the host plant and environmental conditions. Mixed upon soil replacement changes in texture and deposits of salts
mycorrhizal inocula may ensure the persistence of inoculant and heavy metals often result (Allen 1989). The toxicity of
Restoration of Mycorrhizae in Disturbed Arid Ecosystems 487

metals not only depends on their concentration in the soil, but reducing metal concentrations in plant tissues. (Colpaert and
also their availability and transfer to plants. By providing a Van Assche 1992;1993; Dixon and Buschena 1988).
direct link between soil and plants, mycorrhizal function Ectomycorrhizal fungi can differ in their ability to reduce
could be of great importance in heavy metal polluted soils translocation from root to shoot, so the presence of the
(Leyval et al. 1997). As such, there is increasing interest in the appropriate EM fungi may be critical (Denny and Wilkins
potential for mycorrhizal fungi to be used as bioremediation 1987). The role of AM fungi in heavy metal uptake is harder
agents or as bioindicators of heavy metal pollution. to elucidate because of the obligate nature of the fungal
It is generally believed that pollution inhibits the formation symbiont. Regardless of their ability to grow in polluted soils,
of mycorrhizal associations. However, little is known about the extent to which AM fungi confer metal tolerance in their
the viability and activity of AM and EM fungi in soils host plants, or accumulate heavy metals in roots preventing
with different heavy metal concentrations. Rühling and translocation to shoots is not fully understood (Leyval et al.
Söderström (1990) reported that the number of fruiting bodies 1997). Species of both AM and EM fungi differ in hyphal
and species decreased with increasing pollution along a heavy productivity and in their ability to take up and transfer metals.
metal pollution gradient in Sweden. Isolates of P. tinctorius The turnover of fungal tissue could be an important factor in
collected from old mining sites expressed increased the ability of mycorrhizal fungi to protect host plants against
aluminum tolerance and high mycelial growth when prolonged elevated metal concentrations (Colpaert and Van
compared to isolates from rehabilitated and forested sites Assche 1993).
(Egerton-Warburton and Griffin 1995). For AM fungi, there is
evidence to suggesting that at least some AM fungi are
relatively resistant to high metal concentration. Gildon and
Tinker (1981) reported that plant roots growing naturally on 10 MYCORRHIZAE AND BIOTECHNOLOGY
zinc- and cadmium-contaminated soils had significant AM
colonization. Davies et al. (2001) reported high rates of AM Despite the promise (Wood and Cummings 1992; Podila and
colonization even at the most toxic levels of chromium in Douds 2000), there has been remarkably little real break-
soils. And Rao and Tak (2001) found a significant through in using mycorrhizae for applied activities, beyond
improvement in root colonization and spore density of AM the early work of inoculation for nutrient enhancement.
fungi isolated from gypsum mine spoils when used to Technologies for inoculation are available (Brundrett et al.
inoculate five tree species growing in gypsum mine soils. 1996), but selection for desirable characteristics and matching
The mechanism that confers heavy metal tolerance in plant and fungal genotypes may prove to be crucial. In many
mycorrhizal fungi is largely unknown. The survival of AM cases, if not most, agricultural technologies actually select for
and EM fungi in polluted soil may depend heavily on the populations of fungi that may be detrimental for the crops
density of the external hyphae. The absorption of heavy grown (Johnson et al. 1997). Many of the EM and virtually all
metals to the hyphal surface could reduce soil concentrations of AM fungi are generalists, with the ability to invade a wide
and thus accumulation of fungal and plant tissue (Denny and range of host plants, but with highly variable response
Wilkins 1987; Marschner and Dell 1994). Components of the variables (Johnson et al. 1997). Bever et al. (2001) have
fungal cell wall, such as chitin and melanin, can bind heavy shown how less-than-optimal taxa can persist in a population
metals to the extraradical mycelium (Denny and Wilkins and even come to dominate.
1987; Tam 1995). Turnau et al. (1996) found that the EM Large investments are being made in developing trans-
fungal mantle contained the highest levels of heavy metals genic crops. However, no efforts are underway to even
while the Hartig net contained the lowest levels. Glomalin, determine if compatibilities or response matches are even
the glycoprotein that coats AM fungal hyphae, could play an important! Kaldorf et al. (2001) have shown that genetically-
equally important role in protecting AM fungi and host plants modified hybrid aspen showed no differences from non-
from toxic metal concentrations in soils, although this has not transgenic races in AM or EM infection, and only marginal
yet been investigated. Other possible mechanisms conferring changes in the fungal community composition were observed.
heavy metal tolerance in fungi may include intracellular We have also found that transgenic corn had no effect on the
chelation (Martin et al. 1994) and the sequestration of metals species composition or AM infection (Snyder and Allen,
within mycorrhizal sheaths (Egerton-Warburton et al. 1993). unpublished data). Hiremath and Podila (2000) reviewed
The stability of metal tolerance in both AM and EM fungi efforts that demonstrate that genetic transformations of
remains to be examined (Leyval et al. 1997). Despite these mycorrhizal fungi are possible. Certainly, genes are rapidly
uncertainties, isolation of tolerant fungal ecotypes that occur being identified and studied (Maldonado-Mendoza et al.
on polluted soils could have important application to the 2001) and genes can be added to individual fungi. However,
revegetation or inoculation of barren polluted sites (Gildon to our knowledge, no transgenic mycorrhizal fungi have been
and Tinker 1981; Rao and Tak 2001). successfully tested and no effects of differing fungi on host
The benefit of heavy metal tolerance in mycorrhizal fungi responses, either positive or negative, have been evaluated.
could have direct effects on host plant response to metal This remains an important area of study. Experiences with
concentrations in soil (Meharg and Cairney 2000). Several fungal virus’ have shown that extranuclear genetic material
studies have reported the beneficial role of EM associations in can dramatically affect fungal-plant interactions (Roane et al.
488 Snyder and Allen

1986) and mycorrhizal fungi are no different from others in agronomic and horticultural enterprises that developed
this trait (Douhan et al. 2003). practices that reduced or eliminated mycorrhizal benefits in
an oversimplified view of the dynamics of soil systems.
Mycorrhizal fungi are being studied at the molecular to
11 CONCLUSION biochemical level. However, tapping the vast diversity of
fungi and alternative mechanisms for increasing yield
Mycorrhizal fungi are virtually ubiquitous in their evolution- efficiencies, decreasing pest loss, or rendering polluted
ary associations with plants. All trees used for fuel, fiber, and landscapes usable has not been studied or extensively utilized.
food form mycorrhizae with a diverse array of fungi. All More applied research, especially in developing areas of the
crops, excepting a small number of annuals, and all world without the financial resources to purchase energy,
horticultural plants that decorate our homes and gardens pesticides, or fertilizers, would pay large dividends.
form mycorrhizae. Known benefits range from soil stabili-
za?show $132#>tion, to fertilizer and pesticide reduction, to
enhanced tolerance of pollutants. These fungi also sequester
toxins and could be extremely important in land rehabilitation ACKNOWLEDGEMENT
as well as restoration of lands for grazing or conservation.
Disturbance to plant communities and soil systems can This material is based upon work supported by the National
reduce the density, infectivity, and function of mycorrhizae. Science Foundation under Grant No. DEB-9981548.
Without the appropriate fungi present, the natural
re-establishment of this beneficial symbiosis may take
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Index

Absidia [Alternaria]
A. atrospora, 433 A. kikuchiana, 124, 135
A. cylindrospora, 448 A. longipes, 326
Acacia, 111 A. macrospora, 116
Acaulospora, 104 A. mali, 126
A. laevis, 184 A. solani, 153
Acetobacter, 224, 226 Amanita, 482
A. xylimum, 226 A. boletus, 482
Acid-fermented bread, 223 A. muscaria, 450
Acremonium, 304, 445 AM biofertiliser, 199
A. fusidioides, 433 AMF-mediated plant disease control, 183
Actinomadura roseola, 127 Amorphotheca resinae, 443
Actinomucor, 228 Ampelomyces, 162
A. elegans, 228 A. quisqualis, 164, 166
Aegilops tauschii, 3 Amplification-based methods, 300
Aflatoxin biosynthesis, 348 Amylomyces, 226
Aflatoxin-resistance screening tools, 70 Animal-parasitic nematodes, 205
Aflatoxins, 303, 337, 343 Anisoplia austriaca, 79
African fufu, 228 A. couloni, 80
African Kaffir beer, 225 Anricularia spp., 243
Agar plug-TLC method, 31 Antagonism, 106
Agaricus, 220, 234, 237 Antagonist selection, 174
A. bisporus, 44, 233, 237 –240, 401 Antibiosis, 151, 188
A. brunnescens, 41 Antifungal food activities, 291
A. campestris, 433 Antifungal food additives, 291
Agrobacterium rhizogenes, 197 antibiotics, 293
Alcoholic food and beverages, 225 natamycin, 293
Alcohols, 284 nisin, 293
Alpova diplophloeus, 379 metabolites from lactic acid bacteria, 293
Alterations in pathogen biomass, 186 diacetyl, 294
Alternaria, 19, 21, 52, 53, 57, 303, 304, 314, 330, 335, 336, 353 microgard, 294
A. alternata, 5, 153, 163, 186, 319, 335, 336, 353 reuterin, 294
A. brassicae, 8 organic acids, 291
A. cassiae, 116 – 118 medium-chain fatty acids, 293
A. caulina, 117 propionic acid, 293
A. cirsinoxia, 117 senzoic acid, 292
A. destruens, 112 sorbic acid, 292
A. eichhorniae, 117 Antifungal leads, 125

493
494 Index

Antifungal proteins, 8 [Aspergillus]

Anti-oxidants to control quenching, use of, 476 A. fonsecaeus, 329
Aphanomyces euteiches, 184 A. fumigatus, 91, 137, 304, 305, 314, 317, 326, 334, 444 – 446,
Application of individual markers, 61 450
Application of isozyme analysis, 41 A. fusidioides, 435, 436
Arabidopsis thaliana, 4 A. giganteus, 8
Arbuscular mycorrhiza, 104 A. glauca, 228
biofertilizer, 195 A. glaucus, 229
fungi, 183 A. japonicus, 91
Arbuscular mycorrhizal fungi in plant disease control, 183 A. muscaria, 474
challenges and strategies in enhancing efficacy, 188 A. nidulans, 85, 91, 303, 317, 346 – 349, 421
enhanced AMF biodiversity, 189 A. niger, 24, 220, 227, 283, 284, 293, 315, 316, 320, 329, 411,
improved understanding of microbial ecology, 189 413, 423, 432, 433, 436, 437, 445 –449
examples of AMF-mediated plant disease control, 183 A. nominus, 328
phytopathogenic fungi, 183 A. nomius, 303, 343
plant pathogenic bacteria, 184 A. ochraceoroseus, 328, 343
phytopathogenic viruses, 185 A. ochraceus, 24, 69, 316 – 318, 326, 329
modes of mycorrhizae-mediated disease control, 185 A. oryzae, 91, 225 – 228, 253, 286, 303, 304, 334, 432,
host nutritional effects, 185 435, 437
competition, 186 A. parasiticus, 50, 69, 294, 303, 304, 326, 328, 345 – 350
physiological and biochemical alterations of the host, A. phoenicis, 433, 436
186 –188 A. praecox, 474
Archaeospora, 104 A. pseudotamarii, 328
Argyreia nervosa, 91 A. rubrum, 326
Armillaria, 376, 383 A. shiro-usamii, 220
Armillariella mellea, 445, 446 A. soyae, 227, 303, 304, 348, 463, 464
Arthrinium sacchari, 334 A. tamarii, 91, 343
Arthrobotrys, 211 A. tenuis, 320
A. oligospora, 206 –211 A. terreus, 334, 444, 474
Ascochyta, 59, 62 A. usami mut. shirousami, 433
A. fabae, 62 A. usamii, 220
A. fabae f. sp. lentis, 62 A. versicolor, 91, 275, 303, 316, 326
A. pinodes, 62 A. wentii, 334
A. pisi, 62 Aureobasidium, 162, 163, 433, 439
A. pteridis, 117 A. atrospora, 439
A. rabiei, 62 A. pullulans, 163, 164, 175
Aspergillis nidulans, 345, 346 Auricularia, 233, 234, 239, 243
A. parasiticus, 334 A. auricula, 243
A. tamarii, 334 A. fuscosuccinea, 243
A. versicolor, 334 A. polytricha, 243
Aspergilloides, 21 Aurobasicium, 381
Aspergillus, 21, 34, 51, 61, 72, 73, 84, 127, 224, 226, 227, 293,
294, 303, 305, 314 –316, 318, 320, 326, 329, 333, 334, Baccharis megapotamica, 332
432, 433, 444 Bacillus, 175, 198, 227, 228
A. aestivalis, 474 B. mesntericus, 293
A. awamori, 433, 437 B. natto, 228
A. awamori var. kawachi, 436 B. pumilis, 227
A. bombycis, 303 B. subtilis, 127, 174, 177, 227
A. campestris, 436 B. thuringiensis, 69, 74
A. candidus, 314 B. thuringiensis israelensis, 81
A. carbonarius, 24, 411 Balansia
A. cellulosae, 282, 444, 445 B. claviceps, 91
A. chevallieri, 326 B. epichloe, 91
A. citricus, 329 B. obtecta, 91
A. clavatus, 304, 326 B. strangulans, 91
A. cylindrospora, 449 Beauveria, 81, 82, 84, 87
A. dauci, 8 B. bassiana, 79– 86, 445
A. flavus, 8, 50, 69, 70 –74, 91, 225, 227, 294, 295, 303, 317, 320, B. brongniartii, 80, 83, 86
326, 328, 334, 343, 346, 347 – 350 Beltraniella portoricensis, 305
A. foetidus, 463, 464 Bemisia tabaci, 82
Index 495

Betula, 379 [Biological control of fungal diseases on vegetable crops]

B. pendula, 382 gray mold, 162 – 164
B. platyphylla, 105 leaf and stem blights, 164
Bioactives from entomopathogenic fungi, 84 powdery mildews, 164
Biochemical alterations of the host, 186 rot and crown rots, Trichoderma as agent, 158
Biocontrol biotechnological manipulations, 160, 162
of foliar-infecting fungi, 162 biotechnological manipulations of Trichoderma, 160
of gray mold, 162 Gliocladium as a biocontrol agent, 161
of leaf and stem blights, 164 mycoparasites as biocontrol agents, 162
manipulations of Trichoderma, 160 nonpahtogenic fungi as biocontrol agents, 161
of powdery mildews, 164 Trichoderma biocontrol activity, 160
of root and crown rots, 158 Trichoderma mechanism of action, 160
of seed rots, 158 Trichoderma species identification, 158
-specific gene expression, 139 Biological control of plant-parasitic nematodes, 211
Bioconversion of distillery waste, 431 Biomineralization of heavy metals, 409
fungi for bioconversion of distillery waste, 432 fungal-mediated heavy metal removal from aqueous solutions,
filamentous fungi, 432 410
mixed cultures, 433 biosorption, 410
yeasts, 432 commercial applications, 413
Bioconversion process, 433 extracellular precipitation of metals, 413
Biodegradation of azo dyes, 457, 461 heavy metal biosorption, 411
Biodegradation of azo dyes by fungi, 457 metal biosorption by specialist isolated molecules, 413
Biodegradation of lignocellulose, by white rot fungi, 393 special case for uranium, 413
white rot fungi, 394 Biopesticides based on fungi, 79, 81
metabolic studies, 395 Biopulping, 402
screening for lignin degradation, 395 Bioreactor design, 414
ligninolytic system, 395 Bioreactors, 476
lignin peroxidase, 397 Biosorption, 410
manganese peroxidase, 399 Biosynthetic pathways of aflatoxins, 346
laccase, 399 Biosynthetic pathways of Trichothecenes, 352
properties, 399 Biosystematics, 20
versatile peroxidase, role of LAC in lignin degradation, 399 Biotechnological approaches in plant protection, 1
cellulose dehydrogenes, 401 antifungal proteins and peptides, 8
H2O2 generating enzymes, 400 candidate gene validation, 4
reactive oxygen species, 401 defense pathways, 7
Biodegradation of nitrate esters by fungi, 475 differential cDNA-AFLP screens, 3
Biodegradation of RDX/HMX by fungi, 475 from genomes sequence to gene to mutant to function, 9
Biodegradation of TNT by fungi, 472 identification of QTL-associated genes, 4
Biofungicides, 123 integration of molecular biology with classical breeding, 5
microbial metabolites as antifungal leads, 124 marker-assisted breeding, 2
recent success in fungicide development, 125 microarrays, 9
screening of potential leads, 125 molecular maps, 2
new resource of microbial metaboites, 126 new initiatives and prospects, 9
other microorganisms, 127 phytotoxin detoxification, 8
Streptomyces, 125 plant model systems, 10
potential targets for discovery of antifungal leads, 127 proteomics, 10
acetyl-CoA carboxylase, 128 random mutagenesis with transposons, 9
cell-wall biosynthesis, 128 strategies, 2
nucleic acid metabolism, 129 transgenic plants as a tool for plant protection, 5
protein biosynthesis, 129 transient expression of fungal genes in plants, 9
sterol biosynthesis, 128 Biotechnological potential of ergot alkaloids, 91
Biogenesis of ergot alkaloids, 92 industrial production, 93
Bioherbicides, 111, 114 saprophytic cultivation of Claviceps, 93
Biological control, 112 fermentation, 95
Trichoderma, 147 history, 93
host recognition, 148 long-term preservation, 94
host invasion, 148 solid substrate fermentation, 96
Biological control of damping-off diseases, 158 stationary surface cultivation, 95
Biological control of fungal diseases on vegetable crops, 157 strain improvement, 94
foliar-infecting fungi, 162 submerged cultivation, 95
496 Index

Biotechnological techniques applied to yeasts, 164 [Candida]

Biotechnology of entomopathogenic fungi, 79 C. spherica, 275
biopesticide potential, 79, 81 C. steatolytica, 434
novel strategies for biospesticide use, 83 C. stercorus, 474
molecular genetics, 84 C. topicalis, 228, 284, 434, 444, 445
strain improvement, 85 C. utilis, 247, 253, 293, 434, 435, 437 – 439, 444
transformation systems, 85 C. versalitilis, 272
Bipolaris, 333, 345 C. versicolour, 474
B. sorokiniana, 7, 185 C. vortiovaarai, 228
Biverticillium, 21 Caprinus micaceus, 286
Bjerkandera adusta, 382, 397, 448, 449, 462, 465, 467, 473 Carbohydrate degradation, 320
Blastomyces dermatitidis, 300 Carbon allocation, 482
Blumeria graminis f. sp. hordei, 7 Casuarina, 198
Boletus edulis, 286 Cellobiose dehyrogenase, 400
B. indecisus, 433 Cellulose degradation by fungi, 363, 366
Bombyx mori, 85 biomass as a sugar source, 363
Botryosphaeria dothidea, 129 cultivation conditions and nutrient requirements, 364
Botrytis, 52 fungal strains, 364
B. cinerea, 125 – 127, 140, 149, 153, 157, 162, 163, 174, 175, 284 degradation of complex molecules, 366
Bouteloua gracilis, 105 cellulose degredation, 366
Bradyrhizobium, 198 mixed cultures and fermentation, 369
Brettanomyces, 260 process description, 368
Brevibacterium, 273 saccharification and fermentation, 368
Brown rot and soft rot fungi, 393 economic aspects of ethanol production, 369
Button mushroom, 237 costs and benefits of ethanol production, 370
Byssochlamys, 295, 304 ethanol conversion technologies, 369
B. nivea, 334 Cell-wall biosynthesis, 128
Cenococcum graniforme, 106
Caenorhabditis elegans, 210, 211 Cephalosporium, 224, 332, 351
Cambodian Phaak, 229 Ceratocystis, 394
Candida, 127, 129, 176, 224 – 226, 228, 229, 260, 269, 273, 433, C. fimbriata, 285
439, 440, 444 Cercospora, 224
C. acetoacidophilum, 437 C. cucumerinum, 127
C. albicus, 284 C. destructans, 127
C. apicola, 445 C. kikuchi, 127
C. arborea, 434 Cereal-based fermented food, 228
C. brumptii, 434 Ceriporiopsis subvermispora, 394, 399
C. bututyri, 275 Chadosporium, 228
C. catenulata, 270, 271, 276 Chaetomium, 333, 345, 383, 394
C. diffluens, 271 C. globosum, 163
C. dusenii, 474 Challenges and strategies for development of mycoherbicides, 111
C. elegans, 448, 449 biological control, 112
C. famata, 175, 229, 269, 271 biological factors, 112
C. gallica, 448 – 450 environmental factors, 113
C. geotrichum, 271 technological factors, 113
C. guilliermondii, 224 – 226, 228, 271 approaches for overcoming constraints, 114
C. intermedia, 224 application technology, 117
C. kefyr, 225, 228, 271, 272, 276 bioherbicide agents, 114
C. kefyri, 272 formulation, 116
C. krusei, 224, 228, 433 herbicide synergy, 117
C. lipolytica, 253, 271, 275, 445 mass production, 115
C. maltosa, 253, 444, 445 Cheeses, 225, 273
C. membranaefaciens, 434 Chemical fertilizer usage, 199
C. nebularis, 474 Chemical identification of fungi, 19
C. obtuse, 226 mycology and biosystematics, 20
C. odora, 474 classical identification, 20
C. oleophila, 164, 175, 177 cultivation and media for metabolite profiling, 20
C. rugosa, 434 techniques for metabolite profiling, 21
C. saitoana, 164, 175 – 177 agar plug-TLC method, 31
C. sake, 176, 275 atmospheric pressure ionization MS, 27
Index 497

[Chemical identification of fungi] Commercial importance of flavours, 286

direct infusion MS, 29 Commercial wine yeast, 262
extraction, 22 Commercialization of AM biofertilizer, 195
gas chromatography and GC-MS, 24 AM biofertilizers, 198
HPLC, 23 interaction of natural biofertilizers, 198
identification of cereal borne Penicillia by TLC, 33 major constraints and solutions in commercialization, 199
ochratoxin A determination in Aspergillus niger, 24 list of producers and formulators, 200
volatile metabolites for identification of Penicillia, 26 mycorrhizal commercialization techniques, 200
Chemometrics, 34 mycorrhizal associations, 196
Chenopodium album, 117 aeroponic culture techniques, 197
Chinese Chee-Fan, 228 pot-based techniques, 196
Chinese Jiang, 227 root organ culture technique, 197
Chinese Lao-Chao, 226 Commercialization of BCPD, 177
Chinese Meitauza, 228 Competition, 152, 186
Chinese Minchin, 228 Compost preparation, 237
Chinese red rice (Anka), 228 Conidiobolus coronatus, 87
Chinese spirits, 226 Coniothyrium, 158
Chinese Sufu (Tau-hu-yi), 228 C. minitans, 162
Chlamydosporia, 207 Control of fruit diseases, 173
Chlorosplenium aeruginascens, 376 Control of nematodes by fungi, 205
Chondrostereum purpureum, 111, 382 biological control of plant-parasitic nematodes, 211
Chrysosporium, 304 fungal biocontrol delivery systems, 212
Citrobacter, 127 sources of antagonists to nematodes, 212
Cladesporium fulvum, 3, 7 habitat of nematophagous fungi, 207
Cladophialophora, 445 – 447 fungi, 207
Cladosporium, 127, 326, 381 nematodes, 207
C. cucumerinum, 127 roots, 208
C. cladosporioides, 163 soil, 207
C. resinae, 445, 475 mode of action of nematophagous fungi, 208
C. sphaerospermum, 446, 447 fungus-nematodes interaction, 208, 210, 211
Classical breeding, 5 nematodes, 205
Classical identification, 20 animal-parasitic nematodes, 205
Clavaria, 381 plant-parasitic nematodes, 205
Claviceps, 52, 91, 93– 96, 304 Control of postharvest diseases, 173
C. africana, 221 Cordyceps, 79, 84
C. fusiformis, 92, 93, 96 Coriolopsis gallica, 448
C. laeve, 112 Coriolus hirsutus, 424
C. paspali, 93, 95, 304, 335 C. versicolor, 382, 419, 421, 424, 433, 465
C. purpurea, 91– 96, 221, 304, 326 Corticium caeruleum, 91
C. purpureum, 112 Cortinarius, 482
Cleonus punctiventris, 79 Corynebacterium, 224, 273
Clostridium ljungdahlii, 368 Corynespora cassiicola, 281, 446
C. thermocellum, 369 Criconemella, 212
C. thermosaccharolyticum, 369 Crinipellis, 450
C. tyrobutyricum, 275 Cryphonectria parasitica, 448
Clustering of trichothecene biosynthetic pathway genes, 351 Cryptococcus, 127, 162, 271
Cochliobolus sativus, 105 C. albidis, 163
Coelomomyces, 84 C. laurentii, 175
Colletotrichum, 52, 61, 62, 112, 113, 164, 175 C. neoformans, 300
C. cocodes, 176, 177 Culex quinquefasciatus, 81
C. cucumerinum, 126 Cultivation of Claviceps, 93
C. destructivum, 8, 73 Cunninghamella, 444
C. gloeosporioides, 126, 176 C. blakesleeana, 91, 444, 445
C. gloeosporioides f. sp. aeschynomene, 112, 115 C. echinulata var. elegans, 475
C. graminicola, 107 C. elegans, 444, 450
C. magna, 164 Curvularia, 464
C. orbiculare, 27, 105 –107, 117 C. inaequalis, 463, 464
C. truncatum, 115, 116, 117 Cyathus pallides, 475
C. coccodes, 118 Cylindrocarpon destructans, 127
Commercial AM inoculum, 200 Cylindrocladium, 185
498 Index

Dacrymyces hansenii, 275 Elimination of mycotoxins, 336

D. stillatus, 383 Ellipsoideus, 226
Dactylella oviparasitica, 212 Endogone, 482
Dairy flavors, 286 Endomycopsis, 225, 226
Dairy industry, 269 E. fibuliger, 229
Daldinia, 378 Endophytes, 105, 378
D. concentrica, 382 Enhanced AMF-biodiversity, 189
Daucus carrota, 197 Entomopathogenic fungi, 79
Dearyomyces vanrijiae, 448 Entomophaga maimaiga, 83
Debaryomyces, 260, 269 Enzyme activities of non-Saccharomyces wine yeasts, 258
D. hansenii, 223, 228, 229, 270 – 275 Enzymology of azo dye degradation, 464
Decoloration of industrial wastes, 419, 422 Epichloe, 51, 92, 305
degradation of dye water and, 419 E. typhina, 51, 91
Decolorization Epidemiology of plant pathogenic fungi, 57
biodegradation of azo dyes and, 457, 463 Ergoline, 92
of Chicago sky blue, 422 Ergopeptines, 93
of dye in bioreactor, 424, 425 Erwinia ananas, 224
of dyes by microorganisms, 420 Erynia neoaphidis, 83
ligninolysis and decolorization by enzymes, 420 –424 Erysiphe, 157
reactor, in the, 422 E. cichoracearum, 184
of remazol brilliant blue, 424 E. graminnis f. sp. hordei, 128
purified enzyme, using, 424 E. heraclei, 8
Decolorizing organisms, 422 E. polygoni, 7
Defense pathways, 7 Escherichia coli, 127, 276
Degradation of: Essential oils, 294
complex molecules, 366 Ethiopian Enjera, 224
dye water, 419 Ethiopian Tej, 225
grain, 320 Eupenicillium javanicum, 101
hydrocarbons by yeasts and fungi, 443 Eurotium, 314, 318
Degradation pathways, origins and evolution of, 472 E. amstelodami, 316, 319, 320
Dekkera E. repens, 316
D. anomala, 275 Eutypa spinosa, 378
D. bruxellensis, 275 Evolution of wine yeast strains, 263
Delbrueckii, 303 Exidia glandulosa, 382
Deoxynivalenol, 333 Exophiala, 446
Dermolepida albohirtum, 81 E. jeanselmei, 447
Deuteromycota, 394 E. spinifera, 74
Diacetyl, 294 Explosives as substrates for microbial degradation, 471
Dichomitus squalens, 450 Expression of biocontrol genes, 140
Didymella bryoniae, 126, 127, 164 Extracellular precipitation of metals, 413
Differential cDNA-AFLP screens, 3
Dilophospora alopecuri, 44 Factors affecting mycotoxin production, 325
Dioides Fermentation technology and downstream processing, 220
D. immitis, 300 Fermented fish products, 224, 229
D. gossypina, 281, 446 Fermented food, 223
Direct infusion MS, 29 Fermented foods and beverages, 220
Discovery of antifungal leads, 127 Fermented meat products, 229
Distillery waste, 431 Fermented soybean pastes, 227
Disturbed arid ecosystems, 481 Fingerprinting methods, 59
Drechmeria coniospora, 206, 209, 211 Fistulina hepatica, 376
Dyes decolorization, 420 Flammulina, 233, 234
F. velutipes, 233
Ecology of mycotoxin production, 315 Flavoring compounds, 286
Economic aspects of ethanol production, 369 Flavors and aromas, 281
Economic impact of mycotoxin contamenation, 326 production of microbial flavors, 281
Ectomycorrhizae, 104 complex flavors, 285
Edible fungi, 233 dairy flavors, 286
Edible fungi and recycling of wastes residues, 244 de novo synthesis, 285
Egyptian Kishk, 224 mushroom flavors, 286
Eleusine coracana, 4 terpenoids, 281, 282
Index 499

Food activities, 291 [Fungi in food technology]

Food biotechnology-products and processes, 219 fungi and fermented food, 223
Food technology, 218 organic ingredients production, 220
Foodborne fungal pathogens, 221 Fungi in forest ecosystems, 375
Food-use enzyme production, 219 Fungi in seed deterioration, 311
Frankia, 198 Fungicide development, 125
Fraxinius, 378, 381 Fungi-root-nematode interactions, 211
F. excelsior, 380, 382 Fungus-nematode interactions, 208
Fruit-based alcoholic beverages, 226 Fusarium, 19, 21, 51– 53, 59, 62, 69, 104, 160, 161, 224, 300,
Fumonisins, 330, 353 303 – 305, 314 – 316, 321, 326, 332, 364, 381
Funalia trogii, 433, 463 F. acuminatum, 304, 332
Fungal associations and decay, 377 F. annulatum, 330
Fungal biocontrol delivery systems, 212 F. anthophilum, 330, 353
Fungal biotechnology in food production, 218 F. avenaceum, 303
Fungal degradation of explosives, 471 F. beomiforme, 330
explosives as substrates for microbial degradation, 471 F. crookwellense, 333
2,4,6-trinitrotoluence, 471 F. culmorum, 304, 314, 317, 333
nitrate esters and methylene nitramine cyclic esters, 472 F. dlamini, 353
origins and evolution of degradation pathways, 472 F. equiseti, 106
fungi in bioremediation in the field, 476 F. flocciferum, 411
bioreactors, 476 F. formentarius, 474
land farming and soil slurry reactors, 476 F. graminearum, 8, 72– 74, 247, 304, 305, 333, 335, 351 –353
soil piles and windrows, 476 F. moniliforme, 8, 105, 126, 284, 305, 353, 433, 447
future progress, 476 F. nivale, 326
bioremediation requirements, 477 F. nupiforme, 330
screening for new fungal species strains, 477 F. nygamai, 330
use of anti-oxidants to control quenching, 476 F. oxysporum f. sp. cucumeris, 105
use of photocatalysis, 476 F. oxysporum f. sp. cucumerinum, 105
use of sorption strategies, 477 F. oxysporum f. sp. melonis, 105
Fungal diversity and conservation, 384 F. oxysporum sp. f. pisi, 61
Fungal habit, 376 F. oxysporum f. sp. cucumerinum, 106
Fungal mediated heavy metal removal, 410 F. oxysporum, 58, 61, 126, 137, 160 –162, 184, 364, 365
Fungal taxonomy, 37 F. poae, 285, 304, 332, 333
Fungal volatiles, 320 F. proliferatum, 316 – 318, 320, 330, 353
Fungi and yeasts as biocontrol agents, 163, 164 F. roseum, 102, 103
Fungi as disease suppressor, 101, 105 F. sambucinum, 332
endophyte, 106 F. solani, 8, 161, 304, 320, 475
mycorrhizae, 105 F. sporotrichioides, 74, 304, 326, 332, 351 –353
PGPF, 105 F. subglutinans, 330, 335
Fungi as plant growth promoter, 101 F. succisae, 330
Fungi as plant growth promoter and disease suppressor, 101 F. sulphureum, 332
other PGPF, 103 F. syncephalastum, 228
mycelial fungi, 103 F. tricinctum, 326, 332
Rhizoctonia, 103 F. trogii, 439, 250, 251
Trichoderma, 101 F. verticilliodes, 69, 71, 305, 315 – 318, 320, 326, 330, 331, 335,
mechanisms of plant growth promotion by PGPF, 103 353, 447
hormone production, 103 mycotoxins produced by, 335
mineralization, 103
suppression of deleterious microorganisms, 104 Gaeumannomyces, 52
mycorrhiza, 104 G. graminis var. tritici, 105
arbuscular mycorrhiza, 104 Galactomyces geotrichum, 269, 270, 273
ectomycorrhizae, 104 Galleria, 84
endophyte, 105 Ganoderma, 64, 383, 461, 477
Fungi for bioconversion of distillery waste, 432 G. applanatum, 394
Fungi in bioremediation in the field, use of, 476 G. boninense, 64
Fungi in food technology, 217 G. boninense, 61, 62, 64
food biotechnology: products and processes, 219 G. lucidum, 233, 235
enzyme production, 219 Gas chromatography, 24
fermentation technology and downstream processing, 220 Gases and modified atmospheres, 295
fermented foods and beverages, 220 Gene to mutant, 9
500 Index

Gene validation, 4 Glomus, 186

Genetic variability of yeast, 257 G. entrophospora, 104
Genetic variability of yeast in wine fermentation, 257 G. fasciculatum, 105, 184, 185, 197
enzyme activities of non-Saccharomyces, 258 G. intraradices, 184, 197
inter-and intra-specific variability, 258 G. intraradix, 187
structural and functional wine yeast genomics, 263 G. intrardices, 187
adaptive evolution, 263 G. macrocarpum, 185, 197
genomics characteristics, 263, 264 G. mosseae, 106, 107, 184 – 186, 188
yeast biodiversity in wineries, 257 G. proliferum, 185
yeast species diversity during vinification, 257 G. versiforme, 10, 187
characterization of commercial wine yeast strains, 262 Gongronella
future methods, 262 G. butleri, 433, 439
methods for yeast characterization, 260 G. candidum, 433, 435 – 439
methods for yeast identification, 258 G. crusei, 437
RELP of RNA, 259 G. deliquescens, 436, 438
ribosomal RNA gene sequencing, 258 G. lacrispora, 445
yeast population dynamics, 262 Grifola, 234
Genetics and biochemistry of mycotoxin synthesis, 343 G. frondosa, 233
aflatoxins and sterigmatocystins, 343
occurrence and toxicology, 343 Habitat of nematophagous fungi, 207
biosynthetic pathways of aflatoxins, 346 Haematonectria haematococca, 141, 142
clustering of aflatoxin biosynthetic pathway genes, 345 Hanseniaspora uvarum, 257, 262
factors affecting aflatoxin/ST biosynthesis, 348, 349 Hansenula, 227, 432, 434, 438
fumonisins, 353 H. anomala, 223, 225, 228, 229, 433, 437, 438
genetics and biosynthesis of fumonisins, 354 Hawaiian poi, 229
occurrence and toxicology, 353 Heavy metal biosorption, 411
trichothecenes, 351 Heavy metals, 409
biosynthetic pathways of trichothecene, 352 Hebeloma crustuliniforme, 104, 106
clustering of trichothecene biosynthetic pathway genes, 351 Helicobasidium mompa, 186
occurrence and toxicology, 351 Herbicide synergy, 117
regulation of trichothecene biosynthesis, 352 Heterobasidion annosum, 394
Genetics and biosynthesis of fumonisins, 354 Heteroconium chaetosprira, 106
Genome organization, 137 Heterodera, 205
Genome sequence, 9 H. avenae, 212
Genomic characteristics, 263 Hirsutella, 84
Geotrichum, 295 H. rhossiliensis, 212
G. candidum, 225, 228, 229, 263, 275, 283, 295, 433, 475 H. thompsonii, 84
G. geotrichum, 274 – 276 Histoplasma capsulatum, 300
G. separium, 474 Hoplochelus marginalis, 86
Gerlachia nivalis, 129 Hormoconis resinae, 443
Ghanian kenkey, 224 Hormodendrum, 224
Gibberella, 52 Hormonema, 446
G. fuijikuroi, 305, 330, 354 Host invasion, 148
G. zeae, 304, 351, 352 Host nutritional effects, 185
Gigaspora, 104 Host recognition, 148
G. caledonium, 197 Humicola lanuginose, 314
G. clarum, 197 Hybridization-based methods, 299
G. etunicatum, 197 Hydnangium, 482
G. fistulosum, 197 Hydnotrya, 482
G. gigantia, 197 Hydnotryposis, 482
G. margarita, 184, 197 Hydrocarbon degradation by mixed cultures, 450
G. versiforme, 197 Hydrocarbons, 443
Gliocladium, 158, 162, 304 Hydrolases, 187
as a biocontrol agent, 161 Hypholoma fasciculare, 376, 379
G. catenulatum, 161, 164 Hypocrea, 135
G. deliquescens, 433 H. jecorina, 137
G. virens, 161 H. lixii, 137
Globodera pallida, 210 H. aurantius, 91
G. rostochiensis, 4 Hypoxylon, 378
Glomerella cingulata, 446 H. rubiginosum, 382
Index 501

Hypsizygus, 234 Kloeckera apiculata, 226, 257

H. marmoreus, 233 Kloeckera javanice, 226, 269
Kluyveromyces, 260
Identification of: K. fragilis, 368
pathogens, 52 K. lactis, 252, 270 – 273, 275
penicillia, 33 K. lactis var. lactis, 269
plant pathogenic fungi, 49 K. marxianus, 228, 252, 269, 272, 275, 413, 434, 435, 438, 439
Importance of wood-decay fungi, 375 Kochujang, 227
Improved plant nutrition, 185 Korean jeotkal, 229
Improving BCPD, 175 Korean koenjan, 227
Inactivation of mycotoxins, 336 Korean meju, 228
Indicator of deterioration, 319 Korean soy sauce (Kanjang), 227
Indoor composting, 238
Induced resistance, 107, 152 Laccaria, 482
Industrial production of ergot alkaloids, 93 L. laccota, 104
Industrial wastes, 419 Laccase, 399, 421, 423
Ingredients production, 220 characterization of, 424
Inserted elements, 61 production of, 423
Integrating BCPD with other alternatives, 176 Lactobacillus, 293
Interaction L. brevis, 224
between grain fungi, 317 L. plantarium, 224
of factors, 295 L. reuterii, 294
of natural biofertilizers, 198 Lactococcus, 293
Interference with nematode chemoreception, 210 Lactones, 283
Interpretation of: Lagenidium giganteum, 80, 81
isozyme banding patterns, 39 Leaf-litter decomposition, 380
isozyme data, 39 Lebanese labneh, 225
Ionone, 284 Lecanicillium lecanii, 212
Ipomoea argyrophylla, 91 Leccinum scabrum, 376
I. piurensis, 91 Legume-based fermented foods, 227
I. rubro-coerulea, 91 Lentinula, 233, 243
Irpex lacteus, 462, 473 L. edodes, 233, 240, 243, 286, 376, 401, 421
Ischnoderma benzoicum, 284 Lentinus, 239
Isozyme analysis, 37 Lenzites trabea, 91
Isozyme analysis in fungal taxonomy, 37 Lepiota rhacodes, 286
application of isozyme analysis, 41 Leptinotarsa decemlineata, 85
genetics, 41 Leptographium wageneri, 41
population biology, 44 Leptosphaeria, 53
taxonomy and spices identification, 41 L. maculans, 2, 7
interpretation of isozyme data, 39 Leuconostoc, 225, 226, 228
enzyme systems, 40 L. mesenteroides, 223, 224, 227
interpretation of isozyme patterns, 39 Leutinula, 234
isozyme techniques for fungi and oomycetes, 37 Lignin degradation, 395, 401
choice of separation matrix, 38 Lignin peroxidase, 397, 421
enzyme activity staining, 38 Ligninolysis, 420
isozyme analysis of fungi, 38 Ligninolytic system of white rot fungi, 395
Isozyme banding patterns, 39 Lignocellulose, 393
Isozyme techniques, 37 Limits of PCR, 301
ITS region as target, 303 Lintinula edodes, 233, 235
Lipid degradation, 320
Lipolytic activity, 271
Jambal wine, 226 Listeris monocytogenes, 275
Japanese katsuobushi, 229 Litchi wine, 226
Japanese miso, 227 Lycoperdon pyriforme, 44
Japanese sake, 225
Japanese shoyu, 227 Macrophomina, 52
Javan bongkrek, 228 M. phaseolina, 184
Magnaporthe grisea, 2, 5, 7, 44, 124 –127, 137
Kenyan urwaga, 225 Malaysian soy sauce Kicap, 227
Klebsiella, 224, 226 Management of mycotoxin contamination, 336
502 Index

Manduca sexta, 86 Molecular biology of biocontrol Trichoderma, 135

Manganese peroxidase, 399 biocontrol-specific gene expression in Trichoderma, 139
Mango wine, 226 Cis and Trans-acting genetic factors, 140
Map-based cloning of genes, 2 Molecular detection of fungi, 299
Maramius, 450 Molecular detection of fungi in foods and feeds, 299
Marasmiellus, 450 methods used for detection of fungi, 299
Marasmius androsaceus, 381 amplification-based methods, 300
Marker-assisted breeding, 2 hybridization-based methods, 299 –301
Mechanisms of biocontrol, 175 molecular targets used for the detection of fungi, 302
Mechanisms of disease suppressor by fungi, 106 targets based on the ribosomal RNA gene cluster, 302
antagonism, 106 mycotoxin biosynthetic genes as targets for detection of fungi,
induced resistance, 107 303
Mechanisms of plant growth promotion, 103 aflatoxins, 303
Media for metabolite profiling, 20 other mycotoxin biosynthetic genes, 304
Medicago sativa, 8 patulin, 304
Medium-chain fatty acid, 293 trichothecenes, 304
Melampsora, 53 other sequences as targets, 305
Meloidogyne, 205, 206 Molecular genetic basis of biocontrol, 138
M. incognita, 210 Molecular genetics of entomopathogenic fungi, 84
Melolontha, 83 Molecular identification of entemopathogenic fungi, 86
M. melolontha, 80 Molecular markers, 2, 57
Merulins rufus, 286 types, 58
Metabolite profiling, 19 application of individual markers, 61
Metabolites from lactic acid bacteria, 293 combing markers, 61
Metabolomics, 19 fingerprinting methods, 59
Metal biosorption by isolated molecules, 413 inserted elements, 61
Metal ions, 466 mitochondrial sequences, 61
Metarhizium, 81– 84, 86, 87 other total genome approaches, 60
M. anisopliae, 61, 79 –86, 115, 116, 445 protein coding genes, 59
M. anisopliae var. acridium, 81, 85, 86 ribonsomal DNA, 59
M. flavoviride, 86 selection of molecular markers, 62
Method of composing, 237 Molecular methods for identification of fungi, 49
Methods for yeast characterization, 260 assumptions and limitations, 49
Methods for yeast identification, 258 in vitro, 52
Methods in fungal identification, 49 plant parts, from, 52
Methods used for detection of fungi, 299 recent applications in plant pathology, 52
Mexican Tesguino, 225 Molecular targets used for the detection of fungi, 302
Microbial flavors, 281 Molecular tools for biocontrol strains, 136
Microbial metabolites, 124 Mollisia cinerea, 383
Microbial protein products, 247, 253 Monascus
Microbial protein products as food ingredients, 252 M. anka, 229
Micrococcus, 227, 273 M. purpureus, 228
Micromonospora, 126 Monila, 364, 365
M. coerulea, 126 Monilinia, 53, 125
M. echinospora, 126 M. fructigena, 174
M. purpurea, 126 M. pulcherrima, 175, 334
M. spartanea, 127 Monocuus ruber, 334
Mitochondrial sequences, 61 Monocyclic aromatic hydrocarbons, 446
Mixed cultures and fermentation, 369 Monosporascus, 53
Mixed fermented food, 228 Monoterpenses, 281
Mode of action of nematophagous fungi, 208 Morchella crassipes, 286
Modeling fungal community dynamics, 386 Mortierella, 304
Modeling fungal decay communities, 385 Mucor, 224, 226 –229, 283, 304, 364
Modeling the growth of hyphae and mycelia, 386 M. hiemalis, 91
Moisture sorption isotherms, 313 M. racemosus, 225
Mold deterioration of cereal grain, 318 M. rouxii, 150
Molecular biology for control of mycotoxigenic fungi, 69 Muscodor albus, 26
mycotoxin prevention, 70 Mushroom flavors, 286
development of aflatoxin-resistance screening tools, 70 Mushroom production, 236
identification of resistance-associated proteins, 71 –73 Mycelium radicis-atrovirens, 105
Index 503

Mycena Nectria haematococca, 5, 115

M. galericulata, 383 Nematode, 205
M. galopus, 381 -trapping fungi, 208
M. marasmius, 381 Nematoloma frowardii, 448, 449
Myces euteiches, 184 Nematophagous fungus, 206
Mycoderma, 226 Nematophthora gynophila, 212
M. vini, 225 Neotyphodium, 92, 96
Mycoherbicides, 111 N. coenophialum, 91, 107
Mycoparasites, 162 N. lolii, 91, 107
Mycoparasitism related genes, 150 Neurospora, 37, 137, 227
Mycoprotein, 247, 248 N. crassa, 137, 150, 364 – 366, 463, 464
Mycoprotein and related microbial protein products, 247 N. intermedia, 44
food for human consumption, 248 Nicotiana
additional functionalities, 250 N. benthamiana, 4, 5
composition, 248 N. glutinosa, 185
mycoprotein production (Quorn products), 250 N. tabacum, 138
nucleic acid content, 250 Nigerian
protein value, 249 burukutu, 228
texture, 250 gari, 224
related products, 251 lafun, 229
food ingredients, 252 ogi, 224
active formulations, 252 pito, 225
inactive formulations, 252 Nisin, 293
Mycoprotein production, 250 Nodulisporium, 378
Mycorrhizae, 104, 105, 378, 481 Nomuraeu rileyi, 80
associations, 196 Nonpathogenic fungi, as biocontrol agents, 161
bioremediation and, 486, 487 Nucleic acid
commercialization techniques, 200 metabolism, 129
diversity, 482 sequence-based amplification, 300
function, 482 Nutrient uptake, 482
inoculum Nutritional and medical values of mushrooms, 234
benefits of, 197
use of, 485
Ochratoxins, 24, 329
-mediated disease control, 185
Oidium lactis, 223
response to disturbance, 483
Oosporidium margaritiferum, 228
Mycosphaerella, 53
Ophiostoma, 58
Mycotoxicosis, 325
Organic acids, 291
Mycotoxigenic fungi, 69
Organic degrading lignin, 419
Mycotoxins, 325
Ostertagia, 206
biosynthesis, 72
Ostinia nubalis, 80, 81
biosynthetic gene, 303, 304
Ostreatus, 239
contamination, 73
Ostrinia nubilalis, 85
fungi, 72
Oudemansiella mucida, 125
natural occurrence and toxic effects of, 327
Oyster mushrooms, 239
aflatoxins, 327, 328
ochratoxins, 329, 330
fumonisins, 330, 332 Packed-bed bioreactor, 422
selected trichothecenes (TCTCs), 332, 333, 335, 336 Paddy straw mushroom, 242
preventive measures, 70 Paecilomyces, 21, 81, 228, 364
avoiding human exposure, 337 P. farinosus, 85
management of contamination, 336 P. fumosoroseus, 80, 84
production by toxicogenic fungi, 326 P. lilacinus, 85
factor affectings, 326 P. tenuipes, 87
general consideration, 326 Palm wine, 226
produced by Alternaria sp., 335 Pantoea agglomerans, 175
produced by Penicillium, 334 Paraglomus, 104
synthesis, biochemistry of, 343 Pathogens targeted for BCPD of fruits, 173
Myrothecium, 304, 332, 351, 352 Patulin, 304
M. roridum, 304, 352 Paxillus fortinii, 107
M. verrucaria, 326, 433, 436 P. atrotomentosus, 383
504 Index

[Paxillus fortinii] [Penicillium]

P. involutus, 104, 106, 379 P. pulmonarius, 450
P. sylvestris, 379 P. purpurogenum, 334
Pediococcus soyae, 227 P. restrictum, 128
Pellicularia filamentosa, 91 P. rhodozyma, 439
Pencillium aurantiovirens, 91 P. roqueforti, 26, 27, 225, 271, 303, 314, 317, 321, 326, 335
P. camembertii, 91 P. rubrum, 326, 334
P. chermesinum, 91 P. sajor-caju, 450
P. clavigerum, 91 P. tricolor, 30, 31, 33
P. concavorugulosum, 91 P. ulaiense, 20
P. crustosum, 91 P. urticae, 326
P. fumigatus, 91 P. varioti, 435 – 438
P. griseofulvum, 91 P. verrucosum, 24, 30, 33, 314, 318, 329
P. kapuscinskii, 91 P. viridicatum, 30, 31, 33, 69, 326
P. palitans, 91 Peniophora, 378
P. patulum, 91 P. limitata, 382
P. roqueforti, 91 P. lycii, 382
P. rubrum, 91 Peronosclerospora, 53
P. rugulosum, 91 Peronospora parasitica, 4, 10
P. sizovae, 91 Peroxidase, 399
P. viridicatum, 91 Petromyces alliaceus, 24
Penicillium, 19– 21, 32– 34, 104, 105, 158, 175, 220, 224, 227, 282, P. alliceus, 329
286, 292, 294, 295, 303, 305, 314 – 316, 318, 320, 326, 329, Pezicula malicorticis, 175
334, 381, 413, 433, 436, 444, 445 PGPF
P. atraovenetum, 334 in mycelial fungi, 103
P. aurantiocandidum, 30, 33 Rhizoctonia, 103
P. aurantiogriseum, 30 –33, 314, 315, 319 in Trichoderma, 101
P. boydii, 445, 446 Phaffia rhodozyma, 432
P. brevicompactum, 317 Phakopsora, 53
P. camemberti, 225, 334 Phallus impudicus, 376
P. carneum, 26, 27, 321 Phanaerochaete chrysoporium, 472 – 474
P. chrysogenum, 101, 229, 414, P. sordida, 474
P. citreoviride, 326 Phanerochaete, 422, 444
P. citrinum, 101, 225, 293, 326, 334, P. chrysosporium, 411, 419 –423, 433, 444, 457, 459 –467
P. commune, 27 P. laevis, 444
P. crustosum, 334 P. velutina, 376, 379
P. cyclopium, 30, 31, 33, 326, 329, 334 P. chrysosporium, 394, 395, 398 – 401
P. digitatum, 20, 174, 444, 446 P. ostreatus, 401, 402
P. elegans, 436 Phaseolus mungo, 228
P. expansum, 174, 176, 304, 326 Phellinus ferreus, 382
P. freii, 30, 31, 33 Phenolic acids, 294
P. glabrum, 225, 444, 450 Phialocephala fortinii, 105
P. griseofulvin, 334 Phialophora, 394
P. hordei, 33, 317, 325 Philippine Tapuy, 225
P. infestans, 40 Phlebia radiata, 382, 394, 419, 446
P. islandicum, 326 P. rufa, 382
P. italicum, 20, 174, 281 P. tremelosus, 419
P. janthinellim, 101, 444, 450 Pholiota nameko, 233
P. luteum, 333, 345 Phoma, 59, 62, 103 –105
P. melanoconidium, 30, 33 P. exigua, 62
P. mirabilis, 40 P. medicaginis var. pinodella, 62
P. nalgiovense, 229 P. sorghina, 336
P. nordicum, 24, 305 P. subboltshauseri, 62
P. ochro-chloron, 413 Phomopsis, 378
P. ostreatus, 447 – 450, 462 P. convolvulus, 115
P. oxalicum, 162, 433, 436 P. oblonga, 378
P. palltans, 326 Phylloxera, 79
P. paneum, 321 Phytoalexins, 187
P. polonicum, 30, 31, 33 Phytoanticipins, 187
P. puberulum, 326, 334 Phytopathogenic fungi, 183
Index 505

Phytopathogenic viruses, 185 [Potential applications of white rot fungi]

Phytophthora, 41, 57, 58, 61, 126, 184, 207 upgrading agricultural wastes for animal feed, 402
P. cactorum, 8 Potential of fungal biotechnology, 410
P. capsici, 126, 127, 129 Pseudomonas syringae, 7
P. cinnamomea, 44 Preservation of fungal cultures, 237
P. cinnamoni, 186 Prodiplosis longifila, 81
P. infestans, 8, 40, 44 Production of edible fungi, 233
P. megakarya, 44 button mushroom, 237
P. palmivora, 44, 112 cropping, 238
P. parasitica, 7, 107, 187, 188 indoor composting, 238
P. parasitica var. nicotianae, 184 long method of composting, 237
Phytotoxin detoxification, 8 short method of composting, 238
Picea abies, 380 substrate preparation, 237
P. glauca, 383 specialty mushrooms, 239
P. mariana, 105 Auricularia spp. (wood ear mushroom), 243
P. sitchensis, 105 Lentinula edodes (shiitake), 240
Pichia, 226, 379 Pleutotus spp. (oyster mushrooms), 239
P. anomala, 164, 175, 257, 272, 321 Volvariella spp. (paddy straw mushroom), 242
P. farinosa, 275 technology of, 235
P. guilliermondii, 175, 220 maintenance and preservation of fungal cultures, 237
P. membranefaciens, 226, 228, 275, 305 spawn production, 235
P. ohmeri, 226 Production of mycotoxins, 326
P. tainania, 432 Production technology of edible fungi, 235
P. verrucosum, 321 Propionibacterim shermanii, 293, 294
Pisolithus tinctorius, 104, 482 Propionic acid, 293
Pithomyces chartarum, 336 Prostephanus truncates, 83
Plant disease control, 183 Protein biosynthesis, 129
Plant growth promoter, 101 Protein coding genes, 59
Plant growth promotion, 152 Protein degradation, 320
Plant pathogenic bacteria, 184 Proteolytic activity, 270
Plant-parasitic nematodes, 205 Proteome analysis, 72
Plasmodiophora brassicae, 106 Proteomics, 10
Pleurotus, 233, 234, 239, 240, 444, 448, 450 Pseudallescheria boydii, 444
P. flabellatus, 239 Pseudeurotium
P. florida, 239, 240 P. chrysosporium, 446 –449
P. eryngii, 44, 397 P. zonatum, 446
P. laevis, 449 Pseudomonas, 175, 198, 471, 472
P. ostreatus, 206, 239, 376, 419, 420, 424, 444, 462, 466 P. aeruginosa, 127
P. pulmonarius, 420, 444, 448 P. lacrymans, 184
P. sajorcaju, 239, 463 P. pyocyanea, 127
P. tremellosus, 421 P. pyrrocinia, 125
Plum wine, 226 P. syringae, 175 –177, 185
Pluteus cervinus, 383 P. syringae pv. lacrymaus, 105
Pochonia, 210, 211 P. syringae pv. tomato, 7
P. chlamydosporia, 210, 211 Pseudozyma flocculosa, 165, 166
P. rubescens, 210 Puccinia, 41
Podostroma, 135 P. canaliculata, 112
Pogonomrymex occidentalis, 484 P. graminis f. sp. tritici, 44
Polycyclic aromatic hydrocarbons, 448 Pulcherrima, 257
Polymerase chain reaction, 300 Pullaria, 224
Pontoea agglomerans, 177 Pycnidiophora, 128
Popillia japonica, 83 Pycnoporus cinnabarinus, 399, 419, 422 – 426
Population biology, 44 P. sanguineus, 463, 465, 466
Populus tremuloides, 383 Pyricularia oryzae, 336, 423
Postharvest control, 336 P. setariae, 115
Postharvest fungal colonization of cereal, 314 Pythium, 37, 53, 57, 104, 151, 159 – 162
Postharvest technology of mushrooms, 244 P. aphanidermatum, 105, 161, 184
Potential applications of white rot fungi, 402 P. irregulare, 105
biopulping, 402 P. nunn, 162
mushroom production, 402 P. oligandrum, 162
506 Index

[Pythium] Rhodosporidium, 162

P. parasitica var. nicotianae, 184 R. diobovatum, 163, 164
P. periplocum, 162 Rhodotorula, 162, 163, 224, 257, 432
P. ultimum, 105, 138, 151, 152, 161 R. flava, 227
R. glutinis, 175, 271, 448, 449
R. lactosa, 228
QTL-associated genes, 4 R. rubra, 271
Quercus petraea, 380, 382
Rhodotorulla, 127
Q. robur, 382
Rhus vernicifera, 421
Quorn products, 250
Rhynchosporium, 52, 53
R. secalis, 38, 44
Radicislycopersici, 160 Ribosomal DNA, 59
Radulomyces confluens, 382 Ribosomal RNA gene cluster, 302
Rare actinomycetes, 126 Ribosomal RNA gene sequencing, 258
rDNA regions used as targets, 303 Rivea corymbosa, 91
Reactive oxygen species, 400 Role of MRGs in biocontrol, 151
Recovery of mycorrhizal fungi, 483 Role of spoilage fungi in seed deterioration, 311
Regulation of trichothecene biosynthesis, 352 environmental factors, 312
Remediation of azo dye, 466 water availability, 312, 313
Resistance-associated proteins, 71 gas composition, 314
Respiration and dry matter losses, 318 temperature, 314
Restoration of mycorrihizae, 481 postharvest fungal colonization, 314
Restoration of mycorrhizae in disturbed arid ecosystems, 481 factors, 314
mycorrhizal diversity, 482 mycoflora, 314
fungal and plant species richness, 482 ecology of mycotoxin production, 315
mycorrhizal function, 482 interaction, 317
carbon allocation, 482 viability and degradation of grain, 320
nutrient uptake, 482 carbohydrate degradation, 320
soil stabilization, 483 lipid Degradation, 320
managing existing inoculum, 484 protein degradation, 320
maintaining source areas, 484 rRNA gene as target, 303
managing Natural reinvasion, 484 Russian Koumiss, 225
managing plants to enhance recovery, 485
protecting topsoil, 484 Saccaropolyspora spinosa, 84
soil amendments, 484 Saccharification, 368
use of mycorrhizal inoculum, 485 Saccharomyces, 61, 164, 220, 224 – 226, 228, 229, 257, 258, 259,
inoculum diversity, 486 260, 262, 269, 272, 276, 364
isolating and culturing fungi, 485 S. baili, 292
methods for applying inoculum, 486 S. bayanus, 228
Reuterin, 294 S. boulardii, 276
RFLP of rDNA, 259 S. capensis, 227
Rhagoletis pomonella, 84 S. cerevisae, 4, 127, 137, 141, 142, 150, 163, 176, 177, 223 –229,
Rhizoctonia, 41, 52, 59, 103, 107, 140, 160, 162 252, 253, 257, 259, 260, 261, 264, 269, 270 – 275, 293, 295,
R. cerealis, 107 303, 305, 346, 411, 413, 414, 432, 434, 435, 438, 439
R. leguminicola, 336 S. chartarum, 333
R. oryzae, 59 S. chevielier, 226
R. solani, 7, 61, 101, 103, 105, 124, 126, 127, 138, 140, 147, S. commune, 474
149 –151, 157, 158, 161, 162, 186, 207, 448 S. delbrueckii, 272
R. zeae, 105, 107 S. exiguus, 226
R. pusillus, 314 S. granulatus, 474
Rhizopogon, 482 S. lactis, 225
Rhizopus, 224, 226 – 228, 314 S. ludwigii, 226, 227
R. arrhizus, 91, 227, 411, 413, 448 S. marxianus, 226
R. flava, 227 S. odorus, 283
R. nigricans, 91, 102, 103, 473 S. paradoxux, 257
R. niveus, 252 S. rosei, 227
R. oligosporus, 227, 228 S. rouxii, 227
R. oryzae, 225 –227, 229, 252, 283 S. ruguloso-annulata, 474
R. stolonifer, 176 S. sake, 225
Rhodortorula glutinis, 432 S. unisporus, 272
Index 507

[Saccharomyces] Stenotrophomonas maltophilia, 450

S. uvarum, 225, 227, 411 Stereum gausapatum, 382
S. variegates, 474 Sterigmatocystins, 343
S. pastorianus, 225 Sterium hirsutum, 382
Safety of entomopathogenic fungi, 86 Stictocardia tiliifolia, 91
Salmonella, 276 Strain improvement through biotechnology, 85
Saprotrophs of wood and litter, 380 Strategies for biopesticide use, 83
Scedosporium apiospermum, 446 Streptococcus, 228, 293
Schizosaccharomyces pombe, 226, 414 S. faecalis, 223
Sclerocystis dussii, 184 Streptomyces, 86, 125, 126, 128, 130, 462
Scleroderma, 486 S. cacaoi, 124
Sclerospora graminicola, 58 S. flaveus, 126
Sclerotinia, 125 S. griseochromogenes, 124
S. sclerotiorum, 7, 115, 136, 140, 157, 184 S. griseus, 126
Sclerotium rolfsii, 105, 140, 148, 151, 157, 184, 475 S. humidus, 126
Scopulariopsis, 227 S. hygroscopicus, 124
Scutellospora, 104 S. kasugaensis, 124
Selection and screening of mycotoxins, 337 S. kurssanovii, 126
Selection of effective AMF, 189 S. rimofaciens, 124
Selection of molecular markers, 62 S. venezuelae, 471
Separation matrix, 38 S. violaceoniger, 129
Sepedonium sp., 91 Streptoverticillium cinnamoneum, 414
Septoria triticic, 51 Strobilurus tenacellus, 125
Sequences as targets, 305 Stropharia rugosoannulata, 476
Serpula lacrymans, 394 Structure of ergot alkaloids, 91
Serratia marcescens, 151 Structure of the dye, 422
Shigella, 276 Sudanese kisra, 224
Soil amendments, 484 Suillus, 482
Soil piles and windrows, 476 S. bovinus, 379, 380
Soil stabilization, 483 S. granulatus, 105
Solid substrate fermentation, 96 S. plorans, 104
Sorangium cellulosum, 128 S. variegatus, 379
Sorbic acid, 292 Sulfolobus, 414
Sordaria, 126 Syncephalastrum racemosum, 448
S. araneosa, 129 Systemic-induced resistance, 187
Sorghum bicolor, 188
Sources of antagonists to nematodes, 212
South American fermented maize chichi, 225 Talaromyces, 158, 162
Soy sauces, 227 T. flavus, 162
Spawn production, 235 T. thermophilus, 314
Sphaerotheca, 157 Taxonomy and species identification, 41
S. fuliginea, 164 Tempe, 227
Spoilage fungi, 311 Tempe-like foods, 227
Spoilage of yeast in cheese, 275 Terpenes, 445
Sporidesmium, 158 Terpenoids, 281
S. chartarum, 336 Thelephora terrestris, 380
S. sclerotiorum, 162 Thermomyces lanuginosus, 314
Sporidiobolus salmonicolor, 285 Thiobacilli sulfobacilli, 414
Sporobolomyces odorus, 283 Thiobacillus, 198
S. roseus, 175, 176 Thomomys talpoides, 484
Sporotrichum sulfurescens, 447 Tilletia, 52
Stachybotrys, 21, 158, 304, 332, 351 Tilletiopsis, 165, 166
S. atra, 326 T. albescens, 166
S. elegans, 162 T. minor, 166
Stagonospora T. pallescens, 165, 166
S. convolvuli, 115 Tolypocladium, 84, 87, 137
S. nodorum, 51 T. niveum, 85
Staphylococcus, 273 Torula kumiss, 225
S. aureus, 127 Torulaspora, 303
Steinernema, 205 T. delbrueckii, 275
508 Index

Torulopsis, 224, 226, 229, 253 Ungrading agricultural wastes for animal feed, 402
T. candida, 223 Ustilago maydis, 4
T. dattila, 227
T. holmii, 223 Vegetable crops, 157
T. versatile, 227 Venturia, 52
Torulopsisetchellsii, 227 V. inaequalis, 128, 153
Trametes hirsuta, 444, 449, 462, 467 Verrucosa, 33
T. versicolor, 394, 395, 433, 444, 461 –463, 465 –467, 476 Verticicladiella wageneri, 41
Transformation systems, 85 Verticillium, 57, 81, 158, 162, 413
Transgenic plants, 5 V. biguttatum, 162
Tremella fuciformis nameko, 233 V. dahliae, 8, 162, 184
Trichoderma, 21, 101, 103, 104, 106, 107, 135 – 138, 140, V. lecanii, 61, 80, 82, 86, 164, 166
142, 147 – 151, 153, 158, 160, 162, 163, 240, 304, 366, Verticimonosporium, 332, 351
381, 433, Vespula, 83
biocontrol activity, 160 Vietnamese nuoc-mam, 229
biocontrol agent, 147, 158, 163 Viridicata, 31, 32
biocontrol taxa, 135 Volvariella, 234, 239, 242, 243
mechanism of action, 160 V. bombycina, 242
source of genes for crop improvement, 153 V. volvacea, 233, 242
species identification, 158 Vuilleminia comedens, 378, 282
T. asperellum, 136, 137, 158
T. atroviride, 136, 137, 150, 151, 153, 158 Water activity, 313
T. hamatum, 160 Water potential, 313
West African dawadawa, 228
T. harziamum, 41, 101 – 103, 105, 106, 135, 140, 141, 148 – 153,
West African gari, 229
158, 160 – 163, 184, 283
White rot fungi, 393, 394
T. inhamtum, 136
Wine fermentation, 257
T. koningii, 101, 102, 158
Wine yeast genomes evolution, 264
T. longibrachiatum, 136, 152, 160
Wines, 226
T. nudum, 286
Wingea roberstii, 228
T. parceramosum, 136
Wood ear mushrooms, 243
T. reesei, 137, 364, 367, 368, 433
Wood-decay fungi, in forest ecosystems, 375
T. stromaticum, 136
describing the roles of, 377
T. versicolor, 439
community scale, 380
T. virens, 136, 140, 149, 150, 151,
endophytes, 378
T. viride, 101, 102, 137, 283, 334, 364, 411, 436, 437
mycorrihizae, 378
Tricholomopsis
saprotrophs of wood and litter, 380
T. platyphylla, 376 modeling fungal decay communities, 385
T. rutilans, 383 dynamics, 386
Trichosporan pullulans, 228 growth of hyphae and mycelia, 386
Trichosporom beigelii, 223 Woodland ecosystems, 375
Trichosporom spp., 273 World production of mushrooms, 233
Trichosporon, 433
T. beigelii, 228, 284 Xanthium spinosum, 117
T. cutanum, 271 Xanthomonas campestris pv. vesicatoria, 7
T. mucoides, 448 X. oryzae pv. oryzae, 7
T. versicolor, 448, 449 Xylaria, 378
T. pullulans, 223
Trichothecenes, 304, 351 Yarrowia, 269
Trichothecium, 304, 332, 351 Yarrowia lipolytica, 137, 141, 269 –271, 273 –275, 346, 448
T. roseum, 326 Yeasts, 432
Tuber, 482 biodiversity in wineries, 257
Tuber crop-based fermented food, 228 biological control agents, 165
Tylospora fibrillosa, 383 Botrytis cinerea, against, 163
Types of molecular markers, 58 dairy industry, 269
Types of mycorhizae, 482 cheeses, 273, 274
contribution and spoilage of yeast, 275
Ulocladium, 162 cream and butter, 271
U. atrum, 163 farm milk, 271
U. chartarum, 163, 448, 449 kefyr, 272
Index 509

[Yeasts] Zoophthora
fermented milk products, other, 272 Z. neoaphidis, 84
yogurt, 271 Z. radicans, 83
population dynamic, 262 Zygosaccharomyces, 260, 303
probiotics, 276 Z. bailii, 305
species diversity during vinification, 257 Z. rouxii, 275
wine fermentation, 257 Zymononas mobilis, 364