Cambridge Books Online

http://ebooks.cambridge.org/

Exploitation of Fungi

Symposium of the British Mycological Society held at the University of

Manchester September 2005

Edited by G. D. Robson, Pieter van West, Geoffrey Gadd

Book DOI: http://dx.doi.org/10.1017/CBO9780511902451

Online ISBN: 9780511902451

Hardback ISBN: 9780521859356

Chapter

16 - A novel understanding of the three-way interaction between Tricho

derma spp., the colonized plant and fungal pathogens pp. 291-309

Chapter DOI: http://dx.doi.org/10.1017/CBO9780511902451.017

Cambridge University Press

 16
A novel understanding of the
three-way interaction between
Trichoderma spp., the colonized
plant and fungal pathogens
S. L. W O O , M. R U O C C O , R. C I L I E N T O ,
P. A M B R O SI N O , R. M A R R A , F. V I N A L E ,
S. L A N ZU IS E , D. T U R R À, S. F E R R A I O L I ,
I. S O R I E N T E , S. G I G A N T E , F. SC A L A
A N D M. L O R I T O

Abstract
Trichoderma-based biofungicides are a reality in agriculture, with
more than 50 formulations available today as registered products world-
wide. Several strategies have been applied to identify the main genes and
compounds involved in this complex cross-talk between the fungal antag-
onist and the microbial pathogen, as mediated by the plant. Proteome and
genome analysis have greatly enhanced our ability to conduct holistic and
genome-based functional studies. We have identified and determined the
role of a variety of novel genes and gene-products, including ABC trans-
porters, enzymes and other proteins that produce or act as novel elicitors
of induced systemic resistance, proteins recognized by the plant as avir-
ulence factors, as well as molecules that generally activate the antagonistic
activity in Trichoderma spp. We have cloned mycoparasitism-related pro-
moters and used them in combination with GFP and other markers to
study the interaction in vivo and in situ between Trichoderma and the
fungal pathogen or the plant. Finally, we have transgenically improved
the ability of the antagonist to kill other microbes and to activate plant
defence mechanisms.

Introduction
Plant diseases caused by pathogenic fungi infections represent a
major limiting factor for the cultivation and the conservation of agri-
cultural plants of interest. The consequences of parasite attack result in
both quantitative and qualitative reduction of crop production, large

Exploitation of Fungi, eds. G. D. Robson, P. van West and G. M. Gadd. Published

by Cambridge University Press. # Cambridge University Press 2007.

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:33 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
292 S. L. Woo et al.

economic losses and represent a risk for human and animal health due to
the accumulation of residues in the environment and mycotoxin contam-
inants in food products. Chemical control of phytopathogenic fungi is
limited not only by economical cost, but also by concerns about the impact
of continued applications on consumer health and the environment. In
addition, applications of methyl bromide, the most frequently used soil
fumigant (also causing a reduction of the ozone level in the atmosphere),
will be completely banned in Europe in the next few years, which provides
a strong impulse for the development of ‘biological’ methods based on, for
instance, antagonistic microbes and soil solarization for disease manage-
ment. Alternatively, new technologies for plant defence can be proposed
that are based on the utilization of transgenic plants and microorganisms,
the application of antagonist microbes (fungi or bacteria) capable of
controlling important diseases or of useful compounds originating from
these microorganisms.
In particular, fungi of the genus Trichoderma are among the most
effective biopesticides applied for plant protection against fungal diseases,
both in traditional and organic farming. The fungi are commercially
produced, and several patents protect their use (Harman & Kubicek,
1998; Kubicek & Harman, 1998). Biopesticides based on antagonistic
Trichoderma strains are used for biocontrol of phytopathogenic fungi
that cause diseases including root and crown rot of vegetables, seedling
damping-off, vascular wilt, take-all of cereals, root infection of fruit
trees, mildew of grapes and other crops, monilia disease, and soft rot of
pome and stone fruit etc. The availability and diffusion of these products
is much more widespread than commonly known, and Trichoderma is
also listed both in Europe and USA as a pesticide permitted for use in
organic farming. The size of the biopesticide market based on antagonistic
fungi is modest compared to chemicals, but it is steadily growing and
is expected to increase significantly with the introduction of these pro-
ducts in India, China, northern Africa and central Asia. In addition to the
registered products (a few dozen formulations), a large variety of non-
registered bioprotectants, biostimulators and biofertilizers based on
Trichoderma or other antagonistic fungi are being sold everywhere
(Lorito et al., 2004). For instance, commercial products based on the
fungus Trichoderma spp. include Binab TTM (T. harzianum and T. poly-
sporum registered in Sweden, UK, USA), Bio FungusTM (Trichoderma
spp. in Belgium), EcoSOMTM (T. viride in India), RootShieldTM T-22G,
T-22 Planter Box, Bio-Trek (T. harzianum in USA and north Europe),
Root ProTM (T. harzianum in Israel), SupresivitTM (T. harzianum in the

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:33 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
 A novel understanding of the three-way interaction 293

Czech Republic), TrichodexTM (T. harzianum in Italy, Israel and USA),

TriecoTM (T. viride in India), Trichoderma 2000TM (Trichoderma spp. in
Israel), TrichopelTM Trichojet, Trichodowels, Trichoseal (T. harzianum
and T. viride in New Zealand) etc.

Trichoderma as a versatile biocontrol agent

Trichoderma spp. have the remarkable ability to interact both as a
parasite and symbiont with different substrates and living organisms,
including plants and other microbes. These fungi can utilize a variety of
nutrient sources. They are not only some of the most resistant microbes to
natural and man-made chemicals and toxins, but they are also able effec-
tively to degrade some compounds, including hydrocarbons, chlorophe-
nolic compounds, polysaccharides and xenobiotic pesticides (Harman
et al., 2004). Many strains of Trichoderma are strong opportunistic
invaders, fast growing, producers of enormous quantities of spores and
powerful antibiotics. These properties make these fungi ecologically very
successful, which is reflected in the fact that they are widely distributed in
diverse ecosystems of agriculture, native prairie, forest, salt marsh and
desert soils of all climatic zones, as well as in lake water, dead plant
material, living roots of virtually any plant species, seeds and air.
These saprophytic fungi are commonly isolated from the soil, which
is their natural habitat, but they are also present in other surroundings.
The genus Trichoderma, according to recent systematics, is comprised
of more than 30 species, whereby the majority exhibit only the asexual
life-cycle, although in rare cases the sexual form Hypocrea is also found.
Trichoderma fungi have a tremendous amount of genetic diversity and
are biologically versatile with the ability to perform numerous different
biological functions. For example, there are strains that are strong anta-
gonistics towards phytopathogenic fungi, some that are effective soil
colonizers and biodegraders, and others that are producers of important
metabolites. Therefore, Trichoderma spp. are of substantial interest for
studies on the biology of fungi and good candidates for applications in
agriculture and industry.
Many Trichoderma strains are ‘rhizosphere competent’, having the abil-
ity to colonize the plant root, and they may also be capable of promoting
plant growth and root development. Recent studies on the interaction of
Trichoderma with plants and other microbes indicate that these fungi may
induce systemic resistance in plants, thus increasing the plant defence
response to attack from a variety of pathogens (Harman, 2000; Harman
et al., 2004).

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:33 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
294 S. L. Woo et al.

Molecules produced by Trichoderma also have potential for promoting

plant growth (Inbar et al., 1994). Plants treated with T. harzianum showed
improved seed germination, increased overall plant size and augmented
leaf area and weight (Altomare et al., 1999). The possibility of combining
systemic biofungicides and plant growth promoters has a great market
potential if the molecular basis of the mechanisms involved can be
understood.
Natural and genetically modified strains of T. reesei are widely used in
industry, typically for protein production, whereas isolates of T. harzia-
num, T. atroviride or T. virens are used in agriculture as biopesticides,
bioprotectants, biostimulants and biofertilizers on a large variety of
crops in the field and greenhouse. It is possible to use Trichoderma over
a wide range of applications because its propagules can be produced
economically, in high concentrations, both in liquid and dry formulations,
and can be stored for many months. Today, more than 50 different
Trichoderma-based agricultural products can be found registered in differ-
ent countries across five continents, where they are sold as biocontrol
products for protection and yield improvement of numerous crops.
The laboratory techniques that can be applied at present provide the
possibility to genetically modify these fungi in a very specific way, permit-
ting improvement of the ability to secrete given enzymes, inhibit or kill
plant pathogens, or stimulate plant growth and resistance to disease attack
(Brunner et al., 2005). The research conducted on Trichoderma spp. in the
past 20 years has revealed numerous molecular and genetic mechanisms
involved in the many beneficial or useful biological processes involving
these fungi. However, regardless of the obvious potential there are some
difficulties that limit the development and application of these biopesti-
cides. In addition to the lack of effective strains and the availability of
correct formulations, accessibility is often limited in: (1) basic information
required for product registration, such as a sufficient knowledge of the
mechanisms of action; (2) efficacy testing in the geographic regions of
the country where the product is to be registered; (3) methods for monitor-
ing the production of eventual toxic metabolites produced by these fungi
that permit an evaluation of possible risks derived from a large scale
application.

Trichoderma a beneficial fungal antagonist

The mechanisms utilized by Trichoderma in the interaction with
the plant and other microbes are very complex and include mycopara-
sitism (necrotrophic direct fungus–fungus interaction), competition for

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:34 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
 A novel understanding of the three-way interaction 295

nutrients, antibiosis, inhibition of pathogen enzymes, antagonism against

nematodes, colonization of the rhizosphere, phyllosphere and spermo-
sphere, promotion of plant growth and root development, and induction
of systemic resistance against different pathogens (Harman, 2000). Several
cell wall degrading enzymes (chitinases and glucanases) (Limon et al.,
1996; Lorito and Woo, 1998; Lorito et al., 2001; Woo et al., 1999) and
antibiotics have been included among the factors that play a main role in
this complex biological process (Lorito et al., 1996; Schirmböck et al.,
1994). Different types of molecules functioning as ‘biocontrol/antagonism
inducers’ or elicitors for both Trichoderma and the plant have been found
to be released by the antagonist and by the activity of its lytic enzymes on
the cell wall of the fungal host (see below). In addition, Trichoderma sends
a variety of biochemical signals to the plants including avirulence factors,
as discussed below.
Several cell wall degrading enzymes are being used today, both directly
as proteins or through gene expression, for the development of new
defence strategies against phytopathogenic fungi (Lorito et al., 2001;
Lorito et al., 1998). Especially promising is the use of enzyme mixtures
capable of degrading the fungal cell wall that have been found to be active
against a wide range of fungi. To date, no resistant phytopathogenic fungi
have been discovered, and these fungal enzymes are considered to be
amongst the most potent antifungal compounds known (in comparison
to similar plant and bacterial products). Trichoderma spp. produce large
amounts of these lytic enzymes, which are stable at room temperature,
have efficacy levels similar to those of commercial fungicides and maintain
biological activity even if applied ectopically to plants in greenhouse or
produced in post-harvest (see Lorito & Scala, 1999 for a review). Moreover,
these fungal enzymes are strongly synergistic with synthetic fungicides of
different classes, in particular azole and other cell membrane affecting
compounds (Lorito et al., 1996). Moreover, Trichoderma strains produce
a large number of antibiotics, including gliotoxin and viridin, alpha-
pyrones, terpenes, polyketides, isocyanide derivatives, piperacines and
complex families of peptaibols (Sivasithamparam & Ghisalberti, 1998).
Many of these antibiotics are synergistic when combined with cell wall
degrading enzymes (those originating from Trichoderma and other sources)
and produce a strong inhibitory effect on many fungal plant pathogens
(Fogliano et al., 2002; Lorito et al., 1996; Schirmböck et al., 1994).
Competition for space or specific infection sites, nutrients and other
factors necessary for growth in the rhizosphere may also be another
mechanism through which antagonistic microbes control plant pathogens.

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:34 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
296 S. L. Woo et al.

Although competition for space has not been clearly demonstrated in the
case of Trichoderma, there is evidence showing that it plays a role in
biocontrol when these antagonists establish their dominance in a specific
environmental niche. T. harzianum is able to control the attack of Botrytis
cinerea on grapes by colonizing blossom tissue and excluding the pathogen
from its infection site (Gullino, 1992). The mechanisms of the competition
for nutrients such as carbon, nitrogen and iron, root and seed exudates
have been better defined. Competition for carbon and nitrogen between
T. harzianum and F. oxysporum f. sp. melonis on rhizosphere colonization
has been identified as the most important mechanism involved in the
biocontrol of this vascular disease. It was demonstrated that all the other
possible modes of action played little or no role in this case (Sivan & Chet,
1989). Mechanisms by which antagonists parasitize other fungi have been
thoroughly studied by using Trichoderma spp. Many studies, recently
performed by using different gene expression reporter systems, have
visualized the presence and the activity of propagules of the antagonist
in different fungal structures of the pathogen, including sclerotia (Lu
et al., 2004; Mach et al., 1999).
Many studies indicate that Trichoderma spp. act not only as antagonists
against plant pathogens, but also as agents to alter plant disease suscept-
ibility. The ability of Trichoderma species to stimulate defence responses
in the host plant has been clearly observed: in cotton with strains of
T. virens (Howell et al., 2000), in cucumber with some strains of T. harzianum,
in pepper (Sid Ahmed et al., 2000) and in tobacco (Chang et al., 1997)
against various phytopathogens including Rhizoctonia solani and some
Phytophthora species. The Trichoderma treatments to different plant
hosts caused the increase of ex-novo synthesis of compounds involved in
active defence mechanisms, such as the production of terpenoids (Howell
et al., 2000), capsidiol (Sid Ahmed et al., 2000), as well as peroxidases,
beta-glucosidase, cellulase and chitinase activities (Yedidia et al., 2000).
For example, in grape cell suspension cultures, an elicitor produced by
T. viride was able to activate defence responses resulting in cellular
necrosis and an increase in resveratrol concentration, an important phyto-
alexin produced following B. cinerea and Plasmopara viticola infec-
tions (Calderon et al., 1993; Dai et al., 1995).
There are many aspects of the molecular mechanisms that Trichoderma
utilizes in its relationship with plants and other microbes that are
not understood due to the complexity of the processes involved, but
also because many of the recent and potent tools available for investi-
gating molecular-genetic and biochemistry techniques (such as EST

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:34 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
 A novel understanding of the three-way interaction 297

sequencing, DNA array and miniarray or proteome analysis) have yet to

be fully implemented to study the mechanisms of biocontrol of fungal
antagonists.

Interactions among plants, pathogens and antagonists

In comparison to the relationship between plants and antagonists,
the mechanisms involved in the interaction between the plant and fungal
pathogens have been investigated extensively and they are much better
understood. The involvement of different genes and other factors have
been identified as having a role in various aspects of avirulence and
pathogenicity on the part of the microorganisms or in the process of
resistance on the side of the plant. The molecular cross-talk that involves
pathogen elicitors and plant receptors has been very well studied (Dangl &
Jones, 2001). Clearly, in order to identify genetic and molecular factors
that regulate the three-way plant–pathogen–antagonist interaction
requires an holistic approach aimed at investigating the changes in gene
expression that occur in each of the three partners involved in this inter-
action, singly and then in all combinations. This approach parallels the
natural crop conditions where plants are exposed contemporaneously to a
multitude of microorganisms, pathogens and antagonists (in addition to
symbionts), and disease development is a consequence of this complex,
multivariate interaction.
The means for studying this three-way relationship can be determined
by examining the information available from work involving the mecha-
nisms of action in both the plant–pathogen and the plant–antagonist
interactions, and using this information for developing new genetic and
molecular tactics to analyze the multicomponent interaction. Present
techniques used in post-genomic analysis with the development of EST
sequences, gene library, arrays, proteomic maps and transcriptomic anal-
ysis are opportune for identifying and characterizing new genes, proteins
and signal molecules involved in the process, which have potential utility in
developing novel strategies for plant protection (Lorito et al., 2001; Rey
et al., 2004).
Pathogen attack of the plant instigates various mechanisms of resistance
that generally involves the activation of several localized responses at the
infection site, such as the ‘oxidative’ and ‘nitrosative’ burst, characterized
by a rapid accumulation of reactive oxygen species (ROS) and nitric oxide
(NO) (Sandermann, 2000), the induction of genes involved in phytoalexin
biosynthesis or the encoding of several pathogenesis-related proteins (PR
proteins), the production of mechanical structures to reinforce plant

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:35 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
298 S. L. Woo et al.

tissues or block pathogen movement, and the activation of the hyper-

sensitive response (HR) (Keen, 2000).
Plant perception of pathogen presence is mediated by signals produced
by the pathogen (elicitors) that are perceived by specific plant receptors.
It is likely that similar molecular mechanisms to activate specific genes
also occur during the interactions between antagonist–pathogen and
antagonist–plant. Recently, it has been demonstrated that there is a produc-
tion of mycoparasitism inducers that are released from the pathogen or the
plant and are ‘detected’ by the antagonist, although the specific molecules
and the genes involved in the process have yet to be identified. Furthermore,
several preliminary observations suggest that specific oligosaccharides
derived from the pathogen or plant cell wall (or even from Trichoderma
itself) are able to activate the process of biocontrol in Trichoderma spp., and
induce at the same time systemic resistance in the plant. Potentially, these
molecules (proteins and low molecular weight compounds) may be applied
as ‘elicitors’ of biocontrol to stimulate resident fungal antagonist popula-
tions and defence mechanisms in the plant, thus improving the efficacy of
biopesticides in the field as well as the production of enzymes or biomass in
commercial fungal strains at the industrial level.

Interaction between Trichoderma and the plant

Fungal antagonists such as Trichoderma do not perform only, as
previously described, by physically and chemically attacking phytopath-
ogens, but they can also control diseases by interacting directly with the
plant. Recently, numerous studies have demonstrated that there is an
induction of localized and systemic resistance in plants treated with fungal
antagonists, including Trichoderma spp. These plant defence responses to
pathogen attack or other injuries caused by physical or insect damage,
application of chemicals, etc., are very complex and involve signal mole-
cules such as salicylic acid, jasmonic acid, ethylene, nitrous oxide, etc.
These signals activate a cascade of reactions involving the production of
metabolites (phytoalexins etc.) and proteins (PR proteins) that are com-
ponents of plant disease resistance.
Recently, the role of rhizobacteria in plant resistance has been partially
clarified (Pieterse & van Loon, 1999). These microbes activate a specific
type of induced resistance, particular to the rhizobacteria, known as
induced systemic resistance (ISR). In ISR, PR proteins are not expressed
by the plant, and plants react more strongly to pathogen attack because of
an increased sensitivity to jasmonic acid and ethylene (Kuc, 2001). Similarly,
some fungi, such as Fusarium, Rhizoctonia, symbiontic mycorrhizal fungi

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:35 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
 A novel understanding of the three-way interaction 299

and Trichoderma, have this capacity to promote plant growth and to induce
resistance to pathogens. The first report of ISR caused by Trichoderma was
observed on tomato, lettuce, pepper, bean and tobacco plants treated with
T. harzianum strain T39 in the biocontrol of B. cinerea (De Meyer et al.,
1998). Since then, numerous studies on the induction of systemic resistance
in various plants to a variety of pathogens by the application of different
Trichoderma species has been reported, including R. solani and some
Phytophthora spp. (see Harman et al., 2004 for a review). The treatments
with the biocontrol agent were found to alter the production of substances
implicated in the active defence mechanisms; an increase in the production
of substances including peroxidases, beta-glucosidases, cellulases and chiti-
nases activities (Yedidia et al., 2000), or the ex-novo synthesis of compounds
such as terpenoids (Howell et al., 2000) and capsidiol (Sid Ahmed et al.,
2000). In grape cell cultures, an elicitor produced by T. viride activated
defence responses that resulted in cellular necrosis and accumulation of
resveratrol (Calderon et al., 1993; Dai et al., 1995).
In order to discern if the biocontrol effect of T. virens on soilborne
diseases was due to localized or systemic acquired resistance, as an alter-
native to direct mycoparasitism by the antagonist, it was necessary to use
mutants of Trichoderma with different antibiotic or mycoparasitic capa-
bilities. The biocontrol of R. solani on cotton seedlings was found to
depend upon the ability of the antagonist strain to induce the production
of phytoalexins in the plant such as hemoglossypol, deoxyhemoglossypol
and glossypol (Howell et al., 2000). These results provide a new insight into
the mechanisms involved in biocontrol and suggest that induced resistance
may, in some cases, play a more important role than direct action by an
antagonist on pathogen control (Harman et al., 2004). The activation of
induced resistance may confer protection for a longer period of time and
against a wider range of pathogens, including viruses (Harman et al.,
2004). For example, an application of T. harzianum to tomato roots was
found to reduce significantly leaf infection caused by Alternaria solani
almost three months later.
Induced resistance in plants caused by the interaction with Trichoderma
may involve different molecules (Fig. 16.1). It was noted that these fungi
produced a xylanase of 22 kDa that induced a localized resistance reaction
(Lotan & Fluhr, 1990). Several peptides and proteins produced by T. virens
were found to have elicitor activity and to induce the biosynthesis of
terpenoid phytoalexin and peroxidase activity in cotton (Howell et al.,
2000). Our preliminary results indicate that T. harzianum produces pro-
teins that may be homologous to the products of the avirulence genes Avr4

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:36 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
300 S. L. Woo et al.

and Avr9 found in the tomato pathogen Cladosporium fulvum. These pro-
teins, acting as race or pathovar specific elicitors, induce resistance responses
in plants having the matching resistance genes (Dangl & Jones, 2001).
An additional genre of molecules that are capable of inducing resistance
may originate from the interaction Trichoderma has with pathogens and
plants. Cell wall degrading enzymes secreted by the antagonist have activ-
ity both on the cell walls of the fungal pathogens as well as the plants,

Mycoparasitism and ISR

Phase 1: high MW (CWDEs) Phase 2: high and low MW

Antibiotics Antibiotics

Trichoderma
Trichoderma

host

host
CWDEs CWDEs elicitors

The pre- and post-contact events

of mycoparasitism also activate
HR and ISR in the plant

Figure 16.1 Model for a correlation between mycoparasitism and induced

systemic resistance (ISR) during multiple interactions between Trichoderma,
plants and pathogenic fungi. In the fungus–fungus interaction there may be
first a pre-contact phase 1 in which the mycoparasite secretes antibiotics and
cell wall degrading enzymes (ovals) to search the environment for a living or a
dead host/substrate. This phase can be inhibited by blocking the movement
of high molecular weight compounds, found to be chitinases, glucanases and
other lytic enzymes. When a host is found (phase 2), the degradation products
from the host cell walls (triangles) reach the antagonist and mediate the
activation of the mycoparasitism-related genes, which induces stronger
secretion of enzymes and antibiotics, chemiotactic growth toward the host,
coiling and host penetration, etc. This phase can be inhibited by blocking the
movement of low molecular weight compounds, found to be oligosaccharides
with an amino acid moiety (Lorito et al., unpublished). Arrows indicate that
the plant has developed the ability to ‘sense’ these molecular interactions
(especially during phase 2) occurring in the rhizosphere and use these signals
to activate defence responses involving hypersensitive reaction (shown
infiltration of low MW compounds in tobacco leaf) and ISR.

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:36 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
 A novel understanding of the three-way interaction 301

which consequently causes the release of oligosaccharides that may acti-

vate both the mechanisms of antagonism in Trichoderma and defence
responses in plants (Fig. 16.1). Recent results indicate that Trichoderma
culture filtrates contain macromolecules and low molecular weight com-
pounds that induce strong changes in cytosolic Ca2þ levels in plant cell
cultures and may cause programmed cell death (Mariani & Lorito, unpub-
lished data). Rhizosphere competent strains of Trichoderma are able to
colonize plant roots and penetrate the root epidermis, thus causing a
change in plant metabolism, such as an increase in the levels of plant
enzymes involved in the defence mechanisms (Harman et al., 2004). The
means by which Trichoderma limits penetration only to the outer cortex of
the plant tissue are not yet known, although there may be a correspond-
ence to its ability to activate the plant defence system by producing
avirulence (avr) like proteins and other types of elicitors. Furthermore, it
has also been reported that there is production and accumulation of
phytoalexins; in the case of cucumber, root colonization increased the
accumulation of phenolic compounds in the leaves that are inhibitory
towards Pseudomonas syringae pv. lachrymans, Agrobacterium tumefaciens,
F. oxysporum f. sp. melonis, B. cinerea and other fungi (Harman et al.,
2004). It appears that many Trichoderma strains, which maintain close
relationships with the plant, may have the intrinsic ability to act as phyto-
pathogens, and probably have all the required molecular tools to behave as
such, but instead establish beneficial interactions with the plant by stim-
ulating root development and increasing plant resistance to pathogenic
microbes. In exchange, the plant also provides some mutual positive
effects for Trichoderma by increasing the available nutrient base through
plant root exudates and limiting competition with other microbes found in
the rhizosphere.
Accumulating evidence indicates that molecular cross-talk occurs
between Trichoderma and the plant that it colonizes. When the fungus
transfers molecules to receptive plants, these compounds are recognized
and able to activate various processes such as defence to biotic attack by
microorganisms and insects, and accelerate development or resistance to
abiotic stresses caused by drought, nutrient deficiency, etc.
Today, the mechanism by which Trichoderma is able to protect the
plant is considered more important, at least in some applications, than
‘classical’ direct mycoparasitism and competition that occurs with the
phytopathogenic host. Numerous researchers in the field support the
hypothesis that ISR and not antagonistic ability may be the mechanism
that produces the better wide-spectrum performances of some commercial

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:37 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
302 S. L. Woo et al.

biocontrol strains of Trichoderma. However, much is yet to be discovered

about the molecules and genes involved in the process of ISR, and similar
mechanisms involved in plant interactions with Trichoderma, mycorrhizal
fungi, rhizobacteria or microbial pathogens. Different molecular genetic
techniques that have been utilized to study Trichoderma spp., including
homologous recombination, use of reporter genes, use of constitutive and
inducible promoters, and secretion of heterologous proteins, will be very
useful in examining its dual interaction with the plant and pathogens
(Mach et al., 1999; Lu et al., 2004; Woo et al., 1999).

Molecular cross-talk occurring between Trichoderma

and the plant
Only recently, with the advent of new technologies and a con-
certed effort of several research groups, have some of the molecules that
control the Trichoderma–plant interaction, and in particular the induced
resistance effect, been identified. Trichoderma is able to produce at least
three different types of elicitors, those comprising of (1) enzymes or
peptides; (2) avr-like proteins; and (3) oligosaccharides or low-molecular-
weight compounds released by the action of specific lytic enzymes on
fungal and plant cell walls.
The elicitors made up of different peptides and proteins vary in size from
6.2 to 42 kDa (possibly including a serine protease and a xylanase) and
induce the biosynthesis of terpenoid phytoalexins and peroxidase in the
plant (Harman et al., 2004). Data from our laboratory suggest that
among these elicitors there are proteins that Trichoderma uses for its
mycoparasitic and antagonistic activities, such as chitinases and gluca-
nases. These are secreted initially at low levels and then profusely when the
presence of a host is detected (Fig. 16.1). The plant recognizes these
compounds as foreign fungal molecules (regardless of their enzymatic
mode of action) probably by using specific receptors and resistance
genes, similar to those utilized in pathogen recognition. Perhaps the detec-
tion of the Trichoderma metabolites serves as an indicator for the plant
of the ongoing antagonist–pathogen interactions occurring in close proxi-
mity and the necessity to activate the defence system because of the risk
of pathogen attack. This hypothesis is supported by the observation that
the increased resistance mediated by Trichoderma in the plant is based on a
pre-activation of the biochemical response rather than on the over-
accumulation of defence proteins.
Our recent results indicate one possibility of how some Trichoderma
strains elicit plant defences by the use of avirulence proteins. The biocontrol

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:37 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
 A novel understanding of the three-way interaction 303

strain T. atroviride P1 was transformed with the gene encoding for the Avr4
protein of Cladosporium fulvum, a leaf pathogen of tomato, under a strong
constitutive or inducible promoter. The Avr4-secreting Trichoderma mutants
over-expressing the transgene were applied to the seeds of tomato cultivars
containing and not containing the corresponding Cf4 resistance gene.
Fewer plants emerged from the Cf4 containing seeds, and these seedlings
were stunted and less healthy than those emerging from the non-Cf4-
containing seeds, which did not differ from the untreated controls. When
Trichoderma avr mutants were applied to the roots of mature Cf4 plants,
necrotic zones rapidly appeared on the root and leaf surface that resembled
HR (Ruocco & Lorito, unpublished data). From a biotechnological view-
point, avr genes from biocontrol fungi are attractive because they can
potentially provide protection against a large spectrum of pathogens, by
transforming plants or beneficial microbes with the Trichoderma avr genes,
or ectopically applying the avr products to stimulate plant resistance.
The third type of elicitors are molecular compounds released from the
pathogen and the plant cell wall by the action of Trichoderma cell wall
degrading enzymes. These molecules not only stimulate antagonistic activ-
ity in the fungus by activating the mycoparasitic gene expression cascade,
but they also act as elicitors of plant defence when plant cells are exposed
to them or when injected in the root and leaf surface (Woo & Lorito,
unpublished data, Figure 16.1). Some of these compounds were found to
be comprised of short oligosaccharides made of two types of monomers
either with or without an amino acid residue. This provides a novel
possibility of using the enzymes secreted by Trichoderma, rather than the
fungus itself, to induce ISR in the plant. This enzyme treatment may
stimulate the activity of resident or introduced Trichoderma populations
by the release of fungal inducers and activate the ISR system in the plant
by the release of plant elicitors, as well as having an antimicrobial effect
in the application zone.
Finally, the understanding of the mechanism of interaction between
Trichoderma and the plant has provided for the first time the opportunity
to genetically increase the ability of a T. atroviride strain to induce ISR. We
were able to over-express a glucose oxidase gene from Aspergillus niger in
Trichoderma strain P1, and obtained mutants that, in vitro and in vivo
experiments, performed much better than the wild type both as a myco-
parasite and as an ISR inducing agent (Brunner et al., 2005). In this case,
bean seeds treated with the spores from the mutant produced plants that
were more resistant to leaf infection by B. cinerea when compared to those
treated with the wild type Trichoderma. Probably, the high glucose oxidase

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:37 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
304 S. L. Woo et al.

production by the genetically-improved Trichoderma catalyzed the pro-

duction of hydrogen peroxide and reactive oxygen species that systemi-
cally induced the plant defence mechanisms.

Effect of Trichoderma on plant growth improvement

Many microorganisms, such as rhizobacteria, are capable of pro-
ducing beneficial effects to plants when they colonize the roots resulting
in increased growth and yields (Figs. 16.2, 16.3 and 16.4). The promotion
of both root development and growth in a wide range of plants has
been noted with rhizosphere competent strains of Trichoderma (Harman
et al., 2004). In particular, the effects of some commercial strains, such as
T. harzianum T22, have been well studied on important crops like maize,
whereby seeds treated with the antagonist produced plants that had much

T
c
T

Figure 16.2 Plant growth promotion effect of Trichoderma spp. strains on

greenhouse cultivated lettuce. T (and boxed zone) ¼ Trichoderma seed-
treated plants, c ¼ untreated controls.

A B

Figure 16.3 Plant growth promotion effects of Trichoderma spp. strains

on greenhouse cultivation of tomato (A) and zucchini (B). In each case,
left-hand rows contain untreated controls and right-hand rows contain
plants treated with Trichoderma.

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:38 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
 A novel understanding of the three-way interaction 305

deeper rooting compared to untreated controls and were also more toler-
ant to drought (Harman & Björkman, 1998). In addition, it was shown
that this effect is accentuated when other beneficial root colonizing micro-
organisms, such as mycorrhyzal fungi, were also applied. Increased root
development caused by the antagonists may also produce a significant
increase in product yield, as observed in maize (Harman & Björkman,
1998). Trichoderma may provide an advantage to crops that are grown in
less than optimum field conditions with various biotic stresses and
thus substantially improve production. However, it should be noted that
the results differed with the lines of maize tested, and in some cases
Trichoderma even caused a reduction in plant growth and yield (Harman
& Björkman, 1998). This was also found to be the case with tomato in
which the different genetic background of the eight tested plant varieties
reacted differently to a strain of T. atroviride and one of T. harzianum
(Fig. 16.4 C and D) (Ruocco & Lorito, unpublished). This suggests that
more studies are required to clarify the mechanisms involved in the

T c

C D

c
T(P1) T(T22) T(P1) T(T22)
c

Figure 16.4 Plant growth promotion effects of different Trichoderma spp.

strains on pot-grown tomato (T ¼ Trichoderma-treated seeds, c ¼ untreated
controls). (A) and (B), effect of T. harzianum strain T22 on stem and root
development. (C) and (D), effect of T. atroviride strain P1 and T. harzianum
strain T22 on two different tomato cultivars.

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:38 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
306 S. L. Woo et al.

Trichoderma–plant interaction to identify the most useful strains and crop

combinations that produce desirable results.
The mechanisms that Trichoderma uses to increase root and shoot
growth and yields may depend on several physiological processes, such as
its ability to make nutrients more available for plant uptake, or degrade
toxic compounds. Trichoderma are able to degrade hydrogen cyanide
(HCN) either present in soil or produced by some deleterious rhizobacteria
(Harman et al., 2004). They are also able to produce a diffusible metabolite
that has plant hormone characteristics (Windham et al., 1986). Further,
Trichoderma is able to solubilize minor nutrients like Mn4þ, Cu2þ and
Fe3þ by reducing them to the soluble forms Mn2þ, Cu1þ and Fe2þ, making
these minerals available for the plant (Altomare et al., 1999). Similar
results were observed with nitrogen, whereby Trichoderma treated plants
produced maximum yields even when 40% less nitrogen-containing fertil-
izer was supplied. Therefore, Trichoderma may be able to improve nitrogen-
uptake efficiency of plants and, consequently, reduce nitrogen pollution in
soil and water (Harman et al., 2004).

Conclusions
The mechanisms by which Trichoderma spp. are able to control
plant diseases or stimulate resistance in the plant are just starting to be
elucidated. In the complex association between this fungal antagonist, the
plant and pathogens there is continuous molecular cross-talk maintained
among the participants. The presence or absence of a member will alter the
secretion of different compounds, biochemical and biological activities,
perception or receptivity to the released by-products and consequent
responses of antagonism by Trichoderma and/or defence response by the
plant. The beneficial effects to the plant are not limited to disease control,
but also include increased growth and development. The potential of
applications utilizing Trichoderma as a bioagent (fungicide, stimulant,
protectant, etc.), and its by-products, alone or in combination with exist-
ing technology and commercial products, is expanding as our knowledge
increases about the mechanisms involved in this three-way relationship.

References
Altomare, C., Norvell, W. A., Björkman, T. & Harman, G. E. (1999). Solubilization of
phosphates and micronutrients by the plant-growth promoting and biocontrol
fungus Trichoderma harzianum Rifai strain 1295–22. Applied and Environmental
Microbiology, 65, 2926–33.

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:38 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
 A novel understanding of the three-way interaction 307

Brunner K., Zeilinger, S., Ciliento, R., Woo, S. L., Lorito, M., Kubicek, C. P. & Mach, R. L.
(2005). Genetic improvement of a fungal biocontrol agent to enhance both
antagonism and induction of plant systemic disease resistance. Applied and
Environmental Microbiology, 71, 3959–65.
Calderon, A. A., Zapata, J. M., Munoz, R., Pedreno, M. A. & Barcelo, A. R. (1993).
Resveratrol production as a part of the hypersensitive-like response of grapevine
cells to an elicitor from Trichoderma viride. New Phytologist, 124, 455–63.
Chang, P.-F. L., Xu, Y., Narasimhan, M. L., Cheah, K. T., D’Urzo, M. P., Damsz, B.,
Kononowicz, A. K., Abad, L., Hasegawa, P. M. & Bressan, R. A. (1997).
Induction of pathogen resistance and pathogenesis-related genes in tobacco by a
heat-stable Trichoderma mycelial extract and plant signal messengers. Physiologia
Plantarum, 100, 341–52.
Dai, G. H., Andary, C., Mondolot-Cosson, L. & Boubals, D. (1995). Involvement of phenolic
compounds in the resistance of grapevine callus to downy mildew (Plasmopara
viticola). European Journal of Plant Pathology, 101, 541–7.
Dangl, J. L. & Jones, J. D. G. (2001). Plant pathogens and integrated defence responses to
infection. Nature, 411, 826–33.
De Meyer, G., Bigirimana, J., Elad, Y. & Hofte, M. (1998). Induced systemic resistance in
Trichoderma harzianum T 39 biocontrol of Botrytis cinerea. European Journal of
Plant Pathology, 104, 279–86.
Fogliano, V., Ballio, A., Gallo, M., Woo, S. L., Scala, F. & Lorito, M. (2002). Pseudomonas
lipodepsipeptides and fungal cell wall degrading enzymes act synergistically in
biocontrol. Molecular Plant Microbe Interactions, 15, 323–33.
Gullino, M. L. (1992). Control of Botrytis rot of grapes and vegetables with Trichoderma
spp. In Biological Control of Plant Diseases, Progress and Challenges for the
Future, eds. E. C. Tjamos, G. C. Papavizas & R. J. Cook. New York: Plenum
Press, pp. 125–32.
Harman, G. E, Lorito, M. & Lynch, J. M. (2004). Uses of Trichoderma spp. to alleviate or
remediate soil and water pollution. Advances in Applied Microbiology, 56, 313–31.
Harman, G. E. (2000). The dogmas and myths of biocontrol. Changes in perceptions based on
research with Trichoderma harzianum T-22. Plant Disease, 84, 377–93.
Harman, G. E. & Kubicek, C. P. (1998). Trichoderma and Gliocladium, Vol. 2, Enzymes,
Biological Control and Commercial Applications. London: Taylor and Francis.
Harman, G. E. & Björkman, T. (1998). Potential and existing uses of Trichoderma and
Gliocladium for plant disease control and plant growth enhancement. In
Trichoderma and Gliocladium, Vol. 2, eds. G. E. Harman & C. P. Kubicek. London:
Taylor and Francis, pp. 229–65.
Harman, G. E., Howell, C. R., Viterbo, A., Chet, I. & Lorito, M. (2004). Trichoderma
species – Opportunistic, avirulent plant symbionts. Nature Reviews Microbiology,
2, 43–56.
Howell, C. R., Hanson, L. E., Stipanovic, R. D. & Puckhaber, L. S. (2000). Induction of
terpenoid synthesis in cotton roots and control of Rhizoctonia solani by seed
treatment with Trichoderma virens. Phytopathology, 90, 248–52.
Inbar, J., Abramsky, M., Coen, D. & Chet, I. (1994). Plant growth enhancement and disease
control by Trichoderma harzianum in vegetable seedlings grown under commercial
conditions. European Journal of Plant Pathology, 337–46.
Keen, N. T. (2000). A century of plant pathology, a retrospective view on understanding host-
parasite interactions. Annual Review Phytopathology, 38, 31–48.
Kubicek, C. P. & Harman, G. E. (1998). Trichoderma and Gliocladium Vol. 1, Basic Biology,
Taxonomy and Genetics. London: Taylor and Francis.
Kuc, J. (2001). Concepts and direction of induced systemic resistance in plants and its
application. European Journal of Plant Pathology, 107, 7–12.

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:39 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
308 S. L. Woo et al.

Limón, M. C., Llobel, A., Pintor Toro, J. A. & Benı́tez, T. (1996). Overexpression of chitinase
by Trichoderma harzianum strains used as biocontrol fungi. In Chitin Enzymology,
Vol. 2, ed. R. A. A. Muzzarelli. Ancona, Italy: AtecEdizioni.
Lorito, M. & Scala, F. (1999). Microbial genes expressed in transgenic plants to improve
disease resistance. Journal of Plant Pathology, 81, 73–88.
Lorito, M. & Woo, S. L. (1998). Advances in understanding the antifungal mechanism(s) of
Trichoderma and new applications for biological control. In Molecular Approaches
in Biological Control, eds. B. Duffy, U. Rosenberger and G. Défago. Dijon, France:
IOBC wprs Bulletin/Bulletin OILB srop, Vol. 21 (9), pp. 73–80.
Lorito, M., Scala., F., Zoina, A. & Woo, S. L. (2001). Enhancing biocontrol of fungal pests by
exploiting the Trichoderma genome. In Enhancing Biocontrol Agents and Handling
Risks, eds. M. Vurro & J. Gressel. Amsterdam: IOS Press, pp. 248–59.
Lorito, M., Woo, S. L., Garcia Fernandez, I., Colucci, G., Harman, G. E., Pintor-Toro, J. A.,
Filippone, E., Muccifora, S., Lawrence, C. B., Zoina, A., Tuzun, S. & Scala, F.
(1998). Genes from mycoparasitic fungi as a source for improving plant resistance
to fungal pathogens. Proceedings of the National Academy of Sciences of USA, 95,
7860–5.
Lorito, M., Woo, S. L. & Scala, F. (2004). Le biotecnologie utili alla difesa sostenibile delle
piante, i funghi. Agroindustria, 3, 181–95.
Lorito, M., Woo, S. L., D’Ambrosio, M., Harman, G. E., Hayes, C. K., Kubicek, C. P. &
Scala, F. (1996). Synergistic interaction between cell wall degrading enzymes and
membrane affecting compounds. Molecular Plant-Microbe Interactions, 9,
206–13.
Lotan, T. & Fluhr, R. (1990). Xylanase, a novel elicitor of pathogenesis-related proteins in
tobacco, uses a non-ethylene pathway for induction. Plant Physiology, 93, 811–17.
Lu, Z., Tombolini, R., Woo, S. L., Zeilinger, S., Lorito, M. & Jansson, J. (2004). In vivo study
of Trichoderma-pathogen interactions using constitutive and inducible green
fluorescent protein reporter systems. Applied and Environmental Microbiology, 70,
3073–81.
Mach, R. L., Peterbauer, C. K., Payer, K., Jaksits, S., Woo, S. L., Zeilinger, S., Kullnig,
C. M., Lorito, M. & Kubicek, C. P. (1999). Expression of two major chitinase genes
of Trichoderma atroviride (T. harzianum P1) is triggered by different regulatory
signals. Applied and Environmental Microbiology, 65, 1858–63.
Pieterse, C. M. J. & van Loon, L. C. (1999). Salicylic acid-independent plant defense
pathways. Trends in Plant Science, 4, 52–8.
Rey, M., Llobell, A., Monte, E., Scala, F. & Lorito, M. (2004). Genomics of Trichoderma. In
Applied Mycology and Biotechnology, Vol. 4, Fungal Genomics, eds. D. K. Arora &
G. G. Khachatourians. Elsevier Science, pp. 225–48.
Sandermann, H. (2000). Active oxygen species as mediators of plant immunity: three case
studies. Biological Chemistry, 381, 649–53.
Schirmböck, M., Lorito, M., Wang, Y.-L., Hayes. C. K., Arisan-Atac, I., Scala, F., Harman,
G. E. & Kubicek, C. P. (1994). Parallel formation and synergism of hydrolytic
enzymes and peptaibol antibiotics, molecular mechanisms involved in the
antagonistic action of Trichoderma harzianum against phytopathogenic fungi.
Applied and Environmental Microbiology, 60, 4364–70.
Sid Ahmed, A., Sánchez, C. P. & Candela, M. E. (2000). Evaluation of induction of systemic
resistance in pepper plants (Capsicum annuum) to Phytophthora capsici using
Trichoderma harzianum and its relation with capsidiol accumulation. European
Journal of Plant Pathology, 106, 817–24.
Sivan, A. & Chet, I. (1989). The possible role of competition between Trichoderma harzianum
and Fusarium oxysporum on rhizosphere colonization. Phytopathology, 79,
198–203.

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:39 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016
 A novel understanding of the three-way interaction 309

Sivasithamparam, K. & Ghisalberti, E. L. (1998). Secondary metabolism in Trichoderma and

Gliocladium. In Trichoderma and Gliocladium, Vol. 2, eds G. E. Harman & C. P.
Kubicek. London, UK: Taylor and Francis Ltd., pp. 139–91.
Windham, M. T., Elad, Y. & Baker, R. (1986). A mechanism for increased plant growth
induced by Trichoderma spp. Phytopathology, 76, 518–21.
Woo, S. L., Donzelli, B., Scala, F., Mach, R., Harman, G. E., Kubicek, C. P., Del Sorbo, G. &
Lorito, M. (1999). Disruption of the ech42 (endochitinase-encoding) gene affects
biocontrol activity in Trichoderma harzianum P1. Molecular Plant-Microbe
Interactions, 12, 419–29.
Yedidia, I., Behamou, N., Kapulnik, Y. & Chet, I. (2000). Induction and accumulation of PR
proteins activity during early stages of root colonization by the mycoparasite
Trichoderma harzianum strain T-203. Plant Physiology and Biochemistry, 38,
863–73.

Downloaded from Cambridge Books Online by IP 85.122.17.211 on Wed Apr 27 11:15:40 BST 2016.
http://dx.doi.org/10.1017/CBO9780511902451.017
Cambridge Books Online © Cambridge University Press, 2016