JOURNAL OF PLANT PROTECTION RESEARCH

Vol. 53, No. 2 (2013)

DOI: 10.2478/jppr-2013-0019

SCREENING OF ANTIMICROBIAL AND ANTIOXIDANT

SECONDARY METABOLITES FROM ENDOPHYTIC FUNGI
ISOLATED FROM WHEAT (TRITICUM DURUM)

Nouari Sadrati, Harzallah Daoud*, Amina Zerroug, Saliha Dahamna, Saddek Bouharati

University Ferhat Abbas Sétif 1, Laboratory of Applied Microbiology, Faculty of Natural Sciences and Life
Sétif 19000, Algeria

Received: August 31, 2012

Accepted: March 20, 2013

Abstract: The emergence of antibiotic-resistant micro-organisms calls for inventive research and development strategies. Inhibition
of these pathogenic micro-organisms may be a promising therapeutic approach. The screening of antimicrobial compounds from
endophytes is a promising way to meet the increasing threat of drug-resistant strains of human and plant pathogens. In the present
study, a total of 20 endophytic fungi and 23 endophytic actinomycetes have been isolated from wheat (Triticum durum). Mohamed Ben
Bachir variety collected from Bordj Bou Arreridj region (Algeria) during winter 2010. The isolates were screened and evaluated for
their antimicrobial and antioxidant activities. Antimicrobial activity was evaluated for crude ethyl acetate extracts using an agar dif-
fusion assay against twelve pathogenic bacteria, yeast, and two phytopathogenic fungi. All extracts showed inhibitory activity on at
least one or more pathogenic microorganisms, with an average zone of inhibition varied between 7 mm to 25 mm, and the largest zone
was of 25 and 25.3 mm against candida albicans and Escherichia coli respectively. The antioxidant capacity of the extracts was evalu-
ated by β-carotene/linoleic acid assay. Results showed that 60% of these extracts have antioxidant activity, exhibiting 50, 57% to 78,
96% inhibitions. While the inhibitory activity for oxidation of linoleic acid of 40% of them was less than 50%. From the present work
it is possible to conclude that these microorganisms could be promising source of bioactive compounds, and warrant further study.

Key words: endophytic microorganisms, antimicrobial activity, antioxidant activity, Triticum durum

INTRODUCTION and Carroll (1977) reported the presence of endophytes

There is an ever-growing need for new and useful in needles of Pseudotsuga menziesii (Rajagopal et al. 2012).
compounds to provide assistance and relief in all aspects Endophytes are microorganisms that include bacteria
of the human condition. Both human pathogens and fun- and fungi living within plant tissues without causing any
gal phytopathogens are prone to develop ‘‘drug’’ resis- immediate overt negative effects. Endophytes have been
tances. The effectiveness of the older types of antibiotics found in every plant species examined to date. These mi-
can decrease substantially. In addition, because of safety croorganisms are recognized as potential sources of novel
and environmental problems, many synthetic agricul- natural products for exploitation in medicine, agricul-
tural agents have been and still are targeted for remov- ture, and industry. More bioactive natural products may
al from the market. The removal of such agents creates be isolated from the microorganisms (Kumar and Sagar
a need to find alternative ways to control farm pests and 2007). Endophytes are ubiquitous and have a rich biodi-
pathogens. There is an urgent need to work towards the versity. It is noteworthy, that of the nearly 300,000 plant
invention of safer antifungal agents which are expected to species that exist on the earth, each individual plant is the
be renewable, non-petrochemical, naturally eco-friendly, host to one or more endophytes (Strobel and Daisy 2003).
and easily obtainable (Demain 2000; Liu et al. 2001). In view of the special colonization in certain hosts, it is
Natural products are adapted to a specific function in estimated that there may be as many as 1 million differ-
nature. Thus, the search for novel secondary metabolites ent endophyte species. However, only a handful of them
should concentrate on organisms that inhabit novel bio- have been described (Andrew and Hirano 1991). This
types. Endophytic fungi inhabit a biotype that is not well means the opportunity to find new and targeting natural
studied (Nithya and Muthumary 2011). The presence of products from interesting endophytic microorganisms,
endophytic fungi in plant tissues was discovered more among the myriad of plants in different niches and eco-
than 75 years ago when Sampson (1935) reported such systems, is great.
fungi from Lolium grass. The contemporary resurgence Endophytes are the chemical synthesizers inside
of research on endophytic fungi began when Bernstein plants (Owen and Hundley 2004). Many of them are

*Corresponding address:
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 Screening of antimicrobial and antioxidant secondary metabolites from endophytic fungi isolated… 129

capable of synthesizing bioactive compounds that can

be used by plants for defense against pathogens. Some MATERIALS AND METHODS
of these compounds have been proven useful for novel
drug discovery (Guo et al. 2008). The possibility that en- Samples collection and isolation of strains
dophytes can biosynthesize associated plant compounds Roots and leaves of wheat (T. durum) from the Mo-
was first comprehended and published by Stierle et al. hamed Ben Bachir variety were collected from the Bordj
(1993). The publication followed the highly heralded dis- Bou Arreridj region (Algeria) during the winter of 2010.
covery of endophytic Taxomyces andreanae that produces Each sample was placed in a separate sterilized bag and
the multi-billion dollar anticancer compound Taxol® brought back to the laboratory. Samples were processed
(generic name: paclitaxel). This compound was isolated within 24 hours from when they were collected (Tejesvi
from the Pacific yew tree Taxus brevifolia. Inspired by this et al. 2007). Samples from leaves and roots were washed
discovery, numerous efforts have been made to identify under running tap water and cut into several pieces of
endophytes as sources of associated natural plant prod- approx 5 mm diameter. Pieces were surface-sterilized by
ucts. Many scientists have become increasingly interested the immersion sequence of: 96% ethanol for 1 min, so-
in studying fungal endophytes as potential producers dium hypochlorite (2% available chlorine v/v) for 3 min,
of novel and biologically active compounds. In the past 96% ethanol for 30 seconds (Larran et al. 2007), and then
two decades, many valuable bioactive compounds with finally rinsed twice in sterile distilled water. Ten pieces
antimicrobial, insecticidal, cytotoxic, and anticancer ac- per organ were placed in each Petri dish (a dish contained
tivities have been successfully discovered from the endo- 2% Potato Dextrose Agar (PDA) supplemented with
phytic fungi. These bioactive compounds could be classi- 250 mg/l chloramphenicol and Starch Casein Agar (SCA)
fied as alkaloids, terpenoids, steroids, quinones, lignans, to isolate the fungi and the actinomycetes, respectively.
phenols, and lactones (Zhang et al. 2006; Xu et al. 2008). Five replicates (Petri dishes) of each sample organ were
Endophytes producing Podophyllotoxin (PDT), a well- made. Dishes were then incubated at 26±2°C (Larran et al.
known aryltetralin lignan with potent anticancer, antivi- 2007; Zin et al. 2007). The plates were checked each day
ral, antioxidant, antibacterial, immunostimulation, and after inoculation and any fungi or actinomycetes that ap-
anti-rheumatic properties, are obtained from endophytic peared were isolated, purified, and then maintained at
fungus Alternaria sp. isolated from Sinopodophyllum, and 4°C on PDA and Nutrient Agar (NA) slopes, respectively,
endophytic fungus Fusarium oxysporum obtained from Sa- for further identification.
bina recurva (Gao et al. 2007; Kour et al. 2008). Endophytes
producing Camptothecin (CPT), an antineoplastic agent, Antagonistic study and identification of endophytes
are obtained from Entrophospora infrequens (Puri et al. All the 43 pure isolates of endophytic fungi and aci-
2005). Endophytes producing immunosuppressives, for nomycetes were screened for their in vitro antagonism
example, subglutinols A and B, are produced by Fusarium against phytopathogenic fungi, Phytophthora infestans and
subglutinans (Lee et al. 1995). Pestacin and isopestacin as Fusarium oxysporum f. sp. albedinis by dual culture, ac-
antioxidant were separated from Pestalotiopsis microspora cording to the method described by Orole and Adejumo
associated with Terminalia morobensis (Harper et al. 2003). (2009), and Srividya et al. (2012). The percentage reduc-
The most frequently encountered endophytes are tion in radial growth was calculated for each endophyte
fungi (Staniek et al. 2008). Endophytic actinobacteria as follows:
have been also isolated from a variety of healthy plant Percentage of inhibition zones
species ranging from crop plants, such as wheat, rice, po-
tato, carrot, tomato, and citrus. Endophytic actinobacte-
ria are relatively unstudied and are also potential sourc-
es of novel natural products for exploitation in medicine,
agriculture, and industry (Strobel et al. 2004). Endophyte where:
association offers the greatest potential for biocontrol A – radius of the pathogen in the control plate,
programmes because these fungi are integrated into B – radius of pathogen in the dual culture plate.
host systems (Cao et al. 2005; Clay 1989). Dewan and Siv- Primary screening of all 23 pure isolates of actinomy-
asithamparam (1989) reported that a fungal endophyte cetes against pathogenic bacteria was done by the per-
isolated from wheat provides the host with significant pendicular streak method (Yadav et al. 2008). Actinomy-
protection from infection by the “take all” fungus. Endo- cete was streaked on the nutrient agar as a straight line
phytes enhance plant growth (Igarashi et al. 2002), and and incubated at 27°C. After seven days of incubation,
promote plant establishment under adverse conditions test organisms (Bacillus sp., Salmonella typhi, Enterococcus
(Hasegawa et al. 2006). In Algeria, however, endophytes faecalis, Klebsiella pneumoniae, Escherichia coli, Pseudomo-
of crop plants have not been studied. The main aim of nas aerugenosa and Staphylococcus aureus) were streaked
the study was to isolate endophytic fungi and actinomy- perpendicular to the streak line. After 24 hours of incu-
cetes from leaves and roots of wheat (Triticum durum), bation at 37°C, the microbial inhibitions were observed
and extract bioactive secondary metabolites with the use by determining the inhibition distance (in mm) between
of solvent, then determine the antibacterial and antioxi- the endophytic actinomycetes and pathogenic bacteria in
dant activities. dual cultures.
Certain pure cultures of the endophyte fungal isolates
that proved strong antagonism against phytopathogenic
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130 Journal of Plant Protection Research 53 (2), 2013

fungi were selected. Identification at the genus level Antioxidant activity

was based on the morphology of the fungal culture, the In this assay, the antioxidant capacity of the extracts
mechanism of spore production, and characteristics of was evaluated by the β-carotene/linoleic acid test accord-
the spores, by following the standard mycological manu- ing the method described by Dapkevicius et al. (1998).
als (Pitt and Hocking 1985; Botton et al. 1990; Champion A stock solution of β-carotene-linoleic acid mixture was
1997). The isolates of actinomycetes were characterized prepared as follows: 0,5 mg β-carotene was dissolved in
up to genus level according to traditional morphological 1 ml of chloroform (HPLC grade) and 25 μl linoleic acid,
criteria. The characteristics of colonies on the plate, the and 200 mg Tween 40 were added. Chloroform was com-
distinctive reverse colony color, morphology of substrate pletely evaporated using a vacuum evaporator. Then, 100
and aerial hyphae, morphology and mass color of spores ml distilled water saturated with oxygen (30 min 100 ml/
as well as diffusible pigment produced were all taken into min) was added and vigorously shaken. From this reac-
consideration (Holt et al. 1994; Silva et al. 2009; Verma et al. tion mixture, 2,500 μl were dispensed into test tubes, and
2009). 350 μl portions of the extracts prepared at 2 g/l concen-
trations were added. The emulsion system was incubated
Fermentation and antimicrobial assay for 48 h at room temperature. The same procedure was
The strains showing moderate to good activity were repeated with BHT as the positive control, and with a H20
selected for secondary screening, which was performed and methanol as the negative control. After this incuba-
by the agar disc diffusion method. For this reason, Er- tion period, absorbance of the mixtures was measured
lenmeyer flasks (250 ml) containing 100 ml of Potato at 490 nm. Antioxidative capacities of the extracts were
Dextrose Broth (PDB) or Nutrient Broth (NB) were au- compared with those of BHT and H20 and methanol. The
toclaved at 121°C for 15 min. After this, PDB was in- percentage of inhibition of each extract was calculated us-
oculated with mycelium plugs from the margins of ac- ing the following formula:
tively growing cultures on PDA of the seven selected
endophytic fungi (A1W, A2W, A6W, A7W from leaves,
and A3W, A4W, A5W from roots). Nutrient broth was in-
oculated with plugs from three selected actinomycetes
growing on SCA (A8W, A9W and A10W from roots). The where:
flasks were incubated for 3 weeks on a rotary shaker at – AA% – the percentage of antioxidant activity,
150 rpm and 25°C. The fermentation broths were then – Abs test – absorbance in the presence of the extract (test),
filtered through two-folds of cheese cloths. The filtrates – Abs BHT – absorbance in the presence of the positive con-
were extracted twice with equal volumes of ethyl acetate. trol (BHT).
The organic solvent extracts were evaporated in a rotary
evaporator and then stored at 4°C until used (Kwon et Data analysis
al. 2007; Zin et al. 2007). The ethyl acetate extracts of en- Statistical analysis was done using SAS/STAT ® 9.2.
dophytic fungi were individually tested against a panel The results of the antimicrobial activity were analyzed
of microorganisms including a total of 15 microbial cul- statistically by the two-way ANOVA followed by the
tures, 3 gram positive bacteria, (Bcillus sp., S. aureus, E. Student-Newman-Keuls MULTIP-rank test to compare
faecalis), 9 gram negative bacteria (S. typhi, E. coli, Ser- the average inhibition zones of the extracts. Results of the
ratia marcescens, Enterobacter agglomerans, P. aeruginosa, antioxidant activity analysis was made by the one-way
K. pneumoniae, Stenotrophomonas maltophilia, Citrobacter ANOVA followed by the Student-Newman-Keuls MUL-
freundii, Pseudomonas sp.), 2 phytopathogenic fungi (F. TIP-rank test to compare the inhibition percentages of
oxysporum f.sp. albedinis, P. infestans) and a yeast (Candida endophytes extracts and those of the controls. The results
albicans). Test organisms were provided by the Labora- were expressed as mean ±SD, and the measures were re-
tory of Microbiology, Ferhat Abbas University. The dried peated three times (n = 3). The difference was considered
fungal extracts were dissolved in water and DMSO (9:1) statistically significant when the p value was ≤ 0,05. Colo-
to a final concentration of 1 mg/ml. Antimicrobial tests nization and isolation rates were calculated according to
were then carried out by the agar disc diffusion method Yuan et al. (2010) by the following relations:
using 100 µl of suspension containing 108 CFU/ml of Colonization rate = Total number of samples/Total
bacteria, 106 CFU/ml of yeast, and 104 spore/ml of fun- number of samples in that trial
gi spread on NA, Sabouraud Dextrose Agar (SDA) and Isolation rate = Total number of isolates yielding in
PDA medium, respectively. The discs (6 mm in diameter) the given trial/Total number of samples in that trial.
were impregnated with 20 µl of the extracts and placed
on the inoculated agar. Negative controls were prepared
using the same solvents employed to dissolve the ex- RESULTS
tracts. The inoculated plates were incubated at 37°C for
24 h for clinical bacterial strains, 48 h for yeast, and 72 h Endophytes isolation
for filamentous fungi (Baris et al. 2006). Antimicrobial ac- After the isolation, a total of 43 isolates were recovered
tivity was evaluated by measuring the zone of inhibition from 200 samples (100 segments of roots and 100 segments
against the test organisms. Each assay in this experiment of leaves), 20 of them were fungi and 23 actinomycetes.
was repeated three times. The number of isolates, rate of isolation and of colonization
obtained with roots, were higher than those obtained with
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 Screening of antimicrobial and antioxidant secondary metabolites from endophytic fungi isolated… 131

leaves. The number of fungal isolates recovered from roots greatest antagonism activity. The isolates A4W, A8W,
was 12 (60%) with a rate of 0,48% and 32% of isolation and A9W and A10W were more active against P. infestans,
colonization, respectively. Whereas, the number of isolates with an inhibition percentage that reached 54.07% as
from leaves was 8 (40%) with an isolation and colonization the maximum by the isolate A10W. While isolates A2W,
rate of 0,32% and 24%, respectively. Almost, the same re- A3W, A5W, A7W, A8W and A9W were more active
sults were obtained with the actinomycetes (Table 1). against F. oxysporum f. sp. albedinis, with an inhibition per-
centage that reached to 63,89% as the maximum by the
Antagonistic study and identification of endophytes isolate A3W (Table 2). The three isolates of actinomycetes
The isolates were tested for anti-pathogen activity A8W, A9W and A10W, were more effective against most
by the dual culture. Ten isolates (23%) demonstrated the test bacteria, especially E. coli (Table 3).

Table 1. Number of isolates, colonization, and isolation rate from different tissues

Organs of the wheat plant

leaves roots
Endophytes
number of colonization number of isolation colonization
isolation rate
isolates rate [%] isolates rate rate [%]
Endophytic fungi 8 0.32 24 12 0.48 32
Endophytic
7 0.28 20 16 0.64 48
actinomycetes

Table 2. Inhibition percentages of fungal growth by isolated endophytic fungi and actinomycetes with higher activity in the primary
screening

Inhibition percentage [%]

Isolates
Phytophthora infestans Fusarium oxysporum f. sp. albedinis
A1W 8.11 11.11
A2W 5.41 44.44
A3W 21.62 63.89
A4W 45.95 16.67
A5W 29.73 52.78
A6W 5.42 16.67
A7W 00 58.33
A8W 42.96 45.00
A9W 51.85 43.33
A10W 54.07 7.50

Table 3. Antagonistic activity of selected endophytic actinomycetes with high activity against test bacteria in the primary screening

Zone of inhibition [mm]

Pathgenic bacteria
A8W stain A9W strain A10W strain
Bacillus sp. 4 15 14
Staphylococcus aureus 6 6 7
Enterococcus faecalis 00 13 13
Salmonella typhi 8 8 6
Escherichia coli 22 23 15
Pseudomonas aerugenosa 14 7 13
Klebsiella pneumoniae 8 7 00

Table 4. Identity of selected endophytic fungi and actinomycetes isolated from leaves and roots of Triticum durum Desf.

Isolate Code Taxon Plant Organ

A1W Alternaria sp. leaves
A2W Cladosporium sp. leaves
A3W Penicillium sp. 1 roots
A4W Penicillium sp. 2 roots
A5W Aspergillus sp. roots
A6W Chaetomium sp. leaves
A7W Phoma sp. leaves
A8W Streptomyces sp. 1 roots
A9W Streptomyces sp. 2 roots
A 10 W Streptomyces sp. 3 roots
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132 Journal of Plant Protection Research 53 (2), 2013

Table 5. Antimicrobial activity shown by selected endophytic fungi and actinomycetes extracts against tests microorganisms in the
secondary screening

Inhibition Zones [mm] (mean)

ethyl acetate extracts of isolates

Streptomyces sp. 1

Streptomyces sp. 2

Streptomyces sp. 3
Cladosporium sp.

Chaetomium sp.
Penicillium sp.

Penicillium sp.

Aspergillus sp.
Pathogenic

Alternaria sp.

Phoma sp.
Micro-organisms

Gram+ Bacteria
Bacillus sp. 11.7 10.7 13.7 13.0 11.7 12.0 00 12.0 00 16.0
Staphylococcus aureus 12.0 10.7 13.0 10.7 11.7 9.7 00 00 00 00
Enterococcus faecalis 11.7 10.3 12.7 12.0 11.3 11.7 13.0 00 00 00
Gram- Bacteria
Salmonella typhi 14.3 12.3 14.3 11.3 13.3 11.7 10.0 00 00 11.0
Escherichia coli 18.3 16.0 25.3 19.0 21.7 20.0 15.0 24.5 13.0 15.3
Serratia marcescens 14.3 10.7 12.0 12.0 11.3 10.7 11.0 17.0 13.0 14.0
Enterobacter agglomerans 00 11.0 00 00 00 00 10.0 13.0 10.0 9.0
Pseudomonas aeruginosa 00 00 0.0 00 0.0 0.0 00 00 00 17.0
Klebsiella pneumoniae 12.0 9.7 13.0 12.0 11.7 12.7 14.0 00 00 00
Stenotrophomonas maltophilia 00 12.0 12.3 11.7 13.3 00 10.0 00 00 10.0
Citrobacter freundii 00 00 00 00 00 00 00 00 00 00
Pseudomonas sp. 9.3 9.0 9.0 10.3 11.3 00 00 14.0 00 00
Fungi and yeast
Candida albicans 20.7 15.0 23.0 20.0 13.0 15.0 14.0 19.0 25.0 16.0
Fusarium oxysporum f .sp. albidinis 00 00 19.0 11.0 17.0 10.0 15.0 12.0 15.0 00
Phytophthora infestans 8.0 10.0 14.0 17.0 16.0 10.0 00 16.0 14.0 15.3

(mean): average of three replicates (n = 3)

Table 6. Comparison of average inhibitions of extracts obtained by ethyl acetate and their effect on the growth of test microorganisms

Inhibition zones [mm]

Ethyl acetate extracts Test micro-organisms
G- bacteria G+ bacteria fungi all micro-organisms
Alternaria sp. 7.593 a 11.778 a 9.556 cd 8.822 abc
Cladosporium sp. 8.963 a 10.556 a 8.333 d 9.156 abc
Penicillium sp. 1 9.556 a 13.111 a 18.667 a 12.089 a
Penicillium sp. 2 8.482 a 11.889 a 16.00 ab 10.667 ab
Aspergillus sp. 9.186 a 11.556 a 15.333 abc 10.889 ab
Chaetomium sp. 6.111 ab 11.111 a 11.667 bcd 8.222 abc
Phoma sp. 7.778 a 4.333 b 9.667 cd 7.467 bc
Streptomyces sp.1 7.611 a 4.000 b 15.667 abc 8.500 abc
Streptomyces sp. 2 4.000 b 00 c 18.000 a 6.000 c
Streptomyces sp. 3 8.481 a 5.333 b 10.444 cb 8.244 abc

Means with the same letter are not significantly different at (p < 0.05)

Comparison of microscopic and macroscopic charac- Antimicrobial assay

teristics of the ten selected isolates was done by screen- All extracts showed inhibitory activity on at least one
ing with the identification keys. Thus, we were able to or more pathogenic microorganisms. The average zone
identify these isolates at the genus level. The isolate of inhibition varied between 7 mm to 25 mm. The larg-
A5W was found to belong to the Aspergillus sp. genus, est zone was of 25 mm against E. coli and C. albicans by
A1W to the genus Alternaria sp., both A3W and A4W Penicillium sp. 1 and Streptomyces sp. 2, respectively. Six
to the genus Penicillium sp., A2W to the genus Cladospo- isolates were active against all Gram positive: Bacillus
rium sp., A6W to the genus Chaetomium sp., and A7W to sp., S. aureus, E. faecalis, and fungal species F. oxysporum
the genus Phoma sp. The three isolates A8W, A9W, and f. sp. albedinis and P. infestans. From the 9 Gram negative
A10W were found to belong to the genus Streptomyces species utilized, the extracts were more active against
sp. (Table 4). 5 species only, which were: S. typhimurium, E. coli, S. marc-
escens, K. pneumoniae and S. maltophilia. The other 4 spe-
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 Screening of antimicrobial and antioxidant secondary metabolites from endophytic fungi isolated… 133

cies: E. agglomerans, P. aeruginosa, C. freundii, Pseudomonas Antioxidant assay

sp. were not sensitive to most of the extracts (Table 5). The The crude extracts of fungi and Streptomycetes were
comparison of the mean inhibition zones of endophytes evaluated for their capacity of antioxidant activity us-
extracts obtained by ethyl acetate and their effect on the ing the β-carotene/linoleic acid system oxidation. After
growth of different groups of pathogenic microorgan- 24 hours of incubation, the results showed that some of
isms are presented in table 6. The comparison showed the sample extracts discouraged linoleic acid oxidation,
that extracts of Penicillium sp. 1, Penicillium sp. 2 and As- while others were less active. We recorded that each of
pergillus sp. were the most effective, and exhibited broad the extracts of Penicillium sp. 2, and Aspergillus sp. had
spectrum activity on the three different groups of patho- an anti-oxidation activity to linoleic acid, and an inhibi-
genic microorganisms (fungi, Gram positive and negative tion percentage which ranged from 78.961±3.183% to
bacteria). On other hand, the extracts were more effective 73.977±1.102%, respectively. These are high percentages
on Gram positive bacteria and fungi compared to Gram compared to Butylated hydroxytoluene (BHT), which
negative bacteria (Fig. 1). was considered to have a 97% inhibition to the oxidation
of linoleic acid. While, the isolates Phoma sp., Alternar-

Fig. 1. Comparison of average inhibitions of ethyl acetate extracts on the growth of different groups of pathogenic micro-organisms
(fungi, Gram positive and negative bacteria)
Means with the same letter are not significantly different at (p < 0.05)

Fig. 2. Antioxidant activity of crude ethyl acetate extracts of endophytes compared to the controls (BHT, MeOH and H2O) by the test
of β-carotene / linoleic acid after 24 hours
Each point represents the mean ±SD (n = 3), each value of the curve followed by the letters shared no significant difference
among themselves, at (p < 0.05)
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134 Journal of Plant Protection Research 53 (2), 2013

ia sp., and Penicillium sp. 1 had less inhibition, with inhi- tested. With regard to actinomycetes, Rabah et al. (2007)
bition percentages of 65.740±4.486%, 64.218±7.569%, and isolated a number of Streptomyces with antimicrobial ac-
63.012±1.734%, respectively. The inhibitory activity of the tivity. Among them, three isolates (SS15, SS19 and SS7)
rest of the isolates: Cladosporium sp., Chaetomium sp. and demonstrated antimicrobial activity against Gram posi-
three Streptomycetes isolates to the oxidation of linoleic tive and negative bacteria, yeast, and fungi.
acid was less than 50% (Fig. 2). Antagonism might be due to the production of bio-
logically active compounds in media (Castillo et al. 2002).
The reason for the different sensitivity between Gram
DISCUSSION positive and Gram negative bacteria could be ascribed to
Differences in colonization and isolation rates have the morphological differences between these microorgan-
been proven in several other studies and are in agreement isms. Gram negative bacteria have an outer polysaccha-
with our results. The differences were those observed be- ride membrane which carries the structural lipopolysac-
tween plants tissues such as roots, leaves, and stems of charide components. This makes the cell wall imperme-
wheat (T. aestivum) (Sieber et al. 1988; Larran et al. 2007); able to lipophilic solutes. Gram positive should be more
between roots and leaves of rice (Naik et al. 2009; Tian susceptible having only an outer peptidoglycan layer
et al. 2004) as well as between roots, leaves, stems, and which is not an effective permeability barrier (Pandey
flowers of several species of medicinal plants (Gong and et al. 2004; Ogundare et al. 2006). The results obtained in
Gou 2009; Lv et al. 2010). The difference in endophytes our study suggest that these endophytes have the poten-
assemblages in the various tissues indicated that some tial to be a source for novel bioactive products.
fungal endophytes have an affinity for different tissue An enormous variety of plants have been studied for
types. This affinity might be a reflection of their capacity new sources of natural antioxidants. Phenolic and flavo-
for utilizing or surviving within a specific substrate (dif- noid compounds derived from plants were proved to be
ferent tissue texture and chemistry) (Huang et al. 2008). potent antioxidants and free radical scavengers. Significant
Most of the isolated fungal taxa active in this study, correlations between phenolic compounds and antioxidant
belong to the common isolated endophytes and were re- properties of medicinal plants were noted (Baghiani et al.
ported as endophytes in previous studies on crops plants. 2010; Khennouf et al. 2010). The same was seen in stud-
They were isolated from T. aestivum (Larran et al. 2002; ies on endophytes. Each of the Phoma, Cladosporium, and
Larran et al. 2007), and from rice O. sativa (Tian et al. 2004; Chaetomium fungi were found to have antioxidant activity.
Naik et al. 2009). The most frequently isolated actinomy- This activity was greater among the Chaetomium fungus
cetes strains from healthy plants were those belonging to accompanied by a greater proportion in the total phenolic
the genus Streptomyces (Zin et al. 2007). content compared with other active isolates. The same was
As all taxa of endophytes were from healthy tis- with the endophytic fungus A. alternata (Fernandes et al.
sues, it appears that either they were non-pathogenenic or 2009). Furthermore, ethyl acetate is often used as an extrac-
the plants had developed a resistance mechanism against tion solvent with a significant selectivity in the extraction
the pathogens. This suggests that the isolates recovered of low-molecular-weight phenolic compounds and high-
here are either avirulent or hypovirulent or are virulent molecular-weight polyphenols (Scholz and Rimpler 1989).
but in a latent phase (Petrini 1991). It is probable that On the other hand, Conde et al. (2008) have reported that
these taxa are not pathogens for their antagonism against ethyl acetate allowed the highest phenolic content and the
phytopathogens. These fungi could be adapted to this selective removal of nonphenolic compounds. Therefore,
host and be antagonists of their pathogens. Depending it could be that the antioxidant activity of ethyl acetate ex-
on their antagonistic capacity, they would be able to dis- tracts of the endophytes isolated from wheat was caused
place, reduce, suppress or induce resistance against them by the presence of phenolic compounds in the extracts.
(Larran et al. 2007). Those active endophytic fungi inside The present results may lead to the conclusion that
the plants may play an important role in protecting the endophytes are considered to be a potential source for
plant host against pathogenic microorganisms and have novel bioactive products (Strobel 2003). The data present-
an intimate correlation with the development and physi- ed in this study demonstrated that extracts of endophytic
ological activity of wheat (Tian et al. 2004). fungus and Streptomycetes isolated from wheat, have anti-
There are many reports about antimicrobial com- microbial and antioxidant activities, especially Penicillium
pounds produced by endophytes in cultures that were ac- sp. 1, Penicillium sp. 2, and Aspergillus sp. Thus, endo-
tive against plant and human pathogenic microorganisms. phytic fungi and actinomycetes play an important role in
Chareprasert et al. (2006) reported an antimicrobial activ- the search for natural compounds. Endophytic fungi and
ity exhibited by endophytic fungi isolated from teak and actinomycetes might also represent an alternative source
rain trees. These fungi were found to produce some me- for the production of therapeutic agents and bioactive
tabolites active against bacteria and yeast. From 67 endo- metabolites that are not easily obtained by chemical syn-
phytic fungi isolated from Q. variabilis, 19.4% (Aspergillus thesis, and which have a high activity against pathogenic
sp., Penicillium sp., and Alternaria sp.) showed significant microorganisms. However, this work will serve as a pre-
antimicrobial activity (Wang et al. 2007). In accordance lude to more comprehensive studies on the chemistry
with Lin et al. (2007) concerning the study of the medicinal and biology of the bioactive natural products produced
plant C. acuminate, 174 endophytic fungi were isolated and by these endophytes. Further examination can be done
from 18 taxa. Alternaria (12.6%) was dominant, and three to learn if endophytes may have the potential to serve as
showed antimicrobial activity from 22 Alternaria extracts a biological control or as new pharmacological agents.
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 Screening of antimicrobial and antioxidant secondary metabolites from endophytic fungi isolated… 135

Gao L., Huang J., Li J. 2007. Fermentation conditions of Sino-

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