1. AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA
ISSN Print: 2151-7517, ISSN Online: 2151-7525, doi:10.5251/abjna.2014.5.1.15.23
© 2014, ScienceHuβ, http://www.scihub.org/ABJNA
Study of antagonistic capability of Trichoderma harzianum isolates
against some pathogenic soil borne fungi
Sajid Salahuddin AL-Saeedi
Bihar Moqdad AL-Ani
Department of Biology, College of Science, Anbar University, Ramadi, Iraq.
ABSTRACT
In this study in vitro potential of two isolates of T.harzianum (T1, T2) were evaluated against
seven soil borne pathogenic fungi isolates ( Acremonium sp. , Alternaria sp. , Aspergillus sp.,
Penecillium sp. , Pythium sp. , Rhizoctonia sp. and Verticillium sp.) in dual culture teqniques and
through production of volatile inhibitors at two incubation periods (5 and 7 days)and non-volatile
inhibitors collected at four incubation periods (3,6,9 and 12days) and different concentrations
(25,50,75 and 100%) were investigated to evaluate their effects on pathogens mycelia. T2 isolate
showed highest inhibitory effect of mycelial growth of pathogens (45.99%). Maximum effect
noticed in Alternaria-T2 interaction (51.18%). Effect of volatile metabolites at 7day seems to be
more effective on mycelial growth of all pathogens than the 5 day periods. Maximum growth
inhibition noticed against Rhizoctonia sp. by T2 isolates (53.66%). A variability among the
collection periods of T2 cultural filtrates and the fungal pathogens was evident. Generally, the
had concentration (100%) of T2 isolate had highest growth reduction in all pathogens.
Keywords: bio control, Trichoderma harzianum , soil borne fungi .
INTRODUCTION
Soil borne plant pathogens such as bacteria, fungi
and nematodes annually create a major economically
losses in many important crops. Toxicological
environmental and sociological concerns have led to
drastic reduction in the availability of efficient
commercial compounds, and also the use of
fungicides may lead to the appearance of new
resistant strains of pathogens. The biological control
is the best alternative especially against soil borne
pathogens. Biological control of pathogens, i.e., the
total or partial destruction of pathogen populations by
other organisms, occurs routinely in nature
(Hajiehgrari et al, 2008). Among the various
antagonists used for the management of plant
diseases.
Trichoderma spp. have been widely studied as
potential biocontrol agents for controlling many plant
pathogens (Agrios, 2005; Lynch,1990).Most of the
studies on Trichoderma species have been
conducted with respect to their activity as biological
control agents against fungal pathogens
(Papavizas,1992, Sahebani and Hadavi , 2008,
Hanada,2009). The antagonistic activity of
Trichoderma depends on multiple synergistic
mechanisms (Nallathambi et al, 2009; Howell,
2003). The various mechanisms include antibiosis,
parasitism, inducing host-plant resistance,
competition, secretion of chitinolytic enzymes,
mycoparasitism and production of inhibitory
compounds (Harman et al., 2004). Trichoderma
produces a number of secondary metabolites with
biological activity(Harman, 2006; Ghisalberti and
Sivasithamparam, 1991).
Many strains are strong opportunistic invaders, fast
growing, prolific producers of spores and powerful
antibiotic. Consequently, these fungi are ecologically
very successful since strains have been found in all
climatic zones (Monte, 2001).
The aim of this study was to evaluate the biological
potential of Trichoderma harzianum gainst some soil
borne plant pathogens.
MATERIALS AND METHODS:
Isolation and identification of Trichoderma
species :
Two isolates of Trichoderma harzianum were used in
this study. The first one (T1) was isolated from
polluted cultures of the fungus Pleurotus sp. and the
other isolate (T2) was isolated from palm debris, on
Potato Dextrose Agar (PDA) and incubated at 26±

2. Agric. Biol. J. N. Am., 2014, 5(1): 15-23
16
2ºC for 5-7 days. The colonies which appeared were
transplanted to PDA medium for purification using the
method of Raper and Fennell (1965).
Isolation and identification of phytopathogenic
fungi : cremonium sp. , Alternaria sp., Aspergillus
sp., Penicillium sp. were isolated using dilution plate
technique (Johnson et al., 1959) on PDA with Rose
Bengal 0.035 g/L and Streptomycin 1/1000M (3ml/L)
and incubated at 26±2ºC for 10 days .The colonies
were counted and identified. Pythium sp. and
Verticillium sp. were isolated from Cress roots while
Rhizoctonia sp. was isolated from Okra roots.
Diseased root tissues were washed under running
tap water to remove surface soil, dust and other
contaminants. Tissue pieces were cut out from the
leading edge of lesion, and placed in one percent
sodium hypochlorite for five minutes; they washed in
sterile distilled water and dried, sterile filter paper.
Then pieces 1cm long plated onto PDA and
incubated at 26± 2ºC for 5-7 days. The colonies
which appeared were transplanted to PDA medium
for purification. Stock cultures of fungi were
maintained on PDA slants and stored at 4°C in
refrigerator. Fungi were identified based on the
microscopic observations and colony characteristics
following the taxonomic key of Watanabe, (2002).
Microscopic examination was carried out by mounting
the culture in lacto phenol cotton blue.
Evaluation of Dual culture on agar plates: To
evaluate the activity of Trichoderma isolates(T1,T2)
against pathogenic fungi, two discs (5mm) obtained
from one week old culture on PDA, the first one
carrying the stock of the antagonistic agents (T1 and
T2) and the other of the pathogenic agents were
placed at the periphery of Petri plate (9cm in
diameter) at the distance . One disc of each
pathogenic agent was maintained as control (alone
culture). Each replicates have three plates. Both the
dual and alone cultures were incubated at 26±2°c for
one week and measurement of radial mycelial growth
of the fungus was taken every 24 hours. The
percentage inhibition of growth (I) was calculated
using the formula given below:
[I (%) = (1 – T /C) x 100]…...1
Where: I= Inhibition Percentage of pathogen growth
by antagonists. C=Radial growth in control. T=Radial
growth in the treatment (Mokhtar and Aid ,2013).
Evaluation of dual culture using slide methods:
For each pathogen-T.harzianum interaction, a clean
slide was placed in 9 cm diameter plates and
sterilized. Then a small amount of autoclaved melted
potato dextrose agar was spread over the slide to
make a thin PDA film on the slide. The 5 mm discs of
one week old of each pathogen and T. harzianum
isolates were placed on the opposite sides of the
slide 3 cm apart on the PDA surface. Then 5ml of
distilled water was added to the plate to prevent
drying and then incubated at 26 ± 2ºC for a week. At
the end of incubation period, region of contact
between T. harzianum–Pathogen hyphae was
stained with lactophenol and cotton blue and
examined under a light microscope (Hajieghrari et
al., 2008).
Effect of volatile metabolites: The effect of volatile
metabolites produced by the antagonistic
microorganisms on pathogens, mycelial growth was
determined by the method described by Dennis and
Webster (1971) and Goyal et al. (1994). The
antagonistic fungi were centrally inoculated by
placing 5 mm diameter mycelia disc taken from one
week old culture on the PDA plate and incubated at
26±2°C for 2 days. The top of each Petri dish was
replaced with bottom of the PDA plate inoculated
centrally with the pathogen. Two plates were sealed
together with paraffin tape and further incubated at
25ºC. In control treatment, T.harzianum isolates
replaced with inoculum of 5 mm of sterile PDA
medium only . Colonies diameter of the pathogen
was measured at 5 and 7 days after incubation and
the inhibition of mycelial growth was calculated. The
percent growth inhibition was calculated by using
equation 1(Dolatabadi et al., 2011).
Effect of non-volatile inhibitors : To determine the
effects of nonvolatile metabolites of T.harzianum
isolate on mycelia growth of the pathogens, three
discs of mycelial agar plugs (5 mm diameter) were
removed with a cork borer from the edge of the
young culture. T.harzianum isolates were inoculated
in 100 ml sterilized potato dextrose broth (PDB) in
250 ml flasks and incubated at 26 ± 2ºC on a rotary
shaker set at 150 rpm for 3,6,9 and 12 days. The
culture was filtered through Millipore filter to removing
mycelial mats and then sterilized through biological
membrane filter (0.2 μm). Certain numbers of wells
were prepared on PDA cultured with the pathogenic
fungi (after 2 days for Alternaria sp., Pythium sp. ,
Verticillium sp. and 4days for Aspergillus sp. and
5days for Rhizoctonia sp.) with the help of sterile cork
borer (9 mm in diameter), 0.2 ml of the Trichoderma
culture filterate concentrations (25,50, 75,100%) were
introduced into the wells . Controls were made by
using the distilled water. Plates were then incubated
at 26±2°C for 7days. Three replications were

3. Agric. Biol. J. N. Am., 2014, 5(1): 15-23
17
maintained for each treatment. The percent growth
inhibition was calculated by using equation (1)
(Dolatabadi et al., 2011).
Statistical analysis: Complete Randomized Design
(C.R.D.) was used as an experimental design. Data
were analyzed by using statistical analysis system-
IBM SPSS statistics 21. Least significant difference
(LSD) was used to compare the significant difference
between means at P≤ o.o5.
RESULTS AND DISCUSSION:
Both of Trichoderma isolates were identified as T.
harzianum according to the taxonomic key of
Watanabe (2002).
The results of dual culture indicated that both isolates
of T.harzianum inhibited the growth of pathogenic
fungi at varying degrees (Table1). It was found that
T.harzianum2 inhibited the growth of pathogens
(45.99%) more than T.harzianum1 isolate (41.08%).
Hence this isolate was selected for subsequent
investigations.
T. harzianum inhibited the growth of the target
organisms through its ability to grow much faster than
the pathogenic fungi thus competing efficiently for
space and nutrients. Starvation was the most
common cause of death for microorganisms, so that
competition for limiting nutrients resulted in biological
control of fungal phytopathogens ( Siameto et
al.,2011). The variation in fungicidal activity among
the two T.harzianum isolates could be attributed to
the presence of different types of chemical
constituents in different isolates (Wang et al., 2003;
Zhou et al., 2008; Eneyskaya et al., 2009; Yang et
al., 2009).On the other hand the pathogenic fungi
affected by the antagonistic fungi at different rates,
maximum growth inhibition was recorded by
Aletrnaria sp. (51.15%) followed by Pythium sp.
(46.77%) and Verticillium sp. (46.23%) and
Rhizoctonia sp. (45.11%) respectively. Whereas
Acremonium sp. was less affected fungi (25%)
(table1).
The differences in average responses to antagonism
by T.harzianum isolates between the different genera
of pathogens indicated that several gens of both the
antagonist and pathogen must be involved in
regulating the different levels of antagonism observed
in the study (Bell et al., 1980).
Table 1: Inhibition percentage of the mycelia
growth of isolated fungi by Trichoderma
harzianum, in the dual cultures, on PDA medium.
Mean
ebarnrireb nIitIbihnI
Pathogenic
fungi T.harzianum 2T.harzianum 1
25.00
C
28.9521.05*
Acremonium
sp.
51.18
A
52.6549.71
Alternaria sp.
45.11
AB
46.3243.89
Aspergillus sp.
43.74
B
50.3237.15
Penicillium sp.
46.77
AB
47.0646.47
Pythium sp.
46.23
AB
47.2545.20
Rhizoctonia sp.
46.77
AB
49.4144.12
Verticillium sp.
45.99
A
41.08
B
Mean
* values are average of 3 replicates. Means with the
same letter in each column are not significantly
different in L.S.D. at (P ≤.05).
The direct confrontation of T. harzianum against the
different fungal isolates in vitro on PDA medium
showed that when the mycelium of both cultures
came in contact with each other the hyphal growth of
the pathogenic fungus were found to be inhibited by
the hyphae of T. harzianum. A clear zone of
interaction was formed in all Trichoderma-pathogen
combination except in the interacting between
T.harzianum and Aspergillus sp., an inhibition zone
without physical contact between the colonies around
the pathogen colony was observed.
The presence of an inhibition zone in dual culture
without the hyphae contact suggests the secretion of
diffusible non-volatile inhibitory substance by the
T.harzianum isolate. Previous studies have
demonstrated that before mycelia of fungi interact,
Trichoderma sp. produces low quantities of
extracellular exochitinases (Kullnig et al., 2000;

4. Agric. Biol. J. N. Am., 2014, 5(1): 15-23
18
Brunner et al., 2003). The diffusion of these enzymes
dissolves cell fragments of host cells. These cell
fragments in turn induce the production of further
enzymes and trigger a cascade of physiological
changes, stimulating rapid and directed growth of
Trichoderma sp. (Zeininger et al., 1999).
Microscopically observation of hyphal interaction
indicated that antagonistic hyphae coiled around the
hyphae of pathogen, mycelia denatured and inhibited
them. T.harzianum either formed hook or bunch like
structure around the hyphae of pathogens before
penetration or entered directly. No hyphae coiling
was observed in Aspergillus-Trichoderma
interaction. T.harzianum prevented the spore's
formation of the Alternararia sp. and Aspergillus sp.
and seclerotia in Rhizoctonia sp. in compare with
controls (fig.1).
Fig 1:Microscopic observations showed the effect of T. harzianum on mycelia of Pthogenic fungi by:Coiling the
hyphae of T. harzianum (black arrows) around the hyphae of:A,B= Alternaria sp., ,C=Penicillium sp., D,E= Pythium
sp.,F=Verticillium sp. and on spore formation (white arrows) of: G=Alternaria sp. Compared with control (H),
I=Aspergillus sp. Compared with control (J), K=the effect of T. harzianum on cytoplasm (voculation) in Rhizoctonia
sp. (red arrow) as compared with control (L).
Microscopic observation of the interaction region
between T.harzianum-pathogens revealed that
T.harzianum has a high inhibitory effect against the
different isolated fungi, with a several biological
modes like mycoparasitism and antibiosis. This
results has been reported and confirmed by Siameto
et al. (2011); Mokhtar and Aid (2013).
Effect of volatile metabolites on the growth of
pathogens presented in table 2 showed that the
volatile metabolic substances of antagonistic fungus
(T2) inhibited the mycelia growth of the different
pathogenic fungi, with different rates over the five
days of treatment, the percentage inhibition of
mycelia growth reached 43.75% for Rhizoctonia sp.
and decreased to 35.86%, 29.62% and 26.8% for
Aspergillus sp., Alternaria sp. and Pythium sp.
respectively, the minimum ratio was recorded in
Verticillium sp.(23.7%).
After seven days of incubation, more effects of
mycelial growth inhibition were observed, T.
harzianum (T2) caused maximum growth inhibition
against Rhizoctonia sp. (53.66%) followed by
Aspergillus sp. and Alternaria sp. (39.3% and
36.28% respectively), but in the Pythium sp. and
Verticillium sp. have recorded the lowest inhibition
percentages (19.61% and 18.43% respectively)
(table 2).
These observations are similar to those of Ayoubi et
al. (2012) findings. The earlier studies also revealed
that antimicrobial metabolites such as aromatic
pyrone antibiotics (Keszler et al., 2000).
A
E
I
B C D
F G H
J K L

5. Agric. Biol. J. N. Am., 2014, 5(1): 15-23
19
Effect of filterate concentrations of T. harzianum that
collected at different times on mycelial growth of test
pathogenic fungi was revealed that the aqueous
extracts of T. harzianum reduced the mycelial growth
of the target fungal pathogens. However, variability
among the collection times of cultural filtrates and the
fungal pathogens was evident.
Table 2: Inhibition percentage of the mycelia
growth of isolated fungi by the volatile
substances of T. harzianum, in the dual cultures,
on PDA medium.
Inhibition percentage
of mycelia growth after:
Pathogenic
fungi
7days5days
36.28
B
29.62*
AB
Alternaria sp.
39.30
B
35.86
AB
Aspergillus
sp.
19.61
C
26.80
AB
Pythium sp.
53.66
A
43.75
A
Rhizoctonia
sp.
18.43
C
23.70
B
Verticillium
sp.
7.19610.391LSD
* values are average of 3 replicates. Means with the
same letter in each column are not significantly
different in L.S.D. at (P ≤.05).
Maximum growth reductions in all studied
pathogenic fungi were observed with increasing
concentrate- ions (Tables 3,4,5,6,7). A similar effects
were observed by Anita et al. (2012) when studying
the effect of secondary metabolites secreted by T.
atroviride at different concentrations and found rapid
decrease observed in the linear growth of the
pathogen with increased concentration of the
metabolites.
In Alternaria sp. there was remarkable increase in
growth inhibition at the treatment with T.harzianum
filtrates that collected after 6 and 9 days of incubation
which were 58.75% and 56.43% respectively.
Whereas, less inhibition effect was recorded at the
treatment with filtrates that collected after 12 days of
incubation (48.57%) (Table 3).
In Aspergillus sp. highest growth inhibition was
noticed at the treatment with T.harzianum filtrates
that collected after 3and 9 days of incubation
(46.91% and 48.85% respectively). Less inhibition
effect was recorded at the treatment with filtrates that
collected after 6 and 12 days of incubation (42.09%
and 45.47%)(Table 4) .The maximum inhibition
percentage was recorded in Pythium sp. by treated
with collected T.harzianum filtrates after 6 days
(71.39%) and minimum inhibition percentages were
observed by treated the same fungus with collected
T.harzianum filtrates after 9 and 12 days (60.31%
)(table 5), while in Rhizoctonia sp. no significant
differences noticed in response to tested
T.harzianum filtrate(table 6).
The maximum inhibition percentage was observed by
treated Verticillium sp. with filtrate collected at 6 and
3 days (63.53% and 60.94%) and the activity was
decreased at 9 and 12 days which were 58.13% and
57.81% respectively(table 7).
Table 3. Effect of different filtrate concentrations
of T. harzianum grown on P.D medium at different
incubation times on the growth of Alternaria sp.
Mean
Concentration / inhibition
Percentage
Incubation
time
100%
75%50%52%
53.44
B
61.2552.55050*
3days
58.75
A
62.5063.7556.2552.5
6 days
56.43
AB
61.4357.1454.2952.86
9 days
48.57
C
48.5748.5748.5748.57
12 days
58.44
A
55.49
A
52.28
B
50.98
B
Mean
* values are average of 3 replicates. Means with the
same letter in each column are not significantly
different in L.S.D. at (P ≤.05).

6. Agric. Biol. J. N. Am., 2014, 5(1): 15-23
20
Table 4. Effect of different filtrate concentrations of T. harzianum grown on P.D medium at different
incubation times on the growth of Aspergillus sp.
Mean
Concentration / Inhibition Percentage
Incubation time
100%75%50%52%
46.91
A
53.67
a
45.95
b
45.95
b
42.08
b
3days
42.09
B
51.74
ab
45.95
b
40.15
b
30.50
c
6 days
48.85
A
49.81
ab
49.81
ab
49.81
ab
45.95
b
9 days
45.47
B
49.81
b
44.02
b
44.02
b
44.02
b
12 days
51.26
A
46.43
AB
44.98
B
43.58
C
Mean
* values are average of 3 replicates. Means with the same letter in each column are not significantly different in
L.S.D. at (P ≤.05).
Table 5. Effect of different filtrate concentrations of T.harzianum grown on P.D medium at different
incubation times on the growth of Pythium sp.
Mean
Concentration / Inhibition Percentage
Incubation time
100%75%50%52%
66.18
B
68.2468.2464.7163.53*
3days
71.39
A
76.6772.2268.8967.78
6 days
60.31
AB
61.2561.2560.0058.75
9 days
60.31
C
63.7560.0044.2057.50
12 days
67.48
A
65.43
B
63.40
BC
61.89
C
Mean
* values are average of 3 replicates. Means with the same letter in each column are not significantly different in
L.S.D. at (P ≤.05).

7. Agric. Biol. J. N. Am., 2014, 5(1): 15-23
21
Table 6. Effect of different filtrate concentrations of T. harzianum grown on P.D medium at different
incubation times on the growth of Rhizoctonia sp.
Mean
Concentration / Inhibition Percentage
Incubation time
100%75%50%52%
48.0350.9849.0149.0143.13
3days
51.2252.9450.9851.9649.01
6 days
43.1447.0645.1045.0935.29
9 days
47.4049.0247.0646.4447.06
12 days
50.0048.0448.1343.62
Mean
Table 7. Effect of different filtrate concentrations of T. harzianum grown on P.D medium at different
incubation times on the growth of Verticillium sp.
Mean
Concentration / Inhibition Percentage
Incubation time
100%75%50%52%
60.94
A
63.7562.5060.0057.50
3days
63.53
A
64.7164.7162.3562.36
6 days
58.13
B
60.0057.5057.5057.50
9 days
57.81
B
61.2560.0055.0055.00
12 days
62.43
A
61.18
A
58.71
B
58.09
B
Mean
* values are average of 3 replicates. Means with the same letter in each column are not significantly different in
L.S.D. at (P ≤.05).
The results of non-volatile substance against the
pathogens that were more effective support this
hypothesis. The results were in agreement with
Hajieghrari et al. (2008) who evaluated six isolated
of Trichoderma sp. against five isolates of soil borne
phytopathogenic fungi in dual culture techniques and
through production of volatile and non-volatile
inhibitors.
The different effects of T.harzianum culture filtrates
collected at different incubation times between tested
pathogenic fungi due to T.harzianum ability to

8. Agric. Biol. J. N. Am., 2014, 5(1): 15-23
22
produce various defense enzymes and secondary
metabolite containing antibiotics with varied nature,
quantity and quality at different incubation times
(Anita et al,2012; Perveen and Bokhari, 2012).
Selection of biocontrol agents as well as
understanding the mechanisms involved in the
antagonistic effect of Trichoderma spp. on plant
pathogens are important in designing effective and
safe biocontrol strategies. Different isolates of
Trichoderma have different combative ability for
pathogen; their indirect effects may also vary.
Therefore, one of the most interesting aspects of
biology is the study of the mechanisms employed by
biocontrol agents to affect disease control. Possible
mechanisms of antagonism employed by
Trichoderma spp. includes nutrient and niche
competitions, antibiosis by producing volatile
components and non-volatile antibiotics that are
inhibitory against a range of soil borne fungi, as well
as parasite- ism.
ACKNOWLEDGEMENT
We would like to thank Mrs. Mustafa (Department of
Biology, College of Science, Anbar University,
Ramad,Iraq) for contributing to this work.
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