Journal of Taibah University for Science

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tusc20

In vitro antagonistic activity of Trichoderma spp.

against fungal pathogens causing black point
disease of wheat

Mohamed Taha Yassin, Ashraf Abdel-Fattah Mostafa & Abdulaziz

Abdulrahman Al-Askar

To cite this article: Mohamed Taha Yassin, Ashraf Abdel-Fattah Mostafa & Abdulaziz
Abdulrahman Al-Askar (2022) In vitro antagonistic activity of Trichoderma spp. against fungal
pathogens causing black point disease of wheat, Journal of Taibah University for Science, 16:1,
57-65, DOI: 10.1080/16583655.2022.2029327

To link to this article: https://doi.org/10.1080/16583655.2022.2029327

© 2022 The Author(s). Published by Informa

UK Limited, trading as Taylor & Francis
Group

Published online: 07 Feb 2022.

Submit your article to this journal

Article views: 3341

View related articles

View Crossmark data

Citing articles: 4 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=tusc20
JOURNAL OF TAIBAH UNIVERSITY FOR SCIENCE
2022, VOL. 16, NO. 1, 57–65
https://doi.org/10.1080/16583655.2022.2029327

RESEARCH ARTICLE

In vitro antagonistic activity of Trichoderma spp. against fungal pathogens

causing black point disease of wheat
Mohamed Taha Yassin , Ashraf Abdel-Fattah Mostafa and Abdulaziz Abdulrahman Al-Askar
Botany and Microbiology Department, College of Science, King Saud University, Riyadh, Saudi Arabia

ABSTRACT ARTICLE HISTORY

Black point disease of wheat contributes to high economic losses every year. The overuse of Received 7 November 2021
fungicides resulted in pathogenic fungal resistance, health and environmental hazards that Revised 10 January 2022
necessitate the formulation of safe biocontrol agents. Dual cultural assay of Trichoderma viride Accepted 11 January 2022
and Trichoderma harzianum strains proved their highest efficiency against Alternaria alternata KEYWORDS
with inhibition percentages of 75.04 and 67.83%, while the lowest activity was detected against Antifungal; antagonism;
Drechslera halodes with growth inhibition of 51.54 and 43.92%, respectively. Alternaria alter- Trichoderma; Dual culture;
nata and D. halodes strains exhibited carbendazim resistance, while Fusarium proliferatum was mycoparasitism; GC-MS
the most susceptible one. Chemical analysis of T. harzianum and T. viride extracts using gas
chromatography-mass spectrometry indicated that 6-pentyl-α-pyrone and cyclooctanol com-
pounds were the main active constituents representing about 26.43 and 32.74% respectively.
The tested bioagents were highly effective against the fungal strains causing black point dis-
ease of wheat so that these biocontrol agents could be used in formulation of natural fungicides
avoiding the harmful impact of the synthetic fungicides.
Abbreviations: FCZ; Fluconazole Antifungal agent, GC-MS; Gas chromatography-Mass spec-
trometry, MIC; Minimum inhibitory concentration, MFC; Minimum fungicidal concentration, PDA;
Potato dextrose agar, T.H; Trichoderma harzianum, 1. T. V; Trichoderma viride

1. Introduction pathogenic fungal strains including antibiosis, compe-

Wheat (Triticum spp.) is a strategic crop belonging to
the Poaceae family [1]. Globally, wheat is the most pre-
dominant cereal crop, accounting for over 218 million
ha [2]. The annual production yield is evaluated as 735
million tons, with an estimated value of US$ 145 bil-
lion [3]. Wheat has a high nutritional value due to its
carbohydrate, protein, mineral, fat and vitamin con-
tent [4]. Wheat grains are susceptible to infection with
black point disease during grain filling or at milking
stage [5,6]. Black point disease of wheat contributes
to significant economic losses worldwide, with esti-
mated yield losses of 24% [7]. The disease has sev-
eral harmful effects, such as decreased wheat qual-
ity, reduced seed germination and hindered seedling
growth [8,9]. Fusarium spp., Alternaria spp., Bipolaris spp.
and Drechslera spp. are fungal pathogens frequently
associated with black point disease of wheat [10]. How-
ever, frequent use of fungicides leads to environmen-
tal and health problems and results in the develop-
ment of fungicide resistance [11]. The use of antago-
nistic fungi as biological control agents is a potential
alternative to fungicides, as it is an eco-friendly way
to minimize seed fungal infections [12]. These bioa-
gents exhibited different mechanisms of action against
tition for nutrients and mycoparasitism [13]. Tricho-
derma harzianum and T. viride strains were recorded as
biological control agents against the concerned phy-
topathogenic strains and also as plant growth pro-
moters [14]. Furthermore, Trichoderma spp. can pro-
duce a wide range of secondary metabolites that
protect against phytopathogenic fungi through vari-
ous mechanisms, including antibiosis and mycopara-
sitism [15]. These include several volatile compounds,
such as ethylene, acetaldehyde and acetone, which
account for their antibacterial and antifungal activ-
ity [16,17]. Trichoderma spp. exert their antagonistic
effects by secreting antibiotics, such as herzianolide,
trichodermin and trichodermol. Thus, T. viride and T.
harzianum could be used as biological control agents
against phytopathogenic fungal strains, such as Fusar-
ium oxysporum, Alternaria alternata and Fusarium solani
[18]. The high global incidence of black point dis-
ease of wheat and the deleterious effects of chemi-
cal fungicides on the environment and human health
necessitates the development of natural fungicides.
Thus, this study aims to investigate the antagonis-
tic effects of two Trichoderma strains (T. viride and T.
harzianum) against four phytopathogenic fungal strains

CONTACT Mohamed Taha Yassin myassin2.c@ksu.edu.sa Botany and Microbiology Department, College of Science, King Saud University, 2455,
Riyadh 11451, Saudi Arabia
© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.
58 M. T. YASSIN ET AL.
(A. alternata, B. sorokiniana, D. halodes and F. prolifera-
tum) which cause black point disease of wheat.
2. Materials and methods
2.1. Plant fungal pathogens and antagonistic
strains
The four phytopathogenic fungal strains (Fusarium pro-
liferatum ATCC 74149, Alternaria alternata ATCC 66981,
Bipolaris sorokiniana ATCC 32093, Drechslera halodes
ATCC 32198) and two Trichoderma spp. (Trichoderma
viride ATCC 18652 and Trichoderma harzianum ATCC
48131) were obtained from the Botany and Microbi-
ology Department, College of Science, King Saud Uni-
versity. The fungal strains were subcultured on potato
dextrose agar (PDA) slants, incubated at 25 ± 2°C for 5
days and finally stored in the refrigerator at 4°C until
further use.
2.2. Evaluation of fungal antagonistic activity
(Dual culture technique)
The antagonistic activity of Trichoderma viride and Tri-
choderma harzianum against fungal pathogens which
cause black point disease of wheat was evaluated using
the dual culture technique as previously described [19].
Mycelial discs (6 mm in diameter) of fungal antagonis-
tic strains were inoculated in PDA plates and placed
at 1.5 cm from the edge of freshly prepared PDA
plates. Similarly, 6 mm mycelial discs of fungal phy-
topathogens were placed in the opposite direction
of the concerned antagonistic fungal strain at 1.5 cm
from the edge of the prepared PDA plates. The control
plates were inoculated with different fungal pathogens
(Alternaria alternata, Bipolaris sorokiniana or Fusarium
proliferatum) and incubated with the treated plates at
25 ± 2°C for 5 days. Afterwards, the radial growth of
the phytopathogens in the control and treatment plates
was measured and inhibition percentages were calcu-
lated using the following formula:
% inhibition = (A–B)/A × 100
where A = colony diameter of the phytopathogen in
the control plate, B = colony diameter in the dual cul-
ture plate.
2.3. Antifungal potency of culture filtrates of
concerned antagonistic fungi
The percentage of growth inhibition (%) of the phy-
topathogenic fungal strains by the Trichoderma filtrates
was evaluated. The two antagonistic fungal strains were
grown in 50 mL of potato dextrose broth and incu-
bated at 25 ± 2°C for 7 days in an orbital shaker at
150 rpm. Cell-free filtrates were obtained via filtration
using double layers of muslin followed by centrifuga-
tion at 9000 rpm for 10 min to remove the fungal spores
that may hinder membrane sterilization. Finally, the
filtrates were sterilized using Millipore filters (22 µm).
PDA medium supplemented with 25% Trichoderma spp.
filtrates was poured in sterile Petri dishes and inocu-
lated with 6 mm mycelial discs of the phytopathogenic
fungal strains. The control plates were inoculated with
6 mm mycelial discs of different fungal pathogens. The
plates were then incubated at 25 ± 2°C for 5 days. The
radial growth diameters of the phytopathogens were
then measured using a Vernier calliper. The inhibition
of mycelial growth index (IMG %) was calculated using
the following formula:
Inhibition % = (A–B)/A × 100,
where A = radial growth diameter of the phytopatho
gens in the control plates, B = radial growth diameter
of the phytopathogens in the treated plates [20].
2.4. Slide culture technique to detect
mycoparasitism relationship
PDA medium was prepared and poured into sterile
Petri dishes. The medium was then cut with a ster-
ilized blade, placed over a sterilized glass slide, and
inoculated with Trichoderma spp. on one side and a
pathogen strain on the opposite side. The slide was then
incubated at 25 ± 2°C for 5 days, followed by removal
of the PDA medium. The fungal mycelium was then
stained with lactophenol cotton blue and a sterile cov-
erslip was placed over the slide. The hyphal interactions
between the Trichoderma spp. and fungal pathogens
were observed using a light microscope (40×) [21].
2.5. Antimicrobial activity of carbendazim
(commonly used fungicide)
The food poisoning technique was used to detect the
antifungal efficacy of carbendazim, a commonly used
fungicide. Different carbendazim concentrations (50,
100, 150, 200, 250 and 300 ppm) were added to the
PDA medium after sterilization. The plates were then
inoculated with a 6 mm disc of fungal growth and incu-
bated at 25 ± 2°C for 5 days. The growth diameter of the
pathogenic fungi tested was measured using a Vernier
calliper. The growth inhibition ratio (%) was calculated
using the following equation:
% inhibition = (A–B)/A × 100,
where A = growth diameter in the control plates,
B = growth diameter in the treated plates [19].
2.6. Preparation of Trichoderma spp. crude
extracts
The ability of Trichoderma strains to produce active
metabolites which exhibit antifungal activity against

fungal strains causing black point disease of wheat was
investigated. The Trichoderma isolates were cultured
onto PDA medium and incubated at 25 ± 2°C for 7 days.
The fungal growth was then scraped from the agar
surface to obtain a mycelial sample of 5 g. Then, the
mycelial growth was added to 200 mL of 80% ethanol
supplemented with 0.2 M HCl and incubated in a rota-
tory shaker at 25 ± 2°C for 24 h. The samples were then
centrifuged at 9000 rpm to separate the solid compo-
nents. The extraction procedure was performed again
for the solid components, using ethyl acetate as a sol-
vent. Finally, the extracts were combined and partially
evaporated using a rotatory evaporator. The yield was
calculated using the following equation:
Extract Yield % = (R/S) × 100,
where R = extract residue, S = weight of the raw
mycelial sample [22].
2.7. Antifungal efficacy of Trichoderma spp.
extracts against fungal pathogens
The disc diffusion method was used to evaluate the
antifungal activity of Trichoderma spp. extracts against
phytopathogenic fungi which cause black point disease
of wheat. Fifteen mL of PDA medium was poured in ster-
ile Petri dishes as a basal medium, followed by 10 mL
of seeded medium previously inoculated with a suspen-
sion of spores from the fungal phytopathogens tested.
Then, filter paper discs (8 mm diameter) were loaded
with different concentrations of Trichoderma spp. crude
extracts (50, 100, 150, 250, 300 and 400 µg/disc) and
placed over the seeded plates [19]. Filter paper discs
with 25 µg of fluconazole, a reference antifungal agent,
were used as positive controls. The plates were then
incubated at 25 ± 2°C for 5 days. The diameters of the
inhibition zones were measured using a Vernier cal-
liper and the lowest concentration of Trichoderma spp.
extract which exhibited an inhibitory effect against the
phytopathogens tested was considered the minimum
inhibitory concentration (MIC). The minimum fungici-
dal concentration (MFC) was obtained by plating an
inoculum from the inhibition zones into freshly pre-
pared PDA medium. The lowest concentration with no
fungal growth was considered the MFC.
2.8. GC-MS analysis of the T. viride and T.
harzianum extracts
Chemical analyses of T. harzianum and T. viride extracts
were achieved to detect the active constituents exhibit-
ing antifungal activity. GC-MS was performed using an
Agilent 7890 gas chromatograph and an Agilent 5975C
Mass Spectrometer, USA. Conditions of analysis were
optimized as stated by Yassin et al. 23 [23]. Identifica-
tion of active components of Trichoderma extracts was
JOURNAL OF TAIBAH UNIVERSITY FOR SCIENCE 59
achieved by comparison of their mass spectrum with
reference standards in NIST database.
2.9. Statistical analysis
Data were statistically analyzed by GraphPad Prism 5.0
(GraphPad Software, Inc., La Jolla, CA, USA) using one-
way analysis of variance. The experiments were done
in triplicates and the data were presented as a mean of
triplicates ± standard error.
3. Results
3.1. Antagonistic antifungal activity of
Trichoderma strains
Dual culture results showed that both T. viride and T.
harzianum strains limited the growth of different fun-
gal phytopathogens with different growth inhibition
patterns as shown in Figure 1. T. viride strain showed
the highest antagonistic activity against different phy-
topathogenic fungal strains compared to T. harzianum
strain. Alternaria alternata was the most susceptible
strain to the concerned antagonistic fungal strains (T.
viride and T. harzianum) recording inhibition percent-
ages of 75.04 and 67.83%, respectively (Table 1). In con-
trast, the lowest antagonistic activities of T. viride and
T. harzianum strains were estimated against D. halodes
recording growth inhibition percentages of 51.54 and
43.92%, respectively. Furthermore, the results revealed
that T. viride exerted antagonistic action against B.
sorokiniana and F. proliferatum with growth inhibition
percentages of 62.65 and 64.23%, while T. harzianum
showed inhibition percentages of 59.04 and 53.42%,
respectively.
3.2. Antifungal potency of culture filtrates of
concerned antagonistic fungi
Cultural filtrates of the antagonistic fungal strains (T.
harzianum and T. viride) exhibited antifungal potency
against different pathogenic fungal strains. T. viride fil-
trates demonstrated the maximum growth inhibition
against B. sorokiniana (57.97%) followed by D. halodes
(42.90%), A. alternata (30.46%) and F. proliferatum
(19.02%). On the other hand, the cultural filtrates of T.
harzianum exhibited the highest anti-phytopathogenic
activity against B. sorokiniana followed by D. halodes,
A. alternata and F. proliferatum recording growth inhi-
bition percentages of 53.06, 40.81, 28.72 and 6.97%,
respectively, as seen in Table 2.
3.3. Slide culture technique to detect
mycoparasitism relationship
T. harzianum strain exhibited mycoparasitism against
the tested phytopathogenic fungal isolates except D.
halodes strain, while T. viride showed no mycoparasitic
60 M. T. YASSIN ET AL.

Figure 1. Antagonistic activity of T. viride and T. harzianum strains against different phytopathogenic strains.

Table 1. Antagonistic activity of T. harzianum and T. viride against the concerned fungal phytopathogens. (T.h, T. harzianum; T.v, T.
viride).
Mean diameter of mycelial growth of Percentage of mycelial growth
phytopathogens (mm) inhibition (%)

Phytopathogens Biculture (T. h) Biculture (T. v) Control T. h T. v

A. alternata 18.3 ± 0.29a 14.2 ± 0.37a 56.9 ± 0.26a 67.83a 75.04a
B. sorokiniana 20.4 ± 0.38a 18.6 ± 0.24b 49.8 ± 0.31b 59.04b 62.65b
D. halodes 30.9 ± 0.41b 26.7 ± 0.19c 55.1 ± 0.22a 43.92c 51.54c
F. proliferatum 29.3 ± 0.12b 22.5 ± 0.32d 62.9 ± 0.34c 53.42d 64.23a
∗Different letters in the same column indicated that values were significantly different (P < 0.05).

Table 2. Inhibitory effect of the culture filtrates of antagonistic strains (T. harzianum and
T. viride) against wheat fungal phytopathogens.
Mean diameter of mycelial
growth of phytopathogens IMG %

Phytopathogens Control (T. h) filtrates (T. v) filtrates T. h T. v

A. alternata 73.2 ± 0.86a 50.9 ± 0.15a 52.02 ± 1.50a 28.72a 30.46a
B. sorokiniana 55.2 ± 0.26b 23.2 ± 0.34b 25.90 ± 0.40b 53.06b 57.97b
D. halodes 57.1 ± 0.52b 32.6 ± 0.19c 33.8 ± 0.41c 40.81c 42.90c
F. proliferatum 79.4 ± 0.32c 64.3 ± 0.37d 74.05 ± 0.49d 6.97d 19.02d
∗Inhibition Index of Mycelial Growth (IMG %)
∗T.h: Trichoderma harzianum; T.v: Trichoderma viride.
∗Different letters in the same column indicated that values were significantly different (P < 0.05).

action. The antagonistic T. harzianum isolate exhibited
parasitic action against A. alternata, B. sorokiniana and
F. proliferatum strains through different mechanisms of
action as seen in Figure 2. The mycoparasitic behaviour
included coiling of Trichoderma hyphae around the phy-
topathogenic hyphae, formation of penetration peg
and disintegration of pathogenic hyphae.
exhibited carbendazim resistance. Furthermore, car-
bendazim was moderately effective against B. sorokini-
ana strain with inhibition percentage of 69.08% at
1.00 ppm concentration. In addition, the MIC of car-
bendazim against B. sorokiniana was 0.50 ppm and the
mycelial growth was completely suppressed at MFC of
2.00 ppm (Table 3).

3.4. Fungicidal activity of carbendazim (reference 3.5. Antifungal efficacy of Trichoderma spp.
fungicide) extracts against fungal pathogens using disc
diffusion method
Carbendazim as a reference fungicide showed highly
fungicidal activity against F. proliferatum strain at all T. viride extract exerted the highest antifungal activity
tested concentrations, while D. halodes and A. alternata against different phytopathogenic strains compared to
 JOURNAL OF TAIBAH UNIVERSITY FOR SCIENCE 61

Figure 2. Mycoparasitic behaviour of T. harzianum against B. sorokiniana, F. proliferatum and A. alternata.

Table 3. Effect of different concentrations of fungicide (carbendazim) on mycelial growth of wheat phytopathogenic fungi. (A. a,
Alternaria alternata; B.s, Bipolaris sorokiniana; D. h, Drechslera halodes; F. p, Fusarium proliferatum).
Percentage of mycelial growth
Mean diameter of mycelial growth (mm) inhibition (%)

Carbendazim, conc. (ppm) A. a B. s D. h F. p A. a B. s D. h F. p

0.00 (control) 56.9 ± 0.26a 49.8 ± 0.31a 55.1 ± 0.22a 62.9 ± 0.34a 0.00a 0.00a 0.00a 0.00a
0.50 68.6 ± 0.22b 26.8 ± 0.14b 73.1 ± 0.32b 0.00 ± 0.00b 0.00a 46.18b 0.00a 100.00b
1.00 62.7 ± 0.26b 15.4 ± 0.28c 71.9 ± 0.43b 0.00 ± 0.00b 0.00a 69.08c 0.00a 100.00b
1.50 60.8 ± 0.19c 9.8 ± 0.18d 64.7 ± 0.16c 0.00 ± 0.00b 0.00a 80.32d 0.00a 100.00b
2.00 60.2 ± 0.29c 0.00 ± 0.00e 62.1 ± 0.25c 0.00 ± 0.00b 0.00a 100.0e 0.00a 100.00b
2.50 58.9 ± 0.17c 0.00 ± 0.00e 61.6 ± 0.09c 0.00 ± 0.00b 0.00a 100.0e 0.00a 100.00b
3.00 57.9 ± 0.34c 0.00 ± 0.00e 60.4 ± 0.12c 0.00 ± 0.00b 0.00a 100.0e 0.00a 100.00b
∗Different letters in the same column indicated that values were significantly different (P < 0.05).

T. harzianum extract. B. sorokiniana was the most sensi-
tive strain to T. harzianum and T. viride extracts record-
ing suppressive zone diameters of 11.36 and 12.29 mm,
respectively at a concentration of 200 µg/disc as seen
in Table 4. In contrast, A. alternata exhibited the low-
est susceptibility to T. harzianum and T. viride strains
with MIC values of 300 µg/disc, respectively. The MFC
of T. viride extract against different phytopathogenic
strains was 300 µg/disc. Moreover, T. harzianum extract
showed fungicidal activity against B. sorokiniana and
D. halodes with MFC value of 300 µg/disc, while it was
400 µg/disc against A. alternata and D. halodes strains.
3.6. GC-MS analysis of the T. viride and T.
harzianum extracts
GS-MS of T. viride and T. harzianum was performed
to detect different active ingredients exhibiting anti-
phytopathogenic activity. 6-pentyl-α-pyrone (26.43%)
was the predominant active component of T. harzianum
extract followed by hexadecanoic acid (15.98%), acetic
acid (12.84%), 2-phenylethyl alcohol (11.36%), 2-butox
yethyl acetate (8.53%), 1-methoy-2-propanone (7.29%),
1-hexadecanol (6.45%), isopentyl acetate (4.65%),
dioctyl ester, hexanedioic acid (4.24%) and 9-eicosane
(2.23%) as demonstrated in Table 5. In contrast, the
major active component of T. viride was cyclooc-
tanol (32.74%) followed by propyl-benzene (21.65%),
cadinene (11.23%), epizonaren (9.34%), d- limonene
(8.49%), α-bisabolol (7.32%), β-farnesene (6.78%) and
propanoic acid (2.45%) as seen in Table 6.
4. Discussion
Trichoderma spp. showed a potential biocontrol effi-
ciency against fungal phytopathogens either through
direct mechanisms of mycoparasitism and antibiosis or
by indirect mechanisms as competition for nutrients,
enhancing plant defence responses and exhilarating
plant growth [24]. In this regard, Trichoderma bioagents
suppress the growth of fungal phytopathogens by a
mixed action through their metabolites inhibitory effect
and lytic enzymes which disintegrate the fungal cell wall
[25]. Trichoderma spp. have been ascertained as bio-
logical control agents against several phytopathogenic
fungal strains [26]. The current study confirmed the
62 M. T. YASSIN ET AL.

Table 4. Antifungal activity of T. viride and T. harzianum extracts against different phytopathogenic fungal strains.
Inhibition zone diameter of fungal strains (mm)

Treatment Concn (μg/disc) Fungal pathogens 100 μg/disc 200 μg/disc 300 μg/disc 400 μg/disc FCZ (25 μg/disc)
(T. h) extract A. a 0.00 ± 0.00 0.00 ± 0.00 10.57 ± 0.34 11.56 ± 0.51 15.27 ± 0.18
B. s 0.00 ± 0.00 11.36 ± 0.67 13.06 ± 0.23 15.43 ± 0.29 20.56 ± 0.19
D. h 0.00 ± 0.00 10.21 ± 0.00 12.56 ± 0.56 14.21 ± 0.32 18.48 ± 0.51
F. p 0.00 ± 0.00 0.00 ± 0.00 11.23 ± 0.37 12.23 ± 0.12 16.23 ± 0.38
(T. v) extract A. a 0.00 ± 0.00 9.78 ± 0.54 11.26 ± 0.65 13.43 ± 0.11 15.54 ± 0. 23
B. s 0.00 ± 0.00 12.29 ± 0.09 14.69 ± 0.24 16.62 ± 0.64 19.78 ± 0.35
D. h 0.00 ± 0.00 11.89 ± 0.26 13.42 ± 0.49 15.76 ± 0.59 18.65 ± 0.17
F. p 0.00 ± 0.00 10.34 ± 0.73 12.46 ± 0.33 13.29 ± 0.41 16.81 ± 0.06

Table 5. GC-MS analysis of the active components of T. harzianum extract.

Compounds Chemical formula M.W Retention time (min.) % of Total
6-pentyl-α-pyrone C10 H14 O2 166.22 7.846 26.43
2-phenylethyl alcohol C8 H10 O 122.16 8.263 11.36
Isopentyl acetate C7 H14 O2 130.18 9.568 4.65
Acetic acid C2 H4 O2 60.05 11.872 12.84
1-Hexadecanol C16 H34 O 242.44 13.539 6.45
Hexadecanoic acid C16 H32 O2 256.42 15.785 15.98
Dioctyl ester, hexanedioic acid C22 H42 O4 370.60 17.349 4.24
1-methoy-2-propanone C 4 H8 O 2 88.10 18.652 7.29
9-Eicosane C20 H40 282.50 19.743 2.23
2-butoxyethyl acetate C8 H16 O 160.21 21.263 8.53

Table 6. GC-MS analysis of the active components of T. viride extract.

Compounds Chemical formula M.W Retention time (min.) % of Total
Cyclooctanol C8 H16 O 128.21 7.568 32.74
β-farnesene C15 H24 204.35 8.546 6.78
Propyl-benzene C9 H12 120.19 9.378 21.65
α-Bisabolol C15 H26 O 222.37 12.985 7.32
D- limonene C10 H16 136.24 14.653 8.49
Cadinene C15 H24 204.35 16.445 11.23
Epizonaren C15 H24 204.35 21.354 9.34
Propanoic acid C3 H6 O2 74.08 23.456 2.45

potent activity of T. viride and T. harzianum against fun-
gal pathogenic strains causing black point disease of
wheat. Dual cultural assay indicated that T. viride strain
exerted the highest antagonistic potency against A.
alternata (75.04%) followed by F. proliferatum (64.23%),
B. sorokiniana (62.65%) and D. halodes (51.54%), while
T. harzianum exhibited a moderate efficacy against A.
alternata followed by B. sorokiniana, F. proliferatum and
D. halodes with corresponding inhibition percentages
of 67.83%, 59.04%, 53.42% and 43.92%, respectively.
The proved antagonistic efficacies of T. viride and T.
harzianum against fungal strains causing black point
disease were coincident with that reported by Ghor-
banpour et al., 17 and Adnan et al., 27 [27,28]. More-
over, the metabolites produced by Trichoderma spp.
may play a major role in suppressing the growth of phy-
topathogenic fungi as the hyphae of pathogens obvi-
ously collapsed between the interaction zones as clearly
presented by dual culture assay. Several researchers
attributed the formation of clear interaction zone of
hypha disappearance of pathogenic fungi to the action
of secondary metabolites as gliotoxin produced by Tri-
choderma spp which was believed to play an important
role in the antibiosis process [29,30]. On the other hand,
the potent biological activities of Trichoderma strains
may be also assigned to their high colonization rates
resulting in suppression of the competitive microorgan-
isms [28].
In contrast, resistance of A. alternata and D. halodes
to carbendazim fungicide was detected in the current
study at different concentrations. The previous result
was coincident with that of Yang et al., 31 who reported
the resistance of A. alternata to a number of agricultural
fungicides [31]. The development of fungicides resis-
tance may be due to alteration of fungicides target sites
in fungal cell or to the fungal enzymatic breakdown of
these fungicides [32].
In our current study, T. harzianum exhibited myco-
parasitism against A. alternata, F. proliferatum and
B. sorokiniana, while no mycoparasitic action was
detected against D. halodes. In this regard, the myco-
parasitic activity of T. harzianum against B. sorokiniana
was demonstrated by coiling of the Trichoderma hyphae
around the hyphae of pathogenic fungus. Furthermore,
hyphal penetration of T. harzianum into A. alternata,
B. sorokiniana and F. proliferatum hyphae was proven
as another mycoparasitic mode of action [33,34]. On
the other hand, the mycoparasitic action may be pow-
ered by number of extracellular enzymes that disinte-
grate fungal hyphae of phytopathogenic fungi as chiti-
nases and cellulases facilitating hyphal penetration of
Trichoderma bioagents [35,36]. The same explanations

exposing these modes of fungal parasitism against
the concerned phytopathogenic fungi were also doc-
umented by Keswani et al., 37 and Sornakili et al., 38
[37,38].
GC-MS analysis showed that 6-pentyl-α-pyrone
(26.43%) was the main active ingredient of T. harzianum
extract which was recorded to possess antimycotic
activity [39]. The previous concept was ascertained by
Pandey et al., 40 who reported the antimycotic activ-
ity of the previous compound against fungal pathogens
causing plant diseases [40]. The antifungal volatile
constituents as 6-pentyl-α-pyrone produced by Tricho-
derma harzianum were found to suppress the fun-
gal growth of phytopathogenic fungi through myco-
fumigation avoiding side effects of using chemical
fungicides [41,42]. Moreover, 6-pentyl-α-pyrone has the
capability of inhibiting fusaric acid which is a fun-
gal mycotoxin produced by fusarial phytopathogens
[43]. Other volatile active components of T. harzianum
extract including hexadecanoic acid, acetic acid, 2-
phenylethanol, isopentyl acetate, 1-hexadecanol and
eicosane were mentioned to exert antifungal activity in
previous literature [44–48].
On the other hand, the other components as cyclooc-
tanol, propyl-benzene and cadinene were the main
chemical constituents of T. viride extract with relative
percentages of 32.74, 21.65 and 11.23%, respectively.
GC-MS results were in accordance with the previous
study reported by Awad et al., 19 which proved that
cyclooctanol and propyl-benzene were the main active
compounds of T. viride extract recording 8.48% and
41.75%, respectively. Furthermore, the previous con-
stituents were proved to exert antifungal efficiency
against fungal phytopathogens [19]. The sesquiter-
pene compounds of T. viride extract as d- limonene, β-
farnesene, α-bisabolol and cadinene were also reported
to possess antifungal activity [49–52]. Due to the pres-
ence of many bioactive constituents, the antifungal
activities of Trichoderma strains cannot be referred to
one bioactive component only, but also to the syner-
gism among several bioactive constituents of Tricho-
derma extracts [53].
5. Conclusion
Fungal strains (T. viride and T. harzianum) exhib-
ited strong antagonistic activities against fungal phy-
topathogens causing black point disease of wheat.
T. viride exhibited a higher antagonistic activity than
T. harzianum strain against the carbendazim resistant
phytopathogenic fungi. The high antagonistic activity
of Trichoderma strains against fungal pathogens caus-
ing black point disease of wheat encourages perform-
ing further in vivo studies for the application of these
bioagents as natural fungicides avoiding the harmful
impacts of synthetic fungicides.
JOURNAL OF TAIBAH UNIVERSITY FOR SCIENCE 63
Acknowledgement
The authors extend their appreciation to the Researchers Sup-
porting Project number (RSP-2021/362), King Saud University,
Riyadh, Saudi Arabia.quest.
Availability of data and materials
The datasets used and/or analyzed during the current
study are available from the corresponding author on
reasonable request.
Disclosure statement
No potential conflict of interest was reported by the author(s).
quest.
Funding
The study was funded by the Researchers Supporting Project
number [RSP-2021/362], King Saud University, Riyadh, Saudi
Arabia.
ORCID
Mohamed Taha Yassin http://orcid.org/0000-0002-0997-8350
References
[1] Mourad AM, Alomari DZ, Alqudah AM, et al. Recent
advances in wheat (Triticum spp.) breeding. In: Al-Khayri
M, Jain SM, Johnson DV, editors. Advances in Plant Breed-
ing Strategies: Cereals Vol. 5. New York (NY): Springer
International Publishing; 2019. p. 559–593.
[2] Giraldo P, Benavente E, Manzano-Agugliaro F, et al.
Worldwide research trends on wheat and barley: a bib-
liometric comparative analysis. Agronomy. 2019;9:352.
[3] FAO. FAOSTAT database collections. Rome: Food and
Agriculture Organization of the United Nations; 2019.
[4] Carcea M. Nutritional value of grain-based foods. Foods.
2020;9:504.
[5] Xu K-G, Jiang Y-M, Li Y-K, et al. Identification and
pathogenicity of fungal pathogens causing black point
in wheat on the North China plain. Indian J Microbiol.
2018;58:159–164.
[6] Bashyal B, Rawat K, Sharma S, et al. Major seed-borne dis-
eases in important cereals: symptomatology, aetiology
and economic importance seed-borne diseases of agri-
cultural crops: detection, diagnosis & management. Vol.
1. New York (NY): Springer International Publishing; 2020.
p. 371–426.
[7] Figueroa M, Hammond-Kosack KE, Solomon PS. A review
of wheat diseases – a field perspective. Mol Plant Pathol.
2018;19:1523–1536.
[8] Abdullah M, Zulkiffal M, Din A, et al. Discrepancy in germi-
nation behavior and physico-chemical quality traits dur-
ing wheat storage. J Food Process Pres. 2019;43:e14109.
[9] Li Q-Y, Xu Q-Q, Jiang Y-M, et al. The correlation between
wheat black point and agronomic traits in the North
China plain. Crop Prot. 2019;119:17–23.
[10] El-Gremi SM, Draz IS, Youssef WAE. Biological control of
pathogens associated with kernel black point disease of
wheat. Crop Prot. 2017;91:13–19.
[11] Brauer VS, Rezende CP, Pessoni AM, et al. Antifungal
agents in agriculture: friends and foes of public health.
Biomolecules. 2019;9:521.
64 M. T. YASSIN ET AL.
[12] Köhl J, Kolnaar R, Ravensberg WJ. Mode of action
of microbial biological control agents against plant
diseases: relevance beyond efficacy. Front Plant Sci.
2019;10:845.
[13] Sharma PK, Gothalwal R. Trichoderma: a potent fungus
as biological control agent agro-environmental sustain-
ability. Vol. 1. New York (NY): Springer International
Publishing; 2017. p. 113–125.
[14] Meena M, Swapnil P, Zehra A, et al. Antagonistic
assessment of Trichoderma spp. by producing volatile
and non-volatile compounds against different fungal
pathogens. Arch Phytopathology Plant Protect. 2017;50:
629–648.
[15] Sood M, Kapoor D, Kumar V, et al. Trichoderma: The
“secrets” of a multitalented biocontrol agent. Plants.
2020;9:762.
[16] Piechulla B, Lemfack MC, Kai M. Effects of discrete bioac-
tive microbial volatiles on plants and fungi. Plant Cell
Environ. 2017;40:2042–2067.
[17] Ghorbanpour M, Omidvari M, Abbaszadeh-Dahaji P,
et al. Mechanisms underlying the protective effects of
beneficial fungi against plant diseases. Biol Control.
2018;117:147–157.
[18] Ramírez-Cariño HF, Guadarrama-Mendoza PC, Sánchez-
López V, et al. Biocontrol of Alternaria alternata and
Fusarium oxysporum by Trichoderma asperelloides and
bacillus paralicheniformis in tomato plants. Antonie van
Leeuwenhoek. 2020;113:1247–1261.
[19] Awad NE, Kassem HA, Hamed MA, et al. Isolation
and characterization of the bioactive metabolites from
the soil derived fungus Trichoderma viride. Mycology.
2018;9:70–80.
[20] Bunbury-Blanchette AL, Walker AK. Trichoderma species
show biocontrol potential in dual culture and green-
house bioassays against Fusarium basal rot of onion. Biol
Control. 2019;130:127–135.
[21] Naglot A, Goswami S, Rahman I, et al. Antagonistic
potential of native T. viride strain against potent tea
fungal pathogens in North east India. Plant Pathol J.
2015;31:278.
[22] Synytsya A, Monkai J, Bleha R, et al. Antimicrobial activity
of crude extracts prepared from fungal mycelia. Asian Pac
J Trop Biomed. 2017;7:257–261.
[23] Yassin MT, Mostafa AA, Al-Askar AA. In vitro antican-
didal potency of syzygium aromaticum (clove) extracts
against vaginal candidiasis. BMC Complement Altern
Med. 2020;20:25.
[24] Bhardwaj N, Kumar J. Characterization of volatile sec-
ondary metabolites from Trichoderma asperellum. J Appl
Nat Sci. 2017;9:954–959.
[25] Dukare AS, Singh RK, Jangra RK, et al. Non-fungicides-
based promising technologies for managing post-pro
duction penicillium induced spoilage in horticultural
commodities: a comprehensive review. Food Rev Int.
2020: 1–41.
[26] Mazrou YS, Makhlouf AH, Elseehy MM, et al. Antagonis-
tic activity and molecular characterization of biological
control agent Trichoderma harzianum from Saudi arabia.
Egypt J Biol Pest Co. 2020;30:4.
[27] Adnan M, Islam W, Shabbir A, et al. Plant defense against
fungal pathogens by antagonistic fungi with Tricho-
derma in focus. Microb Pathog. 2019;129:7–18.
[28] Bizos G, Papatheodorou EM, Chatzistathis T, et al. The
role of microbial inoculants on plant protection, growth
stimulation, and crop productivity of the olive tree (Olea
europea L). Plants. 2020;9(6):743.
[29] Tomah AA, Abd Alamer IS, Li B, et al. A new species
of Trichoderma and gliotoxin role: a new observa-
tion in enhancing biocontrol potential of T. virens
against phytophthora capsici on chili pepper. Biol Control.
2020;145:104261.
[30] Khan RAA, Najeeb S, Hussain S, et al. Bioactive sec-
ondary metabolites from Trichoderma spp. against phy-
topathogenic fungi. Microorganisms. 2020;8(6):817.
[31] Yang L-N, He M-H, Ouyang H-B, et al. Cross-resistance
of the pathogenic fungus Alternaria alternata to fungi-
cides with different modes of action. BMC Microbiol.
2019;19:205.
[32] Baibakova EV, Nefedjeva EE, Suska-Malawska M, et al.
Modern fungicides: mechanisms of action, fungal resis-
tance and phytotoxic effects. Annu Res Rev Biol. 2019;
32(3):1–16.
[33] Parulekar-Berde CV, Joshi SA, Berde VB. Fungal com-
munities as biological control agents for different phy-
topathogenic organisms Recent Trends in mycological
Research. New York (NY): Springer; 2020. p. 189–201.
[34] Asad SA, Tabassum A, Hameed A, et al. Determination of
lytic enzyme activities of indigenous Trichoderma isolates
from Pakistan. Braz J Microbiol. 2015;46:1053–1064.
[35] Bhat K. A New agar plate assisted slide culture technique
to study mycoparasitism of Trichoderma spp. on Rhizoc-
tonia solani and Fusarium oxysporium. Int J Curr Microbiol
Appl Sci. 2017;6:3176–3180.
[36] Rajani P, Rajasekaran C, Vasanthakumari M, et al. Inhi-
bition of plant pathogenic fungi by endophytic Tricho-
derma spp. through mycoparasitism and volatile organic
compounds. Microbiol Res. 2021;242:126595.
[37] Keswani C, Singh HB, Hermosa R, et al. Antimicrobial sec-
ondary metabolites from agriculturally important fungi
as next biocontrol agents. Appl Microbiol Biotechnol.
2019;103:9287–9303.
[38] Sornakili A, Thankappan S, Sridharan A, et al. Antagonistic
fungal endophytes and their metabolite-mediated inter-
actions against phytopathogens in rice. Physiol Mol Plant
Path. 2020;112:101525.
[39] Ismaiel AA, Ali DM. Antimicrobial properties of 6-pentyl-
α-pyrone produced by endophytic strains of Tricho-
derma koningii and its effect on aflatoxin B1 production.
Biologia. 2017;72(12):1403–1415.
[40] Pandey M, Ahmad S, John SA, et al. Antifungal potential
of native Trichoderma isolates against pythium aphani-
dermatum causing chilli damping-off. Ann Plant Prot Sci.
2019;27:102–106.
[41] Alfiky A, Weisskopf L. Deciphering trichoderma–plant–
pathogen interactions for better development of biocon-
trol applications. Journal of Fungi. 2021;7(1):61.
[42] Degani O, Khatib S, Becher P, et al. Trichoderma asperel-
lum secreted 6-pentyl-α-pyrone to control magnapor-
thiopsis maydis, the Maize late wilt disease agent. Biology
(Basel). 2021;10(9):897.
[43] Sharma VS, Sharma PR. The comparative mechanistic
aspects of Trichoderma and probiotics: scope for future
research. Physiol Mol Plant Pathol. 2017;100:84–96.
[44] Ahsan T, Chen J, Zhao X, et al. Extraction and identi-
fication of bioactive compounds (eicosane and dibutyl
phthalate) produced by streptomyces strain KX852460 for
the biological control of Rhizoctonia solani AG-3 strain
KX852461 to control target spot disease in tobacco leaf.
AMB Express. 2017;7:54.
[45] Mookherjee A, Bera P, Mitra A, et al. Characterization
and synergistic effect of antifungal volatile organic com-
pounds emitted by the geotrichum candidum PF005,

an endophytic fungus from the eggplant. Microb Ecol.
2018;75:647–661.
[46] Liu Q, Yin G, Zhang J. The suitable condition for lactobacil-
lus parafarraginis ZH1 producing hexadecanoic acid and
inhibiting pathogenic and spoilage yeasts. Biol Control.
2020;149:104318.
[47] Debonne E, Vermeulen A, Bouboutiefski N, et al. Mod-
elling and validation of the antifungal activity of DL-
3-phenyllactic acid and acetic acid on bread spoilage
moulds. Food Microbiol. 2020;88:103407.
[48] Zhang H, Du H, Xu Y. Volatile organic compound-
mediated antifungal activity of pichia spp. and Its effect
on the metabolic profiles of fermentation communities.
Appl Environ Microbiol. 2021;87(9):e02992–e02920.
[49] Wang H, Yang Z, Ying G, et al. Antifungal evaluation of
plant essential oils and their major components against
toxigenic fungi. Ind Crops Prod. 2018;120:180–186.
JOURNAL OF TAIBAH UNIVERSITY FOR SCIENCE 65
[50] He X, Zhang L, Chen J, et al. Correlation between Chem-
ical composition and antifungal activity of clausena
lansium essential Oil against candida spp. Molecules.
2019;24:1394.
[51] Cai R, Hu M, Zhang Y, et al. Antifungal activity and mech-
anism of citral, limonene and eugenol against zygosaccha-
romyces rouxii. Lwt. 2019;106:50–56.
[52] de Medeiros CA, ÂdV P, de Oliveira JC, et al. Evaluating the
antifungal activity of α-bisabolol in association with NaCl
on Fusarium oxysporum in Maize grains. Curr Microbiol.
2021;78(2):604–610.
[53] Khruengsai S, Pripdeevech P, D’Souza PE, et al. Biofumi-
gation activities of volatile compounds from two Tricho-
derma afroharzianum strains against Fusarium infections
in fresh chilies. J Sci Food Agric. 2021;101:5861–5871.