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

CONTACT Mohamed Taha Yassin [email protected] 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- that may hinder membrane sterilization. Finally, the
tum) which cause black point disease of wheat. filtrates were sterilized using Millipore filters (22 µm).
PDA medium supplemented with 25% Trichoderma spp.
2. Materials and methods filtrates was poured in sterile Petri dishes and inocu-
lated with 6 mm mycelial discs of the phytopathogenic
2.1. Plant fungal pathogens and antagonistic fungal strains. The control plates were inoculated with
strains 6 mm mycelial discs of different fungal pathogens. The
The four phytopathogenic fungal strains (Fusarium pro- plates were then incubated at 25 ± 2°C for 5 days. The
liferatum ATCC 74149, Alternaria alternata ATCC 66981, radial growth diameters of the phytopathogens were
Bipolaris sorokiniana ATCC 32093, Drechslera halodes then measured using a Vernier calliper. The inhibition
ATCC 32198) and two Trichoderma spp. (Trichoderma of mycelial growth index (IMG %) was calculated using
viride ATCC 18652 and Trichoderma harzianum ATCC the following formula:
48131) were obtained from the Botany and Microbi-
Inhibition % = (A–B)/A × 100,
ology Department, College of Science, King Saud Uni-
versity. The fungal strains were subcultured on potato where A = radial growth diameter of the phytopatho
dextrose agar (PDA) slants, incubated at 25 ± 2°C for 5 gens in the control plates, B = radial growth diameter
days and finally stored in the refrigerator at 4°C until of the phytopathogens in the treated plates [20].
further use.

2.4. Slide culture technique to detect

2.2. Evaluation of fungal antagonistic activity mycoparasitism relationship
(Dual culture technique)
PDA medium was prepared and poured into sterile
The antagonistic activity of Trichoderma viride and Tri- Petri dishes. The medium was then cut with a ster-
choderma harzianum against fungal pathogens which ilized blade, placed over a sterilized glass slide, and
cause black point disease of wheat was evaluated using inoculated with Trichoderma spp. on one side and a
the dual culture technique as previously described [19]. pathogen strain on the opposite side. The slide was then
Mycelial discs (6 mm in diameter) of fungal antagonis- incubated at 25 ± 2°C for 5 days, followed by removal
tic strains were inoculated in PDA plates and placed of the PDA medium. The fungal mycelium was then
at 1.5 cm from the edge of freshly prepared PDA stained with lactophenol cotton blue and a sterile cov-
plates. Similarly, 6 mm mycelial discs of fungal phy- erslip was placed over the slide. The hyphal interactions
topathogens were placed in the opposite direction between the Trichoderma spp. and fungal pathogens
of the concerned antagonistic fungal strain at 1.5 cm were observed using a light microscope (40×) [21].
from the edge of the prepared PDA plates. The control
plates were inoculated with different fungal pathogens
(Alternaria alternata, Bipolaris sorokiniana or Fusarium 2.5. Antimicrobial activity of carbendazim
proliferatum) and incubated with the treated plates at (commonly used fungicide)
25 ± 2°C for 5 days. Afterwards, the radial growth of The food poisoning technique was used to detect the
the phytopathogens in the control and treatment plates antifungal efficacy of carbendazim, a commonly used
was measured and inhibition percentages were calcu- fungicide. Different carbendazim concentrations (50,
lated using the following formula: 100, 150, 200, 250 and 300 ppm) were added to the
% inhibition = (A–B)/A × 100 PDA medium after sterilization. The plates were then
inoculated with a 6 mm disc of fungal growth and incu-
where A = colony diameter of the phytopathogen in bated at 25 ± 2°C for 5 days. The growth diameter of the
the control plate, B = colony diameter in the dual cul- pathogenic fungi tested was measured using a Vernier
ture plate. calliper. The growth inhibition ratio (%) was calculated
using the following equation:
2.3. Antifungal potency of culture filtrates of
concerned antagonistic fungi % inhibition = (A–B)/A × 100,

The percentage of growth inhibition (%) of the phy- where A = growth diameter in the control plates,
topathogenic fungal strains by the Trichoderma filtrates B = growth diameter in the treated plates [19].
was evaluated. The two antagonistic fungal strains were
grown in 50 mL of potato dextrose broth and incu-
2.6. Preparation of Trichoderma spp. crude
bated at 25 ± 2°C for 7 days in an orbital shaker at
extracts
150 rpm. Cell-free filtrates were obtained via filtration
using double layers of muslin followed by centrifuga- The ability of Trichoderma strains to produce active
tion at 9000 rpm for 10 min to remove the fungal spores metabolites which exhibit antifungal activity against
 JOURNAL OF TAIBAH UNIVERSITY FOR SCIENCE 59

fungal strains causing black point disease of wheat was achieved by comparison of their mass spectrum with
investigated. The Trichoderma isolates were cultured reference standards in NIST database.
onto PDA medium and incubated at 25 ± 2°C for 7 days.
The fungal growth was then scraped from the agar 2.9. Statistical analysis
surface to obtain a mycelial sample of 5 g. Then, the
mycelial growth was added to 200 mL of 80% ethanol Data were statistically analyzed by GraphPad Prism 5.0
supplemented with 0.2 M HCl and incubated in a rota- (GraphPad Software, Inc., La Jolla, CA, USA) using one-
tory shaker at 25 ± 2°C for 24 h. The samples were then way analysis of variance. The experiments were done
centrifuged at 9000 rpm to separate the solid compo- in triplicates and the data were presented as a mean of
nents. The extraction procedure was performed again triplicates ± standard error.
for the solid components, using ethyl acetate as a sol-
vent. Finally, the extracts were combined and partially 3. Results
evaporated using a rotatory evaporator. The yield was
calculated using the following equation: 3.1. Antagonistic antifungal activity of
Trichoderma strains
Extract Yield % = (R/S) × 100, Dual culture results showed that both T. viride and T.
harzianum strains limited the growth of different fun-
where R = extract residue, S = weight of the raw gal phytopathogens with different growth inhibition
mycelial sample [22]. patterns as shown in Figure 1. T. viride strain showed
the highest antagonistic activity against different phy-
2.7. Antifungal efficacy of Trichoderma spp. topathogenic fungal strains compared to T. harzianum
extracts against fungal pathogens strain. Alternaria alternata was the most susceptible
strain to the concerned antagonistic fungal strains (T.
The disc diffusion method was used to evaluate the viride and T. harzianum) recording inhibition percent-
antifungal activity of Trichoderma spp. extracts against ages of 75.04 and 67.83%, respectively (Table 1). In con-
phytopathogenic fungi which cause black point disease trast, the lowest antagonistic activities of T. viride and
of wheat. Fifteen mL of PDA medium was poured in ster- T. harzianum strains were estimated against D. halodes
ile Petri dishes as a basal medium, followed by 10 mL recording growth inhibition percentages of 51.54 and
of seeded medium previously inoculated with a suspen- 43.92%, respectively. Furthermore, the results revealed
sion of spores from the fungal phytopathogens tested. that T. viride exerted antagonistic action against B.
Then, filter paper discs (8 mm diameter) were loaded sorokiniana and F. proliferatum with growth inhibition
with different concentrations of Trichoderma spp. crude percentages of 62.65 and 64.23%, while T. harzianum
extracts (50, 100, 150, 250, 300 and 400 µg/disc) and showed inhibition percentages of 59.04 and 53.42%,
placed over the seeded plates [19]. Filter paper discs respectively.
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 3.2. Antifungal potency of culture filtrates of
inhibition zones were measured using a Vernier cal- concerned antagonistic fungi
liper and the lowest concentration of Trichoderma spp. Cultural filtrates of the antagonistic fungal strains (T.
extract which exhibited an inhibitory effect against the harzianum and T. viride) exhibited antifungal potency
phytopathogens tested was considered the minimum against different pathogenic fungal strains. T. viride fil-
inhibitory concentration (MIC). The minimum fungici- trates demonstrated the maximum growth inhibition
dal concentration (MFC) was obtained by plating an against B. sorokiniana (57.97%) followed by D. halodes
inoculum from the inhibition zones into freshly pre- (42.90%), A. alternata (30.46%) and F. proliferatum
pared PDA medium. The lowest concentration with no (19.02%). On the other hand, the cultural filtrates of T.
fungal growth was considered the MFC. harzianum exhibited the highest anti-phytopathogenic
activity against B. sorokiniana followed by D. halodes,
2.8. GC-MS analysis of the T. viride and T. A. alternata and F. proliferatum recording growth inhi-
harzianum extracts bition percentages of 53.06, 40.81, 28.72 and 6.97%,
respectively, as seen in Table 2.
Chemical analyses of T. harzianum and T. viride extracts
were achieved to detect the active constituents exhibit-
3.3. Slide culture technique to detect
ing antifungal activity. GC-MS was performed using an
mycoparasitism relationship
Agilent 7890 gas chromatograph and an Agilent 5975C
Mass Spectrometer, USA. Conditions of analysis were T. harzianum strain exhibited mycoparasitism against
optimized as stated by Yassin et al. 23 [23]. Identifica- the tested phytopathogenic fungal isolates except D.
tion of active components of Trichoderma extracts was 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 exhibited carbendazim resistance. Furthermore, car-
parasitic action against A. alternata, B. sorokiniana and bendazim was moderately effective against B. sorokini-
F. proliferatum strains through different mechanisms of ana strain with inhibition percentage of 69.08% at
action as seen in Figure 2. The mycoparasitic behaviour 1.00 ppm concentration. In addition, the MIC of car-
included coiling of Trichoderma hyphae around the phy- bendazim against B. sorokiniana was 0.50 ppm and the
topathogenic hyphae, formation of penetration peg mycelial growth was completely suppressed at MFC of
and disintegration of pathogenic hyphae. 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- 1-hexadecanol (6.45%), isopentyl acetate (4.65%),
tive strain to T. harzianum and T. viride extracts record- dioctyl ester, hexanedioic acid (4.24%) and 9-eicosane
ing suppressive zone diameters of 11.36 and 12.29 mm, (2.23%) as demonstrated in Table 5. In contrast, the
respectively at a concentration of 200 µg/disc as seen major active component of T. viride was cyclooc-
in Table 4. In contrast, A. alternata exhibited the low- tanol (32.74%) followed by propyl-benzene (21.65%),
est susceptibility to T. harzianum and T. viride strains cadinene (11.23%), epizonaren (9.34%), d- limonene
with MIC values of 300 µg/disc, respectively. The MFC (8.49%), α-bisabolol (7.32%), β-farnesene (6.78%) and
of T. viride extract against different phytopathogenic propanoic acid (2.45%) as seen in Table 6.
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 4. Discussion
400 µg/disc against A. alternata and D. halodes strains.
Trichoderma spp. showed a potential biocontrol effi-
ciency against fungal phytopathogens either through
direct mechanisms of mycoparasitism and antibiosis or
3.6. GC-MS analysis of the T. viride and T.
by indirect mechanisms as competition for nutrients,
harzianum extracts
enhancing plant defence responses and exhilarating
GS-MS of T. viride and T. harzianum was performed plant growth [24]. In this regard, Trichoderma bioagents
to detect different active ingredients exhibiting anti- suppress the growth of fungal phytopathogens by a
phytopathogenic activity. 6-pentyl-α-pyrone (26.43%) mixed action through their metabolites inhibitory effect
was the predominant active component of T. harzianum and lytic enzymes which disintegrate the fungal cell wall
extract followed by hexadecanoic acid (15.98%), acetic [25]. Trichoderma spp. have been ascertained as bio-
acid (12.84%), 2-phenylethyl alcohol (11.36%), 2-butox logical control agents against several phytopathogenic
yethyl acetate (8.53%), 1-methoy-2-propanone (7.29%), 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- resulting in suppression of the competitive microorgan-
gal pathogenic strains causing black point disease of isms [28].
wheat. Dual cultural assay indicated that T. viride strain In contrast, resistance of A. alternata and D. halodes
exerted the highest antagonistic potency against A. to carbendazim fungicide was detected in the current
alternata (75.04%) followed by F. proliferatum (64.23%), study at different concentrations. The previous result
B. sorokiniana (62.65%) and D. halodes (51.54%), while was coincident with that of Yang et al., 31 who reported
T. harzianum exhibited a moderate efficacy against A. the resistance of A. alternata to a number of agricultural
alternata followed by B. sorokiniana, F. proliferatum and fungicides [31]. The development of fungicides resis-
D. halodes with corresponding inhibition percentages tance may be due to alteration of fungicides target sites
of 67.83%, 59.04%, 53.42% and 43.92%, respectively. in fungal cell or to the fungal enzymatic breakdown of
The proved antagonistic efficacies of T. viride and T. these fungicides [32].
harzianum against fungal strains causing black point In our current study, T. harzianum exhibited myco-
disease were coincident with that reported by Ghor- parasitism against A. alternata, F. proliferatum and
banpour et al., 17 and Adnan et al., 27 [27,28]. More- B. sorokiniana, while no mycoparasitic action was
over, the metabolites produced by Trichoderma spp. detected against D. halodes. In this regard, the myco-
may play a major role in suppressing the growth of phy- parasitic activity of T. harzianum against B. sorokiniana
topathogenic fungi as the hyphae of pathogens obvi- was demonstrated by coiling of the Trichoderma hyphae
ously collapsed between the interaction zones as clearly around the hyphae of pathogenic fungus. Furthermore,
presented by dual culture assay. Several researchers hyphal penetration of T. harzianum into A. alternata,
attributed the formation of clear interaction zone of B. sorokiniana and F. proliferatum hyphae was proven
hypha disappearance of pathogenic fungi to the action as another mycoparasitic mode of action [33,34]. On
of secondary metabolites as gliotoxin produced by Tri- the other hand, the mycoparasitic action may be pow-
choderma spp which was believed to play an important ered by number of extracellular enzymes that disinte-
role in the antibiosis process [29,30]. On the other hand, grate fungal hyphae of phytopathogenic fungi as chiti-
the potent biological activities of Trichoderma strains nases and cellulases facilitating hyphal penetration of
may be also assigned to their high colonization rates Trichoderma bioagents [35,36]. The same explanations
 JOURNAL OF TAIBAH UNIVERSITY FOR SCIENCE 63

exposing these modes of fungal parasitism against Acknowledgement

the concerned phytopathogenic fungi were also doc- The authors extend their appreciation to the Researchers Sup-
umented by Keswani et al., 37 and Sornakili et al., 38 porting Project number (RSP-2021/362), King Saud University,
[37,38]. Riyadh, Saudi Arabia.quest.
GC-MS analysis showed that 6-pentyl-α-pyrone
(26.43%) was the main active ingredient of T. harzianum Availability of data and materials
extract which was recorded to possess antimycotic
activity [39]. The previous concept was ascertained by The datasets used and/or analyzed during the current
Pandey et al., 40 who reported the antimycotic activ- study are available from the corresponding author on
ity of the previous compound against fungal pathogens reasonable request.
causing plant diseases [40]. The antifungal volatile
constituents as 6-pentyl-α-pyrone produced by Tricho-
Disclosure statement
derma harzianum were found to suppress the fun- No potential conflict of interest was reported by the author(s).
gal growth of phytopathogenic fungi through myco- quest.
fumigation avoiding side effects of using chemical
fungicides [41,42]. Moreover, 6-pentyl-α-pyrone has the Funding
capability of inhibiting fusaric acid which is a fun- The study was funded by the Researchers Supporting Project
gal mycotoxin produced by fusarial phytopathogens number [RSP-2021/362], King Saud University, Riyadh, Saudi
Arabia.
[43]. Other volatile active components of T. harzianum
extract including hexadecanoic acid, acetic acid, 2-
ORCID
phenylethanol, isopentyl acetate, 1-hexadecanol and
eicosane were mentioned to exert antifungal activity in Mohamed Taha Yassin http://orcid.org/0000-0002-0997-8350
previous literature [44–48].
On the other hand, the other components as cyclooc- References
tanol, propyl-benzene and cadinene were the main
[1] Mourad AM, Alomari DZ, Alqudah AM, et al. Recent
chemical constituents of T. viride extract with relative
advances in wheat (Triticum spp.) breeding. In: Al-Khayri
percentages of 32.74, 21.65 and 11.23%, respectively. M, Jain SM, Johnson DV, editors. Advances in Plant Breed-
GC-MS results were in accordance with the previous ing Strategies: Cereals Vol. 5. New York (NY): Springer
study reported by Awad et al., 19 which proved that International Publishing; 2019. p. 559–593.
cyclooctanol and propyl-benzene were the main active [2] Giraldo P, Benavente E, Manzano-Agugliaro F, et al.
compounds of T. viride extract recording 8.48% and Worldwide research trends on wheat and barley: a bib-
liometric comparative analysis. Agronomy. 2019;9:352.
41.75%, respectively. Furthermore, the previous con-
[3] FAO. FAOSTAT database collections. Rome: Food and
stituents were proved to exert antifungal efficiency Agriculture Organization of the United Nations; 2019.
against fungal phytopathogens [19]. The sesquiter- [4] Carcea M. Nutritional value of grain-based foods. Foods.
pene compounds of T. viride extract as d- limonene, β- 2020;9:504.
farnesene, α-bisabolol and cadinene were also reported [5] Xu K-G, Jiang Y-M, Li Y-K, et al. Identification and
pathogenicity of fungal pathogens causing black point
to possess antifungal activity [49–52]. Due to the pres-
in wheat on the North China plain. Indian J Microbiol.
ence of many bioactive constituents, the antifungal 2018;58:159–164.
activities of Trichoderma strains cannot be referred to [6] Bashyal B, Rawat K, Sharma S, et al. Major seed-borne dis-
one bioactive component only, but also to the syner- eases in important cereals: symptomatology, aetiology
gism among several bioactive constituents of Tricho- and economic importance seed-borne diseases of agri-
derma extracts [53]. cultural crops: detection, diagnosis & management. Vol.
1. New York (NY): Springer International Publishing; 2020.
p. 371–426.
5. Conclusion [7] Figueroa M, Hammond-Kosack KE, Solomon PS. A review
of wheat diseases – a field perspective. Mol Plant Pathol.
Fungal strains (T. viride and T. harzianum) exhib- 2018;19:1523–1536.
ited strong antagonistic activities against fungal phy- [8] Abdullah M, Zulkiffal M, Din A, et al. Discrepancy in germi-
nation behavior and physico-chemical quality traits dur-
topathogens causing black point disease of wheat.
ing wheat storage. J Food Process Pres. 2019;43:e14109.
T. viride exhibited a higher antagonistic activity than [9] Li Q-Y, Xu Q-Q, Jiang Y-M, et al. The correlation between
T. harzianum strain against the carbendazim resistant wheat black point and agronomic traits in the North
phytopathogenic fungi. The high antagonistic activity China plain. Crop Prot. 2019;119:17–23.
of Trichoderma strains against fungal pathogens caus- [10] El-Gremi SM, Draz IS, Youssef WAE. Biological control of
ing black point disease of wheat encourages perform- pathogens associated with kernel black point disease of
wheat. Crop Prot. 2017;91:13–19.
ing further in vivo studies for the application of these [11] Brauer VS, Rezende CP, Pessoni AM, et al. Antifungal
bioagents as natural fungicides avoiding the harmful agents in agriculture: friends and foes of public health.
impacts of synthetic fungicides. Biomolecules. 2019;9:521.
64 M. T. YASSIN ET AL.

[12] Köhl J, Kolnaar R, Ravensberg WJ. Mode of action [29] Tomah AA, Abd Alamer IS, Li B, et al. A new species
of microbial biological control agents against plant of Trichoderma and gliotoxin role: a new observa-
diseases: relevance beyond efficacy. Front Plant Sci. tion in enhancing biocontrol potential of T. virens
2019;10:845. against phytophthora capsici on chili pepper. Biol Control.
[13] Sharma PK, Gothalwal R. Trichoderma: a potent fungus 2020;145:104261.
as biological control agent agro-environmental sustain- [30] Khan RAA, Najeeb S, Hussain S, et al. Bioactive sec-
ability. Vol. 1. New York (NY): Springer International ondary metabolites from Trichoderma spp. against phy-
Publishing; 2017. p. 113–125. topathogenic fungi. Microorganisms. 2020;8(6):817.
[14] Meena M, Swapnil P, Zehra A, et al. Antagonistic [31] Yang L-N, He M-H, Ouyang H-B, et al. Cross-resistance
assessment of Trichoderma spp. by producing volatile of the pathogenic fungus Alternaria alternata to fungi-
and non-volatile compounds against different fungal cides with different modes of action. BMC Microbiol.
pathogens. Arch Phytopathology Plant Protect. 2017;50: 2019;19:205.
629–648. [32] Baibakova EV, Nefedjeva EE, Suska-Malawska M, et al.
[15] Sood M, Kapoor D, Kumar V, et al. Trichoderma: The Modern fungicides: mechanisms of action, fungal resis-
“secrets” of a multitalented biocontrol agent. Plants. tance and phytotoxic effects. Annu Res Rev Biol. 2019;
2020;9:762. 32(3):1–16.
[16] Piechulla B, Lemfack MC, Kai M. Effects of discrete bioac- [33] Parulekar-Berde CV, Joshi SA, Berde VB. Fungal com-
tive microbial volatiles on plants and fungi. Plant Cell munities as biological control agents for different phy-
Environ. 2017;40:2042–2067. topathogenic organisms Recent Trends in mycological
[17] Ghorbanpour M, Omidvari M, Abbaszadeh-Dahaji P, Research. New York (NY): Springer; 2020. p. 189–201.
et al. Mechanisms underlying the protective effects of [34] Asad SA, Tabassum A, Hameed A, et al. Determination of
beneficial fungi against plant diseases. Biol Control. lytic enzyme activities of indigenous Trichoderma isolates
2018;117:147–157. from Pakistan. Braz J Microbiol. 2015;46:1053–1064.
[18] Ramírez-Cariño HF, Guadarrama-Mendoza PC, Sánchez- [35] Bhat K. A New agar plate assisted slide culture technique
López V, et al. Biocontrol of Alternaria alternata and to study mycoparasitism of Trichoderma spp. on Rhizoc-
Fusarium oxysporum by Trichoderma asperelloides and tonia solani and Fusarium oxysporium. Int J Curr Microbiol
bacillus paralicheniformis in tomato plants. Antonie van Appl Sci. 2017;6:3176–3180.
Leeuwenhoek. 2020;113:1247–1261. [36] Rajani P, Rajasekaran C, Vasanthakumari M, et al. Inhi-
[19] Awad NE, Kassem HA, Hamed MA, et al. Isolation bition of plant pathogenic fungi by endophytic Tricho-
and characterization of the bioactive metabolites from derma spp. through mycoparasitism and volatile organic
the soil derived fungus Trichoderma viride. Mycology. compounds. Microbiol Res. 2021;242:126595.
2018;9:70–80. [37] Keswani C, Singh HB, Hermosa R, et al. Antimicrobial sec-
[20] Bunbury-Blanchette AL, Walker AK. Trichoderma species ondary metabolites from agriculturally important fungi
show biocontrol potential in dual culture and green- as next biocontrol agents. Appl Microbiol Biotechnol.
house bioassays against Fusarium basal rot of onion. Biol 2019;103:9287–9303.
Control. 2019;130:127–135. [38] Sornakili A, Thankappan S, Sridharan A, et al. Antagonistic
[21] Naglot A, Goswami S, Rahman I, et al. Antagonistic fungal endophytes and their metabolite-mediated inter-
potential of native T. viride strain against potent tea actions against phytopathogens in rice. Physiol Mol Plant
fungal pathogens in North east India. Plant Pathol J. Path. 2020;112:101525.
2015;31:278. [39] Ismaiel AA, Ali DM. Antimicrobial properties of 6-pentyl-
[22] Synytsya A, Monkai J, Bleha R, et al. Antimicrobial activity α-pyrone produced by endophytic strains of Tricho-
of crude extracts prepared from fungal mycelia. Asian Pac derma koningii and its effect on aflatoxin B1 production.
J Trop Biomed. 2017;7:257–261. Biologia. 2017;72(12):1403–1415.
[23] Yassin MT, Mostafa AA, Al-Askar AA. In vitro antican- [40] Pandey M, Ahmad S, John SA, et al. Antifungal potential
didal potency of syzygium aromaticum (clove) extracts of native Trichoderma isolates against pythium aphani-
against vaginal candidiasis. BMC Complement Altern dermatum causing chilli damping-off. Ann Plant Prot Sci.
Med. 2020;20:25. 2019;27:102–106.
[24] Bhardwaj N, Kumar J. Characterization of volatile sec- [41] Alfiky A, Weisskopf L. Deciphering trichoderma–plant–
ondary metabolites from Trichoderma asperellum. J Appl pathogen interactions for better development of biocon-
Nat Sci. 2017;9:954–959. trol applications. Journal of Fungi. 2021;7(1):61.
[25] Dukare AS, Singh RK, Jangra RK, et al. Non-fungicides- [42] Degani O, Khatib S, Becher P, et al. Trichoderma asperel-
based promising technologies for managing post-pro lum secreted 6-pentyl-α-pyrone to control magnapor-
duction penicillium induced spoilage in horticultural thiopsis maydis, the Maize late wilt disease agent. Biology
commodities: a comprehensive review. Food Rev Int. (Basel). 2021;10(9):897.
2020: 1–41. [43] Sharma VS, Sharma PR. The comparative mechanistic
[26] Mazrou YS, Makhlouf AH, Elseehy MM, et al. Antagonis- aspects of Trichoderma and probiotics: scope for future
tic activity and molecular characterization of biological research. Physiol Mol Plant Pathol. 2017;100:84–96.
control agent Trichoderma harzianum from Saudi arabia. [44] Ahsan T, Chen J, Zhao X, et al. Extraction and identi-
Egypt J Biol Pest Co. 2020;30:4. fication of bioactive compounds (eicosane and dibutyl
[27] Adnan M, Islam W, Shabbir A, et al. Plant defense against phthalate) produced by streptomyces strain KX852460 for
fungal pathogens by antagonistic fungi with Tricho- the biological control of Rhizoctonia solani AG-3 strain
derma in focus. Microb Pathog. 2019;129:7–18. KX852461 to control target spot disease in tobacco leaf.
[28] Bizos G, Papatheodorou EM, Chatzistathis T, et al. The AMB Express. 2017;7:54.
role of microbial inoculants on plant protection, growth [45] Mookherjee A, Bera P, Mitra A, et al. Characterization
stimulation, and crop productivity of the olive tree (Olea and synergistic effect of antifungal volatile organic com-
europea L). Plants. 2020;9(6):743. pounds emitted by the geotrichum candidum PF005,
 JOURNAL OF TAIBAH UNIVERSITY FOR SCIENCE 65

an endophytic fungus from the eggplant. Microb Ecol. [50] He X, Zhang L, Chen J, et al. Correlation between Chem-
2018;75:647–661. ical composition and antifungal activity of clausena
[46] Liu Q, Yin G, Zhang J. The suitable condition for lactobacil- lansium essential Oil against candida spp. Molecules.
lus parafarraginis ZH1 producing hexadecanoic acid and 2019;24:1394.
inhibiting pathogenic and spoilage yeasts. Biol Control. [51] Cai R, Hu M, Zhang Y, et al. Antifungal activity and mech-
2020;149:104318. anism of citral, limonene and eugenol against zygosaccha-
[47] Debonne E, Vermeulen A, Bouboutiefski N, et al. Mod- romyces rouxii. Lwt. 2019;106:50–56.
elling and validation of the antifungal activity of DL- [52] de Medeiros CA, ÂdV P, de Oliveira JC, et al. Evaluating the
3-phenyllactic acid and acetic acid on bread spoilage antifungal activity of α-bisabolol in association with NaCl
moulds. Food Microbiol. 2020;88:103407. on Fusarium oxysporum in Maize grains. Curr Microbiol.
[48] Zhang H, Du H, Xu Y. Volatile organic compound- 2021;78(2):604–610.
mediated antifungal activity of pichia spp. and Its effect [53] Khruengsai S, Pripdeevech P, D’Souza PE, et al. Biofumi-
on the metabolic profiles of fermentation communities. gation activities of volatile compounds from two Tricho-
Appl Environ Microbiol. 2021;87(9):e02992–e02920. derma afroharzianum strains against Fusarium infections
[49] Wang H, Yang Z, Ying G, et al. Antifungal evaluation of in fresh chilies. J Sci Food Agric. 2021;101:5861–5871.
plant essential oils and their major components against
toxigenic fungi. Ind Crops Prod. 2018;120:180–186.