J. Bio. & Env. Sci.

2024

Journal of Biodiversity and Environmental Sciences (JBES)

ISSN: 2220-6663 (Print) 2222-3045 (Online)
Vol. 24, No. 1, p. 23-29, 2024
http://www.innspub.net

RESEARCH
RESEARCH PAPER OPEN ACCESS

In vitro aggression of Trichoderma species against Fusarium

induced wilt disease in cotton

R. Annadurai, M. Kamaraj*

Department of Botany, Jamal Mohamed College (A), Triuchirappalli, Tamil Nadu, India

Article published on January 06, 2024

Key words: Abiotic, Inhibited, Rhizosphere, Temperature, Trichoderma

Abstract
A soil-borne fungus called Fusarium species assaults plants through their roots at all stages of growth and results
in significant economic losses by causing signs of necrosis and wilting. Trichoderma species tested against
Fusarium species under in vitro conditions. In this research, isolate of Trichoderma species was found in
rhizosphere soils of cotton crops using the dual culture approach. It was discovered that various Fusarium
species, including F1, F2, F3 and F4 were negatively affected by local isolates of Trichoderma sp. The studied
Fusarium species growth was inhibited by the isolates of Trichoderma spp. it was also examined how abiotic
stresses including pH, temperature and NaCl affected the development of Trichoderma isolates. The isolates T1,
T4, and T6 were most resilient to abiotic stress. Chitinase generated by local isolates was examined for its
particular activity. In the culture supernatant, the antagonist T4 isolate produced more chitinase activity.
*Corresponding Author: M. Kamaraj  [email protected]

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Introduction soil borne diseases. Pseudomonas species

Cotton (Gossypium spp.) is a valuable cash crop since demonstrated a significant reduction in disease
it produces grain, fibre, and oil, according to incidence and an increase in chickpea growth Khalifa
Sunilkumar et al. (2006). Cotton is the world's most et al. (2022); Mozumder et al. (2022). Meanwhile,
important source of natural fibre, accounting for Trichoderma sp. effectively suppressed Fusarium wilt
around 35% of total fibre production Billah et al. through competition for nutrients and space,
(2021). It is a renewable resource, and cotton farms mycoparasitism, antibiosis, and improved plant
employ more people in planting, processing, and growth, leading to showing well-controlled efficiency
textile production than synthetic fibre. Cotton has Awad-Allah et al. (2022); Do Amaral et al. (2022);
significant environmental and social benefits, and Rao et al. 2022).
following oil extraction, cotton seed may be used as
animal feed by Rathore et al. (2015). However, cotton Trichoderma species are potential fungal bioagents
has been plagued by a variety of pests and diseases, for the management of various economically
the most serious of which are Fusarium and important plant pathogens. This species is usually
Verticillium wilt, which cause significant global preferred due to rapid growth, antagonistic effect,
cotton economic losses Davis et al., 2006; Cun et al. ability to increase nutrient availability, and uptake,
(2002). secretion of cell wall-degrading enzymes, ability to
produce antibiotics and volatile compounds
Fusarium species is a soil-borne fungal disease that responsible for antibiosis properties Harman et al.,
affects plants through their roots at all stages of (2004); Sarrocco et al. (2006). In this study, we
growth, causing significant economic losses by tested selected Trichoderma species for their
generating necrosis and wilting symptoms in many biocontrol ability against the Fusarium wilt of cotton
agricultural plants and having a significant overall under in vitro conditions.
influence on production. The disease caused by this
fungus is characterized by wilted plants, yellowed Materials and methods
leaves and root rot minimal or absent crop yield. Isolation of pathogenic fungi
Fusarium sp. found in its many pathogenic forms, is Evaluation of infected parts of the cotton plant
the most damaging species of the genus where in resulted in isolation and identification of Fusarium
plants are concerned. A number of new disease species based on the examination under microscope.
reports on Fusarium have been submitted to the Parts of plants with symptoms of Fusarium wilt
literature pool on agricultural research Anajat and infection were surface sterilised by immersion in
Kahkashan (2012). 0.3% sodium hypochlorite for 10 minutes, and then in
70% ethanol and later rinsed thoroughly with sterile
Many pathogenic microorganisms have developed distilled water. They were transferred to potato
resistance against chemical fungicides. This seriously dextrose agar (PDA) medium in petriplates and
hinders the management of diseases of crops and incubated at 26 ± 2oC for seven days (Aneja, 2001).
agricultural plants. Considering the deleterious The characteristic growth of the fungus with
effects of synthetic fungicides on life supporting morphological characters of micro-conidia and
systems, there is an urgent need for alternative agents macro-conidia and chlamydospores were observed by
for the management of pathogenic microorganisms. (Agrios, 2005). Pure cultures were maintained on
Biological control is still in its research phase with few PDA slants and stored at 4oC in the refrigerator.
studies reported for bacterial wilt Messiha et al.
(2007). Therefore, it is necessary to explore new Isolation of antagonist
measures for controlling Fusarium wilt. Microbial Trichoderma species were isolated from rhizosphere
fungicides have been confirmed as effective and soils of healthy cotton fields. It was using a
environmentally friendly measures to control crop Trichoderma selective agar (TSA) medium by Elad et

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al. (1982). The isolates were identified to primarily on Poosapati et al. (2014). The influence of different
the macroscopic (pigmentation, growth rate, colour NaCl concentrations (0, 50, 100, 150, 200 and 250
etc.) and microscopic morphology (spore mM) on the growth of Trichoderma sp. isolates was
morphology, formation etc.) according to the method determined on PDA for 5 days Mohamed and Haggag
by Gams et al. (1980); (Rifaii, 1969). (2005). Chitinase activities of isolates were
determined by following the release of 1 mol GLcNAc
Dual culture experiments from chitin Elad et al. (1982).
Competitive interactions between antagonistic
Trichoderma sp. local isolates and plant pathogenic Statistical analysis
fungi were evaluated in dual culture experiments on The experimental design was Completely
petridishes (90 mm diameter) containing 20 ml of Randomized Design (CRD) with three replicates as
potato dextrose agar (PDA). Two 5 mm diameter described by Gomez and Gomez (1984). Test of
mycelial discs cut from 5 day old cultures of variance was calculated using Analysis of variance
pathogenic fungi and Trichoderma sp. were placed at (ANOVA) and statistical F-tests were evaluated at
opposite sides, 30 mm apart in petridishes and P≤0.05. Differences among treatment means for each
incubated in darkness at 30oC. Three replicates were measured parameter were further separated using
prepared for each pairing. fishers Least Significance Difference (LSD) to
determine levels of significance according to Cochran
Radial growth reduction was calculated in relation to and Cox (1992).
growth of the control as follows;
% inhibition of mycelial growth = [(C-T)/C] ×100 Results and discussion
Where, C is the radial growth of pathogenic fungi in Isolates of Trichoderma sp. were used in this study.
control plates; T is the radial growth of pathogen in Trichoderma species were isolated from rhizosphere
presence of Trichoderma Dennis and Webster (1971). soils of healthy cotton during cropping season
collected from the fields. The soil was sieved (< 2
Determination of effects on growth of Trichoderma mm). The some physico-chemical analysis of soil
sp. of abiotic stress factors samples are given in Table 1. The mycelial growth of
The influence of temperature on the growth of isolates of Trichoderma sp. was examined in media
Trichoderma sp. isolates was determined by containing different pH and temperature (Table 2).

Table 1. Physical and chemical properties of the soils isolated of Trichoderma spp.
Isolates Some soil properties
Organic matter (%) N (%) CaCO3 (%) EC (dS m-1 at 25 oC) pH Texture grade
T1 1.33 0.086 29 0.37 8.35 clay
T2 1.46 0.069 34 0.32 8.46 clay
T3 1.99 0.096 26 0.28 8.21 clay
T4 1.57 0.070 19 0.26 8.34 clay
T5 1.37 0.097 39 0.34 8.35 clay
T6 1.99 0.084 30 0.27 7.8 clay

Table 2. The medium mycelial growth (mm) of isolates at different pH and temperature levels.
Isolates pH Temperature (ºC)
5 6 7 15 20 30
T1 130.3 88.4 70.3 192.3 176.1 168.5
T2 109.2 79.0 67.1 170.4 167.1 150.6
T3 102.2 79.2 65.8 172.8 161.5 151.6
T4 132.9 86.5 70.1 181.1 180.2 170.2
T5 111.4 82.1 60.3 164.0 160.7 155.1
T6 129.7 84.7 69.4 180.5 174.3 166.1

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Table 3. Inhibition rate (%) of growth of pathogenic fungi by Trichoderma spp. local isolates in dual culture.
Isolates F1 F2 F3 F4
T1 77.8±2.33b 84.6±2.53a 79.4±2.38c 80±2.4b
T2 73.3±2.19c 68.7±2.06d 93.2±2.79a 56.7±1.70d
T3 80±2.4b 71.4±2.14c 80.3±2.40b 80.8±2.42b
T4 73.3±2.19c 82.8±2.48b 93.2±2.79a 79.4±2.38c
T5 86.4±2.59a 73.3±2.19c 83.8±2.51b 77.9±2.33c
T6 82.2±2.46a 84.6±2.53a 79.4±2.34c 82.3±2.46a
Variance 26.45067 52.23467 44.129 93.23767
Mean 78.833 77.566 84.88 76.183

Table 4. Inhibition (%) of mycelial growth of Trichoderma sp. isolates in NaCl levels.
NaCl (mM)
Isolates
0 50 100 150 200 250
T1 - - 3.3 8.8 13.6 21.5
T2 - 1.6 10 13.7 26.7 72.6
T3 - - 11.1 15.6 25.8 52.7
T4 - - - 8.9 15.3 33.8
T5 - - 8.9 14.2 21.7 61.2
T6 - - 3.3 11.1 11.5 37.8

T1, T4 and T6 isolates were formed quickly the antifungal chemicals. Similarly, Hwang et al. (2017)
mycelia at 15oC. Also, the mycelial formation of reported T. asperellum, T. harzianum as most
isolates with the increase of temperature was reduced effective growth inhibitors of Fusarium species under
at 30oC, respectively and the growths of isolates were in vitro culture. Trichoderma isolates grew much
decreased at increasing different pH levels. T1, T4 and faster on PDA than the tested Fusarium species under
T6 isolates respectively have been observed as the culture conditions.
most acidic pH-resistant isolates. At neutral pH
isolate T1 produced significantly higher biomass and The mycelial growth of local isolates of Trichoderma
radial diameter as compared to others. The isolates sp. was examined in media containing different
were showed different tolerance at different concentrations of NaCl (Table 4). At 50 mM NaCl
temperature and pH treatments. Poosapati et al. growths of T1, T3, T4, T5 and T6 isolates were not
(2014) observations on T. asperellum survived and affected as seen in Table 4. In 250 mM NaCl, the
germinated properly at 28°C and underwent harsh mycelial growths of T2 and T5 were inhibited at the
temperature stress conditions. Also, Petrisor et al. highest rate (72.6 % and 61.2 %, respectively). The
(2016) reported that T. viride strain Td50 grew faster growth of isolates affected at different rate in
at 25-30°C and very slowly at 15°C. increased salt levels. The growth of T4 isolate was not
affected in 100 mM NaCl. The most resistant isolates
Results showed that the antagonistic activity of to abiotic stress were T6, respectively and the most
Trichoderma sp. isolates against the tested Fusarium sensitive isolates were examined as T1, and T4.
species varied, with the highest percentage of Similar results were noted by salinity was identified
inhibition in F3 (93.2%) with T2 and T4 isolates as one of the environmental parameters limiting
(Table 3). Under culture conditions, Trichoderma Trichoderma species antagonistic activity Rawat et al.
isolates grew considerably quicker on PDA than the (2013); Poosapati et al. (2014). It has been explained
tested Fusarium species. T1 and T6 isolates were 84.6 that the isolates antifungal metabolites protect
percent effective against F2. T5 isolate reduced F1 against salt Mohamed and Haggag (2005); Rawat et
growth at an 86.4% rate. T3 isolate was 80.8% al. (2013). Leo Daniel et al. (2011) also reported
effective against F4. These discrepancies, which might similar results while T. viride characterization for
be caused by varied pathogen resistance, are thought abiotic stress. Aside from a decrease in growth rate,
to be obtained from the isolates production of various salt content in medium (salinity stress) caused

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noticeable alterations in morphology and a steady Billah M, Li F, Yang Z. 2021. Regulatory Network
reduction in sporulation with increasing of Cotton Genes in Response to Salt, Drought and
concentration. Wilt Diseases (Verticillium and Fusarium): Progress
and Perspective. Front. Plant Sci. 12, 759245.
Table 5. Chitinase activities by Trichoderma sp.
isolates and analysis of variance Rathore KS, Campbell LM, Sherwood S,
Nunes E. 2015. Cotton (Gossypium hirsutum L.).
Isolates Specific activity (mU mg protein-1)
T1 31±0.93b Methods Mol. Biol. 1224, 11–23.
T2 27±0.81c
T3 9.2±0.27f
T4 43.6±1.30a Davis RM, Colyer PD, Rothrock CS, Kochman
T5 17±0.51e JK. 2006. Fusarium Wilt of Cotton: Population
T6 24±0.72d
Variance 140.012 Diversity and Implications for Management. Plant
Mean 25.3 Dis., 90, 692–703.

We have compared the activity of chitinase of Cun M, Guiliang J, Chuanlin Z. 2002. The
Trichoderma isolates are seen in Table 5. When the advances in cotton breeding resistance to Fusarium
results of each chitinase activity were compared, the and Verticillium wilts in China during past fifty years.
highest enzyme production was observed in T4 (43.6 Zhongguo Nongye Kexue 35, 508-513.
mU mg protein-1). The significance of the difference in
values was determined through ANOVA at a Anajat B, Kahkashan P. 2012. In vitro inhibition
potential of Phoenix dactylifera extracts on the
significance level of 0.01. The lowest chitinase activity
growth of pathogenic fungi. Journal of Medicine
was obtained in T3 (9.2 mU mg protein-1). Similar
Plants Research 6(6)114-123.
findings have been obtained in several Trichoderma
isolates El-Katatny et al. (2006); Kucuk and Kıvanc
Messiha NAS, Van Diepeningen AD, Farag NS,
(2004).
Abdallah SA, Janse Van JD, Bruggen AHC.
2007. Stenotrophomonas maltophilia a new potential
Conclusion biocontrol agent of Ralstonia solanacearum, causal
In conclusion, biocontrol agents could play an agent of potato brown rot. European Journal of Plant
important role in the protection of cotton (Gossypium Pathology 118, 211-235.
spp.). Among the 6 Trichoderma spp. isolate
antagonist which produced the highest inhibitory Khalifa MW, Rouag N, Bouhadida M. 2022.
power against Fusarium species, a pathogen on Evaluation of the antagonistic effect of Pseudomonas
cotton, in vitro with an inhibition power has been rhizobacteria on Fusarium wilt of chickpea.

86.4 and 93.2 %. Further research needs to be Agriculture 12, 429.

carried out field tests (in vivo) to determine the

Mozumder AB, Chanda K, Chorei R, Prasad
antagonism ability of Trichoderma spp. isolates in
HK. 2022. An evaluation of aluminum tolerant
vitro in inhibiting Fusarium fungus that causes
Pseudomonas aeruginosa A7 for in vivo suppression
yellow disease in cotton plants.
of Fusarium wilt of chickpea caused by Fusarium
oxysporum f. sp. ciceris and growth promotion of
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