Isolation and in vitro assessment of native Trichoderma spp. for biocontrol efficacy against major soil-borne plant pathogens

Introduction

Soil borne plant pathogens are one of the major constraints limiting crop production and productivity as they can persist in soil and infect plants at different stages of their growth. Important pathogens from genera such as Fusarium, Pythium, Sclerotium, Rhizoctonia and other pathogenic fungi and oomycetes are associated with economically important diseases and may cause considerable yield losses under favourable environmental conditions. Their management is especially difficult since many soil-borne pathogens can survive for long periods in soil or infected crop debris, making eradication difficult. Historically, chemical fungicides have been extensively used for the control of soil-borne diseases. Fungicides can be effective in suppressing disease, but repeated and indiscriminate application can lead to environmental contamination, the development of populations of resistant pathogens and disturbance of beneficial soil microorganisms. These limitations have increased the interest in environmentally safe and sustainable alternatives for plant disease management (Harman, 2006; Sharma et al., 2014) [1] . Biological control with beneficial microorganisms has become an important part of sustainable disease management. Trichoderma spp. is one of the most extensively studied and widely exploited biological control agents among the microbial antagonists. Their rapid growth, ecological adaptability and ability to suppress a broad range of plant pathogens makes them a good candidate for biological control. Trichoderma spp. has been extensively studied in India against many soil-borne and foliar pathogens (Sharma et al., 2014) [1] . T. harzianum and T. viride are the most widely used species. Trichoderma spp. antagonistic activity involves a number of mechanisms, including competition for nutrients and space, mycoparasitism, antibiosis, production of secondary metabolites and cell-wall-degrading enzymes and induction of plant defense responses (Benítez et al., 2004; Harman, 2006; Vinale et al., 2008) [2,3,4] . These mechanisms can act individually or in combination and their efficacy is dependent on the Trichoderma strain, target pathogen and environmental conditions. Several studies have reported antagonistic potential of Trichoderma against important soil-borne pathogens such as Pythium aphanidermatum, Rhizoctonia solani, Sclerotium rolfsii and Fusarium oxysporum (Hima et al., 2016) [5] . The potential of Trichoderma as an environment friendly alternative to chemical disease management has also been highlighted in reviews (Verma et al., 2007; Vinale et al., 2008) [6, 4] . However, the biocontrol ability of Trichoderma differs with species and isolates due to differences in growth rate, competition, metabolite production, enzyme activity, mycoparasitism and environmental adaptation. Thus, screening of native isolates is important to identify effective strains, which are suitable to local conditions (Benítez et al., 2004; Sharma et al., 2014) [2,1] . The dual culture method is extensively used for initial in vitro screening of Trichoderma isolates against plant pathogens. It gives a simple comparison based on the pathogen growth and percentage inhibition of mycelial growth and helps to identify promising isolates for further evaluation (Hima et al., 2016) [5] . Hence the present study was planned to isolate and characterize native Trichoderma spp. and to test in vitro biocontrol potential of native Trichoderma spp. against major soil-borne plant pathogens. The selected isolate was further tested against several economically important pathogens to assess its wider potential in sustainable plant disease management (Mukherjee et al., 2022; Sharma et al., 2023) [7,8] .

Materials and Methods

Experimental site and materials

Isolation and identification of Trichoderma spp

Rhizosphere soil samples were collected from different agricultural and horticultural crops of Latur, Parbhani and Dharashiv districts of Maharashtra. Native isolates of Trichoderma were isolated by serial dilution plate technique using Potato Dextrose Agar (PDA) media. The obtained colonies were purified by repeated hyphal-tip transfer and maintained on PDA slants at 5 ± 1 °C until further use.

The isolates were identified based on cultural and morphological characteristics such as colony growth, pigmentation, conidiophore branching pattern, phialide arrangement and conidial morphology according to the standard taxonomic descriptions of Nagamani et al., 2015 [9] and Bissett (1991) [10] .

Selection of Trichoderma harzianum for Broad-Spectrum Antagonistic Evaluation

The native Trichoderma isolates were initially evaluated for their antagonistic activity against Pythium aphanidermatum, the causal agent of ginger rhizome rot, by the dual culture technique as described by Dennis et al. (1971) [11] . The most effective isolate of Trichoderma harzianum, identified by previous screening, was selected for further evaluation against a broader spectrum of economically important plant pathogens. The selected T. harzianum isolate was then tested in vitro against eight test pathogens viz., F. oxysporum f. sp. ciceri, Pythium aphanidermatum, Sclerotium rolfsii associated with chili, F. oxysporum f. sp. pisi, S. rolfsii associated with rajma, Mangifera pustulosis, F. oxysporum f. sp. udum and Colletotrichum lindemuthianum. The pure cultures of the pathogens were obtained from Department of Plant Biotechnology, Vilasrao Deshmukh College of Agriculture Biotechnology, Latur, Maharashtra and were used directly for the antagonistic studies.

In vitro antagonistic activity of selected Trichoderma harzianum

The antagonistic activity of the selected isolate of T. harzianum against the eight test pathogens was estimated by the dual culture technique (Dennis et al., 1971) [11] . A 2-mm mycelial disk from the actively growing margin of a 7-day-old culture of T. harzianum and the respective pathogen were placed at opposite ends of a 90-mm Petri plate containing PDA medium. The untreated control plates were inoculated only with the respective pathogen. The experiment was arranged in a Completely Randomized Design (CRD) with three replications. The plates were incubated at 28 ± 1 °C and the radial growth of each pathogen was recorded at the given observation intervals. The antagonism of the selected T. harzianum isolate was quantified through comparison of pathogen growth in dual culture with that of the untreated control.

Assessment of antagonistic activity

The antagonism efficiency of the selected T. harzianum isolate was assessed based on the reduction in radial growth of each test pathogen compared with the untreated control. Percentage inhibition of mycelial growth was calculated using the formula given by Vincent (1947) [12] :

𝐼 = [ ( 𝐶 -𝑇 ) /𝐶] × 100

Statistical analysis

The experiment was laid out in a Completely Randomized Design (CRD) with three replications. Analysis of variance (ANOVA) was used to analyze data on radial growth and percent inhibition of mycelial growth of test pathogens. The means of treatments were compared using the Critical Difference (CD) at 1% level of significance (P ≤ 0.01). The standard error of the mean [SE m ] and the critical difference (CD) were determined using the standard procedures described by Gomez et al. (1984) [13] . Where necessary, percentage inhibition data were arcsine-transformed prior to statistical analysis.

Results and Discussion

Isolation and characterization of native Trichoderma isolates

Six indigenous Trichoderma isolates were effectively isolated and purified on Potato Dextrose Agar (PDA) medium. Considerable variation was noted among the isolates in colony color, texture, pigmentation, zonation and degree of conidial development. T. virens LTv-1 formed a more distinct green to dark-green colony with dense aerial growth, while T. harzianum LTh-1 produced a predominantly whitish to pale green, dense, cottony colony with progressive green pigmentation. T. asperellum LTAs-1 was a relatively uniform green colony with a compact, velvety to powdery appearance. T. virens LTv-2 showed prominent aerial mycelial growth with whitish growth associated with green pigmentation and marked radial development. T. harzianum LTh-2 showed a pale green to whitish growth with distinct peripheral aerial mycelium, while T. koningii LTk-1 developed green colony with pronounced white aerial mycelial growth and irregular radial distribution. These differences in cultural characteristics gave useful phenotypic differences (Bissett, 1991; Gams et al., 1998; Samuels, 2006, Nagamani 2015) [10,14,15] .

In vitro antagonistic activity of selected Trichoderma isolate against major soil borne pathogens

The antagonism activity of the selected native Trichoderma isolate was evaluated against eight economically important plant pathogens using the dual culture technique. Mycelial growth of each pathogen was recorded on the 7th day after inoculation (DAI), when the pathogen colony in the respective untreated control plates had attained complete growth. The extent of antagonism was assessed based on the average radial growth of the pathogen and percentage inhibition of mycelial growth. Considerable variation was observed in the response of the test pathogens to the selected Trichoderma isolate, with average mycelial growth ranging from 13.33 to 27.66 mm and inhibition of mycelial growth ranging from 69.26 to 85.18% (Table 2; Figure 2). Among the pathogens tested, Mangifera pustulosis exhibited the highest susceptibility to the selected Trichoderma isolate, recording the lowest average mycelial growth of 13.33 mm and the highest inhibition of mycelial growth (85.18%). The second highest inhibition was recorded against Sclerotium rolfsii associated with chilli, where the pathogen showed an average growth of 15.16 mm, corresponding to 83.15% inhibition. Fusarium oxysporum f. sp. ciceri was also strongly inhibited, with an average mycelial growth of 17.83 mm and 80.18% inhibition. Likewise, the average mycelial growth and inhibition percentage was 19.33 mm and 78.52% respectively against Pythium aphanidermatum with high level of antagonism. The average growth of F. oxysporum f. sp. udum was 23.33 mm with 74.07 % inhibition and Colletotrichum lindemuthianum was 24.16 mm with 73.15 % inhibition. The average mycelial growth was 25.66 mm with 71.48% inhibition of F. oxysporum f. sp. pisi . The lowest antagonistic activity was comparatively observed against Sclerotium rolfsii associated with rajma, which showed the highest average mycelial growth of 27.66 mm and the lowest percentage of inhibition of mycelial growth (69.26%). Thus, the selected Trichoderma isolate inhibited all the eight test pathogens but the extent of inhibition differed significantly among pathogens indicating different susceptibility to the antagonism of Trichoderma.

The pathogens could be arranged in the order of susceptibility according to the percentage inhibition as follows: M. pustulosis (85.18%) > S. rolfsii (chilli) (83.15%) > F. oxysporum f. sp. ciceri (80.18%) > P. aphanidermatum (78.52%) > F. oxysporum f. sp. udum (74.07%) > C. lindemuthianum (73.15%) > F. oxysporum f. sp. pisi (71.48%) > S. rolfsii (rajma) (69.26%). This variation shows a significant antagonism potential of the selected native Trichoderma isolate against diverse plant pathogens. [3,16,4] . Trichoderma spp. is known to act against plant pathogens through several complementary mechanisms including direct parasitism of pathogenic fungi and production of biologically active compounds and cell-wall degrading enzymes (Harman et al., 2004; Vinale et al., 2008) [3,4] . Previous studies have reported the antagonistic activity of Trichoderma against various groups of plant pathogens such as Fusarium, Pythium, Sclerotium and Colletotrichum, providing support for the potential of Trichoderma as a broad-spectrum biological control agent (Harman et al., 2004; Vinale et al., 2008) [3,4] . The differential inhibition observed among the pathogens could be associated to their growth behaviour, biological characteristics and susceptibility to the antagonistic mechanisms of the selected Trichoderma isolate. The efficacy of Trichoderma varies according to the strain and target pathogen as the antagonistic interactions are influenced by the characteristics of the bio-control organism and pathogen (Vinale et al., 2008) [4] . The native Trichoderma isolate selected showed broad-spectrum in vitro antagonistic activity, inhibiting the mycelial growth of all eight pathogens tested by > 69% overall. Maximum inhibition was recorded against M. pustulosis (85.18%) and minimum against S. rolfsii associated with rajma (69.26%) indicating the considerable potential of the selected isolate for further evaluation as biological control agent.

Conclusion

The present investigation revealed the presence of diverse native Trichoderma isolates with different cultural and morphological characteristics supporting their preliminary identification as T. harzianum, T. virens, T. asperellum and T. koningii. The selected native Trichoderma harzianum isolate showed appreciable in vitro antagonistic activity against all the eight plant pathogens tested. But the magnitude of inhibition varied among the pathogens. Maximum mycelial growth inhibition was recorded against Mangifera pustulosis (85.18%) followed by Sclerotium rolfsii associated with chili (83.15%) and Fusarium oxysporum f. sp. ciceri (80.18%) while minimum inhibition was observed against S. rolfsii associated with rajma (69.26%).

The findings indicate that the selected native Trichoderma isolate possesses promising in vitro bio-control potential against diverse plant pathogens and may serve as a candidate for further biological control research. However, the present study was conducted under laboratory conditions; therefore, greenhouse and field evaluation, together with further investigation of the mechanisms responsible for pathogen suppression, are necessary before practical disease-management recommendations can be made.

Acknowledgement

AcknowledgementsThe authors gratefully acknowledge the Vilasrao Deshmukh College of Agricultural Biotechnology, Latur, and the CM Fund sponsored laboratory, Department of Plant Biotechnology for providing the essential laboratory facilities and institutional support required to conduct this study. The authors also extend their sincere thanks to the faculty members and technical staff of the Department of Plant Biotechnology for their valuable assistance and support throughout the experimental work and data analysis.

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19. How to cite this article: Pawar P, Dharbale K, Bhadke S, Hajgude S, Bharose A, Ambadkar C, et al. Isolation and in vitro assessment of native Trichoderma spp. for biocontrol efficacy against major soil-borne plant pathogens. International Journal of Advanced Biochemistry Research. 2026;10(9):1530-1535. doi: https://www.doi.org/10.33545/26174693.2026.v10.i9S.10038