Isolation, characterization and evaluation of native trichoderma isolates for biological suppression of fusarium wilt and growth promotion in pea (Pisum sativum L.)

Introduction

Modern agricultural practices, while successful in enhancing crop productivity, have considerably disturbed the ecological balance of agro-ecosystems. The excessive and continuous use of chemical fertilizers and pesticides, coupled with mono-cropping systems, has led to soil and water contamination, increased incidence of pests and diseases, and accumulation of harmful chemical residues in agricultural produce (Bardin et al., 2015) [1] . In view of the growing demand for safe food and environmentally sustainable farming systems, there is an urgent need to develop eco-friendly and efficient alternatives for plant disease management. Biological control has emerged as a promising strategy that employs beneficial microorganisms and their metabolites to suppress plant pathogens, thereby reducing dependence on chemical inputs (Harman et al., 2021) [2] . Biological control agents (BCAs) serve a dual function as bio-pesticides and bio-fertilizers.

In addition to suppressing plant pathogens, they enhance soil health through bioremediation and improved nutrient availability, thus contributing to ecological balance and sustainable agricultural production (Baker and Paulitz, 1996) [3] . BCAs suppress pathogens through diverse mechanisms, including mycoparasitism, production of antibiotics and lytic enzymes, competition for nutrients and space, and induction of systemic resistance in plants (Pal and Gardener, 2006) [4] . Beneficial microorganisms, particularly plant growth-promoting fungi and bacteria, further stimulate plant growth by enhancing nutrient uptake, synthesizing phytohormones, and effectively colonizing plant roots, thereby strengthening plant defense systems (Islam et al., 2016) [5] . Among the various biological control agents, species of the genus Trichoderma have received considerable attention owing to their adaptability and broad-spectrum antagonistic activity against numerous plant pathogens. This fungal biocontrol agent belongs to the family Hypocreaceae, are commonly found in soil and rhizosphere environments (Kubicek et al., 2019) [6] . Several species, including T. harzianum, T. viride, T. asperellum, and T. atroviride, have been extensively investigated for their effectiveness in biological disease management (Sánchez-Montesinos et al., 2021; Sun et al., 2022) [7,8] . They produce bioactive secondary metabolites and cell wall-degrading enzymes that suppress pathogens, enhance nutrient uptake, promote plant growth, and improve tolerance to abiotic stresses. Numerous studies have demonstrated successful management of diseases such as seed rot, damping-off, wilt, and sheath blight through application of Trichoderma spp. (Jogaiah et al., 2013; Luo et al., 2010) [29,30] . Pea (Pisum sativum L.), a cool-season legume belonging to the family Fabaceae, is widely cultivated for its high nutritional value. It serves as an important source of proteins, carbohydrates, vitamins, and essential minerals, contributing significantly to human nutrition (Sharma, 2010) [9] . Globally, pea occupies millions of hectares and plays a vital role in ensuring food and nutritional security. India ranks among the leading pea-producing countries (Mohanty et al., 2020) [10] . However, pea cultivation is constrained by several fungal diseases, particularly damping-off and wilt caused by pathogens such as Rhizoctonia, Pythium, Phytophthora, and Fusarium, which adversely affect seed germination, seedling establishment, and overall crop productivity (Lamichhane et al., 2017) [11] . High-quality seed is fundamental for successful crop establishment and yield enhancement but biological seed treatment, especially seed bio-priming with beneficial microorganisms, has gained prominence as an environmentally safe approach for managing seed-and soil-borne pathogens while improving germination and seedling vigor (Harman and Taylor, 1988) [12] . Bio-priming ensures rapid colonization of beneficial microbes on seed surfaces, enhances plant tolerance to environmental stresses, and strengthens resistance against pathogens. Among microbial agents, Trichoderma species have been widely reported to enhance germination percentage, seedling growth, chlorophyll content, and overall plant health in pea and other crops (Singh et al., 2015) [3] . The natural habitat of Trichoderma is predominantly soil and the rhizosphere, where it plays a crucial role in plant protection and growth promotion. Beyond disease suppression, these fungi produce phytohormones, solubilize nutrients, decompose organic residues, and improve soil fertility. Owing to these multifaceted benefits, Trichoderma represents a major component of the global fungal biocontrol market (Swain and Mukherjee, 2020) [14] . Considering increasing environmental concerns and the necessity for sustainable disease management, exploration and utilization of native Trichoderma isolates are essential for developing region-specific, effective, and adaptable biocontrol strategies. Therefore, in light of the importance of eco-friendly disease management and improvement of seed quality, the present investigation was undertaken on ecofriendly and sustainable management of Fusarium wilt disease in pea by application of native Trichoderma spp., in mid hills of Uttarakhand.

Materials and Methods

The present investigation was conducted in the Biocontrol Laboratory, Department of Plant Pathology, College of Hill Agriculture, Ranichauri, Tehri Garhwal, Uttarakhand during 2024-2025. The laboratory experiments were laid out in a Completely Randomized Design (CRD) with eight treatments and three replications.

Collection of soil samples and isolation of trichoderma isolates

A total of seventy rhizospheric soil samples were collected from different farming situations of mid hill regions of Uttarakhand. From each field, soil samples were collected from 4-5 healthy plants, including one plant from the centre and four from the corners of the selected plot. Approximately 10 g of soil from each location was collected in labelled sterile sampling bags. Isolation of Trichoderma spp. from soil samples was carried out using the serial dilution plate technique on Trichoderma Selective Medium (TSM) (Waksman, 1922) [15] . The isolated cultures were characterized based on cultural, morphological and microscopic characteristics.

In vitro evaluation of Trichoderma isolates

The antagonistic potential of isolated Trichoderma spp. was evaluated against major plant pathogens viz., Fusarium oxysporum, Sclerotium rolfsii, Rhizoctonia solani, Botrytis cinerea, Aspergillus niger and Alternaria solani using Dual Culture Technique on PDA. Isolated Trichoderma isolates were assessed against fungal pathogens by the inverted plate assay technique on PDA for antibiosis test (Dennis and Webster 1971) [16] . Percent inhibition of mycelial growth was calculated using the standard formula (Wonglom et al., 2019) [17] .

Where, C = Mycelial growth of pathogen in control plate T = Mycelial growth of pathogen in treatment (Test Plate) I= Per cent mycelial growth inhibition of pathogen.

Organic acid production, change in pH and phosphate solubilisation

Organic acid production by different Trichoderma isolates was evaluated using Pikovskaya's broth supplemented with bromophenol blue indicator. Five mm mycelial discs from 72-hour-old cultures were inoculated into broth and incubated at 28 °C for 21 days Prasad (2023) [18] . To measure pH, the electrode was rinsed with distilled water, and the meter was set to pH mode at 25 °C. The electrode was then placed in the media, and the pH reading was noted after 30 seconds of stabilization. Phosphate solubilization potential was assessed using both Pikovskaya's agar and broth media. In Pikovskaya'sagar assay, isolates were inoculated on medium containing tri-calcium phosphate and halo zone formation was recorded. In broth assay, cultures were incubated for 21 days, followed by filtration, centrifugation and reaction with Bray's extract and chloromolybdic acid reagent and absorbance was measured spectrophotometrically at 660 nm with a standard curve generated using a standard K2HPO4 solution for reference.

Development of bioformulation from selected promising Trichoderma isolates

Selected promising Trichoderma isolates were mass multiplied using barnyard millet (Echinochloa frumentacea L.) grains. The grains were soaked in water for 12 hr (50 g/ 250 ml flask), autoclaved (15 lbs psi for 30 minutes), inoculated with mycelial discs of selected Trichoderma isolates and incubated at 28 °C for 12 days. Colonized grains by Trichoderma were air-dried in open shade, finepowdered with the help of a Willy Mill grinder, sieved through a 50-mesh sieve, and mixed with sterilized talcum powder (1:3 ratio) along with 1% carboxymethyl cellulose (CMC) to prepare a talc-based bioformulation (Mohiddin et al., 2017) [19] .

Plant material

Seeds of pea (Pisum sativum L.) variety 'Arkel' were procured from the local market of Ranichauri, Tehri Garhwal, Uttarakhand.

Seed treatment

Seven promising Trichoderma isolates were evaluated for their effect on the seed. The details of treatments used in the present study for evaluation of Trichoderma isolates toward plant growth promotion activities using a pot experiment under glass house conditions are depicted in 5. Seeds were surface sterilized using 1% sodium hypochlorite solution for 3 min, then they were rinsed with sterile distilled water. Seeds were treated with a talc-based formulation of Trichoderma isolates @10 g/kg seeds.

Evaluation of Trichoderma isolates under glasshouse conditions for wilt disease suppression in pea caused by

Fusarium oxysporum

Pathogen inoculum of Fusarium oxysporum was prepared using sterilized barnyard millet grains and incubated at 28°C for 14 days. Sterilized sandy loam soil mixed with 0.5% compost was filled in plastic pots (30 cm diameter) containing 5 kg of soil per pot. Soil was inoculated with fungal inoculum @10 g per pot and incubated for 8 days. Sand and sandy loam soils were autoclaved at 121 °C for 1 h. Sterilized soil amended with 0.5% pathogen-free matured compost was filled in sterilized plastic pots (30 cm diameter; 5 kg soil/pot). Each pot was inoculated with 10 g fungal inoculum mixed up to 5 cm soil depth and incubated under warm, humid conditions for 8 days.

Observations

Percent disease incidence (PDI) was calculated using (Shiva et al. 2020) [20] .

Percent disease reduction (PDR) over control was calculated as:

Where, DIT=Disease incidence in treatment DIC=Disease incidence in Untreated control

Statistical analysis

Experimental data were analyzed using a Completely Randomized Design (CRD). Analysis of variance (ANOVA) was performed using MS Excel and OPSTAT software at a 5% level of significance (Gomez, 1984) [21] .

Results and Discussion

Isolation and characterization of isolates

Thirty native Trichoderma isolates were isolated from seventy soil samples collected from mid and high hills of Uttarakhand. Maximum isolates were obtained from Tehri district (13 isolates) followed by Chamoli (3 isolates), whereas Nainital, Pauri, Bageshwar, Uttarkashi and Almora districts yielded comparatively fewer isolates. These isolates were screened for their cultural and morphological characteristics on PDA medium (Table 1). Considerable variation was observed among the isolates with respect to colony growth rate, mycelial colour, colony pigmentation, texture, concentric ring formation, and radial growth. Significant variation was recorded in radial growth among the isolates. At 4 days after inoculation (DAI), colony diameter ranged from 56.00 mm (TS10) to 90.00 mm (TS18, TS26, TDG, TS32, TS40, TB1, and TCH6). Fastgrowing isolates such as TS18, TS23, TS26, TDG, TS32, TS40, and TB1 covered the full Petri plate (90 mm) within 4 days, indicating rapid mycelial extension. At 7 DAI, all isolates attained 90.00 mm colony diameter, indicating uniform final growth potential but differing growth kinetics. The rapid radial growth observed in certain isolates may reflect higher metabolic activity and competitive ability, which are important attributes for effective rhizosphere colonization and biocontrol potential. The initial mycelial colour of most isolates was white, except TS15, TS32, TS45, TB4, and TCH3, which showed yellow or yellowishgreen mycelium. With increasing incubation time, colony colour varied from white to green, dark green, yellowish green, and pale green. Green pigmentation, characteristic of conidial production in Trichoderma, was prominent in several isolates such as TS26, TS22, TS29, TDG, and TCH5 (Figure 1). Similar characteristics of plant growthpromoting microorganisms from rhizospheric soils using dilution plating techniques has been reported earlier by Yeole and Dube (1997 and Hadar et al. (1984) [22,23] . Significant variability was observed among isolates with respect to cultural and morphological characteristics. Eight isolates exhibited significantly faster radial growth and covered the entire Petri plate within four days (90 mm), whereas three isolates required five days and nineteen isolates required seven days. Colony colour on the upper surface includes Green, White, Yellowish green, Dark green and Light green and on the lower surface includes Offwhite, Yellow, Pale green and Pale yellow in different Trichoderma isolates. With respect to colony texture majority of isolates exhibited smooth colony texture, while nine isolates showed coarse/ rough texture. With respect to concentric rings, 6 Trichoderma isolates had concentric rings type growth, whereas 24 Trichoderma isolates lacked concentric rings, as shown in Table 1. Under microscopic observations, isolates of Trichoderma were found to produce conidia of various shapes, ranging from globose to sub-globose and oval to ellipsoidal (Table 2 and Figure 2). Similar variability among Trichoderma isolates has been reported previously (Sharma and Singh, 2014) [24] .

In-vitro evaluation of Trichoderma isolates

In-vitro antagonistic evaluation revealed statistically significant differences among isolates. In the dual culture assay, isolate TDG showed significantly higher mycelial inhibition against Fusarium oxysporum (100%), which was statistically superior to other isolates as shown in [25,26] . In the inverted plate assay, isolate TDG recorded significantly higher mycelial inhibition against F. oxysporum (93.33%), S. rolfsii (85.92%), B. cinerea (87.45%), A. solani (88.52%) and A. niger (85.55%) and was statistically at par with TB1. However, against R. solani, isolate TB1 recorded significantly higher inhibition (85.55%), which was statistically similar to TDG (85.18%) as shown in Table 4. Similar inhibition of pathogens through volatile metabolite production by Trichoderma spp. has been reported earlier by Kumar et al. (2019) [27] . During biochemical evaluation, isolates TDG and TB1 recorded significantly higher pH reduction (3.2), indicating higher organic acid production potential. In phosphate solubilization assay on solid medium, seven isolates tested positive, whereas the remaining isolates showed no solubilization. In Pikovskaya broth medium, isolate TU3 recorded significantly higher phosphate solubilization (0.12 mg ml⁻¹), which was statistically superior to other isolates, whereas isolate TB4 recorded the lowest solubilization (0.07 mg ml⁻¹). Similar prressive phosphate solubilization activity among Trichoderma isolates has been reported earlier by Prasad et al. (2023) [18] .

Effect of Trichoderma isolates on wilt disease suppression under glasshouse conditions

Evaluation of Trichoderma isolates applied through seed bio-priming under glasshouse conditions revealed significant differences among treatments for seed germination, percent disease incidence (PDI), and percent disease reduction (PDR) against wilt of pea caused by Fusarium oxysporum (Table 5 and Figure 3). Among the treatments, T4 recorded the highest seed germination (99.00%), which was significantly superior to all other treatments. This was followed by T6 (95.52%) and T8 (93.33%). The untreated control (T1) exhibited the lowest germination percentage (53.33%). With respect to disease incidence, T4 showed the minimum PDI (1.00%), which was highly significantly lower than all other treatments and resulted in the highest disease reduction (99.00% 6 and Figure 3. Disease suppression might be attributed to antagonistic, parasitic and competitive behaviour of Trichoderma spp., along with production of antibiotics, volatile metabolites and hydrolytic enzymes such as glucanases, chitinases and pectinases. Similar mechanisms of pathogen suppression by Trichoderma spp. have been reported earlier by Howell (2003) [28] .

Conclusion

The present investigation revealed substantial variability among native Trichoderma isolates with respect to their cultural, morphological, microscopic, antagonistic, and plant growth-promoting attributes, highlighting the rich diversity of indigenous Trichoderma populations. Based on cultural characteristics and microscopic observations, the isolates were tentatively identified as belonging to different Trichoderma species known for their biocontrol potential against a wide range of phytopathogens. Among the isolates evaluated, TDG and TB1 exhibited superior antagonistic activity against most of the test pathogens, while TS40 demonstrated remarkable efficacy against Rhizoctonia solani and Botrytis cinerea. The enhanced inhibitory activity exhibited by these isolates may be attributed to a combination of mechanisms, including rapid colonization, competition for space and nutrients, mycoparasitism, and the production of diffusible and volatile antifungal metabolites. Furthermore, isolate TU3 displayed the highest phosphate-solubilizing ability, indicating its potential role in nutrient mobilization and plant growth enhancement. Under glasshouse conditions, seed bio-priming with selected Trichoderma isolates significantly improved seed germination and effectively suppressed Fusarium wilt of pea compared with the untreated control. Among the treatments, isolate TDG proved to be the most promising, recording the highest seed germination (99%) and disease reduction (99%), followed by TB1. The strong correspondence between in vitro antagonistic performance and in vivo disease suppression underscores the reliability of laboratorybased screening for the identification of effective biocontrol candidates. Collectively, the findings demonstrate that native Trichoderma isolates, particularly TDG and TB1, possess multifunctional traits encompassing pathogen suppression, plant growth promotion, and nutrient mobilization. These isolates hold considerable promise as environmentally sustainable alternatives to chemical fungicides and warrant further validation under diverse agro-climatic field conditions. Their successful development into stable and efficient bioformulations could contribute significantly to the integrated and eco-friendly management of Fusarium wilt in pea cultivation.

Statements & Declarations

• Funding: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. • Competing Interests: The authors have no relevant financial or non-financial interests to disclose.

• Authors' contributions: All authors contributed to the study's conception and design. Material preparation, data collection and analysis. • Data availability: The datasets generated during and analysed during the current study are available from the first author upon reasonable request. • Ethics approval: This research article does not contain any studies with human participants or animals performed by any authors. • Consent to participate: This manuscript is new and not considered elsewhere for publication. All authors have approved the submission of this manuscript. • Conflict of Interest: The authors declare no conflict of interest.

Acknowledgements

The authors thank the Vice Chancellor, Veer Chandra Singh Garwali Uttarakhand University of Horticulture and Forestry, Ranichauri, Tehri Garhwal (249199), India for providing research facilities.

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