Chickpea (Cicer arietinum L.) is a vital pulse crop, particularly in semi-arid regions, with India accounting for the largest share of global production (ICAR-IIPR, 2022). In addition to being rich in protein and essential nutrients, chickpea contributes to soil fertility through biological nitrogen fixation. In India, it is cultivated over 10.91 million hectares, producing approximately 13.75 million tonnes annually (ICAR-IIPR, 2022). Among the Indian states, Maharashtra contributes about 26% of the national chickpea output; however, productivity in the Marathwada region remains low due to several biotic and abiotic stresses (Kumari and Malik, 2024, Sontakke et al., 2020).
       
Among these, Fusarium wilt caused by Fusarium oxysporum f. sp. ciceris is considered the most devastating disease, causing yield losses ranging from 10% to 90%, depending on environmental conditions and pathogen virulence (Muche et al., 2022). Although the deployment of resistant cultivars and the application of fungicides offer partial control, these strategies often prove inadequate and raise ecological concerns due to their long-term impacts (Singh et al., 2022).
       
Biological control presents a sustainable and eco-friendly alternative, with Trichoderma spp. gaining increasing attention for their broad-spectrum antagonistic mechanisms. These include mycoparasitism, antibiosis, competition for nutrients and space and the induction of systemic resistance in host plants (Harman et al., 2004; Waghunde et al., 2016; Trivedi et al., 2020). Additionally, Trichoderma species are known to promote plant growth by producing phytohormones and enhancing the uptake of micronutrients, particularly iron, zinc and manganese (Aishwarya et al., 2020; Illescas et al., 2021; Siddiqui et al., 2025).
       
Despite the rising interest in Trichoderma based biocontrol, limited studies have explored the efficacy of native strains adapted to specific agro-ecological zones, such as the chickpea rhizosphere of Marathwada. Locally adapted isolates may offer superior effectiveness due to their compatibility with local soil and climatic conditions (Reddy et al., 2024; Kumari et al., 2025). Notably, lesser-studied species like T. yunnanense and T. pleuroticola have demonstrated promising biocontrol potential globally (Yu et al. 2007). Pathogenic invasive microbes Trichoderma pleuroticola transform bacterial and fungal community diversity in Auricularia cornea crop production system (Ye et al., 2023). however, their role in chickpea disease suppression, plant growth promotion and micronutrient dynamics under Indian conditions remains largely unexplored.
       
Therefore, the present study aimed to (i) isolate and characterize native Trichoderma spp. from chickpea-growing soils of Marathwada, (ii) evaluate their antagonistic activity against F. oxysporum f. sp. ciceris and (iii) assess their influence on chickpea growth, yield and micronutrient uptake under controlled pot culture conditions. It was hypothesized that native Trichoderma isolates would exhibit strong antagonistic activity and enhance chickpea growth and micronutrient uptake more effectively due to their adaptation to local edaphic and climatic conditions.

The experiment was conducted during the rabi season of 2023-24, at the Department of Plant Pathology, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani, Maharashtra.
 
Collection and Isolation of Trichoderma spp.
 
Rhizosphere soil samples were collected from healthy chickpea plants across three agro-climatic zones of the Marathwada region. Serial dilutions (10^-4 to 10^-7) of the soil samples were prepared and plated on Trichoderma Selective Medium (TSM) as described by Elad et al., (1981). Plates were incubated at 28±2^oC for 5-7 days. Colonies exhibiting typical Trichoderma morphology were purified using the hyphal tip method (Rifai, 1969; Bisset, 1991) and maintained on potato dextrose agar (PDA) at 4^oC for further use.
 
Antagonistic Activity In Vitro
 
The antagonistic potential of Trichoderma isolates against Fusarium oxysporum f. sp. ciceris (Foc) was evaluated using the dual culture technique (Dhingra and Sinclair, 1985). PDA plates were inoculated with 5 mm mycelial discs of both the pathogen and the Trichoderma isolate placed opposite each other. Control plates were inoculated with the pathogen alone. After 7 days of incubation at 28 ± 2^oC, the percentage inhibition of pathogen growth was calculated using Vincent’s formula (Vincent, 1947):

 
Where
C = Radial growth of the pathogen in control (mm).
T = Radial growth of the pathogen in dual culture (mm).
 
Molecular characterization
 
Genomic DNA was extracted from selected Trichoderma isolates and the internal transcribed spacer (ITS) region was amplified using primers ITS1 and ITS4 (White et al., 1990). PCR products were sequenced and species identification was confirmed through BLAST analysis. Gel electrophoresis images and culture plate photographs were documented with high resolution and appropriate captions.
 
Development of Trichoderma formulations
 
For mass multiplication, selected Trichoderma  isolates were cultured in 1 L of potato dextrose broth (PDB), each inoculated with five 5 mm mycelial discs. Cultures were incubated at 28±2^oC for 14 days on a rotary shaker at 100 rpm. The biomass was harvested and mixed with sterilized talc powder in a 1:2 ratio, along with 5 g/kg of carboxymethyl cellulose to prepare talc-based formulations containing approximately 10⁶ cfu/g. Shelf-life and viability were monitored over a 90-day storage period, showing stable colony-forming unit (10⁶ cfu/g) counts.
 
Pot experiment
 
A pot culture experiment was carried out under greenhouse conditions during the rabi season of 2024 using chickpea cultivar BDNGK-798 (sourced from Agricultural Research Station, Badnapur, Maharashtra). Surface-sterilized seeds were treated with talc-based Trichoderma formulations at 10 g/kg seed rate. Additionally, soil in each pot was inoculated with 25 g of the formulation. Foc inoculum was applied to all treatments except the healthy control at 2% w/w, standardized based on the weight of colonized substrate (Sand maize media) mixed with soil.
       
The experiment was laid out in a completely randomized design (CRD) with three replications. Micronutrient content (Zn, Mn, Cu, Fe) in 30-day-old seedlings was analyzed using atomic absorption spectrophotometry following wet digestion (Rashid, 1986). At 65 days after sowing, plant growth parameters, yield attributes, nodulation and wilt incidence were recorded. Disease incidence (DI) was calculated as:

 
Statistical analysis
 
Data were statistically analyzed using R software under a completely randomized design. Percentage data were arc-sine transformed and treatment means were compared using standard error of the mean (SEm) and critical difference (CD) at P = 0.01 (Panse and Sukhatme, 1978).

Isolation and Identification of Trichoderma spp.
 
Nineteen Trichoderma isolates were obtained from chickpea rhizosphere soils across eight districts of Marathwada and identified based on morphological and cultural characteristics using Potato Dextrose Agar (PDA) medium, as described by Gams and Bissett (2002). The isolates were designated by location: Parbhani (CTP1-CTP4), Nanded (CTN1-CTN3), Jalna (CTJ1-CTJ2), Aurangabad (CTA1-CTA2), Latur (CTL1-CTL2), Osmanabad (CTO1-CTO2), Beed (CTB1-CTB2) and Hingoli (CTH1-CTH2) (Fig 1 and 2). A high isolation frequency (86.36%) indicates the widespread occurrence and diversity of Trichoderma spp. in chickpea-growing soils, consistent with findings from Harman et al., (2004), who reported the dominance of Trichoderma in agricultural rhizospheres. This highlights their potential for further use in biological control and plant growth-promotion (Reddy et al., 2024; Kumari et al., 2025).




 
Biocontrol efficacy
 
Dual culture assays showed that all nineteen Trichoderma isolates inhibited Fusarium oxysporum f. sp. ciceri mycelial growth, with inhibition ranging from 39.64 to 86.30%. Eight isolates demonstrated significant inhibition (71.90-86.30%) (Fig 3), with T. yunnanense (CTA2) showing the highest (86.30%), followed by CTP4 (84.81%) and CTJ1 (84.08%). Other isolates such as CTN2, CTB2 and CTB1 also showed strong activity. These results confirm the strong biocontrol potential of Trichoderma spp., especially T. yunnanense and T. pleuroticola, consistent with recent studies highlighting their efficacy in chickpea wilt management (Joshi and Sunkad 2025; Parmar and Gohel, 2024). Variability in inhibition may be due to differences in antibiotic, siderophore and enzyme production (Syed et al., 2023; Reddy et al., 2024).


 
Molecular identification
 
Eight selected isolates were molecularly confirmed using ITS1 and ITS4 primer sequencing (Fig 4). PCR amplification and ITS sequencing (~650 bp) validated species-level identification as T. yunnanense (CTP4, CTN2, CTJ1, CTA2, CTO2), T. rifai (CTL2), T. simmonsii (CTB1) and T. pleuroticola (CTB2), showing 97.93-99.83% similarity. Sequences were deposited in NCBI GenBank (OP781938-OP781945). Combined morphological and ITS analyses revealed significant diversity among isolates, highlighting their prevalence in chickpea cropping systems and potential as valuable microbial resources (Surma et al., 2025).


 
Micronutrient uptake enhancement
 
Data tabulated in Table 1, revealed that seed treatment and soil application of Trichoderma isolates significantly enhanced micronutrient uptake in chickpea at 30 DAS. T. yunnanense isolate CTA2 exhibited significantly highest uptake of zinc (26.57 ppm), copper (8.27 ppm), iron (46.03 ppm) and manganese (16.50 ppm), compared to control and pathogen control. Other effective isolates included T. yunnanense (CTP4, CTJ1, CTN2) and T. pleuroticola (CTB2), which also significantly improved micronutrient content. These increases are attributed to siderophore and organic acid production by Trichoderma, enhancing micronutrient solubility and plant availability, thereby improving soil fertility and chickpea nutrition. Similar findings have been reported by Aishwarya et al., 2020; Ali et al., 2022; Syed et al., 2023; Chalie-U Rokozeno et al., 2025, who observed improved nutrient uptake in chickpea, respectively, following Trichoderma application.


 
Plant growth promoting attributes and wilt incidence
 
Data on growth promoting characters of chickpea (Table 1) revealed that Trichoderma-treated plants showed improved germination (73.67-86.33%), vigour index (2800-4241), number of branches (3.77-5.33), nodules (7-12), pods (10.33-16.33), biomass and yield per plant (4.00-7.17 g), with CTA2 producing the maximum. Trichoderma yunnanense (CTA2) recorded the highest germination% (86.33%), vigour index (4241), number of branches per plant (5.33), nodules per plant (12.00), pods per plant (16.33) and yield per plant (7.17 g), significantly exceeding both the control and pathogen control. These values were statistically at par with treatments involving T. yunnanense (CTP4, CTJ1, CTN2) and T. pleuroticola (CTB2). Similarly, fresh and dry weights of shoots and roots were significantly enhanced under T. yunnanense (CTA2) and T. yunnanense (CTP4). Observations on wilt incidence 65 days after sowing, showed T. yunnanense (CTA2) had the lowest wilt incidence (16.39%), followed by T. yunnanense (CTJ1) at 22.50% and T. pleuroticola (CTB2) at 24.02%, indicating superior disease suppression by these isolates.
       
These findings are consistent with reports by Chohan et al., (2024) and Parmar and Gohel (2024), who demonstrated the plant growth-promoting and biocontrol potential of Trichoderma spp. in legumes. Additionally, similar improvements in growth and yield were observed in legume crops treated with Trichoderma isolates, as reported by Chalie-U Rokozeno et al., 2025; Syed et al., 2023. These dual benefits reinforce the role of Trichoderma as both biocontrol agents and biofertilizers (Venkataramanamma et al., 2022; Haque et al., 2025).
 
Implications and future directions
 
The superior performance of T. yunnanense (CTA2, CTP4) and the promising results from T. pleuroticola (CTB2) show strong potential as dual-purpose bio-inoculants for managing Fusarium wilt and enhancing chickpea yield. While these results are encouraging under controlled conditions, extensive field validation across diverse environments is essential to confirm their efficacy and consistency. Moreover, multi-gene phylogenetic analyses and detailed biochemical studies will provide deeper insights into their mechanisms and genetic stability. Further research on formulation stability and scalability will be crucial to support their commercialization and practical use. This integrated approach will facilitate the development of robust, scalable bioformulations to aid sustainable intensification of chickpea production.

This study successfully isolated and characterized indigenous Trichoderma spp. from chickpea rhizosphere soils of the Marathwada region, identifying T. yunnanense (CTA2, CTP4) and T. pleuroticola (CTB2) as the most effective isolates in suppressing Fusarium wilt and promoting chickpea growth under controlled conditions. These isolates significantly enhanced seed germination, seedling vigour, yield attributes and micronutrient uptake, demonstrating their dual potential as biocontrol agents and biofertilizers. The findings underscore the value of native Trichoderma strains adapted to local agroclimatic conditions for sustainable disease management and yield improvement in chickpea cultivation.

The present study was supported by Department of Plant Pathology and Department of Soil Science and Agril Chemistry of Vasantrao Naik Marathwada Krishi Vidyapeeth University, Parbhani, Maharashtra. This study is derived from the doctoral thesis of Varala Krishnaveni.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.