Bio-control agents are the best alternative to chemicals for the successful management of plant diseases. Among them, Trichoderma is commonly used as a biological control agent in plant disease management due to its ability to suppress soil-borne plant pathogens. In the present study, 20 Trichoderma asperellum isolates were collected from different geographical locations and confirmed using morphological characteristics and molecular phylogenetic inferences based on combined ITS and β-tubulin sequences. All twenty isolates were screened for their antagonism against the collar rot pathogen under in vitro and in planta conditions. The isolates were evaluated through dual culture and volatile methods in an in vitro study. Isolate A10 inhibited the test pathogen Agroathelia rolfsii at 94.66% in a dual culture assay and 70.95% in a volatile assay, followed by the isolates A11 and A17, which recorded 82.64% and 81.19% in dual culture assay and 63.75% and 68.27% in the volatile assay respectively. An in planta study was conducted under greenhouse conditions in tomato var. pusa ruby by pre- and post-inoculation of T. asperellum isolates in the A. rolfsii infected soil to evaluate their antagonistic potential against the disease. The A10 isolate was found effective under both pre- and post-inoculation conditions, with a disease inhibition percent of 86.17 and 80.60, respectively, followed by the isolates A11 and A17, which exhibited inhibition of 77.80% and 75.00% in pre-inoculation and 72.22% and 69.44% in post-inoculation, respectively. Further, biochemical analysis was conducted to determine the specific activity of hydrolytic enzymes produced by T. asperellum during interaction with A. rolfsii. We found that isolate A10 produces more hydrolytic enzymes with the specific activity of 174.68 IU/mg of β-1,3 glucanase, 183.48 IU/mg of β-1, 4 glucanase, 106.06 IU/mg of protease, followed by isolate A17, A11 respectively. In GC-MS analysis, we observed maximum anti-microbial volatile organic compounds from the isolate A10, including 2H-Pyran-2-one (17.39%), which was found to be most abundant, followed by dienolactone (8.43%), α-pyrone (2.19%), and harziandione (0.24%) respective retention time of 33.48, 33.85, 33.39, and 64.23 min, respectively, compared to other isolates. In the TLC assay, we observed that a greater number of bands were produced by the A10 and A17 isolates in the Hexane: Ethyl Acetate (1:1) solvent system than in the 9:1 solvent system, which represents the presence of major metabolites in the ethyl acetate extract.
