Optimisation of Culture Conditions Enhances Antifungal Activity and Reshapes Extracellular Metabolite Profiles in...

Botrytis cinerea is a major phytopathogenic fungus responsible for substantial economic losses in horticultural crops, underscoring the need for sustainable alternatives to synthetic fungicides. This study investigated the influence of physical, chemical and biological culture parameters on the antifungal activity of culture filtrates produced by Trichoderma harzianum BOL-12QD. Culture conditions were sequentially optimised by evaluating light-filter exposure, carbon and nitrogen source composition, potato ecotype selection, co-cultivation with Botrytis cinerea, and volatile-mediated interactions. Antifungal activity was assessed using mycelial growth inhibition assays against Botrytis cinerea. Among the individual factors, violet-filter illumination, a medium containing 5 g L^-1 glucose and 250 g L^-1 potato extract, the Leke Pek'e potato ecotype, ammonium nitrate as nitrogen source, and co-cultivation with Botrytis cinerea at 10⁴ conidia mL^-1 produced the highest inhibitory effects. Sequential integration of these optimised conditions resulted in enhanced antifungal activity, reaching up to 62% inhibition. Volatile organic compounds produced by Trichoderma harzianum BOL-12QD exhibited only minimal antifungal activity under the conditions tested, suggesting that volatile-mediated antagonism plays a limited role in this system. In contrast, culture-dependent modulation of extracellular metabolite profiles was evidenced by comparative ¹H NMR fingerprinting, which revealed condition-specific spectral differences, with the optimised treatment displaying a distinct metabolic signature relative to all other conditions. Cytotoxicity assays in murine peritoneal macrophages showed no significant reduction in cell viability at concentrations up to 200 μg mL^-1. In vivo exposure to the optimised culture filtrate (250 mg kg^-1 d^-1 for 10 days) induced transient treatment-related clinical observations without mortality, indicating a need for further detailed toxicological characterisation. Overall, these findings demonstrate that the antifungal activity of Trichoderma harzianum BOL-12QD is strongly modulated by interacting environmental, nutritional and biological culture parameters. The results support the potential of optimised culture filtrates as a source of bioactive metabolites for biocontrol applications, while highlighting the importance of integrated biochemical and toxicological evaluation.