Bioprospection of Metschnikowia pulcherrima as biocontrol agents against gray mold on grapes with their potential modes of action.

Gray mold in grapes during the postharvest period is primarily caused by Botrytis cinerea, with synthetic fungicides being the main method of control. Antagonistic yeasts offer a viable alternative to conventional fungicides for the management of pathogens post-harvest. The study analyzed the effectiveness of 14 Metschnikowia pulcherrima yeast strains against gray mold in grapes and examined their potential mechanisms of action. In vitro analyses demonstrated that all evaluated yeast strains (100 μL, 1 × 104 CFU/mL) significantly inhibited the mycelial growth of B. cinerea, with inhibition rates ranging from 7 % (MP13 strain) to 81 % (MP07 strain). Subsequent in vivo antifungal assays demonstrated MP07's efficacy in preventing gray mold on grapes. Critically, a comprehensive and systematic mechanistic analysis revealed, for the first time, distinct variations in antimicrobial pathways among strains of the same species. MP07 exhibited the highest levels of biofilm formation (OD590 of 0.68; 100 μL, 1 × 107 cells/mL) and pulcherrimin production (halo diameter of 2.35 cm; 10 μL, 1 × 108 cells/mL). In contrast, MP14 (20 μL, 1 × 108 cells/mL) demonstrated superior antifungal activity (100 %) through the production of volatile organic compounds (VOCs), while MP11 (20 μL, 1 × 108 cells/mL) exhibited the highest β-1,3-glucanase activity (80.78 U1), which further increased to 100.96 U1 upon the introduction of B. cinerea. These findings underscore that screening for biocontrol agents must extend to the strain level, necessitating precise evaluation of their antifungal mechanisms. By leveraging strain-specific inhibitory pathways, targeted combinatorial strategies can be devised. In conclusion, this study lays a theoretical groundwork for developing highly effective, safe, and stable biocontrol agents through precision-based intra-species strain selection, offering a viable alternative to complex multi-species formulations.