Enhanced Antifungal Activity of Bacillus velezensis R22 Against Botrytis cinerea Through Medium and Process Optimization

Botrytis cinerea, the causal agent of gray mold disease, is a major phytopathogen responsible for substantial losses in horticultural crops. In this study, cultivation conditions for Bacillus velezensis R22 were optimized to maximize overall antifungal activity against B. cinerea. A Plackett–Burman design was used to identify medium components affecting antifungal activity in flask cultures, followed by response surface methodology based on a central composite design (CCD) to optimize sucrose concentration, temperature, and agitation speed in a stirred bioreactor. Maximum antifungal activity was obtained at 17.45 g/L initial sucrose, 31.8 °C, and 293 rpm. The biological relevance of the optimized culture was confirmed in a tomato infection model, in which gray mold severity was reduced by 85.3% relative to the untreated control and by 59.9% relative to the non-optimized R22 culture. The same CCD approach was subsequently applied to determine cultivation conditions that maximize the concentration of R22 viable cells. The optimal parameters for 24-h growth (35.46 g/L sucrose, 36.5 °C, and 454 rpm) differed markedly from those identified for maximal antifungal activity. When evaluated on uninfected tomato plants, cultures produced under conditions favoring higher cell density showed enhanced plant growth-promoting activity compared to the non-optimized culture. Mass spectrometric analysis of lipopeptide extracts revealed that the enhanced antifungal activity was accompanied by an increased abundance of long-chain homologs across all major lipopeptide families, particularly surfactins. Thus, our results indicate that maximizing overall antifungal activity may be of greater practical significance than optimization of the individual fungicidal agent.