Antifungal Agents of Bacillus velezensis 5RB: Complex Action of Lipopeptides, Polyketides, and Subtilosin A

Bacillus velezensis is a promising candidate for the biocontrol of plant pathogens due to its production of broad-spectrum antimicrobial metabolites. The present study evaluates the antifungal potential and metabolic profile of the non-pathogenic rhizobacterial strain Bacillus velezensis 5RB. Modifying the mineral composition across three different growth media (A, B, and C) resulted in distinct secondary-metabolite profiles of B. velezensis 5RB. Notably, the free-cell supernatant of strain 5RB cultivated in medium A and C demonstrated the most potent antifungal activity against the gray mold pathogen Botrytis cinerea. For the first time, the antimicrobial metabolites produced by B. velezensis 5RB across these three media were comprehensively characterized using ultra-performance liquid chromatography-quantitative time-of-flight mass spectrometry (UHPLC-QTOF-MS). Mass spectrometric analyses (LC-ESI-MS) identified predominantly lipopeptides from the surfactin and fengycin families, alongside three classes of polyketides: macrolactins, difficidins, and bacillaenes. Multiple lipopeptide homologues, including a novel surfactin isoform (B*), were confirmed by tandem mass spectrometry (LC-ESI-MS/MS). Additionally, this study provides the first experimental evidence for subtilosin A production by B. velezensis 5RB. The multicomponent fermentation matrix of 5RB conferred robust biocontrol protection to tomato leaves against B. cinerea and Phytophthora infestans in an in vitro disease model, underscoring its strong potential for sustainable agricultural applications.