Glucose-driven metabolic reprogramming enhances fengycin biosynthesis and antifungal activity in Bacillus amyloliquefaciens FH1-RS1 against Verticillium dahliae V991

Verticillium dahliae causes destructive cotton Verticillium wilt, and the extracellular metabolites underlying Bacillus -mediated antagonism can differ among biocontrol strains. Although genomic analysis of Bacillus amyloliquefaciens FH1-RS1 revealed diverse antimicrobial biosynthetic potential, the principal extracellular metabolite responsible for its antifungal activity against V. dahliae V991 had not been defined. Systematic medium screening and glucose titration identified Luria-Bertani (LB) medium supplemented with 2% d -glucose (LB20) as a condition supporting maximal antifungal activity, which peaked at 48 h. Transcriptomic profiling of LB20 relative to LB revealed coordinated upregulation of nonribosomal peptide synthetase (NRPS) machinery, fatty acid biosynthesis, oxidative phosphorylation, the tricarboxylic acid cycle, and precursor-supply pathways. Bioactivity-guided fractionation and quantitative chemical analysis showed that fengycin, iturin, and surfactin production increased by 36.2, 7.5 and 3.8-fold, reaching 962.5, 75.19, and 237.89 mg L −1 , respectively, with fengycin exhibiting the most pronounced relative increase. Fraction-level bioassays identified the fengycin-enriched fraction as the dominant source of antifungal activity under the tested conditions. Ultra-high-performance liquid chromatography–high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) further supported a series of fengycin A homologues and isobaric fengycin B counterparts, together with low-abundance fengycin-related variants, while the strongest inhibition was associated with later-eluting, fengycin-enriched fractions containing relatively long-chain homologues. Collectively, these findings link a defined glucose-supplemented culture condition with a high-fengycin production state and provide a basis for optimizing FH1-RS1-derived antifungal metabolite production for biocontrol applications.