Condition dependent variation in bioactive metabolites of Bacillus siamensis during interaction with fungal pathogens

Interactions between antagonistic bacteria and fungal pathogens influence extracellular metabolite production and contribute to the efficacy of antagonistic agents (AGAs). However, comprehensive characterization of condition dependent metabolite variation during bacterial-fungal interactions remains limited. Here, we investigated the extracellular metabolite profiles of Bacillus siamensis SSVP1 during interactions with live and processed heat-killed biomass of Fusarium oxysporum ITCC-5330 and Macrophomina phaseolina ITCC-5519 using untargeted UHPLC-MS/MS-based metabolomics. Comparative analysis across four interaction conditions-Live Fusarium (LF), Live Macrophomina (LM), Dead Fusarium (DF), and Dead Macrophomina (DM)-revealed distinct condition-associated metabolite abundance patterns. Exposure to processed heat-killed fungal biomass was associated with a greater number of detectable extracellular metabolite features than live fungal interactions.Notably, the Fusarium -associated conditions contained 107.43% and 148.27% more metabolite features with significantly negative log 2 FC values than the corresponding Macrophomina -associated conditions under live and processed fungal biomass, respectively. Venn analysis identified both conserved and condition-specific metabolite signatures. Putatively annotated antifungal-, antibacterial-, signalling-, and siderophore-associated metabolites exhibited distinct abundance patterns, with individual members of the iturin, surfactin, and fengycin families displaying contrasting responses across interaction conditions. Peptide/non-ribosomal peptide synthetase-derived compounds (56.95%) and polyketides (25.17%) dominated the differential metabolite pool. Extracellular extracts from all interaction conditions retained antifungal activity despite marked differences in metabolite composition. Collectively, these findings demonstrate that fungal species identity and interaction state are associated with distinct extracellular metabolite profiles of B. siamensis SSVP1 and identify candidate metabolites for future targeted validation and development of AGAs.