Biocontrol Potential of Bacillus Amyloliquefaciens JDF-1 Against Aspergillus Flavus During Cigar Tobacco Fermentation: Antifungal Activity, Aflatoxin Suppression, and Metabolomic Associations.

Mould contamination during cigar tobacco fermentation can cause spoilage and aflatoxin accumulation. This study isolated culturable fungi associated with mouldy cigar tobacco and evaluated Bacillus amyloliquefaciens JDF-1 as a candidate biocontrol strain under controlled fermentation conditions. A total of 215 fungal isolates were obtained using six culture media and identified by colony morphology and ITS sequencing. Antagonistic bacteria isolated from healthy tobacco leaves were screened by dual-culture assays and identified using physiological-biochemical characteristics and 16 S rRNA gene sequencing. Candidate compounds tentatively assigned from the cell-free JDF-1 fermentation supernatant were evaluated in a comparative antifungal screening assay against Aspergillus flavus. Four treatments-sterile-water control (CK), JDF-1 inoculation (TA), A. flavus inoculation (TH), and co-inoculation with JDF-1 and A. flavus (THA)-were compared after 35 days of simulated fermentation. (A) flavus was the predominant culturable fungal species, accounting for 48.84% (105/215) of all isolates. JDF-1 produced the largest inhibition zone (9.5 mm) among the screened bacterial isolates and was preliminarily identified as (B) amyloliquefaciens. Among the tested candidate compounds, cyclo(Leu-Pro) showed the lowest EC₅₀ against A. flavus (101.66 ± 6.55 µg/mL), but its contribution to the activity of the native supernatant was not established. The TH treatment showed pronounced mould development and the highest concentrations of aflatoxins B1, B2, G1, and G2. Co-inoculation with JDF-1 substantially reduced visible mould development and aflatoxin accumulation relative to TH, whereas TA showed a mould incidence of 0.71% and no detectable aflatoxins. JDF-1-containing treatments were also associated with changes in major tobacco chemical constituents and metabolite profiles. Untargeted metabolomics and KEGG enrichment identified treatment-associated LC-MS features and pathway annotations but did not establish pathway regulation, metabolic flux, or causal mechanisms. These findings support JDF-1 as a candidate biocontrol strain for reducing A. flavus contamination and aflatoxin risk during cigar tobacco fermentation under controlled conditions. Further targeted metabolite validation, fungal-biomass quantification, and pilot-scale evaluation are required.