Biocontrol of Aspergillus flavus in stored wheat using Bacillus sp. B3 and Bacillus sp. Mn1 with antagonistic capacity through volatile compounds production

Abstract Background Contamination of stored wheat grains by Aspergillus flavus poses serious food safety risks due to aflatoxin production. This study aimed to identify and evaluate wheat-associated bacterial isolates for their potential to biologically control A. flavus . Two promising strains, Bacillus sp. B3 and Bacillus sp. Mn1, were selected based on a strong antifungal activity against A. flavus , on dual culture assays. Results Dual-culture and co-cultivation assays confirmed that both strains significantly inhibited A. flavus mycelial growth and sporulation. Additionally, both strains exhibited facultative mycophagy, utilizing fungal biomass under nutrient-limited conditions. Volatile organic compounds (VOCs) produced by these bacteria suppressed fungal growth in vitro and on wheat grains without direct contact. Gas chromatography–mass spectrometry (GC–MS) profiling revealed that Bacillus sp. B3 primarily emitted nitrogenous and oxime-related compounds, while Bacillus sp. Mn1 produced diverse volatiles including fatty acids, hydrazine derivatives, and trigonelline. Both strains significantly reduced fungal colonization on treated grains. The strains were characterized through 16 S rRNA gene sequencing, alongside biochemical and enzymatic profiling, to aid taxonomic placement and provide insight into their functional potential. Conclusion Bacillus sp. B3 and Mn1 demonstrated robust antifungal potential through multiple antagonistic mechanisms including diffusible metabolites, mycophagy, and VOC-mediated inhibition. These findings support their use as sustainable, residue-free biocontrol agents to mitigate A. flavus contamination and enhance the safety of stored wheat.