C 15 -bacillomycin D Produced by Bacillus amyloliquefaciens 4-9-2 suppress Fusarium graminearum infection and mycotoxin biosynthesis

Zhongliang Liu¹Yijia Luo¹Rongxin Lin¹Chengming Li¹Hanjun Zhao¹Haqmal Mohammad Aman¹Wisal Muhammad Asif¹Huifeng Dong²Dingkuo Liu²Xiaona Yu³Kong Lingcong^1*Hong-xia Ma^1*

• ¹Jilin Agriculture University, Changchun, China
• ²Tianjin Key Laboratory of Biological Feed Additive Enterprise, S&E Burgeoning Biotechnology (Tianjin) Co., Ltd, Tianjin, China
• ³Liaoning, He Medical College, No. 66, Sishui Street, Hunnan District, Shenyang, China

Fusarium graminearum threatens global food security through crop diseases and mycotoxin contamination, presenting significant challenges in controlling this toxigenic pathogen. This study isolated and cultured bacteria from the soil and screened for antagonistic strains against F. graminearum using a plate confrontation method. The results showed that strain 4-9-2 exhibited excellent antifungal capabilities. 16S rDNA sequencing indicated that the strain had a high homology with Bacillus amyloliquefaciens. Whole genome sequencing revealed a genome size of 3,957,046 bp and a GC content of 46.5%. AntiSMASH analysis predicted 12 biosynthetic gene clusters (BGCs). HPLC and ESI-IT-TOF/MS characterization confirmed the antifungal metabolites as C15-bacillomycin D. In vitro assays demonstrated that purified bacillomycin D significantly inhibited F. graminearum spore germination and hyphal growth, with a minimum inhibitory concentration (MIC) of 64 μg/mL and a half-maximal inhibitory concentration (IC50) of 26.10 μg/mL. Biocontrol experiments validated bacillomycin D's efficacy in suppressing maize kernel infection by F. graminearum and reducing deoxynivalenol (DON) and zearalenone (ZEN) production. The lipopeptide exhibited robust environmental adaptability, maintaining antifungal stability across temperatures (25-100°C), pH (2-12), and in the presence of metal ions or hydrolytic enzymes. Mechanistic studies have revealed that bacillomycin D exert antifungal effects by disrupting the cell membrane structure of F. graminearum. Microscopic observations showed that after interaction with bacillomycin D, F. graminearum exhibited morphological abnormalities and changes in membrane permeability. Further research indicated that bacillomycin D treatment induces an increase in intracellular reactive oxygen species (ROS) accumulation and membrane potential dissipation in F. graminearum. The overall results of this study highlight the potential of bacillomycin D as an effective biocontrol agent against F. graminearum infection. It indicates the crucial role of B. amyloliquefaciens 4-9-2 in inhibiting the F. graminearum.