C15-bacillomycin D produced by Bacillus amyloliquefaciens 4-9-2 suppress Fusarium graminearum infection and mycotoxin biosynthesis

Abstract

Introduction: Fusarium graminearum threatens global food security through crop diseases and mycotoxin contamination, presenting significant challenges in controlling this toxigenic pathogen. Methods: Soil bacteria were isolated and screened for antagonism using plate confrontation. Active strain 4-9-2 was identified by 16S rDNA and whole-genome sequencing. Antifungal metabolites were characterized via AntiSMASH, HPLC, and ESI-IT-TOF/MS. MIC and IC₅₀ against F. graminearum spores/hyphae were determined. Biocontrol efficacy was tested on maize kernels, measuring infection suppression and mycotoxin reduction. Compound stability was assessed under varying temperatures (25–100°C), pH (2–12), metal ions, and enzymes. Mechanisms were investigated through microscopy, membrane permeability, ROS, and membrane potential assays. Results: Strain 4-9-2 (Bacillus amyloliquefaciens) showed potent antifungal activity. Its genome (3,957,046 bp, GC 46.5%) harbored 12 BGCs. The metabolite was identified as C15-bacillomycin D, inhibiting F. graminearum at MIC 64 μg/mL and IC50 26.10 μg/mL. It suppressed maize kernel infection and reduced deoxynivalenol (DON) and zearalenone (ZEN) levels. Bacillomycin D maintained activity across tested temperatures, pH, and stressors. It disrupted membrane integrity, causing morphological defects, increased permeability, ROS accumulation, and membrane depolarization. Discussion: Bacillomycin D from B. amyloliquefaciens 4-9-2 is a promising biocontrol agent against F. graminearum, combining potent antifungal activity, mycotoxin reduction, environmental resilience, and membrane-targeting action.