Bacillus velezensis ATC-AL suppresses mycotoxin-producing fungi by disrupting membrane integrity and inducing mitochondrial dysfunction

Abstract

Aspergillus flavus is a predominant fungal contaminant, producing aflatoxin B1 (AFB₁), one of the most potent mycotoxins posing serious risks to global food safety. In this study, we evaluated the antifungal efficacy of Bacillus velezensis ATC-AL and its active antifungal substances (AAS) against A. flavus and other mycotoxin-producing fungi. The AAS treatment displayed broad-spectrum antifungal activity, completely suppressing AFB₁ synthesis at a minimum inhibitory concentration of 8 μL/mL. It also significantly reduced deoxynivalenol (DON) accumulation by Fusarium graminearum, indicating multi-target antitoxin potential. Mechanistic analyses revealed that AAS disrupts fungal membrane integrity and induces oxidative stress, as evidenced by elevated reactive oxygen species (ROS) levels, lipid peroxidation, and structural damage to the cell membrane and cell wall in A. flavus. Metabolomic profiling further revealed perturbations in the TCA cycle, glutathione metabolism, and nucleic acid biosynthesis, consistent with mitochondrial dysfunction and impaired energy metabolism. Correspondingly, AAS treatment caused a collapse in mitochondrial membrane potential and decreased ATP levels, suggesting apoptosis-like cell death. In grain kernel assays, AAS significantly inhibited fungal colonization and eliminated mycotoxin production, effectively protecting grains from infection by A. flavus and F. graminearum. Collectively, these findings demonstrate that B.velezensis ATC-AL functions as a promising biocontrol agent acting through multiple antifungal and anti-mycotoxin pathways, offering a sustainable strategy for mitigating fungal contamination and mycotoxin risk in grain storage systems.