Abstract: To study the mechanism by which ferrous sulfate (FeSO4) induces ferroptosis in Aspergillus flavus, its ferroptosis characteristics and microstructure were observed after being treated with different concentrations of FeSO4. The results showed that all tested concentrations of FeSO4 significantly inhibited the growth of A. flavus. Additionally, Fe treatment significantly increased the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in the mycelium of A. flavus (P < 0.05), significantly decreased the levels of glutathione/oxidized glutathione (GSH/GSSG) (P < 0.05), and promoted lipid peroxidation in A. flavus spores. Scanning electron microscopy (SEM) revealed that Fe disrupted the morphological structure of A. flavus, leading to significant changes in the permeability of the cell membrane and the release of cellular contents (nucleic acids and proteins) and ions (Ca2+, K+ and Mg2+). Transcriptomics analysis indicated that FeSO4 treatment affected metal ion homeostasis in A. flavus and down-regulated the gene expression related to GSH production. All above findings indicated that Fe could induce ferroptosis in A. flavus. Meanwhile, the synergistic mechanism of FeSO4 and Bacillus methylotrophicus BCN2 in inducing ferroptosis in A. flavu was studied. The results indicated that the combined treatment had stronger antifungal activity against A. flavus and its biofilm. The combined treatment significantly reduced the mitochondrial function of A. flavus, thereby leading to a decrease in the activities of ATPase and key enzymes in the tricarboxylic acid cycle. In addition, it significantly inhibited the growth of A. flavus inoculated in blueberries. This study provides a new idea for the effective prevention and control of A. flavus.

Key words: ferrous sulfate; Aspergillus flavus; ferroptosis; Bacillus; mitochondria