Mycotoxin contamination of nuts, frequently attributed to inappropriate storage, causes substantial economic losses and health concerns globally. Biological control using beneficial microorganisms has emerged as an environment friendly method for efficient mitigation of Aspergillus flavus pollution and consequent mycotoxin elimination in foodstuffs. Nevertheless, the exact mechanisms by which these biocontrol microbes protect nuts from this toxigenic fungus remain largely unknown. Using a fungal infection assay, we observed a remarkable inhibitory effect of Enterobacter ludwigii AA4 against the growth of A. flavus colonizing walnut kernels and aflatoxin B1 production. Mutant E. ludwigii AA4 strains, generated by genetically modifying five biofilm-related genes, notably fadR (which encodes a transcriptional regulator), exhibited significantly impaired biofilm development and reduced efficacy in suppressing A. flavus. These results indicated that biofilm establishment is indispensable for the inhibitory effect of E. ludwigii AA4 against A. flavus. We further investigated the kernel colonization of fadR knockout mutant, which exhibited the most pronounced reduction in biofilm formation, via colony counting and laser scanning confocal microscopy. We found that fadR contributed to the suppression of A. flavus by influencing bacterial biofilm production and kernel settlement. Gene expression analysis and site-directed mutagenesis revealed that fadR modulated biofilm development by negatively regulating the transcription of rcsA, an auxiliary protein gene within the Rcs phosphorelay system, potentially by influencing acetyl phosphate-mediated RcsB phosphorylation. These findings highlight the potential of AA4 in the biological control of A. flavus contamination in walnut kernels.
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