Bacillus subtilis inhibits Aspergillus carbonarius by producing iturin A, which disturbs the transport, energy metabolism, and osmotic pressure of fungal cells as revealed by transcriptomics analysis.

The contamination of Aspergillus carbonarius causes decreases and great decay of agricultural products, and threatens the human and animal health by producing mycotoxins, especially ochratoxin A. Bacillus subtilis has been proved to efficiently inhibit the growth of A. carbonarius. Revealing the major active compound and the mechanisms for the antifungal of B. subtilis are essential to enhance its antifungal activity and control the quality of antifungal products made of it. In this study, we determined that iturin A is the major compound that inhibits Aspergillus carbonarius, a widespread fungal pathogen of grape and other fruits. Iturin A significantly inhibited growth and ochratoxin A production of A. carbonarius with minimal inhibitory concentrations (MICs) of 10 μg/mL and 0.312 μg/mL, respectively. Morphological observations revealed that iturin A caused swelling of the fungal cells and thinning of the cell wall and membrane at 1/2 MIC, whereas it inhibited fungal spore germination and caused mitochondrial swelling at higher concentrations. A differential transcriptomic analysis indicated that the mechanisms used by iturin A to inhibit A. carbonarius were to downregulate the expression of genes related to cell membrane, transport, osmotic pressure, oxidation-reduction processes, and energy metabolism. Among the down-regulated genes, those related to the transport capacity were most significantly influenced, including the increase of energy-related transport pathways and decrease of other pathways. Notably, the genes related to taurine and hypotaurine metabolism were also decreased, indicating iturin A potentially cause the occurrence of osmotic imbalance in A. carbonarius, which may be the intrinsic cause for the swelling of fungal cells and mitochondria. Overall, iturin A produced by B. subtilis played important roles to inhibit A. carbonarius via changing the fungal cell structure and causing perturbations to energy, transport and osmotic pressure metabolisms in fungi. The results indicated a new direction for researches on the mechanisms for lipopeptides and provided useful information to develop more efficient antifungal agents, which are important to agriculture and biomedicine.