Metabolic and Proteomic Shifts in Macrophomina phaseolina Induced by Bacillus subtilis Cell-Free Extract

Macrophomina phaseolina (MP), a fungal phytopathogen, causes charcoal rot disease in soybean. This pathogen's ability to form microsclerotia makes it difficult to control and thus poses a major threat to soybean production. The present study focused on effective charcoal rot disease management using Bacillus subtilis M-4, which exhibits strong biocontrol potential against MP. This study evaluated the antifungal efficacy of B. subtilis M-4 cell-free filtrate (BS-CFF) and bacterial pellet (BS-BP) against MP under in vitro conditions. The results showed that BS-CFF exhibited significantly greater inhibition of MP growth, with higher concentrations yielding stronger mycelial suppression and reduced host plant infection. Moreover, an RT-qPCR assay was performed to evaluate the gene expression in MP after BS-CFF and BS-BP treatment. The results indicated that treatment of BS-CFF downregulated essential fungal mitochondrial genes ( nad5, atp6, cob, rps3, and rnl) that are involved in the growth and pathogenicity of MP. Proteomic analysis further revealed substantial downregulation of fungal proteins associated with genetic information processing (26.34%), energy/carbohydrate metabolism (19.16%), signaling pathways (14.11%), and defense/stress responses (12%) after BS-CFF treatment, compared with BS-BP. Additionally, a phenotype microarray assay confirmed that BS-CFF suppresses the utilization of 18 crucial substrates by 100%. These substrates belong to amino acid and nitrogen categories that are essential for fungal metabolism. These findings elucidate the molecular, proteomic, and metabolic mechanisms underlying BS-CFF's biocontrol efficacy, providing valuable insights for effective fungal disease management in agriculture.