A novel antifungal potential protein of Bacillus subtilis-fermented Gracilaria agar-processing wastewater: identification, mechanism, regulation and applications in citrus protection.

The management of phytopathogenic fungi is essential for ensuring food security and mitigating postharvest losses. In this study, we explored the potent antifungal component derived from Bacillus subtilis by utilizing Gracilaria agar-processing wastewater (GAW) as an innovative fermentation substrate. The key bioactive protein was subsequently isolated, purified and identified as the ABC transporter ATP-binding protein YtrB. Meanwhile, the exogenous regulator 6-benzylaminopurine (6-BA) significantly enhanced the antifungal activity of the fermentation broth, increasing the inhibition rate against Alternaria alternata BNCC 123548 by approximately 22%. Mechanistic studies revealed that YtrB exerts its antifungal effect by disrupting fungal cell membrane integrity, as directly observed by laser confocal microscopy. This disruption leads to leakage of electrolytes and nucleic acids, followed by malondialdehyde accumulation indicative of oxidative stress, and thereby inhibiting mycelial growth. Furthermore, it resulted in a 54.1% reduction in citrus leaf lesions, indicating synergistic broad-spectrum efficacy and highlighting it as a potent antifungal agent. This work demonstrates a promising strategy for the high-value utilization of GAW and provides a potential protein-based candidate for the management of plant fungal diseases.