Bacillus aerophilus A5 suppresses tobacco root rot and gene expressions involved in fungal chitin metabolism

Tobacco root rot is a destructive soil-borne disease that constrains sustainable tobacco production in Yunnan. Fusarium oxysporum and Fusarium solani species complex are currently considered the principal pathogens associated with this disease. Therefore, effective biocontrol agents are needed, and understanding their pathogen-side modes of action is essential for improving biological management strategies. This study screened 100 bacterial strains isolated from the tobacco rhizosphere using plate confrontation assays and identified A5 as a broad-spectrum antagonistic strain. A5 consistently inhibited four tobacco root rot-associated Fusarium species, with hyphal growth inhibition rates of 63.31%, 59.42%, 69.64%, and 68.82%, respectively. Phylogenetic analyses of 16S rRNA and gyrB sequences placed A5 close to Bacillus aerophilus, supporting its classification as a B. aerophilus-related biocontrol candidate. To investigate the fungal response to A5, F. solani was exposed to 10% sterile A5 fermentation supernatant and analyzed using transcriptomic sequencing. GO and KEGG enrichment analyses showed differentially expressed genes (DEGs) involved in UDP-N-acetylglucosamine biosynthesis and associated cell wall precursor metabolic processes. Because F. oxysporum has a more established genetic manipulation system in tobacco root rot research, this species was used for downstream functional validation. RT-qPCR confirmed the significant expression responses of FOXG_19023, FOXG_10443, and FOXG_12345 after the treatment with A5 supernatant. Subsequent construction of knockout mutants demonstrated that deletion of FOXG_19023 significantly restricted colony growth, suggesting that FOXG_19023 may contribute to vegetative growth of F. oxysporum, although its specific role in UDP-N-acetylglucosamine-related cell wall metabolism remains to be explored. Overall, A5 was identified as a tobacco root rot biocontrol bacterium with activity against Fusarium pathogens. These results further suggest that A5 may affect expression of genes involved in pathogen UDP-N-acetylglucosamine-related metabolism, and this response may be associated with impaired Fusarium hyphal growth. This study provides a potential biocontrol resource for sustainable tobacco root rot management and offers candidate pathogen-side targets for future studies on the molecular mechanisms underlying Bacillus-mediated suppression of Fusarium.