Identification and functional characterization of an endophytic Bacillus velezensis strain against Foc TR4 via targeting fungal cell wall integrity

The global banana supply has long been severely threatened by Fusarium wilt of banana (FWB), a devastating disease caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). However, effective strategies for managing this destructive pathogen still remain in their infancy. The newly isolated endophytic bacterium Bacillus velezensis strain EB7 in this study has shown considerable potential for management of FWB, yet the cellular mechanisms underlying its biocontrol activity remain largely unclear. Comprehensive morphological, phylogenetic, and genomic characterization confirmed EB7's taxonomic status and identified abundant biocontrol-related secondary metabolite biosynthetic gene clusters (BGCs). In vitro antagonism assays showed that EB7 exhibited strong inhibitory activity against Foc TR4 with an inhibition rate of 83.60%, by targeting fungal cell wall integrity. This was evidenced by hyphal deformation, cell wall disruption, and abnormal Calcofluor White (CFW) staining in Foc TR4. RNA-seq and qRT-PCR validation demonstrated that EB7 downregulated the chitin synthase gene FOIG_07229 and disrupted the expression of genes involved in Foc TR4 cell wall synthesis and metabolism. Colonization dynamics analysis indicated that EB7 could successfully colonize banana roots and pseudostems endophytically, forming stable populations and migrating systemically without inducing any pathological symptoms in the host. Scanning electron microscopy (SEM) observations revealed that EB7 attached to Foc TR4 hyphae in banana pseudostems, inducing hyphal shrinkage and reducing pathogen invasion of vascular bundles. Pot experiments verified that EB7 significantly alleviated FWB severity, with the average disease index decreasing from 2.6 to 0.9. Our findings demonstrate that strain EB7 is a promising biocontrol agent for FWB, exerting its antifungal effects through targeting Foc TR4 cell wall integrity and establishing stable endophytic colonization in banana plants. This work thus provides novel insights into the biocontrol mechanism of endophytic bacteria against Foc TR4, as well as high-potential microbial resources to support the sustainable management of FWB.