Isolation and characterization of novel banana rhizosphere bacteria for the control of Fusarium oxysporum f. sp. cubense TR4

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense race TR4 (Foc), is one of the most destructive diseases threatening global banana production, particularly the Cavendish cultivar. Conventional control strategies, including chemical treatments and quarantine, remain largely ineffective and unsustainable, underscoring the urgent need for alternative approaches. Biological control using rhizosphere-associated microorganisms offers a promising and environmentally friendly strategy. In this study, we isolated 436 bacterial strains from the rhizosphere of healthy banana plants and screened them for antifungal activity against Foc. Out of the screened isolates, 93 exhibited significant in-vitro inhibitions, and 64 of these were subsequently evaluated in greenhouse assays. We found that 22 strains reduced Fusarium wilt severity by 45-85% compared to untreated controls. Among them, two isolates, DDC20 and DDC_NEW2, consistently demonstrated strong biocontrol activity. In addition, cell-free culture media (CFCM) and crude extracts inhibited spore germination in fluorescence-based assays, indicating the involvement of secreted antifungal metabolites. Microscopy and confocal observations of GFP-tagged Foc revealed hyphal abnormalities in the presence of bacterial treatments, including swelling, irregular branching, and distortion, accompanied by excessive sporulation characterized by abundant microconidia, macroconidia, and chlamydospores. Whole-genome sequencing and comparative analyses placed both isolates within the genus Bacillus. Genome mining using antiSMASH identified multiple biosynthetic gene clusters encoding known antifungal compounds such as surfactin, fengycin, bacillibactin, and difficidin, as well as putative novel clusters. LC-MS confirmed the presence of surfactin and fengycin in bacterial extracts, supporting the genomic predictions. Collectively, these findings highlight the potential of DDC20 and DDC_NEW2 (related to Bacillus spp.) from the banana rhizosphere as effective biocontrol agents against Foc TR4. This integrated approach, combining phenotypic assays, microscopy, and genome mining, provides a strong foundation for the development of sustainable strategies to manage Fusarium wilt in banana cultivation.