BACKGROUND: The development of biocontrol agents represents a promising strategy to manage banana Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). Although many traditional approaches have isolated beneficial microorganisms from soil or the rhizosphere, studies seeking biocontrol resources from the perspective of banana root endophytes remain scarce. RESULTS: Endophytic microbiome analysis revealed significant enrichment of Bacillota in the wilt-resistant cultivar. Among the isolated strains, Bacillus velezensis JDB15 exhibited the best inhibitory effect against Foc TR4. The fermentation broth of JDB15 significantly inhibited spore germination and caused hyphal membrane damage in pathogens. Mechanistic studies indicated that the lipopeptide surfactin C is a candidate active antimicrobial metabolite produced by JDB15, which disrupts pathogen cell membrane integrity, increases membrane permeability, and induces electrolyte leakage. Another isolated endophytic fungus, Trichoderma harzianum strain JDL4, also exhibited strong antagonistic activity against Foc TR4 probably through mycoparasitism. Combined application of cell-free filtrate from JDB15 and JDL4 demonstrated effective control against multiple plant diseases including banana Fusarium wilt, tomato Fusarium wilt, corn southern leaf blight, and rice blast under controlled conditions. CONCLUSION: We suggest that the antimicrobial activity of JDB15 and JDL4 is most probably attributable to the metabolite surfactin C and likely mycoparasitism, respectively. Co-application of the fermentation filtrates of these two strains exhibited broad-spectrum disease control efficacy and significantly improved disease suppression compared with either strain alone. These findings provide novel biological agents for the control of banana Fusarium wilt and other plant diseases. © 2026 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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