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The soil-living bacterium <i>Bacillus subtilis</i> is able to form architecturally complex cell communities, known as biofilms. <i>B. subtilis</i> has long been used in the field of agriculture as a biocontrol reagent to protect plants against soil-borne plant pathogens. The exact mechanisms for plant biocontrol have not been clearly addressed. In this study, we established <i>B. subtilis</i>-tomato interaction system to explore the biocontrol mechanisms. We isolated many <i>B. subtilis</i> strains form various natural environments in China, and obtained 6 wild stains demonstrated above 50% biocontrol efficacy against the <i>R. solanacearum</i> under greenhouse conditions. They also showed robust biofilm formation both in biofilm-inducing media and on tomato plant roots, and exhibited strong antagonistic activities again various plant pathogens <i>in vitro</i>. We knocked out 7 key genes related to <i>B. subtilis</i> biofilm formation by transformation and evaluated the biocontrol efficacy of these mutants. Results indicated the biofilm formation ability of<i> B. subtilis</i> is positively correlated to biocontrol efficiency against tomato bacterail wilt disease. Meanwhile, the antimicrobial capability is also necessary to inhibit <i>R. solanacearum</i>, especially surfactin. The surfactin mutant strains seriously decreased biocontrol efficacy. Finally, we suggested <i>B. subtilis</i> biofilm formation and production of antimicrobial agents may act synergistically to enhance biocontrol efficacy during the plant diseases management. <p><p>Keywords: Bacteria-Phytoplasma-Spiroplasma-Fastidious Prokaryote, Vegetables, Tomato