| Summary: | Verticillium wilt caused by <i>Verticillium dahliae</i> is a threatening disease of cotton, causing economic loss worldwide. In this study, nine endophytic <i>Bacillus</i> strains isolated from cotton roots exhibited inhibitory activity against <i>V. dahliae</i> strain VD-080 in a dual culture assay. <i>B. altitudinis</i> HNH7 and <i>B. velezensis</i> HNH9 were chosen for further experiments based on their high antagonistic activity. The secondary metabolites of HNH7 and HNH9 also inhibited the growth of VD-080. Genetic marker-assisted detection revealed the presence of bacillibactin, surfactin, bacillomycin and fengycin encoding genes in the genome of HNH7 and HNH9 and their corresponding gene products were validated through LC-MS. Scanning electron microscopy revealed mycelial disintegration, curling and shrinkage of VD-080 hyphae after treatment with methanolic extracts of the isolated endophytes. Furthermore, a significant reduction in verticillium wilt severity was noticed in cotton plants treated with HNH7 and HNH9 as compared to control treatments. Moreover, the expression of defense-linked genes, viz., <i>MPK3</i>, <i>GST</i>, <i>SOD</i>, <i>PAL</i>, <i>PPO</i> and <i>HMGR</i>, was considerably higher in plants treated with endophytic <i>Bacillus</i> strains and inoculated with VD-080 as compared to control.
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