The Lethal Effect of Bacillus subtilis Z15 Secondary Metabolites on Verticillium dahliae

Bacillus subtilis Z15 (BS-Z15), isolated from cotton rhizosphere soil, inhibits Verticillium dahliae and suppresses cotton Verticillium wilt in pot experiments. We investigated the influence of environmental factors, pH, temperature, ultraviolet light, protease, and incubation time on the stability of BS-Z15 secondary metabolites (SMs), and the mechanism underlying the cytotoxicity of BS-Z15 SMs on V. dahliae. BS-Z15 and its fermentation broth inhibited V. dahliae, and this effect was mediated by its SMs. These were shown to be stable to the influence of the above environmental factors. BS-Z15 SMs decreased the viability of V. dahliae conidia in a time-dependent manner. Scanning electron microscopy showed that BS-Z15 and its SMs resulted in flattened and depressed conidia. BS-Z15 SMs induced morphological abnormalities in the hyphae, which showed rough aberrant structures, reduced conidiophore production, and accelerated aging. Flow cytometry using Hoechst/propidium iodide double staining revealed that BS-Z15 SMs induced necrosis in V. dahliae in a time-dependent manner. Fluorescence microscopy showed that BS-Z15 SMs did not induce apoptotic bodies in the conidia of V. dahliae but caused significant changes in karyotypes, accompanied by nuclear lysis and nucleic-acid diffusion, which may play important roles in necrosis. In addition, 0.3 mg/mL BS-Z15 SMs had no effect on either the mitochondrial membrane potential or the synthesis of proapoptotic proteins, indicating that the SMs did not induce apoptosis in V. dahliae. Their lethal effect on V. dahliae was by inducing necrosis in its conidia and hyphae. BS-Z15 SMs thus have potential as biological pesticides to control Verticillium wilt in cotton.