Culture Optimization of Endophytic and Rhizosphere Bacteria and Exploration of Antifungal Substances

Verticillium wilt is a soil borne disease caused by Verticillium dahliae, which affects the growth, development, yield, and quality of cotton. Using rhizosphere and endophytic bacteria are considered promising methods for biological control of Verticillium dahliae. In this study, the culture conditions of the endophytic and rhizosphere bacteria (Bacillus vanillea SMT-24, Bacillus subtilis SHT-15, Bacillus velezensis BHZ-29 and Bacillus atrophaeus SHZ-24) screened from cotton rhizosphere soil and cotton endosphere were optimized to promote the production of antifungal substances. The obtained culture filtrates of four strains showed the high tolerance to ultraviolet light (2–8 h), pH (1.0–9.0) and temperature (80–100°C). To explore the antifungal substances, the protein crude components were obtained by stepwise salt precipitation using saturated ammonium sulfate. Protein precipitates with saturation gradient of 0–40% were collected from the culture filtrates of SHT-15 and SHZ-24 strains, showing a positive correlation between protein concentration and the size of the inhibition zone. The antifungal components of SHT-15 strain were further proved to lipopeptides (47.00%), lipids (22.35%), and proteins (30.65%), and those of SHZ-24 were composed of lipids (74.84%) and proteins (25.00%). Using the liquid chromatograph-mass spectrometer (LC-MS) method, it was speculated that BHZ-29 and SMT-24 strains exert bacteriostatic effects through pectin lyase and a novel unknown protein. The bacteriostatic peptides of SHT-15 are mainly C12-C15 surfactants; the SHZ-24 bacteriostatic lipids are mainly activated by hydroxyl fatty acids. This study was designed to isolate more potential antagonists’ substances from Bacillus for the control of Verticillium wilt in cotton and to gain a better understanding of the overall modes of biocontrol of Bacillus species.