Screen Out Highly Efficient Mutant Strains Of Bacillus Amyloliquefaciens Ht-q6 And Study Of Its Biological Control Effect

Endophytic bacteria is a potential group of biocontrol materials, with the study on the group broadened and deepened, it has become the focus of research on biological control at home and abroad. Our team has isolated and identified a strain of endophytic bacteria from walnut, named Bacillus amyloliquefaciens Ht-q6. It has a broad spectrum antimicrobial activity. In this study, we used the Bacillus amyloliquefaciens Ht-q6 as the original strain, mutated by UV and nitrous acid, got a stronger antibacterial activity of mutant strains. Then, we studied the colonization, antimicrobial effect and metabolites of the mutant strain, which lay a theoretical foundation for development and application of it. The methods and conclusions are as follows:1. Used Ht-q6 as the starting strain for mutation by ultraviolet, nitrous acid and compound mutation of nitrous acid and UV. Calculated lethality rate and positive mutation rate of each treatment. Finally determined the best dose of UV mutation was 40 s. Furthermore, colligated the antifungal activity of screening and rescreening, the efficient mutant strain GHt-q6 was obtained. The antibacterial activity of GHt-q6 was 41.67% higher than Ht-q6, and the growth curve of them was basically consistent.2. By induced GHt-q6 we obtained the strain GHt-q6-Rif, which can against 300?L·mL-1 of rifampicin. It also can stably inherit and has antibacterial activity. The bacterial suspension of strain GHt-q6-Rif was used to spray the walnut and tomato seedlings respectively, and soak the walnut seedlings. Through monitoring the strain GHt-q6-Rif, the colonization dynamics of this strain in walnut and tomato were definite. In addition, by using scanning electron microscopy, the colonizing dynamics, location and distribution in the interior of the walnut and tomato become visual displayed. In short, the strain could be successfully colonized and transported in plants, and the colonization stability takes about 21 days.3. The antifungal proteins were isolated by ammonium sulfate precipitation and the crude lipopeptides were separated by acid precipitation. Results of bacteriostatic test about antifungal proteins and crude lipopeptides showed that the proteins and lipopeptides all had extensive spectrum of antimicrobial activity and disease resistance. The antimicrobial proteins were isolated and identified by tandem mass spectrometry (LC-MS-MS), simultaneous the lipopeptides were identified by high performance liquid chromatography tandem mass spectrometry (HPLC-ESI-MS). The result of LC-MS-MS showed that 5 kinds of enzymes isolated from the antifungal proteins were credible:bacillopeptidase F?subtilisin?YnfF? AmyE alpha-amylase and beta-1,3-1,4-glucanase precursor protein. On the other hand, the result of HPLC-ESI-MS showed that the crude lipopeptides contained 3 kinds of homologous compounds:iturins (Bacillomycin D?Bacillomycin L and Iturin C)?fengycin and surfactin.