Screening,fermentation And Biocontrol Characteristics Of Bacillus Spp.antagonistic To Xanthomonas Oryzae Pv.oryzicola

Bacterial leaf streak is a quarantine disease in China,which caused by Xanthomonas oryzae pv.oryzicola.Usually,it will reduce the rice yield by 5%-10%,and in severe cases,it will reach more than 50%,and even the yield will be lost.At present,the disease mainly relies on chemical control.However,the long-term,blind,and unreasonable application of chemical pesticides has led to increased pathogen resistance,unsatisfactory control effects,and adverse effects on food safety and the ecological environment.In recent years,screening microorganisms from various ecological environments to prevent and control plant diseases has become a research hotspot.Microbial pesticides have the advantages of safety,residue-free,and environmental friendliness,which is one of the key directions of green pesticide research and development in China.Bacillus spp.has become one of the research objects of microbial pesticides due to its ability to produce stress-resistant spores,strong environmental adaptability,biosynthesis variety of secondary metabolites,and easy storage.Therefore,this study aims to screen out 1 or 2 Bacillus spp.strains with excellent biocontrol effects against bacterial leaf streak,and provide theoretical basis and experimental basis for the development and application of new biological pesticides.To this end,this study collected 36 soil samples from 9 regions including Shijiazhuang,Dangshan,Xinjiang,Nanchang etc.,and 549 bacterial isolates were obtained.Using Xanthomonas oryzae pv.oryzicola Rs105 as the indicating bacterium,20 isolates were found to have a significant antagonistic effect against Rs105（antagonistic circle diameter≥2 cm）through plate confrontation experiments.The 16S r DNA identification results showed that there were 10 strains of Bacillus,among them,the antagonistic effect of ST15is the best,and the diameter of the antagonistic circle is（2.88±0.06）cm.Based on the colony morphology,physiological and biochemical characteristics,and the partial sequences 16S r DNA and gyr B gene,the strain ST15 was identified as Bacillus altitudinis.The antagonistic spectrum of ST15 was determined,and it was found that ST15 had a strong antagonistic activity against X.oryzae（1.50 cm-3.97 cm）.In addition,ST15 also showed certain antagonistic activity against X.campestris pv.campestris and Erwinia amylovora.Field experiments were conducted to verify the biocontrol effect of the bacteria,and it was found that the ST15 biocontrol agent had a（63.51±3.28）%control effect against bacterial leaf streak at a dose of 15 L/hm²,which was similar to the chemical agent3%benziothiazolinone suspension concentrate at a dose of 1.5 L/hm².At the same time,the growth-promoting effect of the bacteria was studied,and the test results showed that the bacterial suspension of ST15 can significantly promote the growth of rice roots（P<0.0001）.In order to further explore the bio-control potential of the bacteria,a preliminary analysis of the antibacterial substances of ST15 was carried out,and it was found that the bacteria is more prone to produce antibacterial substances in the 1/2 NB medium whose nutrition is relatively poor,and its sterile filtrate has good antagonistic activity against X.oryzae pv.oryzicola and X.oryzae pv.oryzae,and its antagonistic activity was 1.54-1.64times that of 1 mg/m L zinc thiazole solution and 1.32-1.52 times that of 10μg/m L benziothiazolinone solution.The antibacterial substance of ST15 has good stability to high temperature,protease,p H（3.0-11.0）and ultraviolet light treatment.In order to further understand the types of antibacterial substances synthesized by ST15,the whole genome of Bacillus altitudinis ST15 was sequenced.The results showed that the total length of the genome was 3.78 Mb,which did not contain plasmid.Using anti SMASH to predict and analyze the genome of ST15,the results show that ST15contains 11 gene clusters related to the synthesis of secondary metabolites.Among them,Cluster 9,10,and 11 have a high degree of similarity（>40%）with the gene clusters in Bacillus spp.which can synthsize polymyxin,tridecaptin and fusaricidin.The supernatant which contains antibacterial substances was prepared by large-scale fermentation,and the crude antibacterial substances was precipitated by n-butanol,ethyl acetate and ammonium sulfate precipitation.The antagonistic ability of the antibacterial substances obtained by n-butanol extraction is significantly higher than that The antibacterial substance obtained by ethyl acetate extraction and ammonium sulfate precipitation（P<0.0001）indicates that the target active substance in the supernatant is mainly present in the n-butanol phase.Preliminary analysis of the antibacterial substances in the n-butanol phase by LC-MS revealed that ST15 can synthesize 8 compounds in fusaricidin and polymyxin A1.In addition,the shake flask fermentation medium of ST15 was optimized through single factor experiment and orthogonal experiment,and the medium formula of ST15 with high cell density was obtained:fructose 10 g/L,yeast powder 6 g/L,soybean powder 4 g/L,Mn SO₄0.05 g/L,Mg SO₄0.5 g/L.The above research shows that Bacillus altitudinis ST15 is an excellent biocontrol strain with the potential for commercial application.