Genomic Analysis Of A Bacillus JCL16 Antagonizing Nocardia And Exploration Of Its Antimicrobial Substances

With the expansion of Micropterus salmonides aquaculture in China,diseases occur from time to time,resulting in heavy economic losses for fishermen.Bacillus has simple nutritional requirements,strong stress resistance,and can secrete a variety of antimicrobial substances.It has good control effect on fish diseases.Therefore,as a good probiotic,Bacillus should be widely used in fishery culture.In this paper,we selected the naturally diseased fish from the aquaculture Institute of Huai’an City.Through the observation of the diseased fish focus,morpHological observation of suspected pathogenic bacteria,pHysiological and biochemical experiments,macromolecule metabolism experiments and drug sensitivity tests,we determined the strain.The DNA was extracted and amplified by PCR.The 16 Sr DNA gene was sequenced by Shanghai Biotechnology.It was finally identified as Nocardia seriolae.86 trains of Bacillus were isolated and screened from soil samples collected from Huaian Aquaculture Institute.The strain JCL16 with good antagonistic effect against Nocardia seriolae was selected by Oxford cup method with Nocardia seriolae as indicator bacteria and Bacillus as test bacteria.In order to follow-up practical application,the shaking flask fermentation conditions were optimized by single factor experiments.The optimum conditions were as follows: liquid loading16%,inoculation 8%,pH 7.0,incubation temperature 36 C,shaking speed 250 rpm.The diameter of inhibition zone of fermentation broth was increased from 15.5 mm to 18.5 mm after optimization,it will lay a foundation for in vivo control experiments.The results of whole genome sequencing were analyzed from three aspects:genome profile,genome components and gene function annotation,focusing on the analysis of its metabolite genes and mapping the secondary metabolic gene circle.It will lay a foundation for further excavation of more coding genes,secreted proteins and secondary metabolites with special functions,and construction of genetically engineered strains.The total length of his genome is 4101682 bp,the GC% content is 44.52,and there are 4166 genes.The average length of his genome is 867.79.The total length of his genome is 3615213 bp,accounting for 88.14% of the total length of its genome.The secondary metabolites of JCL16 were predicted and 11 gene clusters were obtained,involving polyketide synthase,non-ribosomal peptide synthase,terpenes and other secondary metabolites.Surfactin and fegycin were analyzed by high performance liquid chromatography and mass spectrometry.In vivo control experiment of Nocardia seriolae against Micropterus salmonides was carried out with Bacillus subtilis JCL16.The infection group without Bacillus subtilis JCL16 died on the 16 th day,and the mortality rate of the experimental group with Bacillus subtilis JCL16 was only 40%.The incidence rate of Bacillus subtilis JCL16 in the experimental group was much slower than that in the infected group,which indicated that Bacillus subtilis JCL16 had a good antagonistic effect on Nocardiosis of Micropterus salmonides and had broad application prospects.