Whole Genome Analysis And Key Antimicrobial Genes Of Bacillus Subtilis H2

Plant disease is one of the important factors affecting agricultural production and agricultural ecological stability.Bacillus subtilis is widely used in biocontrol because of its strong stress resistance and the production of a variety of antimicrobial molecules.In the early stage of the laboratory,a strain of B.subtilis H2 with good biocontrol activity was isolated and identified,which showed broad-spectrum antimicrobial activity against plant pathogenic fungi and bacteria,and had good field control effect on rice blast.With the rapid development of genome sequencing technology,microbial genome mining of antimicrobial substances and their genes has gradually become a research hotspot.Therefore,this study takes B.subtilis H2 as the research object,aiming to mining antimicrobial secondary metabolites and their biosynthetic gene clusters BGCs and various genes involved in plant-bacterial interactions from its genome.The main results are as follows:(1)Broad-spectrum antimicrobial activity:B.subtilis H2 showed broad-spectrum antifungal activity against A.alternata,Phlebiopsis crassa,Pestalotiopsis sydowiana,F.asiaticum,F.oxysporum,C.gloeosporioides,C.destructivumi,C.scovillei,C.musae,C.lindemuthianum,Phytophthora nicotianae,Phomopsis sp,Sclerotinia sclerotiorum,M.oryzae.In addition,strain H2 had good antibacterial activity against the plant pathogenic bacteria A.tumefaciens T-37 and E.carotovora RF.(2)Field efficacy:the control effect of 1×108 CFU/m L H2 wettable powder on rice blast reached 70%,which had good control effect and could significantly promote the growth of rice plants.(3)Whole genome analysis:the full-length 4,160,635 bp of B.subtilis H2 genome,the average GC content is 43.78%,does not contain plasmids,and 4,111 protein coding genes are predicted.Through functional annotation of H2 genome,it was found that its biological function was mainly related to metabolism.The anti SMASH predicted that there were 10 secondary metabolite biosynthetic gene clusters BGCs in the genome of strain H2,which were specifically participated in the synthesis of antimicrobial molecules such as sporulation killing factor,surfactin,bacillaene and fengycin.In addition,many bacterial-plant interaction genes were excavated from the genome of strain H2,including more than 94 colonization-related genes,57 chemotaxis and movement-related genes and 37 biofilm formation-related genes,more than 9 genes involved in siderophore synthesis,more than 7 population response-related genes,more than 25 genes involved in cellulase and protease synthesis,and more than 13 plant growth-promoting genes.(4)Comparative genomic analysis:comparative genomic analysis was conducted between H2 and strains B.subtilis 168~T,B.subtilis W23,B.velezensis FZB42~T,B.amyloliquefaciens DSM 7~T,B.subtilis KR2-7 and B.velezensis ZF2.The results showed that H2 had more coding genes(94.8%)than 168~T,W23,FZB42~T and DSM 7~T.The genome phylogenetic analysis and the collinearity analysis between 168~T,W23,FZB42~T and DSM 7~T comparative genomes showed that H2 had the closest genetic relationship with 168~T.The secondary metabolite palmitic acid,PA and its BGCs with antimicrobial activity were identified by comparative genomic analysis of H2,KR2-7and ZF2,and the accuracy of genome mining was verified by GC-MS quantitative detection of PA content.RT-q PCR and antimicrobial mechanism showed that the expression of genes related to PA was significantly up-regulated,and its antimicrobial activity mechanism targets included cell membrane and metabolic activities.