Physical Mutation Breeding,Antibacterial Mechanism And Application Of Biocontrol Bacillus Velezensis Against Bacterial Angular Spot Of Cucumber

Bacterial angular spot of cucumber is caused by Pseudomonas amygdali pv.Lachrymans.In the production of greenhouse cucumber in China,the occurrence area and severity of thedisease are expanding year by year,endangering the production of cucumber.Biological control has become a hot spot in the prevention and control of the disease.The genetic transformation of natural microbial resources can further improve the effect of biologicalcontrol.In this study,a biocontrol B.velezensis ZF145 screened by our research group in the early stage was used as the starting strain.The biocontrol function of the strain was excavated by UV mutation and ARTP mutation technology.The biocontrol function of the strain wasanalyzed by whole genome sequencing and whole genome re sequencing,and the mutation mechanism was also analyzed.In addition,the research on the processing and application technology of microbial agents has been carried out,which provides a theoretical basis for the industrialized application of mutant strains.The main research contents are as follows:（1）A higher activity B.velezensis A561 was obtained.1249 and 663 mutant strains were obtained by UV and ARTP respectively.12 positive mutant strains and 1 ineffective strainwere obtained by UV mutagenesis;71 positive mutant strains were obtained by ARTPmutation.The mutant strains were screened by paper disk method and in vivo test,and a strain of B.velezensis A561 with significantly improved control effect on cucumber bacterialangular spot was obtained.Compared with the wild strain,the control effect was increased by21.83%;A strain of ineffective B.velezensis U279 was obtained,which had no control effect on bacterial angular spot of cucumber in vitro and in vivo.（2）The genomes of wild strain B.velezensis ZF145 and mutant strains B.velezensis A561 and U279 were determined,and the mechanism of mutation site was preliminarilyclarified.The genome size of B.velezensis ZF145 is 3929800 bp and the GC content is 46.5%.There is a total of 3866 genes,including 3748 coding genes.Through comparative genomicanalysis,B.velezensis ZF145 has 8 gene clusters of secondary metabolite synthesis such as fengycin,which has rich biocontrol potential.resequencing analysis showed that anonsynonymous mutation SNP occurred in the positive mutant B.velezensis A561,located in the gene deg S,which may improve the colonization ability of the strain.2 SNPs were detected in the genome of the reverse mutant strain B.velezensis U279,in which the nonsynonymous mutation occurred in the gene rfb B,which encodes d TDP-glucose dehydratase.After themutation,the production of precursors for the synthesis of secondary metabolites decreased,resulting in the loss of antibacterial activity.（3）The fermentation system of B.velezensis A561 was optimized and the processing technology of A561 suspension was established.it was determined that the components of fermentation medium were starch 25 g/L,corn flour 25 g/L,soybean meal 40 g/L,Na₂HPO₄1g/L and Na H₂PO₄1 g/L.The optimum fermentation p H was 6.0,28℃was selected as the optimum temperature and the fermentation time was 36 h.The formula of 50%B.velezensis A561 suspension was determined by single factor test,which contains 50%fermentationbroth of B.velezensis A561,IVF-2 2%,IVF01 3%,xanthan gum 0.3%,magnesium aluminum silicate 0.5%,preservative sodium benzoate 0.10%and the rest was supplemented with water.Various tests show that the process can be used for the processing of microbial agents,allindexes of 2.43×10~8CFU/m L B.velezensis A561 SC met the standardand.