Screening And Breeding Of Soil Transmission Diseases And Genome Analysis

Pepper is one of the most important vegetable crops in the world,which has a wide range of uses,not only for fresh food,but also for seasoning.However,some soil-borne diseases seriously threaten the yield and quality of pepper and other cash crops.The long-term use of chemicals to control soil-borne diseases of hot pepper has not only caused the drug resistance of pathogens,but also caused environmental pollution and other problems.With the international food safety and environmental safety problems becoming more and more serious,the agricultural departments of various countries also pay more and more attention to the use of biological fertilizers and biological pesticides.Therefore,finding safe and effective alternatives that can reduce the use of chemical pesticides has become an international research hotspot.In recent years,plant rhizosphere growth-promoting bacteria(PGPR)and its bioactive antimicrobial compounds can antagonize or promote plant growth,and are generally regarded as an environmentally friendly and biodegradable antimicrobial agent.1.Sclerotium rolfsii,Phytophthora capsici,Fusarium lini and Fusarium oxysporum 4 plant pathogens were used as target bacteria respectively,26 strains of antagonistic bacteria with biocontrol potential were isolated from the rhizosphere soil of pepper in different areas.The biocontrol potential of strain IBFCBF-5 was the strongest.The diameter of inhibition zone on white silk disease of pepper was 18.6 mm,and the inhibition rate was 46.5%.It also had obvious inhibitory effect on other three kinds of pathogenic fungi,and the diameter of bacteria circle was above 10mm.In order to explore the mechanism of biocontrol ability of the strain,a mutant strain TBFCBF-6 was screened by radiation mutagenesis of 60Co-γ rays on the basis of wild bacteria.The bacteria had obvious control effect on Sclerotium rolfsii,Phytophthora capsici,Fusarium lini and Fusarium oxysporum,and the diameter of inhibition zone on Fusarium oxysporum was 22.00 mm,which was 18.28%higher than that of the original strain IBFCBF-5.The identification of the two strains,combined with morphological characteristics,physiological and biochemical characteristics and 16SrDNA sequence analysis,can preliminarily determine that the two strains are Bacillus amyloliquefaciens.2.Then the whole genome of wild strain IBFCBF-6 and mutant strain TBFCBF-6 were sequenced and re-sequenced.Whole-genome sequencing analysis showed that the wild bacteria IBFCBF-5 has a 4.338.658bp circular DNA with a base G+C content of 46.05%;a total of 4546 genes are predicted,including 4341 coding DNA sequences,205 The predicted genes are submitted to the basic database for functional annotation.The Nr database results further verify that the strain IBFCBF-5 belongs to Bacillus amyloliquefaciens.The GO database shows that the metabolic process of the strain IBFCBF-5 accounts for 57%of the "biological process".percent,and antagonistic metabolism-related genes accounted for 0.8%;COG and KEGG database annotation display maximum strain IBFCBF-5 metabolism genes accounted for,indicating that the strain amino acid synthesis and metabolic activity,may be beneficial strains IBFCBF-5 production antagonistic substances.3.Through the re-sequencing analysis of the mutant strain TBFCBF-6,the length of the sequence located on the reference strain IBFCBF-5 reached 93.09%.In the compilation type SNP,the number of CDS region variants reached 36749,of which 28955 were synonymous coding mutations,accounting for 78.97%,and the base substitution method T:A>C:G accounted for the largest proportion;in the Small Indel variant type,the total The number of mutations in the CDS coding region is 460.The largest number of mutations in the CDS coding region is frameshift mutations,accounting for 68.70%of the total.The resequencing genome results show that large structural mutations do not exist,indicating that there is no significant difference between the mutant strains.4.Comparative analysis of differences showed that the number of genes annotated by wild bacteria IBFCBF-5 in all databases was 4305,and that of mutant TBFCBF-6 was 4189;the number of gene annotations in each database also showed that wild bacteria were more than mutant bacteria.COG database differential analysis shows that differences in amino acid,nucleotide,coenzyme,and carbohydrate transport and metabolism processes may be one of the reasons for the changes in bacterial colony morphology.The changes in the number of genes related to the synthesis and secretion of secondary metabolites may also be one of the factors that affect the strain’s antagonistic ability to pepper white sclerosis.Differential analysis of secondary metabolite synthesis gene clusters shows that 14 secondary metabolite gene clusters are predicted.9 gene clusters have antibacterial activity,namely:Plantazolicin,difficidin,Fengycin,Bacillaene,macrolactin H,butirosin A/butirosin B,Bacillibactin,Bacilysin and Surfactin.There are also five gene clusters with unknown functions,indicating that there is a high possibility that there are gene clusters that synthesize new antibacterial substances in the two strains.The large number of Fengycin and Bacillaene genes in the 14 metabolite gene clusters may be an important factor influencing the bacterial strain to antagonize capsicum white Juan disease,Provide a reference for the further study of the biocontrol bacteria Bacillus amyloliquefaciens.