| In order to make better use of biological control methods to control plant diseases,we isolated and screened an endophytic bacterium from walnut,which has a good inhibitory effect on a variety of pathogens.It was identified and named Bacillus amyloliquefaciens Htq6;At the same time,it was found that the strain can be successfully colonized in tomato plants,and a bio-wettable powder was developed accordingly.It has a good control effect on Botrytis cinerea in the field and has great potential in the field of biological control.In order to further study the biological control mechanism of the bacteria,we sequenced the entire gene of the biological control Bacillus Htq6,and performed a comparative genomics analysis with various Bacillus species.At the same time,the transcriptome analysis of the response mechanism of Botrytis cinerea after treatment with the fermentation liquid of bacillus Htq6,The specific research results are as follows:1.Sequencing the whole genome of Bacillus Htq6,the genome size is 3888123 bp,the GC content is46.54%,the number of non-coding t RNA is 85,the total length is 6580 bp.Encode 3841 genes and 146 carbohydrate active enzymes(CAZyme).In addition,we predicted that there are 14 antibacterial gene clusters,which can produce known Surfactin,Bacilysin,Bacillibactin,Difficidin,Fengycin,Bacillaene,Macroactin,Butirosin and nine secondary metabolites.The resulting gene sequence has been uploaded to the Gene Bank database with accession number CP050462.2.333 strains of Bacillus downloaded from NCBI based on whole-gene conserved protein and average nucleotide homology(ANI)method were used for Phylogenetic analysis,and 59 strains of Bacillus amyloliquefaciens including the Htq6 were found Because of the evolution of the taxonomic status of Bacillus amyloliquefaciens and Bacillus velezensis,the classification and name is still wrong,and it should be corrected to Bacillus velezensis;At the same time,the identification and differentiation of B.velezensis,B.amyloliquefaciens and B.siamesis with 16 Sr RNA sequence,gyr A,gyr B and rpo B gene sequences.The results show that based on the 16 Sr RNA sequence and rpo B gene sequence,the three Bacillus species cannot be distinguished in detail.It is recommended to use gyr A or gyr B gene sequences as the basis for identification of the three Bacillus species.3.By comparing and analyzing the distribution of different secondary metabolite gene clusters in 36 Bacillus strains,the secondary metabolite gene clusters of all strains are divided into 35 categories,which 6gene cluster categories are shared by all Bacillus amyloliquefaciens and Bacillus velezensis.;Bacillus velezensis with the most antibacterial secondary metabolic gene clusters,and difficidin is an antimicrobial active substance produced by the unique gene cluster of B.velezensis;macrolactin is mainly produced by by B.velezensis;Lanthipeptide is an antibacterial substance produced by the unique gene cluster of B.amyloliquefaciens.These substances play an important role in the application of B.velezensis and B.amyloliquefaciens as biocontrol bacteria.4.The RNA-Seq technology was used to determine the expression profile of the transcriptome after the treatment of tomato broth fungus(Botrytis cinerea)with Htq6 fermentation broth.Through screening of transcriptome data,1416 differentially expressed genes were obtained,involving 744 up-regulations genes and 672 down-regulations genes;COG annotation and KEGG pathway enrichment analysis were performed on the differential genes,and it was found that the differential genes were mainly concentrated in the metabolism of carbohydrates and amino acids Wait for the process.The results analyzed and proved that the tomato ash carbon source and energy utilization,cell wall,cell membrane and antioxidant-related genes were affected after fermentation broth treatment,and further understanding of the possible bacteriostatic mechanism of biocontrol bacteria and the stress response mechanism of Botrytis cinerea was further understood.In this study,the high-quality biocontrol bacteria Htq6 genome was obtained through second-generation high-throughput sequencing technology,and it was predicted that it may produce 9secondary metabolites,and 60 strains that should be renamed as B.velezensis were identified and transcribed.The analysis clarified that the biocontrol bacteria Htq6 can affect the carbon source utilization and energy production,cell wall synthesis and degradation,biofilm synthesis,and antioxidant-related genes of Botrytis cinerea. |
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