Isolation And Whole Genome Sequencing Of Bacillus Velezensis Zk1 And Metabolomics Study Of The Strain Infecting Postharvest Olecranon Peach

Infection by pathogenic bacteria is one of the important causes of rapid spoilage of olecranon peach after harvesting.In this study,a bacterial pathogen derived from olecranon peach was isolated for the first time and the whole genome was sequenced.The genes related to the pathogenic olecranon peach in the whole genome sequencing results of this strain were analyzed.And the enzymology characteristics of enzyme related to rotting olecranon peach of the strain were studied.Finally,the metabolites of the pathogenic olecranon peach during the 4-day storage period were detected and analyzed using non-targeted metabolomics method based on LC-MS technology;and differential metabolites,major metabolic pathways and related genes of the strain were integrated to discuss the pathogenic mechanism of the strain on olecranon peach.The main findings were as follows:(1)The 23 pathogenic fungi and bacteria isolated were tested for pathogenic ability,and11 dominant pathogens were selected.Under different culture conditions,the pathogenic ability of these dominant pathogens showed individual differences.Among them,the bacterial GH-1strain had the strongest pathogenic ability,and it had many potential hosts.Molecular biology identification found that the strain was Bacillus velezensis strain.The ultrastructural observation results showed that after infection with this strain,the structure of the olecranon peach cells was destroyed.(2)The bacterial GH-1strain was named Bacillus velezensis zk1 strain,and the whole genome was sequenced using three-generation sequencing technology.932602 reads were obtained with a data volume of 5.51G.After assembly,a contig was obtained with a length of 4132269 bp and a GC content of 46.27%.A total of 3997 ORFs(open reading frames)were predicted by genes,with a total length of 3661683 bp,an average length of 916.11 bp,and a GC content of 47.11%.In the KEGG database,2162 protein-coding genes were obtained,of which 232 involved carbohydrate metabolism.Annotated genes in the PHI(pathogen host interaction)database indicated that the strain can cause disease in apple,pear and tomato.In the CAZy(Carbohydrate-Active en ZYmes)database,there were 42 genes of glycoside hydrolases and were related to 20 families;there were 3 genes of polysaccharide lyases,and were related to 2 families;these family genes may be related to amylase,cellulase,xylanase and pectinase.(3)Infection by Bacillus velezensis zk1 strain could lead to a reduction in the content of starch,cellulose,hemicellulose and pectin in the olecranon peach.The enzymology characteristics of amylase,cellulase,xylanase and pectinase secreted by this strain were studied.The results showed that the optimal reaction temperature of amylase was 60?,the optimal reaction p H was 7,and Mn²⁺could inhibit amylase activity;the optimal temperature of cellulase was 60?,the optimal reaction p H was 6,Mn²⁺could also inhibit it;the optimal reaction temperature of xylanase was 40?,the optimal reaction p H was 8,and all kinds of metal ions in this study were activators;the optimal reaction temperature of pectinase was50?,the optimal reaction p H was 6,and Mn²⁺can promote the activity of pectinase.(4)A total of 14203 peaks were detected in the pulp of the olecranon peach infected by the Bacillus velezensis zk1 strain,and 3452 peaks were identified.Combining the FC(Fold Chang)and t test,with FC>2,P<0.05,PLS-DA VIP>1 as the standard,155 high credibility(credibility=P)differential metabolites were identified,of which 68 differential metabolites were significant changed,mainly including acids,sugars,amino acids,amines and purines.Among the acidic substances,there were 8 substances related to the metabolism of the strain,including maleic acid,fumarate,D-malic acid,L-pipecolate,etc.;Kojibiose?2-?-mannobiose and sucrose were found in sugars;8 kinds of amino acids and amines showed significantly changes,some amino acids were involved in cell metabolism,and N,N-Dimethyl-1,4-phenylenediamine was detected on the 4th day.Adenine and hypoxanthine in purines are detected.Metabolites with significantly altered levels were mapped into the KEGG database,and the results of the whole genome annotation of Bacillus velezensis zk1 strain were integrated.It was found that after infecting the olecranon peach,the strain will consume extrinsic sucrose and glucose through the starch and sucrose metabolism pathway.It can also participate in many metabolic activities such as purine metabolism,pyruvate metabolism,butanoate metabolism,and phenylalanine metabolism.