| Pectobacterium carotovorum subsp.carotovorum(Pcc)is a phytopathogen causing soft rot disease world widely.Pcc produces a series of virulence factors such as pectinase,cellulase,protease,for degrading the host plant cell wall,and then obtaining noutrition to growth and reproduction.Some strains of Pcc produe antibiotic such as carbapenem for environmental competition.The previous studies showed the diversity of the strains of Pectobacterium isolated from different hosts originating different sites in the secretion of virulence factors.In this work,15 strains of Pectobacterium isolated from the plants sampled in Jiangsu,Hubei and Yunnan were collected,and the assays of pathogenicity,activities of pectinase,cellulase,protease and carbapenem were performed,and the activity of carbapenem were measured only with the strains from Beijing,the United States,Brazil,Britain.In addition,an alignment of the gene clusters of carbapenem synthesis and its regulation in the strains were carried out basing on the results of the whole-genome sequencing.Furthermore,the strains of the gene deletion,complementary and overexpression of carR(the gene reulates the synthesis of carbapenem)were constructed for analysis of each strain in the some biological phenotypes to explore the carbapenem antibiotic synthesis regulation mechanism.15 strains isolated from different plants collected from Jiangsu,Hubei,Yunnan and other places in China,such as Bush lily(junl,jun2,jun4,jun5,jun6,jun7.jun8,jun9,jun10),Holy Thistle(SFJ),cabbage(Eg5-1),Chinese cabbage(BC),Seleng wormwood(LH),konjac(MY),Pinellia(BX)were identified as Pectoboeterium carotovorum subsp.carotovorum,by fragment-sequenceing of 16S rRNA and four housekeeping genes(recA,mdh,rpoH,fliA),sequence alignment and phylogenetic tree,and using Biolog technology.The results from assays of pathogenicity,activities of pectinase and cellulase enzyme showed that all the strains identified as Pcc above exhibited non differences in both the capabilities of the virulence on the leaves of Chinese cabbage and the activities of pectinase and cellulase compared to that of PccS1,isolated from soft rot tissue of the local color calla lily,the activities of protease were higher in the strains of Bush lily(junl～jun10),Chinese cabbage(BC),konjac(MY),Pinellia(BX)than that of PccS1 and the strain of cabbage(Eg5-1)showed no ability to the produce of protease.Meanwhile,the capabilities of the production of carbapenem in the strains of junl～jun10 and LH were higher than that of PccS1,and in strains of SFJ,Eg5-1,BC,MY,BX,no production of carbapenem.Among the other 15 strains,6 strains supported by Beijing Academy of Agriculrue and Forest(Ecc71,S7,BC1,QC47,QC142,QC166)and 9 strains supported by the James Hutton Institute of England(252,253,254,255,256,257,258,99,109),only 258 had no significant difference in the production of carbapenem,and the other 14 strains produced no carbapenem.To explore carbapenem antibiotic synthesis regulation mechanism,the abilities of the strains derived from PccS1 to produce carbapenem revealed that the synthesis of carbapenem in this bacterial genera was also regulated by carR.It was interesting,the strains producing no carbapenem naturally turned to produce carbapenem even in higher level than that of the wild type of PccS1 except the strain of BX,overexpression of carR gene,and the antibiotic showed effective inhibition on the growth of some bacterial pathogens,such as DC3000,Erwinia amylovory,Erwinia pyrifoliae,Ralstonia solanacearum,Xanthomonas oryzae pv.oryzicola.The whole-genome sequences showed that the strains producing carbapenem(PccS1,junl,LH)contained carR-H gene,all the genes of the synthesis and regulation gene cluster,and that of Eg5-1,BC only contained carF-H genes,In the wholegenome of SFJ,MY,BX presented non of the carbapenem gene cluster.Meanwhile,it was found that the nucleotide sequence of the carbapenem gene cluster of PccS1,junl and LH were at least 91%of identity to the homology.The sequences of nucleotide of carF-H in the strains of PccS1,junl,LH,Eg5-1 and BC were at least 87%in homology.In conlusion,among the 15 strains of Pcc isolated from different hosts in different geographical positions,there are no differences in the virulence,but diversities in the production of carbapenem,and results in three groups:synthesis intrinsical,synthesis recombination and non-synthesis recombination. |
