Identification And Diversity Of Peach Brown Rot Pathogens And Cloning And Expression Of PG Gene

Peach brown rot is one of the most important diseases in peach production and can cause serious yield losses.In order to clarify the species,diversity and main pathogenic factors of peach brown rot pathogens from different peach-growing areas in China,I identified and analyzed the genetic diversity of peach brown rot pathogens from different peach-growing areas in China,and determined the pathogenicity and cell wall degrading enzyme activities of the isolates from different peach-growing areas.At the same time,the main pathogenic factor polygalacturonase(PG)gene was cloned and expressed.Therefore,the pathogenic mechanism and differences of peach brown rot pathogen isolates from different peach-growing areas were explored,which will provide a theoretical basis for the control of peach brown rot in different producing areas.Twenty-three peach brown rot pathogen isolates were obtained from the diseased fruits with brown rot symptoms from from 14 different peach-growing areas of 12 provinces,municipalities and autonomous regions in China The PDA plate morphological characteristics of the isolates were slightly different from each other,but the isolate hfyn from Yunnan Province was significantly different from other isolates.Based on its morphology,rDNA-ITS sequence and phylogenetic analysis,the isolate hfyn was identified as Monilia yunnanensis,and the other 22 isolates were identified as Monilia fructicola.Multiple pairs of specific primer PCR and multiplex PCR amplification also confirmed the above identification results.The genetic diversity analysis of SNP single nucleotide polymorphism,RAPD and ISSR molecular markers showed that the Yunnan isolate hfyn had the farthest relationship with other isolates,and the isolate hfzl-3 from Zhejiang Province also was clustered into farther branch i the phylogenetic tree.Further cluster analysis showed that most of the isolates with close geographical distance or in the same region were usually clustered into the same branch in the phylogenetic tree,but there also had a phenomenon which the isolates in the same region were not clustered into the same branch.Inoculation tests showed that the pathogenicities on peach fruits of 23 isolates from different peach-growing areas were diiferent.The isolates from Lishui,Zhejiang Province,Shijiazhuang,Hebei Province,Qingdao,Shandong Province,and Lishui,Jiangsu Province had more virulent,and the virulences of isolates from Tai'an,Shandong Province,Kunming,Yunnan Province,and Zhuangxing,Shanghai Municipality were relatively weak.The pathogenicities and sporulation abilities of these isolates on peach fruits were significantly stronger than those of apple,but they did not infect non-host tomato.The activity assays of cell wall degrading enzymes(CWDE)showed that each isolate could produce four different cell wall degrading enzymes,in which polygalacturonase(PG)and polymethylgalacturonase(PMG)had the highest enzymatic activities,with the highest enzymatic activities of 357.601 U/mL and 342.066 U/mL,respectively.However,the enzymatic activities of cellulase(Cx)and ?-glucosidase(BG)were generally lower,and the highest enzymatic activities of the isolates were only 21.869 U/mL and 23.378 U/mL,respectively.In addition,the extracted crude enzyme solution had a certain pathogenic effect on peach leaves,which made the leaves soft,chlorotic and yellow,and lost toughness.By PG1 gene sequence alignments,there were 1-50 bp nucleotide differences in each peach brown rot pathogen isolate.In addition to Yunnan isolate hfyn,the PG1 gene sequence of the most pathogenic isolate hffj-1 was the most different from other isolates,and was also clustered into farther branch in the phylogenetic tree.Subsequent experiments were performed on the representative isolate hfhs-3,which had medium pathogenicity.Bioinformatics analysis indicated that the PG1 gene was 1095 bp in length and the encoded protein contained 365 amino acid residues,with a molecular weight of 36.77 kDa and a pI value of 8.55.The N-terminus of the protein had a signal peptide sequence at a position of 1 to 20 AA.After signal peptide(1-20 AA)was truncated,the recombinant plasmid was induced by IPTG.SDS-PAGE electrophoresis showed a band at 36.7 KDa,which was consistent with the predicted results,indicating that the PG1 gene could be heterologously expressed in Escherichia coli.The band was verified by Western blotting hybridization experiments.The activity of the prokaryotic expressed protein was determined,and no activity was detected in the pure protein after renaturation of the inclusion body.