| Apple ring rot caused by Botryosphaeria dothidea Ces&De Not.is one of the important diseases affecting the safe production of apples in China.It mainly affected the stems and fruits of apple trees and caused great yield loss.Because the pathogenic mechanism of the B.dothidea is still unclear,which brings some difficulties in controlling the disease.Therefore,studies on the interaction mechanism between B.dthidea and their host will provide a theoretical basis for further disease control.In this study,an isolate of PGLW-I-5 was used as the research object,and the GPASR(Genealogical Concordance Phylogenetic Species Recognition)method was used to identify the species of pathogen.The pathogenicity related genes were analyzed and filtered by transcriptome technology during B.dothidea infected apple twigs.The functions of pectinase genes Bdpll and Bdpgl in the pathogenesis were verified by gene knockout.The main results obtained from this study are described as follows:1.The combined gene sequence data consisting of the rDNA-ITS and EF-la gene fragments of PGLW-I-5 and the four gene fragments rDNA-ITS,EF-la,His and Hsp are The phylogenetic tree of MP(Maximum Parsimony)was constructed,the results showed that PGLW-I-5 isolate was Botryosphaeria dothidea Ces&De Not.2.The differentially expressed genes of apple ring rot fungi in the stage of infecting apple branches and PDA culturing phase were screened by transcriptome technology.The results of data showed that 1381 genes were up-regulated and 1823 genes were down-regulated in the phase of B.dothidea infecting apple branches.GO and KEGG enrichment analysis revealed that genes associated with hydrolase and pectinase activities,as well as differential expression of genes involved in transmembrane transport,protein translation,and carbohydrate metabolism,were significantly enriched.3.Two pectinase genes Bdpll and Bdpgl were selected from the differentially expressed genes.The method of PEG-mediated protoplast transformation was used to obtain the gene Bdpll and Bdpgl deletion mutants △Bdpll-3 and △Bdpgl-2.4.The colony morphology of the mutants △Bdpll-3 and △Bdpgl-2 on PDA was no significantly difference with the wild-type strain PGLW-I-5,while the colony diameter on the pectin culture was significantly smaller than the wild-type strain,and mutant △Bdpll-3 and △Bdpgl-2 extracellular enzyme activity decreased significantly.The pathogenicity of the two mutants on the fruit was significantly reduced,and the pathogenicity of △Bdpl1-3 on the excised one-year-old twigs in vitro was no significantly difference with the wild type.The expression of the gene in the PL family in the mutant ABdpll-3 was detected by qRT-PCR,and the result showed that three genes in the PL family were significantly up-regulated in the mutant ABdpll-3,which indicated the expression of other genes in the family was partially compensated for the function of the gene Bdpl1. |
