Etiology,Comparative Genomics Analysis And Construction Of Genetic Manipulation Method Of Dickeya Fangzhongdai

Bleeding canker is a bacterial disease of pear(Pyruspyrifolia L.)which occurs in recent years and the disease has been only reported in China.The disease was firstly observed by Yin and Xu in Xuzhou in 1970s.As the disease become more prevalent and aggressive,it has caused a lot of loss in pear industry in Jiangsu,Anhui,Shanghai and Zhejiang provinces.Preliminary studies of our laboratory had identified the pathogen of bleeding canker and classified it as D.fangzhongdai.However,there is no further and systematic study on the disease now.In this study,we researched on etiology,comparative genomics analysis and construction of genetic manipulation method of D.fangzhongdai pear isoaltes and obtained results as follow:Firstly,our study updated the symptoms and epidemiological regularities of bleeding canker of pear trunk and determined some etiology characteristics of its pathogen.Pear rust water disease mainly damaged the trunks of pear trees with a rapid development.The typical symptom of the disease was rust water were suddenly oozed from lenticels,leaf scars,wound oozes.After being whittled,the bark appeared to be light red with small brown spots or red streaks.Infected bark was soft and water-filling,with strong yeasty smell.At the later stage of the disease,a large amount of sticking liquid oozed from the wounds or natural openings,and the color changed from colorless at the beginning to milky white,reddish brown,and finally to rust color within two to three hours.The pathogenicity and biological characteristics of the pathogen were systematically studied in this study.In previous studies,no obvious symptoms had been reported before typical symptoms,making it difficult to recognize in the field.Therefore,the update of symptoms and epidemiological regularities of bleeding canker of pear trunk and etiology characteristics of D.fangzhongdai pear isolate would be helpful for recognition and control of the disease.Stunted flowers and leaves on the infected branches before bleeding which can be regarded as the early symptoms of the disease.Etiology studies on D.fangzhongdai pear isolate showed that the pathogen could grow at the temperatures of 18 to 44? temperature,the cellulase and pectinase exhibt high activity at 28 and 37??What's more,the pathogen could cause serious diseases in potatoes,cabbage and tomatoes.This study laid a foundation for the futher study on the pathogenic mechanism of D.fangzhongdai pear isolate and control of bleeding canker of pear.Secondly,the genomic characteristics of the pathogen were revealed by comparative genomic analysis and some factors that might influence the pathogenicity were found.In order to study the genomic characteristics of D.fangzhongdai pear isolate,the whole genome of three strains(JS5,LN1 and QZH3)were sequenced,and carried out comparative genomic analysis with part of the genome of Dickeya.The results showed that the genome homology of three strains was more than 99%and showed a high degree of collinearity.Intraspecific and intraspecific comparison showed that there were four specific pathogenic islands in the genomes of D.fangzhongdai pear isolate.Some information related to pathogenicity was found in the functional annotation of the genes on the specific pathogenic islands,indicating that the specific pathogenicity island in the genomes might be related to the unique pathogenicity of the pathogen.Specific plasmids,type IV secretory system(T4SS)and type IV pili(T4Ps)were found in the genomes of D.fangzhongdai pear isolate.These specific chromosome fragments may affect the pathogenicity of the bacteria directly and inderectly.In addition,specific type III effector factor(T3SE),plant cell wall degrading enzymes and transporter proteins in the genomes of D.fangzhongdai pear isolates might related to the specific pathogenicity of the pathogen.Comparative genomics analysis of D.fangzhongdai pear isolate provided a direction for the study of pathogenic mechanism of the pathogen.Finally,the genetic manipulation method of D.fangzhongdai pear isolate was constructed.To determine the functions of unique genes in the genome of D.fangzhongdai pear isolate,our study constructed the genetic manipulation method of the pathogen,and the function of T4Ps and T3SS clusters of D.fangzhongdai was determined.In this study,hrpN in T3SS cluster was used as the target gene,and two suicide vectors were used to construct the knockout mutant of this gene by two transformation methods.pEX18GM and biparental mating method were found to be efficient to construct delete mutants for D.fangzhongdai pear isolate.Complementary and overexpressed mutants of D.fangzhongdai pear isolate were constructed by complementary vector pBBRIMCS-5.Pathogen characteristics of delete mutants of T4Ps and T3SS clusters(?T4Ps and ?T3SS)exhibited decreased.It was observed that the T4Ps gene cluster in the D.fangzhongdai pear isolate positively regulated the transcripton of hrpG gene in the T3SS gene cluster,suggesting that T4Ps could affect the pathogenicity by upregulate the transcription of hrpG.Construction of genetic manipulation method of D.fangzhongdai pear isolate laid a foundation for gene function researches of the pathogen.With the accumulation and spread ofD.fangzhongdai,diseases caused by the pathogen might breakout in the near future.So,the study on the pathogenic mechanism and control of D.fangzhongdai is extremely urgent.This work systematically studied characteristics of D.fangzhongdai by etiology,comparative genomics analysis and construction of genetic manipulation method.This work provided a theoretical basis for the early field recognition and control of bleeding canker of pear trunk and laied a foundation for the study on gene function and pathogenic mechanism of the pathogen.