Pathogenicity And Comparative Genome Analysis Of The Pathogens Of Cucumber Bacterial Flow Gum Disease Based On Whole Genome Sequencing

Cucumber flow gum(CFG)disease is a serious bacterial disease on cucumber.In recent three years,CFG was outbreak in northern China,which caused serious economic losses to local cucumber production.Previous studies showed that the pathogen causing CFG are Pseudomonas amygdali pv.lachrymans(pal)and Pectobacterium carotovorum subsp.brasiliense(Pcb).Currently,the studies about Pal and Pcb mainly focus on the bacterial genetic resistance,disease resistance breeding,gene structure and rapid detection of bacteria.However little research has been performed on the pathogenicity,infection and host genetic adaptation of pathogens at the genomic level.In addition,there is still lack of experimental data to support the selection of suitable reference genes during infection of cucumber with Pcb,This will not be conducive to the transcriptome analysis of plant-pathogen interaction.In this study,the strong virulent strains Pal NM002 and Pcb SX309 were screened,the host range of NM002 and SX309 were determined.Then,the virulence genes,infection factors and host genetic adaptation determinants of NM002 and SX309 were systematically revealed by using the third generation sequencing technology and comparative genome analysis.Meanwhile,the optimum reference gene for bacteria-plant during infection of cucumber with Pcb were also determined.The main results are as follows:(1)The pathogenicity of 14 Pal and 14 Pcb strains isolated from different regions was determined by spray and needling inoculation.The results showed that Psl-2 and HG1501290309 were strong virulent strains,which named Pal NM002 and Pcb SX309,respectively.Pathogenic differentiation studies showed that NM002 and SX309 could infect 12 vegetables such as cucumber,pumpkin and so on.These results indicated that Pal and Pcb had a wide host range.(2)To further delineate traits critical to virulence,invasion and survival in the phyllosphere,we reported the first complete genome of P.amygdali pv.lachrymans NM002.Analysis of the whole genome in comparison with three closely-related representative pathovars of P.syringae identified the conservation of virulence genes,including flagella and chemotaxis,quorum-sensing systems,two-component systems,and lipopolysaccharide and antiphagocytosis.It also revealed differences of invasion determinants,such as type III effectors,phytotoxin(coronatine,syringomycin and phaseolotoxin)and cell wall-degrading enzyme,which may contribute to infectivity.The aim of this study was to derive genomic information that would reveal the probable molecular mechanisms underlying the virulence,infectivity and provide a better understanding of the pathogenesis of the P.syringae pathovars.(3)As its name indicated,plant cell wall degrading enzymes is usually regarded as the critical factor in Pectobacterium infection.However,other virulence factors and its genetic adaptation mechanisms to the host are still need to be studied in detail.In this study,the complete genome of P.carotovorum subsp.brasiliense strain SX309 was compared with other pathogenic bacteria belonging to the Pectobacterium genus,isolated from distinct host plants.Genome comparison reveals that most virulence genes are highly conserved in the Pectobacterium strains.Especially,the key virulence determinants involved in the biosynthesis of extracellular enzymes and others including the type II and III secretion systems,quorum sensing system,flagellar and chemotactic genes.In addition,the type VI secretion system and two subtype CRISPR-Cas immune systems may play an essential role in the process of pathogen infecting and adapting to new host species.The extensive comparative genomics analysis revealed virulence genes are highly conservative in the Pectobacterium strains.However,several variable regions of type VI secretion system and two subtype CRISPR-Cas immune systems possibly contribute to the host genetic adaptation.(4)In order to better reveal the molecular mechanisms of cucumber-Pcb interactions,qRT-PCR is a powerful and sensitive technique widely used to determine the fold change of the expression of the genes of interest.In this study,14 reference genes of cucumber and ten reference genes of Pcb were selected,and their expression stabilities were assessed during cucumber infection using four algorithms(deltaCt method,BestKeeper,NormFinder and geNorm).The results revealed that CsARF(ADP-ribosylation factor 1)was the optimum reference gene for transcript normalization for cucumber,and rho(transcription termination factor Rho)was recommended as the most suitable reference gene for Pcb during the process of pathogen-host interaction.Furthermore,to confirm the reliability of the reference genes,we evaluated the relative expression of CsCAT,CsPR5,CsRPMl,CsBIK1 and CsWIPK,which encode proteins that are involved in plant resistance,as well as luxS,carI,hrcC,peh and Ion,which may be important virulence factors involved in the pathogenesis of P.carotovorum subsp.brasiliense.These study not only revealed the genome information of Pal and Pcb,but also explored the optimal reference genes specific for in bacteria-plant pathosystems,which could provide scientific foundation for future studies of the pathogenisity of pathogen and molecular mechanism of the bacteria-plant interaction.