Genome Comparison Between Groups And Unique Gene Function Analysis,Development Of Groups Distingusih Assay And PCR-based Detection Assay Of Acidovorax Citrulli

Bacterial fruit blotch(BFB)caused by the seed-boren pathogen Acidovorax citrulli is a devastating disease of melon,watermelon and other cucurbits.Contaminated seeds are the most important source of primary inoculum for A.citrulli and infested seed can readily initiate BFB epidemics under transplant house conditions,while current specific primers for detecting of A.citrulli lack specifity and sensitivity.A.citrulli strains can be divided into at least two genetically distinct groups with one group(?)being associated with a range of cucurbit hosts and the other group(?)being associated with watermelon.Despite this,the pathogenicity assays used for A.citrulli,including infiltration of seedling cotyledons and mature fruit rind tissues,and spray-inoculation of seedlings,lack the sensitivity to consistently distinguish strains of the two groups.In this study,we elucidated the differences betwween group ? strain AAC00-1 and group ? strain M6 of A.citrulli on genome level by conducting comparative genome analysis,and explored the function of a unique gene in M6.And also,we developed a fruit pathogenicity assay which can distingusih group ? and group? strains and a conventional PCR-based assay for detecting A.citrulli.The key achievements in this study are as follows:In this study we assembled and annotated the genome sequences of A.citrulli group ?strain M6 and yield the draft genome.The M6 draft genome sequences consists of 157 contigs,4321 ORFs and 53 tRNAs with an overall approximate size of 4.85 Mb.The G+C content of the draft genome of M6 was 68.8%.Comparative genome analysis revealed that AAC00-1 was well linear with M6,and there are nine insertion and deletion regions with eight regions in AAC00-1 and one in M6 were found and located.Further investigation of the nine insertion and deletion regions showed that AAC00-1 has 544 unique genes which were absent in M6,and M6 has 113 unique genes which were absent in AAC00-1.There were eight and two bacteriophage regions in AAC00-1 and M6 respectively based on the analysis,which indicated that some of the insertion and deletion regions may recombined to the genome by horizontal gene transfer Moreover,the G+C content analysis also indicated that some of the insertion and deletion regions were introduced into the genome by horizontal gene transfer.Meanwhile,we explored the function of a unique gene AcopE in M6 which encoded a putative T3SS effector.We compared the nucleotide sequence of AcopE to other genome sequences of group ? and ? A.citrulli strains and confirmed that gene AcopE was unique to all group ? strains tested in this study.We investigated the function of gene AcopE by generating deletion mutant with group ? strain Xj112 as background.Pathogenicity experiment on melon and watermelon seedlings showed that the deletion mutant of AcopE was more virulent on both melon and watermelon cotyledons than the wildtype.Our results indicated that the unique gene AcopE contributed to virulence but not host range of A.citrulli.The pathogenicity assays used for A.citrulli,including infiltration of seedling cotyledons and mature fruit rind tissues,and spray-inoculation of seedlings,lack the sensitivity to consistently distinguish strains of the two groups.In this study,we developed an immature,detached melon cv.Joaquin Gold fruit assay that clearly and reliably distinguished strains of the two A.citrulli groups.Based on this assay,four group ? strains induced typical water-soaked lesions in melon fruit rind tissue 7 to 10 days after pin-prick inoculation.In contrast,four group ? strains did not induce water-soaked lesions on detached melon fruits during the same period.We also confirmed that T3SS is critical for A.citrulli to infect immature melon fruits.We concluded that group ? A.citrulli strains have a specific capacity to infect immature melon cv.Joaquin Gold fruit while group ? strains do not.This study provided further evidence for host preference among A.citrulli groups.A new target gene for sepecific dectecting Acidovorax citrulli was found by local comparing the genome sequences of A.citrulli AAC00-1 and close related strain A.avenae ATCC19860.A conventional PCR was developed for detecting Acidovorax citrulli.The specific primers AC-F/AC-R were designed based on the pilYl gene of A.citrulli strain AAC00-1 and tested for specific DNA amplification.The primers amplified a 487 bp DNA fragment from 80 A.citrulli strains isolated from different hosts and different locations,no amplicons were yield from all the other strains including several closely related strains.The threshold of A.citrulli detection for conventional PCR was 103 cfu/ml.In addition,using the conventional PCR assay,3 out of 13 commercial cucurbits seedlots were positive for A.citrulli.The conventional PCR represents an efficient and sensitive approach to detecting A.citrulli in cucurbits seedlots.