Establishment And Application Of Molecular Detection For Citrus Huanglongbing And Study On The Function Of Core Gene ClpV In Erwinia Amylovora

Citrus Huanglongbing is a bacterial disease of systemic infection,which caused by the difficult cultivation of bacterial phytophthora species(Candidatus Liberibacter asiaticus),the symptoms of this disease are complex,easy confused with the deficiency disease,viral diseases and other symptoms.In China,the disease is also very serious,it caused severe harm to the citrus industry.At present,there is no effective means for the management of Huanglongbing.It mainly relies on strict quarantine measures to prevent the spread of the disease,so the establishment of efficient,fast,operation simple detection technology for early diagnosis of suspected Citrus Huanglongbing and providing a stable and reliable inspection and quarantine technology are effective means to prevent the spread of this pathogen.Polymerase chain reaction technology is the main means of the detection of citrus Huanglongbing,it is fast,sensitive and accurate.However,the pathogen of citrus Huanglongbing is a kind of bacteria difficult to cultivate,its distribution is uneven and content is small in plant tissue.So the DNA acquisition process is cumbersome,coupled with the current majority of Huanglongbing pathogens molecular detection using target without genome comparison,therefore there is sometimes a false negative or positive problem in conventional PCR.In this study,the whole genome sequence of the target strain Ca.L.asiaticus was compared with the whole genome sequence of each of the other species by the local Blast software,using the single sequence ratio multi-sequence method.A whole genome comparison technique was used to select the appropriate core gene as the molecular target on the basis of whole genome comparison,and specific probes and primers were designed.The detection methods of conventional PCR and TaqMdan probe q-PCR were established.The experimental results showed that both of the two means can distinguish the Ca.L.asiaticus from Ca.L.africanus,Ca.L.americanus and Ca,L.solanacearum.The highest susceptibility of these two was up to 6 copies/μl,which corresponds to 0.01 fg/μL of DNA,showing good specificity and high susceptibility.In the detection of the actual samples,qRT-PCR can detect 8 strains from 24 samples of suspected Huanglongbing in Hunan,and have high detection rate.In addition,Erwinia amylovora can cause fire blight of pear,apple and many of the Rosaceae family.In this study,two homologous genes of clpV were identified and cloned in Erwinia amylovora for the first time.The clpV mutants AclpV-1 and △clpV-2 were successfully generated by homologous recombination.The effects of △clpV-1 and △clpV-2 on the pathogenicity and the production of toxin were analyzed.The results showed that the mutant △clpV-2 displayed the reduced virulence compared to the wild type,while △clpV-1 was not significantly different from the wild type.