Genetic Diversity Of Prophage Region From ’Candidatus Liberibacter Asiaticus’ Of Diaphorina Citri In Jiangxi

“Candidatus Liberibacter asiaticus”(CLas)is an uncultureable α-proteobacterium associated with citrus Huanglongbing(HLB,yellow shoot disease),It is a highly destructive disease affecting citrus production worldwide.Due to the lack of disease-resistant cultivars and effective chemicals,At present mainly through digging out the HLB-infected tree timely to eliminate pathogens.HLB was first observed in Guangdong Province of China and remains endemic there.It has caused more than 50 million citrus trees susceptible or death in more than 50 countries and regions.And HLB mainly relying on bacteria seedlings(scion)to spread.Due to the content of “Candidatus Liberibacter asiaticus” in Diaphorina citri is much higher than that of the citrus and other plant host cells.Although in the field had the existence of “Candidatus Liberibacter asiaticus”,there is no Diaphorina citri,the disease will not spread at all.Therefore,controlling Diaphorina citri strictly and cutting off the spread of disease are an effective measure to control citrus Huanglongbing.However,Koch’spostulates have not yet been completed because “Candidatus Liberibacter asiaticus” is non-culturable in vitro,which greatly limits researching the mechanism of Diaphorina citri transmit HLB.Recent a lot of researches have shown the presence of phage/prophage in “Candidatus Liberibacter asiaticus”.Phage / prophage are important components of biological entities that directly contribute to the bacterial evolution and population diversity.Therefore,the study of the genetic diversity of phage/prophage in “Candidatus Liberibacter asiaticus” of Diaphorina citri can help to analyze the mechanism of Diaphorina citri.In this study,we identified piercing-sucking pest species that collected from Jiangxi Ganzhou Than investigated these pests whether carry Candidatus Liberibacter asiaticus.And researching phage types and the genetic diversity of the prophage types in different “Candidatus Liberibacter asiaticus” strains of Diaphorina citri from different geographical sources in Jiangxi Ganzhou.The main results are as follows:1.In this chapter we first based on the morphological identification and mitochondrial COI gene to identify piercing-sucking pest species that collected from Ganzhou area of Jiangxi Province.The blast results show that we collected nine kinds of piercing-sucking pests on the citrus from Jiangxi Ganzhou,namely,Diaphorina citri 、Dialeurodes citri,Aleurocanthus spiniferu,Unaspis yanonensis,Iceryapurchasi maskell,Scirtothrips citri,Scirtothrips dorsalis,Toxoptera citricidus,Aphis gossypii and Myzus persicae.By using PCR amplification of O11/O12 c primers on 16 S rDNA marker gene detected whether the nine kinds of piercing-sucking pests carry “Candidatus Liberibacter asiaticus”,The study has been found that only detected “Candidatus Liberibacter asiaticus”in Diaphorina citri,And Dialeurodes citri,Aleurocanthus spiniferu,Unaspis yanonensis,Iceryapurchasi maskell,Scirtothrips citri,Scirtothrips dorsalis,Toxoptera citricidus,Aphis gossypii and Myzus persicae are not objective a stripe of Candidatus Liberibacter asiaticus.Thus it can be seen that collected piercing-sucking pests in the citrus garden,apart from the Diaphorina citri can spread Huanglongbing,other pests does not,and don’t carry “Candidatus Liberibacter asiaticus” in the body too.And in this chapter,it was confirmed that O11 / O12 c primers could be used to detect the positive samples of“Candidatus Liberibacter asiaticus”,which provided the experimental basis for detect prophage in the “Candidatus Liberibacter asiaticus” of Diaphorina citri.2.Though it’s reported that the presence of phage/prophage in “Candidatus Liberibacter asiaticus”,but the dynamic change of prophage of Diaphorina citri on the process of spreading huanglong disease is no clear yet.In order to clarify the phage types of “Candidatus Liberibacter asiaticus” in Diaphorina citri from Jiangxi Gannan,For this experiment,targeted two hypervariable genomic regions hyvⅠand hyvⅡof phage,were used for PCR detection and sequence analysis of samples of Diaphorina citri from seven locality in Jiangxi Gannan.PCR amplicon polymorphisms were observed,the prophage types were classified into four types,single SC1 type,single SC2 type,both SC1-SC2 type and no prophage types in “Candidatus Liberibacter asiaticus” strains of Diaphorina citri in Jiangxi Gannan,but single prophage occupies a certain advantage.And the study found that prophage types in “Candidatus Liberibacter asiaticus” strains of Diaphorina citri in Jiangxi Gannan have difference to other provinces in China,Such as Guangxi strains and Guangdong strains mainly carry SC2 type of prophage,and only detected a single SC1 type in Yunnan strains.3.In order to research on the genetic diversity of prophage region in “Candidatus Liberibacter asiaticus” of Diaphorina citri,and the genetic differenty between Diaphorina citri and citrus strains.For this experiment,using the PCR primers to detect positive samples of Diaphorina citri were collected from seven locality in Jiangxi Gannan.Initial result indicated that five kinds of prophage were detected in this region,namely,A,A2,B,C and C1,among which the percentage of A and B was relatively high.Further the analysis of Neighbor-joining tree showed that “Candidatus Liberibacter asiaticus” strains of Florida in the United States,“Candidatus Liberibacter asiaticus” strains of Chinese on citrus are all have significant genetic differences to “Candidatus Liberibacter asiaticus” strains of Diaphorina citri in Jiangxi Gannan.And suggested that different location of prophage in Diaphorina citri populations have larger genetic differences in this region,different prophage type has differences too.But its litter homology differences in same type of prophage from different geographical regions.It was found that the prophage region of“Candidatus Liberibacter asiaticus” had abundant polymorphism,and the investigation on prophage in Gannan may help analyzing the genetic diversity of prophage in “CandidatusLiberibacter asiaticus”.The study on the genetic diversity of prophage in Diaphorina citri is helpful to further research the toxic mechanism of Diaphorina citri.