Nested-PCR Detection Of Lianjiang Hongjiang Orange Huanglongbing And Endophytic Culturable Bacterial Community Diversity Analysis

The rapid detection methods of Lianjiang Hongjiang Orange huanglongbingwere screened and optimized in this paper and the disease infection rate of threeseedling breeding nurseries and fifteen orchards were determined with Nested-PCRmethod. In addition, the distinctionof the species and quantities of the endophyticculturable bacteria communities in Candidatus Liberibacter sp. pathogen-infected andhealthy Hongjiang Orange were analyzed by traditional tissue separation andmolecular biology. In this study, the main results are as follows:1. Screening and optimizing the detection methods of Huanglongbing. Thespecific primers sets for disease detection were selected with conventional PCRreaction system using rTaq and Mighty Amp DNA polymerase and the Nested-PCR,respectively. The experiments have proved the conventional PCR with rTaq DNApolymerase detection results of repeatability, specificity and accuracy were not stable,while the other two methods could amplify the target fragments, and the describedmethods with band bright, specificity and stable reproductivity can be effectivelyapplied to high speed detect infected citrus trees. Comparatively speaking, thereaction components and DNA polymerase in Nested-PCR system would not affectthe results and its cost was lower. In conclusion, Nested-PCR was screened forinvestigating Huanglongbing infection rate in different nurseries and orchards.2. The tissues of leaves, roots, stems, peels and fruits were collected fromsymptoms of suspected Hongjiang Orange and detected with Nested-PCR. The resultsconfirmed that the pathogen could exist in different organizations and leaves positivedetection rate were higher than other tissues. For this reason, different symptomsleaves such as mottled etiolation, uniform etiolation, narrow lacklustre and other coloranomalies were used for further detection analysis, and mottled etiolation leavespositive detection rate was100%, this characteristic symptom could be served as theevidence of Citrus huanglongbing field diagnostic.3. A total of92samples including grafted seedlings, scion trees and rootstocksfrom3nurseries were detected by Nested-PCR and test results showed that thepathogen had already invaded in tested nurseries and the average infection rate of grafted seedlings and scion trees were corresponding to23.9%(10.0%-36.4%) and21.2%(14.3%-25.0%). Furthermore, one rootstock had appeared the pathogenspecific target fragment. The infection rate of nurseries is relatively common, whichmight be one of the main sources of orchards pathogen.4. In order to know huanglongbing infection rate in the planting areas, a total of461samples collected from qingping town and Hongjiang Farm were detected byNested-PCR. It turned out that the former plant infection rate were11.11%-28.89%and the latter rate were25.93%-30.76%. Besides that, there was a positive correlationbetween the orchard and huanglongbing infection rate, and the huanglongbinginfection rate had exceed20%in more than7years age orchards.5. The roots, stems and leaves from infected or not Hongjiang Orange detectedby Nested-PCR were used for culturable endophytic bacteria isolation using tissueseparation method. The results showed that the quantity of bacteria isolated fromhealthy plants were richer than infected huanglongbing plants. The amount of bacteriawas different in various parts and maximum in healthy root,and min in infected stems.According to bacterial16S rDNA sequence analysis,17strains isolated from healthyplant belonged to10different species, and another15strains isolated from infectedplant also belonged to other10different species, and endophytic bacteriacommunities existed obvious differences in healthy and infected huanglongbing plant.In addition, the amount and species of the dominant genus in roots, stems and leaveshad been significant impacted due to invasion of huanglongbing pathogen.