Isolation And Characterization Of Endophytic Bacteria From Huanglongbing’s Host Plant-Murraya Paniculata And Catharanthus Roseus

Huanglongbing (HLB) is as serious disease found in citrus and hinder our nationaldevelopment of citrus industry. HLB is caused by a negative phloem-restricted,nonculturable bacterium that belongs to the genus “Candidatus Liberibacter,” whichbelongs to the Alphaproteobacteria.It is by far the biggest difficult to culture the pathogen of HLB in vitro. In order toovercome the thought of the traditional pure culture and found the new method aboutco-culture of HLB, we analyze microbial diversity varied in the host plants-Catharanthus roseus and Murraya paniculata and look for the association between theendophytic bacteria and the HLB pathogen.Thus, Our objective in this study was toanalyze, under manual-grafting conditions, how the microbial diversity varied inasymptomatic and symptomatic C. roseus or M. paniculata and delineation of thecultivated endophytic bacterial isolates and characterization of their salient metabolicfeatures. We hope find the endophytic bacteria that have potential interaction with theHLB pathogen.①The healthy and HLB-affected Catharanthus roseus under manual-graftingconditions were used as research material. The endophytic bacterial communities weredelineation by using the traditional culturable approach and cultivation-independenttechniques based on16S rRNA gene. The endophytic bacteria were isolated fromsurface-sterilized C.roseus midribs of leaves and phloem of stems and roots by platingand restriction fragment length polymorphism (RFLP). By the artificial anaerobicculture, we obtained67strains that were identified29genus by GenBank.Curtobacterium sp., Erwinia sp., Bacillus cereus and Brevundimonas sp., Bacillus sp.were the dominant bacterial population in diseased and healthy C.roseus, respectively.However, Staphylococcus equorum was the common dominant isolate in both C.roseus.By the methods of physiological biochemical identification, The number ofbacterial isolates producing IAA and amylolysis activity was18and5. The frequency ofendophytic bacteria abling to produce ACC deaminase, siderophore and protein activitywas8,11and7. The screened for N-fixation ability of Bacterial isolates were4byusing N-free medium and nifH gene. The amount of endophytic bacteria producingantibiotic synthesis (phlD) was1. A total of6bacterial isolates show a minimum of four traits.6functional bacterial strains belonged to5genus that included Staphylococcusequorum、Bacillus sp.、Curtobacterium sp.、Morganella morganii and Lysobacter sp.。We picked188and182positive clones in16S rDNA libraries of diseased andhealthy C.roseus that were respectively restricted by the HaeIII, MspI, RsaI restrictionendonuclease. Based on the similarity of the RFLP banding profiles in diseased andhealthy C.roseus, we obtained16,23OTUs (Operational Taxonomic Units, OTUs)respectively. In addition to Candidatus Liberibacter asiaticus, Candidatus Liberibactersp. was the dominant bacterial population only in diseased C.roseus.②We used M. paniculata that were growth in the cirtus garden of HLB. M.paniculata were seen as huanglongbing’s hidden host plant and we analyze theendophytic bacteria in M.paniculate that have potential to suppress the HLB pathogen.The endophytic bacteria were isolated from surface-sterilized M. paniculata midribs ofleaves and phloem of stems by plating and restriction fragment length polymorphism(RFLP). By the artificial anaerobic culture, we obtained26strains that were groupedinto9genus by GenBank. Enterobacter sp.(IF=19.23%), Bacillus sp.(IF=38.46%) werethe dominant bacterial population in M. paniculata. However, the bacterial colonynumber of endophytic bacteria in the stems was higher than in the leaves.By the methods of physiological biochemical identification, The number ofbacterial isolates producing IAA and amylolysis activity was9and3. The frequency ofendophytic bacteria abling to produce ACC deaminase, siderophore and protein activitywas1,8and2. The screened for N-fixation ability of Bacterial isolates were3by usingN-free medium and nifH gene. The amount of endophytic bacteria producing antibioticsynthesis (phlD) was4. A total of4bacterial isolates show a minimum of four traits.4functional bacterial strains belonged to3genus that included Bacillus sp., Paenibacillussp. and Sphingomonas sp.We chose179positive clones in16S rRNA gene clone library of M. paniculatathat were respectively restricted by the HaeIII, MspI, RsaI restriction endonuclease.20OTUs (Operational Taxonomic Units) were observed based on the similarity of theRFLP banding profiles. Coverage C of the clone library was94.97%and the rarefactioncurve of the clone library tended to plateau, which indicate that the library are largeenough to reflect the bacterial endophytic diversity of the respective samples.63.69%ofthe observed clones were Serratia sp. in M. paniculata.The above the results in our study suggest the following two conclusion: ①With the infection of ‘Ca. L. asiaticus’, the diversity in diseased C.roseusdecreased. The endophytic bacteria isolated from diseased and healthy C.roseus areabundant and have remarkable differences in the composition and function, suggestingthat its endophytic bacteria might be inhibited by the HLB pathogen in a certain degree.②The results suggest that the endophytic bacteria isolated from M. paniculatahave abundant diversity and genetic characterization.