Separation And Analysis Of Endophytic Bacteria From Two Kinds Of Huanglongbing’s Host Plant-citrus And Clausena Lansium (lour.) Skeels

Due to heavy damage, wide distribution area, spread fast and no effectivemeasures to control, citrus Huanglongbing (HLB) became a hot topic in citrus diseaseresearch. HLB is caused by a kind of nonculturable bacterium belongs to the genus“Candidatus Liberibacter” and belongs to Alphaproteobacteria, which survived in thephloem of hosts only. Now, there are many reports about the mutual antagonism andmutualism between bacteria.Based on the previous studies, We selected two kinds of citrus HLB host rutaceae(citrus and Clausena lansium （Lour.）Skeels), understanding microflora from the“Candidatus Liberibacter” living environment. Comparative analysis of the endophyticbacteria in symptoms and symptomless tissues of single HLB-affected citrus plan and ofthe diseased and healthy wampee, that is the foundation to recognize the effects betweenCandidatus Liberibacter and the endophytic bacteria. The endophytic bacteria isolatedfrom wampee may antagonize the HLB pathogen, and also may be mutualism, which isthe foundation of pathogenesis and biological control.1. The population structure and bacterial diversity of endophytic bacteria insymptoms and symptomless tissues of single HLB-affectedcitrus plant wereinvestigated based on16S rDNA sequences analyzed by PCR-DGGE (PCR-denaturinggradient gel electrophoresis). Quantitative analysis of Ca.Las, dominant bacteria andtotal amount of bacterial population in symptoms and symptomless tissues of branches,leaves and fruits was carried out by quantitative PCR.The results showed that the amount of Ca.Las is different significantly insymptoms and symptomless tissues; bacterial population in symptoms citrus tissues wassignificantly higher than in symptomless tissues. The PCR-DGGE analysis resultshowed that endophytic bacterial structures are basically same in symptoms andsymptomless tissues. Extraction, cloning and sequencing of17obvious bands in DGGEelectrophoretogram revealed that8of17bands belonged to the genus Serratia, whichaccounted for47.06%. Sequence alignment showed that they belongedto different strains of Serratia marcescens with the99.63%similarity. Quantitativeanalysis of the total bacteria and Serratia marcescens showed that there is nosignificant difference in total bacterial count between symptoms and symptomlesstissues while the amount of Serratia marcescens showed negatively correlation with amount of Las.2. The diversity of endophytic bacteria flora of Clausena lansium, which is a kindof Huanglongbing’s rutaceae host, was investigated based on the facultative anaerobicculture technique and amplified ribosomal DNA restriction analysis (ARDRA). By theplate facultative anaerobic culture isolation, we obtained41species of endophyticbacteria, belonging to18genera according GenBank. Pseudomonas sp. andAcinetobacter sp. are the dominant bacteria population in healthy Clausena lansium.Chryseobacterium sp., Bacillus sp. and Sphingomonas sp. are the dominant bacteriapopulation in diseased Clausena lansium. The endophytic bacteria species aresignificant difference between healthy and diseased Clausena lansium. However, thereare8genera endophytic bacteria isolated from both Clausena lansium, IncludingAcinetobacter sp., Pseudomonas sp., Bacillus sp., Chryseobacterium sp., Sphingomonassp., Serratia sp., Stenotrophomonas sp. and Brevundimonas sp., while their isolatedfrequency is varies greatly.ARDRA fingerprint analysis showed27and17Operational Taxonomic Units(OTUs) by investigated healthy and diseased Clausena lansium, respectively. However,Pseudomonas sp.was the dominant isolate in both Clausena lansium. γ-proteobacteria isthe main flora in Clausena lansium by the two isolated methods.Based on the functional identification of culturable endophytic bacteria, such asprotein hydrolysis, starch hydrolysis ability, ability of its nitrogen fixation, antibioticbiosynthetic ability, ACC dehydrogenase synthesis ability, whether to produce indole-3-acetic acid (IAA) and blood cell synthesis, we have screened out the strains having atleast5functions including Serratia proteamaculans (Cl-06), Pantoea agglomerans(Cl-17), Pantoea sp.(Cl-18), Arthrobacter sp.(Cl-19), Enterobacter ludwigii (Cl-24),which are belonging to4genera. Serratia proteamaculans was isolated from bothhealthy and diseased Clausena lansium, but the relevanct ratio is very low. Theremaining strains were isolated in healthy plants only.The above the results in our study suggest the following conclusions:The Ca.Las is heterogeneously distributed among various tissues of HLB-affectedcitrus plant. The amount of Ca.Las was negtive correlation with Serratia marcescensand total amount of endophytic bacteria have no relevance with symptom of HLBdisease. How the interaction of Serratia marcescens and Ca.Las in tissue of citrusneeds further study.There are abundant endophytic bacteria in both healthy and diseased Clausena lansium. Edophytic bacteria flora of Clausena lansium was changed by CandidatusLiberibacter, and the diversity declined. The results has shown that CandidatusLiberibacter affect the endogenous bacteriain in some degree. The bacteria isolated fromClausena lansium have multiple functions, which is the foundation to understand therelationship between Candidatus Liberibacter and them.