Isolation Of Facultative Anaerobic Entophytic Bacterial With Ca Las Infected Citrus Tissues And Determination Of Dominant Bacterial Populations

Huanglongbing (HLB) is the most destructive disease of citrus that represent a major threat to the citrus production in the world. The causative agent is phloem-restricted, non stable cultured, Gram-negative bacterium named Candidatus Liberibacter spp. Until now the'Ca. Liberibacter spp.'has been cultivated from Huanglongbing-infected citrus but the cultivation could not be repeated or completed Koch's postulates. Nowdays, many Biological scientific researchers devoted themselves to the study of companion microbe in Huanglongbing pathogen-infected plant tissues, and reported that some endosymbiotic bacterial associated with pathogen were present in diseased plant tissue. The companion microbe may cause intricately and multiformity symptom of disessed citrus. The aim of this study was to analyze the diversity of cultivable and nonculturable entophytic bacterial communities in Candidatus Liberibacter sp. pathogen-infected and healthy citrus plant and find the companion microbial associated citrus plant tissues for decipher the artificial cultivation of HLB pathogen. We selected varied parts of citrus tissues collected from different locations of citrus planted area; the facultative anaerobic entophytic bacteria were isolated and purified based on bacterial morphology, physiology, biochemistry characteristics and the molecular method of PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis) analysis based on the sequence of 16S rRNA V6-V8 fragment.By the directional isolation of the facultative anaerobic and entophytic bacteria from Huanglongbing pathogen-infected and healthy citrus plant tissues. The samples came from different origins included Guangdong, Guizhou, Fujian, Yunnan, Zhejiang, Guangxi provinces. By the directional isolation of the facultative anaerobic entophytic bacteria on the basis of colonial morphology, mycelium differences and 16S rDNA amplification methods, total 12 genera of bacteria were identified from 19 cultivable bacterial populations. The morphological and sequence analysis showed that: GX21 and GX34 belonged to Kluyvera sp., GX22, GX24, GX35, GX36 belonged to Bacillus sp., GX23 belonged to Brevibacillus Shida, GX25 belonged to Planococcus sp., GX26 belonged to Pseudoclavibacter helvolus, GX27 belonged to Microbacterium sp.,GX28 belonged to Tatumella sp., GX29,GX39 belonged to Pseudomonas sp., GX30, GX31, GX33 belonged to Curtobacterium sp., GX32 belonged to Pantoea sp., GX37 belonged to uncultured Klebsiella sp., GX38 belonged to Enterobacter sp., The dominant bacterial population in infected citrus plants were Curtobacterium sp. (IF: 29.07%), Bacillus sp. ( IF: 23.12%), Microbacterium sp. (IF: 21.09%), while in healthy citrus tissues belonged to Bacillus sp.( IF: 21.03%), Planococcus sp.(IF: 20.69%), Pseudomonas sp.(IF: 17.44%).The molecular method of PCR-DGGE analysis based on the sequences of 16S rDNA V6-V8 region was used to the micro flora analysis of citrus tissue from Guangxi province. We selected a pair of bacterial 16S rDNA (799F-1492R) to amplify bacterial sequences directly from citrus tissues by PCR for exclusion of chloroplast DNA. The total DNA of microorganisms was extracted from the different tissue of Huanglongbing pathogen-infected and healthy citrus, and then amplified the 16S rDNA V5-V9 fragment about 730 bp. Then we selected a pair of 968FGC-1378R to amplify V6-V8 region of 16SrRNA by Nested PCR. Purified PCR amplification products were separted by PCR-DGGE method. By 16S rRNA V6-V8 region gene DGGE method, fifty distinct bands were obtained from 16S rDNA amplificons. The bands were purified and sequenced .The sequences were aligned with GenBank database and the result showed that: fifty bands were detected; included 9 genera of bacteria: Serratia sp. (28%), Pantoea sp. (14%), and Acinetobacter sp. (10%), Arthrobacter sp. (8%), and Nocardia sp. (10%), Xanthomonas sp. (10%), and Pseudomonas sp. (8%), Pectobacterium betavasculorum(6%),Candidatus Liberibacter Asiati(c2%),Uncultured bacterium clone (4%)。The dominant bacterium population belonged to Serratia sp.(IF: 28%)and Pantoea sp. (IF: 14%)followed by it. Candidates Liberibacter asiaticus was found only in tangerine pith of deformed orange fruit, which suggested that the content(>1%)of Huanglongbing was more in diseased fruits and other tissues of citrus had low abundance percentage. The density and species of entophytic bacteria were also observed in remarkable difference between infected and healthy citrus plant from the DGGE profiles. Nocardia sp.and Arthrobacter sp. exists stable in all the samples.In this study, we adopted two methods including directional isolation and PCR-DGGE to comprehensively analyze the entophytic bacterial diversity of citrus, to analyze the entophytic bacterial diversity of citrus and find the companion microbe in Huanglongbing pathogen-infected and healthy citrus plant tissues for decipher the artificial cultivation of HLB pathogen, the density and species of entophytic bacteria of the two methods were different. By the culture-dependent method, Curtobacterium sp. and Bacillus sp. were the dominant groups, but the two groups couldn't find by PCR-DGGE method. The results indicated that many of the dominant bacterial species could not be isolated or cultured by artificial cultivation method. Nevertheless, studies on entophytic bacterial diversity by 16S rDNA cloning and sequencing have some limitations. Therefore, combining the traditional directional isolation and PCR-DGGE method, may explore the dominant populations more effectively.