Endophytic Bacterial Diversity Analysis Of Huanglongbing Pathogen-infected Citrus Phloem

Citrus is an important economic crop in the world, the major commercial citrus growing areas include southern China, the Mediterranean Basin (including southern Spain), South Africa, Australia, the southernmost United States and parts of South America. In China, Guangxi, Guangdong, Fujian, Sichuan, Hunan, Hubei and Zhejiang provinces are major production areaes, while smaller plantings are present in other provinces. Huanglongbing in China, is probably the one of worst diseases of citrus industry, which caused by a tiny bacterial pathogen. The causative agent is a motile bacterium, Candidatus Liberibacter spp. Field transmission is mainly by Asian citrus psyllids (Sternorrhyncha: Psyllidae, Diaphorina citri).The disease was first described in 1929 and first reported grafting transmission in China in 1956. The African variation was first reported in 1947 in South Africa, where it is still widespread. There is only few reports available about accepted pure culture of the pathogenalthough recently breakthrough in co-culture of HLB causal agents indicated that some company microorganisms are present in theHuanglongbing infected host tissue, and it may causes complex and diverse symptom of Huanglongbing pathogen-Infected citrus. The morphological, physiological and biochemistry characteristics combined with 16S rRNA-RFLP (Restriction Fragment Length Polymorphism) analysis method were applicated for researching the flora of endophytic bacterial populations,especially the microbial diversity in the healthy and Huanglongbing pathogen-infected citrus. All of these aimed to find associated microorganisms of huanglongbing pathogen were investigated.By the traditional isolation identification and 16S rDNA amplificons methods, 10 genera of bacteria were identified from 21 isolated bacterial populations. The sequences aligned with GenBank database and showed that they were belonged to 10 different genera of bacterium. The morphological and sequence analysis showed that: WJ01, WJ02, WJ03, WJ05, WJ08, WJ12, WJ13, WJ16, WJ17, WJ18, WJ21 belonged to Bacillus sp., WJ04 and WJ14 belonged to Pseudomonas sp., WJ06 belonged to Staphylococcus sp., WJ10 belonged to Kocuria sp. Some strains were also reached the level of species: WJ07, WJ09, WJ11, WJ15, WJ19, WJ20 were respectively consistent with Acinetobacter johnsonii, Serratia marcescens, Lysinibacillus graminis, Miccrococcus sedentarius, Microbacterium laevaniformans, Curtobacterium pusillum. The dominant bacteria in healthy citrus belonged to Bacillus sp., Pseudomonas sp., Kocuria sp.; and in Huanglongbing pathogen-infected citrus were Bacillus sp., Pseudomonas sp., Kocuria halotolerans, Microbacterium sp.By the PCR-RFLP analysis, two 16S rRNA libraries of Endophytic bacteria were constructed. The total DNA of microorganisms was extracted from the phloem of health and huanglongbing pathogen-infected citrus, and then amplified the 16S rRNA partial sequence: the highly variable region V5, V6, V7, V8 and V9. Primers were designed to generate about a 700 bp of product, corresponding to nucleotides 799–1492 of the 16S rRNA. Purified PCR amplification products were ligated into the pMD19-T cloning vector and transformed into competent Escherichia coli JM109 cells using electric-shock. For T-A cloning, healthy plants and diseased plants randomly picked up 229, 228 positive clones were carried out by PCR. The plasmid, containing the target fragment, were extracted from the positive clonesand used to digested by MspⅠ, RsaⅠ, HaeⅢrestriction enzyme. Those samples with different profiles were considered as different clones and represented a different bacterial strain. A representative from each of the different profile groups observed was sequenced. 16S rRNA sequences were compared with sequence data deposited in GenBank, using the BLAST alignment with sequence data held at GenBank. The results showed that: 29 restriction endonuclease types were detected, 10 genera of bacteria and 8 genera of uncultured bacterium were identified. Different genera of RFLP OUTs in healthy plants (HP) and Infected (IP) plants of the 16S rDNA clone library,the proportion of different analysis results showed that: Dyella sp. (HP 27.08%; IP 25%),Enterobacter sp . (HP 3.93%; IP 0%),Serratia marcescens (HP 16.16%; IP 18.86%),Enterococcus sanguinicola (HP 3.06%; IP 3.95%), Achromobacter sp. (HP 1.75%; IP 1.32%), Bosea sp . (HP 1.31%; IP 0.44%), Lysinibacillus fusiformis (HP 0%; IP 0.88%), Candidatus Liberibacter asiaticus (HP 0%; IP 3.07%), Bacillus sp. (HP 1.31%; IP 1.31%), Pseudomonas sp. (HP 17.03%; IP 17.54%), Uncultured bacterium clone (HP 28.38%; IP 27.63%).The results showed that: By traditional cultivation, 16S rDNA secquenceing alignment and the PCR-RFLP analysis, there are plenty of microbiota existed in both the healthy and Huanglongbing infected citrus phloem. But the density and species were not exactly same. This suggested that the changes of microbiota flora were maybe assiaciated with the infection of huanglongbing pathogen in citrus phloem. There are a lot of unculturable microorganisms can not be isolated and uncultured andby artificial cultivation method. Therefore more information of microorganism diversity only can be obtained by combining the molecular methods based on the 16S rRNA sequences with traditional culturing methods.