Genetic Diversity Of Prophage Of ’Candidatus Liberibacter Asiaticus’

Citrus Huanglongbing （HLB） is one of the most destructive diseases of citrus worldwide, which has been reported in nearly 50 countries or regions located in Asia, Africa, Oceania, North America and South America. All known commercial citrus species are susceptible to HLB. HLB affected trees typically develop symptoms of blotchy-mottled or yellow shoot, sometimes accompanied with Zn-deficiency appearance in newer growths, and produce red nose fruits. As the disease progresses, tree vigor declines and fruit yield are significantly reduced, making the orchard economically unviable. The pathogen of HLB belongs to the genus Candidatus Liberibacter. So far, three species of the bacterium have been identified to be associated with HLB: ’Ca. L. asiaticus’（CLas）,’Ca. L. africanus’（CLaf）, and ’Ca. L. americanus’（CLam）, among which,’Ca. L. asiaticus’ is widely distributed worldwide including in China and the United States. The HLB-associated bacterium can be transmitted by grafting between citrus or by dodder from citrus to periwinkle. In field, three species of psyllids （Diaphorina citri, Trioza erytreae and Cacopsylla citrisuga） are the major transmission. Due to the lack of disease-resistant cultivars and effective chemicals, removing HLB-infected trees timely, planting HLB-free citrus nursery trees, and controlling psyllids strictly in a large scale are the three most effective ways to control HLB.Because of the inability to successfully culture the pathogen in vitro, Koch’s postulates have not been completed so far. Little is know about the bacterial biology and population structure. Information about the genetic diversity and population differentiation of CLas in China is currently limited, inhibiting the scope of HLB research and control of the disease. In this study, some novel molecular markers in prophage region of CLas were selected for differentiating CLas population structure based on the sequence alignment of four available genomes （Florida Psy62, Guangxi GX-1, Guangdong A4, California HHAC） and previous studies by our research group, the main results are as follows:1. Detecting of miniature inverted-repeat transposable elements （MITEs） from CLas. MITEs are a type of non-autonomous transposons that play important roles in gene regulation, genome evolution and biodiversity. Recently, two MITEs named MCLas-A and MCLas-B were identified from CLas. MCLas-A was suggested as an active MITE because of its mobility. In order to explore the current occurrence, distribution and transposition of the two non-autonomous transposons,222 samples with HLB were used in the study. The results showed that the bands of B720 （MCLas-B） and the B350 （putative transposition product of MCLas-A） were dominated whereas the bands of B630 （MCLas-A） were only detected in one sample from Guizhou Province, suggesting that the high MCLas-A transposition frequency occurred in recently collected Chinese samples. The biological role of the high transposition frequency remains to be determined. Sequence analysis of representative B350 amplicons showed that the sequence types were consistent with those in previous study.2. Sequence analysis of MITEs and putative transposase. Two MITEs, MCLas-A and MCLas-B, were recently identified from CLas known to be associated with HLB. MCLas-A was suggested as an active MITE because of its mobility. The immediate upstream gene of the two MITEs was predicted to be a putative transposase. The goal of this study was to analyze the sequence variation in the upstream putative transposase of MITEs and explored the possible correlation between sequence variation of transposase gene and MITE activity. PCR and sequence analysis showed that 12 sequence types were found in six major amplicon types from 43 representative CLas isolates from China, the United States and Brazil. Out of the 12 sequence types, three （T4, T5-2, T6） were firstly reported. Potential recombination events were found in the two unique sequence types （T5-2, T6） detected in all Brazilian isolates. Notably, no sequence variation or recombination events were detected in the upstream putative transposase gene of MCLas-A, suggesting that the conservation of the transposase gene might be closely related with the MITE activity. Phylogenetic analysis demonstrated that the two well supported clades including five subclades were identified, clearly reflecting the geographical origins of isolates, especially that of Ruili isolates, Sao Paulo isolates and a few Florida isolates. The California isolate shared higher similar with those from coastal provinces in China than those from Florida. Further characterization on CLas populations worldwide using these genomic loci may be helpful for constructing a global transmission and epidemiology networks of HLB and provide new insight into genome evolution and biology of CLas.3. Flanking sequence analysis of large fragment deletion in CLas. Large fragment deletion in CLas prophage region （CLIBASIA₀5640-CLIBASIA₀5660） was identified from CLas samples from southwestern China （Yunnan, Sichuan and Guizhou）. To further explore the the flanking sequence polymorphisms, primer sets were designed based on multiple genomic comparison. Eight CLas isolates collected from southwestern China were analyzed with a previous designed primer set Lap5640f/Lap5660r. A previously identified 550 bp molecular marker （S550） could be found from some isolates in southwestern China. Unexpectedly, primer set Lap5640f/Lap5660r also generate an amplicon of about 1000 bp, designated as S1000, in one Yunnan sample （YNYJ-E）. Primer set TR1222067f/TR1224135r was designed for the amplification of upstream area （CLIBASIA₀5625-CLIBASIA₀5660）. The result showed that the primer set could amplify the specific band from isolate YNYJ-E. Results of sequence alignment showed that the sequences of S1000 were similar to the corresponding Psy62 genomic region but with sequence insertion/deletion including four fragment insertion and one deletion in CLIBASIA₀5645, three deletion in CLBASIA₀5650 and its downstream gene, one deletion in CLBASIA₀5655. Future studies on the specific genomic locus and the special isolate will further dissect genetic diversity and genome plasticity of CLas.