Establishment Of Loop-mediated Isothermal Amplification Assay And Analysis Of Population Differentiation Of ’Candidatus Liberibacter Asiaticus’

Citrus huanglongbing （HLB） is one of the most destructive diseases of citrus industry worldwide. As a domestic and international quarantine organism, it has been reported in nearly50countries or regions in Asia, Africa, Oceania, North America and South America. In China,11out of the19citrus producing regions have been suffered from the damage of HLB, which has hampered the development of the citrus industry. Currently, three species of the bacterium that belong to the genus’Candidatus Liberibacter’—’Ca. L. asiaticus’,’Ca. L. africanus’and’Ca. L. americanus’—’have been found to be associated with HLB. In China, this disease is caused by’Ca. L. asiaticus’. Typical field symptoms affected by HLB are yellow shoot and/or blotchy-mottled, lopsided fruit with small and aborted seeds. As the disease progresses, the quality and yield of fruits are seriously reduced. Due to lack of effective chemicals and disease-resistant varieties, removing HLB-infected trees, controlling psyllids and planting disease-free citrus seedlings are conducted as the most effective ways to control HLB. Therefore, strengthening the early diagnosis of HLB has been particularly significant. Assessment of genetic diversity provides a framework to study the taxonomy, population structure and occurrence dynamics of phytobacteria. It also provides a key to develop sensitive, specific and rapid methods for disease diagnosis, pathogen detection and disease risk management. Distinguishing’Ca. L. asiaticus’strains is important and necessary for HLB biological and epidemiological studies.Loop-mediated isothermal amplification （LAMP） assay for the detection of HLB has not been reported in China, and information about population differentiation of’Ca. L. asiaticus" is currently limited in high altitude regions. In this study, a LAMP technology for rapid detection of HLB was established, which could be applied to the batch detection of suspected HLB samples and rapid diagnosis of the disease. Meanwhile, population diversity was investigated on CLIBASIA₀5640-CLIBASIA₀5660locus which has been reported on the genome of’Ca. L. asiaticus’. This helps to further assess the origin and epidemiology of HLB in gene level. This research was supported by MOA’s Public Benefit Research Foundation of China （201003067-02;200903004-06） and Innovative Research Team in University （ERT0976）. The main results are as follows:1. Based on the species-specific conserved region of’Ca. L. asiaticus’, a LAMP assay for rapid detection of HLB was developed and evaluated. The results showed that ladder-like products were found from those HLB-positive samples by LAMP. Alternatively, amplification products can be visualised by turbidity or the form of a colour change when SYBR Green I, a fluorescent dsDNA intercalating dye, is employed. The lowest detection limit of the LAMP and Real-time PCR method was3.9×101copies,100fold higher than that of conventional PCR.65of120field samples showed a positive result for HLB by LAMP method, which rated54.2%, while47samples showed positive by the conventional PCR method and it rated39.2%. The positive result of PCR was consistent with that of LAMP. All these results showed that this method was rapid, simple, specific and sensitive, it offered a new technique and alternative for rapid detection of citrus HLB.2. CLIBASIA₀5640-CLIBASIA₀5660locus was used to evaluate the intra-specific differentiations of China’Ca. L. asiaticus’populations,222HLB isolates/strains have been studied. Some strains from Yunnan and Sichuan produced a small DNAband with1033nucleotides deletion, which composed of two ORFs （CLIBASIA₀5650and CLBASIA₀5655） and parts of one ORF （CLIBASIA₀5645）. Compared with Yunnan and Sichuan strains, these strains from other regions only had an expected large DNA band. Downstream primers were designed in the flanking regions of the locus, and sequence analysis showed that P4family phage/plasmid primase locus （CLEBASIA₀5660） had highly sequence variations among all strains analyzed. Primers TR1220342f/TR1222657r was designed in the upstream sites, and the amplification results of Yunnan and Sichuan strains produced two DNA bands. Sequences of the small fragments were highly similar to Liberibacter phage SC2and FP2, indicating that it may be a new sequence of prophage. The difference was mainly caused by the nucleotide insertion, deletion, recombinant, and multiple SNPs. The results showed that’Ca. L. asiaticus’ population from a high altitude region seem to be more genome plastic than those from low altitude regions.In summary, the LAMP assay for rapid detection of HLB was developed and evaluated. This method was rapid, simple, specific and sensitive, which could be applied to the large-scale detection of field samples, In addition, CLBASIA₀5625-CLIBASIA₀5660locus was utilized to further identify’Ca. L. asiaticus’ population of different geographical origin. High altitude strains were mainly analyzed, which was expected to provide a molecular evidence for the origin, biology and epidemiology research of citrus HLB.