HLB Etiology Survey, Population Differentiation Of ’Candidatus Liberibacter Asiaticus’ In China And Lysozyme Protein Prokaryotic Expression Of Its Prophage

Citrus Huanglongbing (HLB) is one of the most destructive citrus diseases worldwide, and has been reported in more than40countries 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 leaves blotchy mottle, uniformly yellowing, sometimes accompanied with Zn-deficiency symptom, and produce color inversion or lopsided fruit with small and aborted seeds. Once infected, tree vigor declines and fruit yield is significantly reduced, making the orchard economically unvaluable. According to incomplete statistics, more than40million citrus trees have been removed for this reason in Guangdong, Guangxi and Fujian, China. During2004-2007, more than2million HLB-infected trees were killed in Sao Paulo State, Brazil. Because of the unsuccessfully culture the pathogen in vitro, Koch’s postulates have not been completed. Currently, three species of the Liberibacter bacterium,’Candidatus Liberibacter asiaticus’(CLas),’Ca. L. africanus’(CLaf), and ’Ca. L. americanus’(CLam), were considered as the pathogens related with HLB. In China, CLas is regarded as the putative pathogen of HLB. Due to the lack of disease-resistant cultivars and effective chemicals, removing HLB-infected trees in a timely fashion, planting disease-free citrus trees, and strictly controlling psyllids are used as the most effective ways to control HLB. In China, CLam and ’Ca. Phytoplasma spp.1associated with HLB were also reported in recently years. In this study, CLas, CLam, CLaf as well as Phytoplasma were detected, and their occurrence and distribution in commercial groves in China was evaluated. In addition, assessment of genetic diversity provides a framework for studying the taxonomy, population structure, and distinguish different isolates strains. It also provides a key to devise sensitive, specific, and rapid methods for pathogen detection, disease diagnoses, and disease risk management. Distinguishing different CLas strains is important and necessary for HLB ecological and epidemiological studies. Information on genetic diversity and population differentiation of CLas is currently limited, especially multi-loci evaluation in China, inhibiting the scope of HLB research and the understanding of CLas. In this study,79loci in CLas genome were screened by comparative genomics assay. Of the79loci,10were selected for preliminary evaluation of genetic diversity of CLas from9provinces of China. The effective chemical control of HLB was unavailable currently, so Lysozyme fusion protein from CLas prophage was expressed, which it might be used to control Liberibacter in the future. The main results are as follows:1. A new formula of CTAB-Triton DNA extraction solution with extra synergist and anti-low temperature precipitation agent was obtained. Time frame consumed on DNA extraction with this formula was largely shortened due to the steps cut down. The quality and yield of DNA were much better than those extracted from micro-extraction method, or even some commercial DNA extraction kits. Petiole and leaf vein of the citrus leaves were recommended to be the main sampling tissues for HLB pathogen detection, through a systematic comparision within different tissues of the CLas-infected pomelo leaves.2. Total629citrus samples with yellowing symptoms collected from10provinces of China, were detected by PCR-based approach. Among them,357samples were positive for CLas and all samples were negative for CLaf, CLam and ’Ca. Phytoplasma’. Our results confirmed that CLas was the main cause agent occurred in the commercial citrus planting region in China. Based on symptom observations in field and greenhouse, blotchy mottle is the main leaf symptom caused by CLas, which could be used as an accurate diagnosis index in the fields. Fruit color inversion and small lopsided fruits could be used as assistant diagnosis index.3. Total14genetic variation loci were found from79screened differentiation loci by comparative genomics analysis. Among which6loci were used for evaluating the CLas population structure of306samples in China. The multi-loci cluster analysis showed that these samples were divided into two groups. Group I contained those samples collected from Yunnan, Guizhou and Sichuan, and Group II contained those samples collected from Guangdong, Guangxi, Fujian, Jiangxi, Zhejiang. Consequently, we hypothesized that Yunnan and Guangdong were two major original centers of CLas in China.4. The CLas prophage lysozyme gene was successfully expressed in E.coli.41kDa CBD-Lysozyme fusion protein was observed by SDS-PAGE, and fusion protein was soluble in the supernatant. CBD tag on-column cleavage was induced by DTT and19kDa lysozyme protein was obtained. It was the same with that predicted by bioinformatics.In conclusion, modified DNA extraction method was used in sample preparation in the study. PCR detection showed that only CLas were found in samples with yellowing symptoms collected from10provinces of China suggesting CLas was the main cause agent in China. Among79variation loci in CLas genome,10molecular markers were selected for the evaluation of genetic diversity of CLas from9provinces of China. It enriched the sequence information, population structure and epidemiological studies of CLas in China. In addition, prokaryotic expression of the lysozyme protein was a good starting point for understanding the function of lysozyme and for the application of prophage lysozyme to resist/kill the Liberibacter bacterium.