| Citrus Huanglongbing (HLB), known as citrus greening disease, is one of the most destructive citrus diseases. HLB is widely distributed in nearly50countries or regions located in Asia, Africa, Oceania, North America and South America. In China,11of19citrus prodution provinces have been suffering damage from HLB. This disease is characterized by the development of yellow shoots and stunted growth of infected trees, especially by the typically blotch-mottled leaves and abnormally-pigmented fruits, which are applied in preliminary diagnosis of HLB. There are three insect-vectored, phloem-limited and Gram-negative pathogen species of ’Candidatus Liberibacter’,’Ca. L. asiaticus’,’Ca. L. africanus’ and ’Ca. L. americanus’. Of which ’Ca. L. asiaticus’ is the most prevalent and draws most research attention worldwide.Because the pathogen of HLB has never been successfully isolated, so Koch’s postulates have not been completed by now, the understanding of morphological, physiological and biochemical characteristics of pathogen is still very limited. Assessment of genetic diversity becomes another important research orientation and help to reveal the population structure and epidemiology of the pathogen. In this study, multiple prophage gene loci were used to evaluate genetic diversities of ’Ca. L. asiaticus’ strains from China, and also used for discussing the origins and epidemiology of HLB on molecular level. This research was supported by MOA’s Public Benefit Research Foundation of China (201003067-02) and Program for Changjiang Sholars and Innovative Research Team in University (IRT0976). The main results were as follows:1. One bacteriophage repressor protein Cl gene locus and two hyper variable genomic regions, both including tandem repeat sequences, were applied for evaluating population differentiation of’Ca. L. asiaticus’ from China. The results showed that the amplification frequency and the tandem repeat numbers (TRNs) in targed regions of the ’Co. L. asiaticus’ population from different geographical origins were obviously different at these gene loci. Especially, more complexity, compared with other regions, was observed in Southwestern regions on the hyper variable genomic regions. Thus, tandem repeat sequences used as molecular markers provided a potential tool to detect genetic diversities of’Ca. L. asiaticus’.2. Based on the date of two prophage terminase genes, two hyper variable genes and one hypothetical protein gene of’Ca. L. asiaticus’ strains from8provinces in China, there found two widespreading prophages (SCI and SC2) in China. According to the differences of prophage types and variation levels, all strains were divided into three different geographical origins. Among which Southwestern region and Guangdong-Guangxi region contained the predominant types SCI and SC2, respectively, and no dominant types was found in Fujian-Jiangxi-Zhejiang region. In addition, a prophage T3, significantly distinguished from prophages SCI and SC2, was disclosed by hypothetical protein genes, which was defined as a new prophage and distributed in Southwestern region and Fujian-Jiangxi-Zhejiang region.3. As shown by researches on six prophage gene loci, different degree polymorphisms existed in these prophage genomic loci. Two prophage terminase genes were comparatively conserved, with a few randomly distributed single-nucleotide polymorphisms. Bacteriophage repressor protein Cl gene had a tandem repetive sequence region with VNTRs. Hyper variable genes and the hypothetical protein gene presented highly variable, which included both nucleotide polymorphisms and VNTRs, especially gene rearrangement and recombination were discovered in the two genes, respectively. Different degrees of variation in prophage genomic loci reflected evolution at different rates, therefore, hyper variable genomic loci could be applied for studying of population differentiation, and with potential for providing furthermore information on origins and epidemiology of HLB.In summary, multiple prophage gene loci were used to evaluate genetic diversities of’Ca. L. asiaticus’ strains from different geographical origins in China. Prophage types and corresponding distribution had been defined preliminary. Meanwhile, this study revealed that nucleotide polymorphisms, VNTRs, rearrangement, and recombination were all the resources of diversities of prophage genes of HLB. |
