| Citrus huanglongbing (HLB, ex greening) is one of the most serious diseases of citrus that greatly reduces production, destroys the economic value of the fruit, and can kill trees. The disease is mainly transmitted by grafting or by psyllid vector and now is wide spread in Asian countries, the Indian Subcontinent and Ocean, Southern Africa and recently in America, Brazil, New Guinea and Cuba. The causal agent is a fastidious prokaryote that lives in the sieve tube elements of infected plants, is systemic in infected insects and is taxonomically in theα-subdivision of the Proteobacteria. HLB exists in nature in three forms that differ by a combination of environmental conditions and insect vectors. The disease caused by Candidatus Liberibacter asiaticus (Las) is a heat-tolerant form vectored by Diaphorina citri, caused by Ca. L. africanus (Laf) is a heat-sensitive form vectored by Trioza erytreae, and caused by Ca. L. americanus (Lam) is a heat-tolerant form vectored by D. citri. However, the pathogenic organisms have not been cultured, nor have Koch's postulates been satisfied.Due to lack of high effective bactericides and resistant citrus varieties, many countries taking action mainly rely on uprooting and burning diseased trees from quarantine area to prevent the introduction and establishment of this disease, therefore, it is important to investigate HLB disease epidemiology. In this study, a genetic diversity study was conducted using PCR-RFLP based on the outer membrane protein gene (omp) sequences. The Las isolates were collected from plant hosts and Asian citrus psyllid (Diaphorina citri) in the main area of HLB in China such as Guangxi municipality, Guangdong, Fujian, Hainan, Jiangxi, and Zhejiang province from 2005 to 2008. The amplicons of omp gene were digested with different enzymes. The difference of restriction patterns shows that Las may comprise several different omp strains, even among strains within a given region (Guangxi municipple). The omp gene of Las from different geographical province was cloned and sequenced. DNA sequences and the deduced amino acid sequences of omp gene were compared to those in GenBank database with BLAST and the results were used to construct phylogenetic tree. The omp gene of Las from China were highly homogeneous. The omp genes of Las exhibited 99% identity, but other species (Las and Laf) were not introduced or developed in china so far. The Sequences were used to construct phylogenetic tree by the maximum-parsimony method. All Las isolates were divided into two subgroups, The Laf belong to one subgroup. The Las isolates from different geographical regions belong to another subgroup. The result is important on explorer HLB disease epidemiology and can help the scientific community develop more efficient methods to control the diseases.The other part of this research work was to develop a multiplex PCR (mPCR) detection system for citrus HLB and citrus bacterial canker disease (CBCD). HLB and CBCD have long been reported to be present overlapped areas of the world like Southeast Asia. With the new discovery of Candidatus Liberibacter americanus (Lam) in Brazil, recent confirmation of HLB in Florida and abnormally massive diffusion of CBCD caused by unprecedented hurricane activities in 2004 and 2005 in Florida, there is a growing concern for implementation of earlier diagnosis for HLB and CBCD and rapid identification of the quarantine pathogens. In this section, a mPCR detection system was developed using a ribosomal protein gene-based primer set and another unique protein gene-based primer specific for Candidatus Liberibacter asiaticus (Las) and Xanthomonas axonopodis pv. citri (Xac), respectively. Besides, an internal positive control (IPC) was also included to help evaluate the performance of our detection system which was designed based on the conserved 18S rDNA sequences of citrus plant. Three different fragments (193, 253 and 382bp) were simultaneously amplified using a single PCR reaction. The consistent results of the mPCR were compared with simplex PCR for detection of each pathogen and confirmed with sequencing analysis. From the field samples detection, the discovery of co-infection of Las and Xac was reported for the first time in the field citrus plants in Beihai, Guangxi municipality, China. This mPCR assay should be useful for diagnosis of HLB and CBCD especially for quarantine purpose, identification of Las and Xac, and certification and plant improvement program for obtaining HLB-free or CBCD-free planting materials. This method should also be applicable for confirmation of visual method upon routine symptom.
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