The Specific Gene Fragment Cloning And Detection And Quarantine Techniques Of Citrus HLB Disease (Candidatus Liberibacter Asiaticus)

Citrus Huanglongbing Disease (HLB), caused by an uncultured phloem-restricted, G-bacterium, is one of severe quarantine diseases domestically and abroad. After the citrus trees are infected, the yield and quality are affected, even the whole is dead. At present the appropriate method is to eradicate and burn infected plant material. In order to prevent the disease from spreading in citrus plant area of China we should develop disease-free area of citrus production. Therefor it is important to do research on detection technique. There are some disadvantages i.e. low specificity and sensitivity in the routine detection techniques. It is hard to meet the Quaratine requirement and conduct presentation diagnosis of HLB. The detection according to nucleic acid molecule is rapid, sensitive and special and can meet modern plant quarantine requirement domestically and abroad. Objective: This thesis aims at developing detection system of DNA molecule with specificity, rapidness, accuracy and stabilization and providing practical tool of disease diagnosis for establishment of disease-free area and precautionary monitoring of HLB through the research on the molecule detection techniques of HLB and diagnosis kit stored at room temperature systemically. Methods: First of all, DNA of Candidatus Liberibacter asiaticus was extracted by the methods of CTAB,reagent kits and soaking-filtration. Primer pairs and a probe were designed using software Primer Premier 5.0. The specificity was validated through PCR detection of the saprophytic microorganism of citrus leaf surface, Tristeza virus and bacteria of Xac etc plant pathogens or G-/ G+ bacteria. The sensitivity of PCR was determinated by amplification of the bacteria DNA and target sequence inserted in recombinant plasmid solution with ten-fold serial dilution. PCR profiles were adjusted by using different type of Thermocycler in different temperature control model, which ensured the stability of PCR detection. After being inserted into clone vector and recombinant plasmid transformed into the E. coli strain (JM109), the target DNA fragment was sequenced, and amplification fidelity of PCR was verified by sequences alignment using BLASTn. Then a harmless positve reference system was constructed with recombinant plasmid DNA. Solidifying PCR reagent kit was produced by adding macromolecular stabilizer (MS) into PCR mixed reagents and vacuum freeze-drying treatment. Field samples with or without disease symptom of citrus collected from Guangxi, Zhejiang, Jiangxi, Sichuan provinces and Chongqing area were detected by PCR assay. The primary study on quantitative detection of HLB pathogen (Ca.L.asiaticus) was made by the iCycleriQ Realtime detection system. Experiment Results: ⑴DNA of Ca.L.asiaticus extraction methods were compared. Among them the soaking-filtration is the best one, which remove plant pigment, protein and polysaccharide totally, pure target DNA was obtained and false negativity of PCR reaction was avoided effectively. ⑵Specific primers (CQULAF3/LAR3) which have good specificity, sensitivity and stability for HLB detection, were designed based on published sequence encoding a ribosome protein gene and detection system of DNA molecule with rapidness, accuracy, stabilization have been developed. ⑶CQULAF4/LAR4 primers and TaqMan probe were designed based on the sequence of specific target fragment of HLB pathogen. Then HLB were detected by the Real time Quantitative PCR technique. It is more sensitive, simpler, faster but more expensive than the conventional PCR. ⑷Candidatus Liberibacter asiaticus is uncultured G-bacterial species. The whole DNA of both the bacteria and the citrus plant is not ideal positive control for its short storage time. Recombined plasmid of HLB target fragment not only was used as ideal positive control with safty but also can be used as a norm for quantitative detection of Ca. L. asiaticus. ⑸The adding of macromolecular stabilizer (MS) can promote the stabilization of pre-mixed PCR reagents. By vaccum freeze-drying treatment, it can be preserved at least six months at room temperature. Conclusion: Detection system of Candidatus Liberibacter asiaticus with rapidness, accuracy, and stabilization was established. Detection Kit based on detection system as a practical tool can apply to disease diagnosis for establishment of disease-free area of citrus production, identification of citrus diseases with suspicious lesions, and precautionary monitoring of HLB dynamically.