| 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 and Brazil. 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; or enhancement of quarantine regulations in non-quarantine area to prevent the introduction and establishment of this disease, therefore, an accurate, rapid and robust detection method is needed for a regulatory response and effective management of HLB through the detection and removal of infected trees and development of a Ca. Liberibacter-free nursery system. The first part of my work is to develop a detection system to fulfill those requirements above: two real-time polymerase chain reaction (RTi-PCR) methods were developed and evaluated, using the primer pairs CQULA04F/CQULA04R and TaqMan probe CQULAP1 designed from a species-specific sequence of the rplJ/rplL ribosomal protein gene. The specificity and sensitivity of the two protocols for detecting'Candidatus Liberibacter asiaticus'in total DNA extracts of midribs collected from infected citrus leaves with symptoms in Guangxi municipality, Jiangxi province and Zhejiang province, were compared with the conventional PCR system established before. Sensitivities using extracted total DNA were 439.0 (1.30×105 CNμL-1), 4.39 (1.30×103 CNμL-1) and 0.44 fgμL-1 (1.30×102 CNμL-1) for conventional PCR, TaqMan and SYBR Green I (SGI) real-time PCR, respectively. SGI RTi-PCR was the most sensitive, but its specificity needed to be confirmed by running a melt-curve assay. The TaqMan real-time PCR assay was rapid and had the greatest specificity. The optimized molecular-based assays should prove useful for pre-symptom diagnosis of HLB disease, monitoring and identification of'Ca. L. asiaticus', and field epidemic regulation.The RTi-PCR is important because it is not only an ultra-sensitive, fast, specific and robust detection technology, but also can provide us a new method to explore the unknown field of this disease and give us a quantitative understanding. Without pure culture of the HLB pathogen, the fundamental research can still be propelled by this quantitative method. TaqMan Probe RTi-PCR system is the premier tool in the second part of work because of its robustness and high specificity comparing with the other system SYBR Green I Fluorescent Dye RTi-PCR, and with this reliable technique, the following work was accomplished:①uneven distribution of HLB bacteria all over a citrus plant;②dynamic change of HLB bacteria population in leaf midrib throughout a year;③reliability of different symptom-based diagnosis;④dynamic change of risk degree caused by poisonous psyllid vector throughout a year;⑤a model for the transmission mechanism vectored by psyllid based on anatomic research. These quantitative results support many conclusions or hypotheses gained by former researchers such as the reliability of symptom-based diagnosis part, and they also challenge some former ideas, for example, it was believed that the HLB pathogens did not reproduce themselves inside the psyllid, but the quantitative results shows that the great difference of bacteria titer between different psyllids can not be explained unless admitting the reproduction in psyllid.According to"the 2nd International Citrus Canker and Huanglongbing Research Workshop"(Orlando, Florida, 2005), the hotspot and orientation of citrus HLB research could be summarized in the following 15 aspects: economics; alternative hosts; detection of disease and vector; characterization taxonomy of HLB; resistance and breeding; culturing HLB; pathogen/vector/host interactions; chemical control; biological control; cultural control; epidemiology; transgenics; genomics; vector biology; fruit yield and quality. Of all the 15 orientations, this research covered at least 4 aspects as follows: molecular detection methods (detection of disease and vector); assessment of citrus relatives for resistance/susceptibility (resistance and breeding); pathogen population in citrus and psyllid vector (pathogen/vector/host interactions); effect of insect population dynamics on disease spread (epidemiology). However, the actual effect of this research should be far more extensive than those aspects listed above. |
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