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Title:   Author  item
                                            LI, WENBIN - USDA APHIS BELTSVILLE MDitem
                                            LI, DAYAN - HARVARD UNIV CAMBRIDGE MAitem
                                            TWEIG, ELIZABETH - USDA APHIS BELTSVILLE MDitem
                                            Hartung, Johnitem
                                            LEVY, LAURENCE - USDA APHIS BELTSVILLE MD
  Submitted to: Plant Disease
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 1/15/2008
                                    Publication  Date: 6/11/2008
                                    Citation: Li, W., Li, D., Tweig, E., Hartung, J.S., Levy, L. 2008. Optimized Quantification of Unculturable Candidatus Liberibacter Species Causing Citrus Huanglongbing in Host Plants by Real-Time PCR. Plant Disease. 92(6):854-861.Interpretive Summary: Citrus ‘huanglongbing’ or ‘greening’ disease is the most significant disease threat to the global citrus industry, and it has recently become established in the principle citrus producing areas of both Brazil and the United States. The pathogen is a bacterium that so far has not been isolated in the laboratory, and as a result there are no quantitative data on the pathogen in citrus hosts. We have previously developed a quantitative assay for the pathogen based on the detection of DNA sequences specific to the pathogen. In this work we show that the assay can be applied on citrus tissues from various species collected worldwide and that the sensitivity of the assay varies with both the species and plant tissue tested and with the geographical origin of the samples tested. These variations in the assay results have been summarized statistically and methods and equations are presented which can be used to accurately quantify the pathogen in any citrus host or tissue of any origin.  Our results will be useful to a growing community of researchers interested in this important plant disease problem.
                                Technical Abstract: Citrus huanglongbing (HLB) caused by the phloem-limited and psyllid-vectored Candidatus Liberibacter spp. is a destructive disease of citrus that is rapidly increasing in importance. The disease was recently reported in the principle citrus producing areas of São Paulo, Brazil in 2004 and of Florida in 2005. A variety of methods have been developed to confirm a symptom-based disease diagnosis or for the detection and/or identification of the pathogen, but no quantitative information has been available on either the pathogen or host vector interactions because the pathogen remains unculturable in artificial media. We have previously developed a quantitative PCR-based assay for Ca. Liberibacter spp. In this study, we evaluated the effects of sample composition on quantification of the pathogen in citrus plants by TaqMan® real-time PCR. Various standard curves were established using cloned plasmids containing target DNA from the pathogen and total DNA samples from field-grown HLB-infected citrus plants. Regression analysis showed that a standard curve established with DNA extracted from naturally infected field-grown plants was more accurate than a standard curve constructed from plasmids containing the amplification targets as cloned inserts. Non-target DNA and putative PCR inhibitors from citrus plants decreased the sensitivity and the amplification efficiency of real-time PCR when plasmids provided the template target in ‘spiked’ healthy citrus extracts. This effect varied among plant tissues, citrus species and geographic locations. Based on these sample effects, a universal standard curve has been established for quantification of the pathogen in various tissues of different citrus species planted in geographically different locations. Sample storage at 4o C for two months prior to PCR assay did not affect subsequent quantification of the pathogen. The validated quantitative real-time PCR method and the universal standard curve will be very useful for studies of host-pathogen interactions, epidemiology and in the development of control strategies for the disease.


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