|Yokomi, Raymond - Ray|
Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/13/2009
Publication Date: 3/6/2010
Citation: Yokomi, R.K., Saponari, M., Sieburth, P.J. 2010. Rapid Differentiation and Identification of Potential Severe Strains of Citrus tristeza Virus by Real-Time Reverse Transcription Polymerase Chain Reaction Assays. Phytopathology. 100:319-327. Interpretive Summary: Citrus tristeza virus (CTV) is transmitted and spread by aphid vectors. Management of CTV in central California is by survey, detection and removal of all CTV-infected citrus trees by a grower-funded program implemented by the Central California Tristeza Eradication Agency (CCTEA). Disease incidence in some areas has reached levels where spread is occurring faster than the capacity to suppress CTV by eradication. Research conducted by ARS has shown that most of the CTV isolates in central California have a mild genotype which is not virulent in virus index tests in the greenhouse. Therefore, a need exists to rapidly identify severe strains of CTV and target trees infected with these strains for removal. A multiplex quantitative real-time polymerase chain reaction (mqPCR) was developed to distinguish potentially severe isolates from mild isolates simultaneously in one reaction tube. To achieve strain differentiation in a multiplex reaction, three different probes were successfully mixed and the assay optimized so competition between isolates did not affect the ability to differentiate each isolate in the mixture at different dilutions. Several methods were also compared to evaluate preparation of the sample’s genomic template prior to PCR. High quality templates were obtained by three different methods. The efficiency of the multiplex assay was evaluated with diverse CTV isolates from an international collection of CTV strains. Subsequently, the multiplex assay was validated with various field isolates from California and Florida. The mqPCR assay can be completed in 1-2 days. This assay is now being used by the CCTEA, in collaboration with ARS, to identify potentially severe CTV strain sources for removal. Growers have endorsed this revised CCTEA program for removal of severe sources of CTV.
Technical Abstract: A multiplex Taqman®-based real-time reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to detect all strains of Citrus tristeza virus (CTV) and to identify potentially severe strains of the virus. A CTV TaqMan probe (CTV-CY5) based on the coat protein (CP) gene sequences was designed for universal CTV strain detection. Three strain-specific probes were developed targeting the intergene sequences between the two coat protein genes (CPi) for CTV strain differentiation in a multiplex reaction. Probe CPi-SP was designed for VT and T3 genotypes to detect potentially severe seedling yellows, orange and grapefruit stem pitting strains of CTV; probe CPi-T36 for T36 genotypes; and probe CPi-T36-NS to identify isolates in an outgroup clade of T36-like genotypes which are mild in California. Total nucleic acids extracted by chromatography on silica particles and SDS-potassium acetate or CTV virions purified by immunocapture all yielded high quality templates for real-time PCR detection of CTV. These assays successfully differentiated CTV isolates with distinct biological characteristics from California, Florida and from an international collection maintained in Beltsville, MD. The utility of the assay was validated using field isolates collected in California and Florida.