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United States Department of Agriculture

Agricultural Research Service

Research Project: CHARACTERIZATION & EPIDEMIOLOGY OF CITRUS TRISTEZA VIRUS & OTHER INVASIVE & EMERGING GRAFT-TRANSMISSIBLE DISEASES OF CITRUS IN CALIFORNIA Title: Use of the Coat Protein (CP) and minor CP Intergene Sequence to Discriminate Severe Strains of Citrus tristeza virus

Authors
item Saponari,, Maria - INSTIT. DI VIROLOGIA
item Yokomi, Raymond

Submitted to: International Organization of Citrus Virologists Proceedings
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 18, 2008
Publication Date: May 14, 2011
Citation: Saponari,, M., Yokomi, R.K. 2011. Use of the Coat Protein (CP) and minor CP Intergene Sequence to Discriminate Severe Strains of Citrus tristeza virus. In: Proceedings of the International Organization of Citrus Virologists. p.43-57.

Interpretive Summary: The biological diversity of Citrus tristeza virus (CTV) is attributed to complexity and variability of its genome. Symptoms produced by CTV strains range from asymptomatic to induction of severe stem pitting in the scion regardless of rootstock. The objective of this study was to develop rapid and sensitive assays to distinguish mild (non-economic) versus severe (economic) CTV isolates. A sequence analysis was performed on the coat protein (CP) and the minor coat protein (CPm) intergene sequences from different CTV isolates and revealed that orange stem pitting (OSP), grapefruit stem pitting (GSP) and seedling yellows (SY) isolates shared a common sequence absent in mild isolates with a T30-like genotype and decline isolates with a T36-like genotype. A multiplex one-step real-time reverse transcription polymerase chain reaction (RT-PCR) assay and a restriction fragment length polymorphism (RFLP) analysis were developed to differentiate these isolates. The assays provide independent confirmation of CTV strain identification. The multiplex real-time RT-PCR assay consisted of universal probe (Cy5-labeled) to detect all CTV isolates and a specific probe (FAM-labeled) to detect severe strains. The RFLP assay was based on primers which amplified a CP sequence which is digested by DdeI enzyme and conserved among the OSP, GSP and SY isolates but not in mild and decline isolates. In addition, the SspI enzyme digested the CP amplicon of mild isolates but not severe isolates. Both assays were validated using natural or artificial combined infections from a panel of known CTV isolates from virus collections at the Central California Tristeza Eradication Agency, Tulare, CA, and USDA, ARS, Parlier, CA and Beltsville, MD. In conclusion, both the real-time RT-PCR assay and the RFLP analysis successfully differentiated between biologically different CTV strains. The real time RT-PCR assay is now being used to evaluate CTV genetic diversity in central California and Florida.

Technical Abstract: A rapid assay is a needed to differentiate mild vs severe strains of Citrus tristeza virus (CTV). Multiple alignment performed on the coat protein (CP) and the minor coat protein (CPm) intergene sequences (~80-100 bp) from different CTV isolates revealed that severe strains generally associated with orange stem pitting (OSP), grapefruit stem pitting (GSP) and seedling yellows (SY) share a conserved sequence which is absent in mild isolates (T30-like genotype) and decline isolates (T36-like genotype). A multiplex one-step real-time reverse transcription polymerase chain reaction (RT-PCR) assay and a restriction fragment length polymorphism (RFLP) analysis were developed to differentiate such isolates. The assays are independent and provide confirmation of strain identification. The multiplex real-time RT-PCR assay consisted of universal primers/Taqman probe (Cy5-labeled) and primers/MGB-Taqman probe (FAM-labeled) to detect severe isolates. The assay simultaneously detected all CTV isolates and differentiated between mild and severe strains in our tests. The RFLP analysis was based on primers which amplified a target sequence containing unique DdeI or SspI restriction sites. The DdeI site was conserved among the OSP, GSP and SY isolates and absent in mild and decline isolates. The SspI site, in contrast, was conserved in mild isolates and absent in the severe strains. Both assays were validated using natural or artificial combined infections from a panel of known CTV isolates from the Central California Tristeza Eradication Agency, Tulare, CA and the USDA, ARS, Parlier, CA and Beltsville, MD locations. In conclusion, both assays successfully differentiated between biologically different CTV strains.

Last Modified: 10/31/2014
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