Use of the Coat Protein (CP) and Minor CP Intergene Sequence to Discriminate Severe Strains of Citrus tristeza virus (CTV) in three U.S. CTV Isolate Collections

A rapid assay is needed to distinguish potentially 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 (~91-99bp) 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 (T30-like genotype) and decline isolates (T36-like genotype). Two assays were developed to differentiate such isolates: i) multiplex one-step real-time RT-PCR; ii) restriction fragment length polymorphism (RFLP) analysis. The real-time assay consisted of broad spectrum detection using a universal primers/Taqman probe (Cy5-labeled) and a primers/MGB-TaqMan probe (FAM-labeled) specific for VT and T3 genotypes of CTV. The multiplex real-time assay simultaneously detected of all CTV isolates and differentiated VT and T3 genotypes in our tests. The RFLP assay is based on primers which amplified a target sequence containing unique restriction DdeI or SspI sites. The DdeI restriction site was conserved among VT and T3 genotypes and absent in T30 and T36 genotypes. In contrast, the SspI restriction site was conserved in mild isolates and absent in the severe strains. Both assays were validated with a panel of local and international CTV isolates from different CTV collections and using natural or artificial combined infections. Both new assays differentiated between genetically and biologically different CTV strains.