|Saponari,, Maria - INSTITUTE DI VIROLOGIA|
Submitted to: International Organization of Citrus Virologists Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: August 20, 2007
Publication Date: October 17, 2007
Citation: Saponari,, M., Yokomi, R.K. 2007. Characterization of Citrus Tristeza Virus Isolates by Single-strand Conformation Polymorphism Analysis of the Coat Protein Gene. In: Proceedings of the 17th International Organization of Citrus Virologists, October 22-26, 2007, Adana, Turkey. p. 139 Technical Abstract: A method is needed to rapidly assess Citrus tristeza virus (CTV) strains and to identify mixed populations in tristeza-infected trees. Single-strand conformation polymorphism (SSCP) can detect point mutations in DNA fragments and determine the structure of viral populations. Previous reports utilized SSCP to detect CTV intra population variability and follow its variation after aphid transmission or host change. Our objective was to standardize a SSCP protocol for typing of CTV isolates of unknown biological activities. Amplicons from the coat protein gene from reference CTV isolates with a SY-, T30-, VT -and T36-like genotype were subjected to SSCP analysis. Each reference isolate had a unique, simple and repeatable SSCP profile and suggested that the protocol could separate phylogenetically and biologically different strains. This hypothesis was tested with different isolates from our collection and that of the Exotic Citrus Pathogen Collection, USDA, ARS – Beltsville, MA. The resultant SSCP profiles and nucleotide sequence analyses confirmed that: (i) the use of a large amplicon (672bp) was not sensitive to single point mutations; (ii) isolates with 97-99% nucleotide sequence identity showed the same SSCP profile; (iii) isolates with the same SSCP profile were phylogenetically and biologically related. Examination of single or mixed infections and their aphid transmitted sub-isolates by SSCP readily showed when the aphid separated the mixture. Aphid-transmitted sub-isolates from single infections had SSCP profile identical to the parent isolate. In conclusion, SSCP analysis of the CP gene differentiated CTV strains and revealed whether infection was by a single or mixture of CTV strains.