Further Characterization studies on Citrus Tristeza Virus (CTV) isolates collection from Mediterranen countries and their interaction with citrus viroids

The biological properties of a collection of citrus tristeza virus (CTV) isolates from 13 Citrus-growing countries were investigated, these lasts were mostly from the Mediterranean area that was thought for long time free from severe CTV isolates. These study confirmed the presence of such isolates in the area, as a lot of biologically indexed isolates were able to produce even stem pitting on Madam vinous Sweet Orange (SwO) considered the most dangerous strain. The symptoms expressed on the standard set of CTV indicator plants; Mexican lime (M.L), Duncan grapefruit (Gft), sour orange (SO) seedlings, Madam Vinous SwO grafted on sour orange, sour orange (SO) seedlings, and Madam Vinous SwO seedlings enables the establishment of the various Biogroups that are present in the Mediterranean Area ranging from the severe Biogroups 4 and 5 mostly in the Middle East region to and the Mild Biogroup 1 in other regions. Interestingly, all the isolates belonging to the Biogroups 4 and 5 gave a positive reaction with the MCA13 MAbs that was produced to discriminate between mild and severe CTV isolates in Florida, but it failed to detect others elsewhere. Such rapid tests are of important concern and are needed to identify isolates that have potential economic impact in commercial citrus groves and to adopt a sound CTV management strategy. But discriminative analysis needed have to be, as rapid as reliable, and this was another aim of this work; finding potential molecular markers that could be used for sensitive and quick identification of virulent CTV isolates through the study of a Mediterranean CTV collection. For this purpose 27 CTV isolates were compared for symptom expression and by conventional Single Strand Conformation Polymorphism (SSCP) and Capillary Electrophoresis CE-SSCP analysis of four 3àƒ àƒ à,¢ àƒ àƒ à,² terminal genes (p18, p20, p27 and p23) and the analysis genetic variation by sequencing. The conventional SSCP is a relatively robust and easy to perform procedure, and is a good primary molecular differentiation for CTV isolates, but it has as main limitation the non quantification of profile differences in terms of the genetic distance between the corresponding DNA fragments providing therefore only a qualitative picture of the variation. CE-SSCP, as alternative to gel slab techniques has become a promising alternative to gel based SSCP methods, presents the advantages of high sensitivity, high specificity and is easy to perform. The technique was able to distinguish between a set of already known sequence differences and the repeatability of the results within and between different runs was also evaluated. A great diversity of SSCP profiles was observed among isolates for the genes studied for both the methods and some isolates of the same biogroups displayed the same haplotype, with both conventional and F-SSCP analysis, mostly for the p20 gene, even if they were from different geographical origin. Other field CTV isolates have to be phenotyped and subjected to other conventional and Fluorescent SSCP tests so that the proposed models can be verified, confirmed or modified.