Developing molecular diagnostic tools for large-scale detection of Vitiviruses and study on their genetic population structure and transmission in grapevine
Résumé
Eight sets of degenerate primers from the RdRp gene were developed for qualitative RT-PCR simultaneous detection of Grapevine vitiviruses GVA, GVB, GVD, GVE and GVF. Other species-specific primers were designed from two different genes for an accurate identification of isolates to test their genetic variability. The first were developed from the RdRp gene, whereas the second from different sequences of the entire CP of several isolates that are present in the gene bank. Moreover, trials of transmission of GVD, GVE and GVF were performed under screen house conditions to test their transmissibility by Planococcus ficus. The study has shown that the developed RdRp primers were able to detect up to 100% of the positive control samples, proving their reliability in preliminary first screening of vitiviruses in general. However, species-specific primers from the same gene were less successful.
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The use ofspecific primers from the CP gene provided information on the high genetic variability of GVA, GVB and GVE, while GVD and GVF had the most conserved CP sequences of the isolates. They also demonstrated that the clusters obtained from the phylogenetic trees’ analyses present a geographical grouping of isolates. The transmission trials resulted in the transmission of GVE by P.ficus.