First report of grapevine leafroll-associated virus 9 in Spain.

Abstract

Grapevine leafroll is one of the most important viral diseases of grapevines (Vitis vinifera). To date, 11 grapevine leafroll-associated viruses (GLRaVs) from the family Closteroviridae have been described. A recent taxonomic revision, however, suggests the existence of only five valid species (GLRaV-1, -2, -3, -4, and -7), whereas GLRaV-4, -5, -6, -9, -Pr, -De and Grapevine leafroll-associated Carnelian virus (GLRaCV) form a uniform phylogenetic clade and represent strains of GLRaV-4 (Martelli et al., 2012). In Spain, IMIDA performs all sanitary tests for certification of candidate clones from all Spanish autonomous communities. The detection of GLRaV-4, -5, -6, -7 and -9 relies on the use of commercial ELISA kits (Bioreba, Switzerland), and the confirmation of positive results by real-time RT-PCR to assess the health status of the clones. In 2011, GLRaV-9 was detected using DAS-ELISA and TaqMan in a candidate clone of Mantua (Man086), a traditional cultivar from Extremadura. To confirm the presence of this virus, conventional RT-PCR and sequencing were performed using total RNA from Man086 as template and specific primers designed in the HSP70h domain of GLRaV-9 (Alkowni et al., 2004). Pairwise comparisons of the corresponding sequence (351 bp, GenBank accession No. KC660077) with GLRaV-9 sequences available in GenBank showed the highest nucleotide (97.2%) and amino acid (95.7%) identities with an isolate from Washington state, USA (EU252530). Another set of primers was designed in the RNA-dependent RNA polymerase domain based on accession AY297819 from California, USA. Alignments between the sequence obtained (533 bp, GenBank accession No. KC660076) and AY297819 showed 96.1% and 97.2% nucleotide and amino acid sequence identities, respectively. This is the first report of GLRaV-9 in Spain.