Identification of potyviruses infecting vanilla by direct sequencing of a short RT-PCR amplicon

Abstract

A simple one-tube one-step RT-PCR assay with degenerate primers followed by direct sequencing of a 327 bp coat protein gene fragment was used to identify the potyviruses infecting vanilla. With this technique, unambiguous species allocation was achieved for 34 potyvirus-infected vanilla samples collected in the Indian Ocean and the Pacific areas between 1997 and 2005. Virus identification relied on blast homology and nucleotide identities of 162 to 327 nt fragments with known potyvirus sequences. Species allocation was confirmed by neighbour-joining of the 149 nt common to 32 vanilla sequences and 51 known potyviruses. Subject to further identification, these data revealed four additional Potyvirus species that may infect vanilla: Bean yellow mosaic virus, Cowpea aphid-borne mosaic virus, Ornithogalum mosaic virus and Wisteria vein mosaic virus. The procedure was rapid, cost-effective, easy to use and showed a good taxonomic discriminating value. It also enabled the identification of potyviruses in adjacent weeds and should thus aid the understanding of outbreaks of potyviruses occurring in varied epidemiological circumstances.