|Next Generation Sequencing for the optimal detection of viral pathogens in Grapevine|
M. AL RWAHNIH (1), S. Daubert (2), D. Golino (1), A. Rowhani (1). (1) University of California-Davis. Department of Plant Pathology, Davis, CA, U.S.A.; (2) University of California, Department of Plant Pathology, Davis, CA, U.S.A.
A bioassay is the regulatory standard used to determine the viral phytosanitary status of commercial grapevine propagation material in the U. S. and many other countries around the world. That test is based on the symptoms developed in the field by specific indicator host plants that are graft inoculated from the vines being tested. We compared the bioassay against Next Generation Sequencing (NGS) analysis of grapevine material. NGS is a laboratory procedure that catalogs the genomic sequences of the viruses and other pathogens extracted as DNA and RNA from infected vines.
NGS analysis was found to be superior to the standard bioassay in detection of viruses of agronomic significance, including virus infections at low titers. Unlike the bioassay, NGS was not effected by environmental conditions, and was effective in the detection of asymptomatic viral strains. NGS was also found to be superior to the bioassay in its accuracy and comprehensiveness, and in the cost of its analysis. Because the analysis can be completed in a number of weeks, as opposed to years for the bioassay, NGS would also be preferred for the discovery and characterization of novel, uncharacterized viruses.
Grapevine reovirus (GReV) has been discovered in grapevine from California by deep sequencing analysis. The source plant (<i>Vitis vinifera</i>) was a Cabernet Sauvignon grapevine (accession CS-Tok3). The GReV genome was characterized from a total nucleic acid extract of bark scrapings that was enriched for double-stranded RNA. The viral genome was found to make up 18% of the deep sequencing reads from this extract. The sequence homologies of its genomic components with those of other phytoreoviruses ranged from 66 to 30% for Raspberry latent virus, and from 57 to 22% for <i>Rice ragged stunt virus</i>, indicating that the novel grapevine reovirus is a distinct viral species.