Comparison of Next-Generation Sequencing Versus Biological Indexing for the Optimal Detection of Viral Pathogens in Grapevine

Abstract

A bioassay is routinely used to determine the viral phytosanitary status of commercial grapevine propagation material in many 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. NGS was also found to be superior to the bioassay in its comprehensiveness, the speed of its analysis, and for the discovery of novel, uncharacterized viruses.

Molecular characterization of GSyV-1 and GLRaV-3 and prevalence of grapevine viruses in a grape-growing area

“…Some of the agents associated with GRWD are Grapevine rupestris stem pitting-associated virus (GRSPaV) and Grapevine virus A and B (GVA and GVB, Betaflexiviridae) (Martelli, 2014). Grapevine Cabernet Sauvignon reovirus (GCSV, Reoviridae) was recently discovered infecting grapevines in the USA (Al Rwahnih et al, 2015).…”

“…The reaction data were analyzed in terms of presence/absence assays and graphically, using the StepOne Software program v.2.3 (Applied Biosystems), by determining the Cq (quantification cycle). The primers and probes used for viruses and viroid detections by real-time RT-PCR have been previously described (Osman et al, 2007;Bianchi et al, 2015) or designed in this work for GCSV, primers Ctg 468F (5'ACGTTGGAT-CAACTAGCCGAAG3') (Al Rwahnih et al, 2015) and GCSV-CS103r…”

Screening of some Croatian autochthonous grapevine varieties reveals a multitude of viruses, including novel ones

“…Another advantage is that NGS may provide insights into the overall sanitary status of a particular vine, which is especially helpful when plants harbor multiple viruses of economic importance. In the case of grapevines, subjecting one sample to NGS is cheaper, faster, and more reliable than using a panel of other pathogen detection approaches [2].…”

Extracción y purificación de dsRNAs largos de plantas infectadas con virus y hongos; aplicación en la detección e identificación de virus

“…Recently, new technologies for virus detection and identification such as next-generation sequencing (NGS) have become available. NGS is a method with great potential for the detection and identification of plant viruses (Al Rwahnih et al, 2015;Bonham et al, 2014;Ho and Tzanetakis, 2013). For any nucleic acidbased method for RNA virus detection, efficient and practical RNA extraction protocols are needed.…”

“…Recientemente han surgido nuevas tecnologías para la detección e identificación de virus, como la secuenciación de próxima generación (NGS). La NGS es un método con gran potencial para la detección e identificación de fitovirus (Al Rwahnih et al, 2015;Bonham et al, 2014;Ho y Tzanetakis, 2013). En todo método basado en áci-do nucleico para la detección de virus ARN se necesitan protocolos de extracción de ARN eficaces y prácticos.…”