Application of high-throughput sequencing to detect residual virus and viroid infections in South African nuclear grapevine material

Abstract The use of high-throughput sequencing technology (HTS) has allowed for greater elucidation of viruses infecting grapevine, which number around 100. Many of these viruses can result in disease—it is therefore vital to the industry that it be supplied with virus-free (VF) plant material. This is generally done through grapevine certification schemes. In this study, 97 nucleus grapevine plants of the South African Virus Improvement Association (VIA) were subjected to total RNA extraction and high-throughput sequencing using the Ion Torrent platform to determine the presence of any viruses and viroids. Nucleus plants are the initial source of all propagation material of Vitis clones in South Africa, and are subjected to a virus elimination process, tested for several disease-inducing viruses, and maintained under vector-excluded conditions. Of the 97 nucleus accessions, 21 were free of any detectable viruses or viroids. Five viruses were detected in eight nuclear accessions; grapevine Rupestris stem pitting-associated virus was identified in six nuclear accessions, while grapevine fleck virus, grapevine Syrah virus 1, grapevine leafroll-associated virus 4, and grapevine Rupestris vein feathering virus were identified in one nuclear accession each. Four viroids and one putative viroid were identified in 76 accessions by HTS analysis. Hop stunt viroid was identified in 63 of the 97 samples, followed by grapevine yellow speckle viroid 1, identified in 52 samples, grapevine yellow speckle viroid 2, identified in 17 samples, and Australian grapevine viroid in three samples. The putative viroid grapevine hammerhead viroid-like RNA was identified in two samples. All virus and viroid infections were confirmed using virus/viroid-specific RT-PCR and Sanger sequencing. These findings highlight the need for more diverse testing virus/viroid detection techniques in nuclear plants of the grapevine certification scheme, and suggest that some viruses, especially GRSPaV, and viroids are recalcitrant to currently employed virus elimination techniques.