Detection of grapevine leafroll associated virus 3 in South African rootstock clones

Grapevine leafroll disease (GLD) is recognised as a global, economically significant viral disease of grapevines. The complex of viruses associated with GLD are termed the Grapevine leafroll associated viruses (GLRaVs) and are sequentially numbered in order of discovery. The main etiological agent of GLD is GLRaV-3. In South Africa, a grapevine certification scheme exists to ensure grape growers have access to leafroll-free planting material. Unlike red Vitis vinifera cultivars, rootstocks infected with GLD are asymptomatic and cannot be identified through visual diagnosis. To complicate matters even further, GLRaV-3 is thought to be poorly detected in rootstock tissue due to uneven distribution of virus and erratic viral titres. Difficulties in GLRaV-3 detection could result in asymptomatic carriers of GLD being grafted onto healthy vines. To assess the supposed poor detection of GLRaV-3 in rootstocks, a survey was conducted in the Western Cape, South Africa, where various rootstock types and corresponding scions were sampled from 60 vines located in former mother blocks. Samples were tested using a reverse transcription-PCR directed against the conserved GLRaV-3 helicase gene. Detection of GLRaV-3 was significantly higher in scion (83%) than in corresponding rootstock tissue (15%). Several graft transmission experiments were conducted to gain a better understanding of the nature of the poor detection of GLRaV-3 in infected rootstocks. Detection of GLRaV-3 varied depending on the rootstock cultivar used, with Richter 110, Paulsen, and Salt Creek/Ramsey rootstocks appearing to be the least resistant to GLRaV-3 infection and subsequent replication. The variable GLRaV-3 detection in 101-14 Mgt and Richter 99 were suggested to be the result of genetic heterogeneity amongst rootstock clones. The possibility of passive movement of virus in phloem tissue cannot be ruled out and further studies will need to be done to understand this phenomenon. The optimal time of sampling and tissue region of rootstocks most suited for GLRaV-3 detection was investigated using real-time reverse transcription-PCR. Detection of GLRaV-3 was found to be significantly higher in basal tissue than in apical tissue of rootstocks Salt Creek/Ramsey and Richter 110. Rootstock US 8-7 was used as a representative control for V. vinifera and, with the exception of R110, was observed to have a significantly higher detection rate over any other rootstock cultivar. The most suitable time of sampling was determined to be late summer/beginning of autumn. A colourimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay, aided by a crude RNA extraction protocol, was adapted from Walsh and Pietersen (2013) and optimised for possible on-site GLRaV-3 detection in rootstocks. The assay was shown to be rapid (70 minutes), specific, more sensitive than conventional reverse transcription-PCR, and holds promise for detection of the virus in rootstocks. This comprehensive study on GLRaV-3 in rootstocks in South Africa will help bridge the gap in knowledge on the largely unknown GLD dynamics seen in rootstocks.Dissertation (MSc)--University of Pretoria, 2019.Microbiology and Plant PathologyMScUnrestricte