Improved virus detection and identification for the Wine Grape Certification Scheme
ARC Plant Protection Research Institute. Plant Pathology and Microbiology
The South African Wine Grape Certification Scheme only tests for the most important of the total range of viruses infecting Vitis worldwide. In this project, we intended expanding the number of viruses that can be tested within the certification scheme. We developed a protocol in which vines can be tested via multiple parallel PCR systems, each capable of detecting a range of viral species within a specific genus, along with some virus specific systems. Should a requirement exist for the identity of the individual virus species to be determined from genus wide PCR system this will be done by Illumina sequencing of pooled preparations of the amplicons from the PCR’s of any given vine, and by doing a number of such vines in parallel by indexing such amplicon pools separately. We have implemented PCR to the following genera to achieve this: Tombusviruses, Maculaviruses, Marafiviruses, Geminiviruses, Reoviruses, Nepoviruses (clades A, B, and C), Vitiviruses, Foveaviruses, Trichoviruses, Vealriviruses, Ampeloviruses, Closteroviruses, Ilarviruses, Alfamoviruses, Cucumoviruses, Potexviruses, Badnaviruses, Potyviruses, Tobamoviruses and Luteoviruses. While the protocol was not be evaluated against all viruses of grapevines, it is theoretically capable of detecting 41 viruses reported to infect this host. Because of the unexpected phenomenon of index-leaching during Illumina sequencing, it was necessary to develop a method of determining the positive/negative threshold for the systems along with a protocol for read analysis. We tested the protocol successfully on a number of vines from nuclear material to symptomatic field-collected material. The protocol and appropriate reagents was transferred to the Virus diagnostic unit of the ARC-PPRI for routine testing of viruses of grapevines. It is anticipated that utilisation of this system by the Wine industry to test nuclear material, will lead to the production of vine planting material of the highest virus-free status in the world, and will have a positive impact on South African winegrape production which unfortunately will be difficult to quantify.
Wayland, J, Jooste, A E C, Pietersen, G. 2015. The optimization of a high-throughput sequencing-based diagnostic system for the detection of viruses of grapevines. Paper presented at Virology Africa. 30 November – 3 December. Cape Town, South Africa.
Walsh, H A, Pietersen, G. 2013. Rapid detection of Grapevine leafroll-associated virus type 3 using a reverse transcription loop-mediated amplification method, Journal of Virological Methods, Mnth December v. 194 (1-2) (p. 308-316)