Technical Yearbook 2023

material tested for trueness of type of the cultivar/clone and tested free of specific viruses (figure 1). Accurate, sensitive virus diagnosis is a critical component for effective control of grapevine viruses through either quarantine or certification. Widely accepted techniques like ELISA and PCR, and the more recently introduced high-throughput sequencing (HTS) tests, are commonly applicable in practice. ELISA is a technique that makes use of antibodies to detect viruses. The ELISA protocol is explained in figure 2. It is a robust technique, requiring relatively cheap apparatus and reagents, and is capable of being used to test thousands of samples. It is therefore very widely used in certification schemes where large numbers of tests of propagation material are required. It however lacks the sensitivity of both PCR and HTS. The latter two techniques are relatively complex and are not expanded upon in this article. Furthermore, they range from moderately expensive (PCR) to very expensive (HTS), and are more commonly utilised at the apex of certification schemes (nuclear material) or in quarantine settings. During the past five years Winetech has funded two projects, conducted at Stellenbosch University, addressing control in propagation material of a locally found virus, grapevine fleck virus (GFkV) and in a second instance of a virus of quarantine importance, grapevine pinot gris virus (GPGV). The first project was also co-funded by SATI. Validation of ELISA for the detection of grapevine fleck virus (GFkV) in the Vine Improvement Organisation (VIA) grape certification scheme In the Vine Improvement Organisation (VIA) grape certification scheme, all rootstock and scion must be visually

free from virus-like diseases known to occur in South Africa. This includes grapevine fanleaf disease, grapevine fleck disease, grapevine leafroll disease, grapevine corky bark disease, grapevine stem pitting disease, grapevine stem grooving disease, grapevine vein necrosis, grapevine vein mosaic and shiraz disease, or test negative for specific viruses associated with these diseases. All viruses of the fleck complex, grapevine fleck virus (GFkV) and a further few related viruses, can be identified with reasonable certainty by grafting them onto the hardwood indicator species, Vitis rupestris St George. This indicator vine elicits symptoms of grapevine fleck disease. These include mild foliar symptoms such as vein clearing and a mosaic pattern of translucent spots in older leaves, through to wrinkled, twisted, and upwards curling leaves with intense flecking. Stunting may occur following infection by severe strains. Hardwood indexing however is time consuming, as symptoms need to be observed over two to three years. Furthermore, it is an expensive technique, requiring vector-free greenhouse space, and regular maintenance and recording of symptoms by skilled personnel. The technique is also affected by environmental factors and is seasonal with symptoms often only observed for short periods of time. The availability of commercial GFkV-specific ELISA tests internationally prompted the VIA to consider testing for GFkV by ELISA. Only ELISA tests that have been evaluated for local use, and approved by Department of Agriculture, Land Reform and Rural Development (DALRDD), may be used within the VIA certification scheme. The sensitivity of four commercially available GFkV ELISAs were determined, along with the relative specificity (ability to

FIGURE 3. Graph of ELISA results of three grapevine fleck virus-infected vines, representing a high, medium and low level of the virus in the plant, tested at different times of the growing season in order to determine the best time to sample vines for tests.

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TECHNICAL YEARBOOK 2023