The aim of the two studies* presented were: 1) to assess the usefulness of commercial ELISA kits against local grapevine fleck virus (GFkV) isolates, validate it for routine use in the certification scheme, and submit recommendations to Department of Agriculture, Land Reform and Rural Development (DALRDD); and 2) to determine whether an emerging exotic virus, grapevine pinot gris virus (GPGV), occurs in South Africa.
Introduction
Vegetative propagation is the process of increasing the number of plants of a particular species or cultivar via asexual means. Many plants reproduce this way naturally (e.g., potatoes via tubers, or strawberries via stolons), but vegetative propagation can also be utilised by humankind. Modern viticulture is based primarily on the vegetative propagation of planting material derived from a mother plant of desirable properties using cuttings.
The main advantage of vegetative propagation methods is that the new vines contain the genetic material of only the parent from which they were derived, so they are essentially clones of the parent plant. This means that, once you have a vine with desirable traits, you can reproduce the same traits indefinitely under constant environmental conditions. This is especially important for grape growers who want to reproduce the highest-quality plants and berries that have consistent properties for use as table grapes or in winemaking. A further advantage of vegetative propagation is that plants bypass the immature seedling phase and therefore reach the mature phase sooner. This can save a lot of time and money in commercial plant production.
The main disadvantage of large-scale vegetative propagation is the danger of narrowing the biodiversity of a species. Also, if a particular plant clone is susceptible to certain diseases, there is potential to lose entire crops, as all the plants would have the same level of susceptibility. Furthermore, the virus disease status of a mother plant is passed onto most, if not all the plantlets derived through vegetative propagation, as viruses would often be systemically distributed throughout the original plant. The international trade in grape propagation has been a very common means of international spread of viruses historically, and of the more than 80 viruses infecting grapevines, most are globally distributed. Furthermore, the process of grafting grapevine scions onto rootstocks, along with vegetative propagation of grapevines, have in the past resulted in grapevine plants having multiple virus infections. Over the past three decades, grapevine vegetative propagation has however generally been accompanied by virus elimination and virus management processes to ensure healthy planting material. The most common means of grapevine virus elimination is the use of heat therapy and meristem tip culture (Figure 1).
Control of viruses in a vegetatively propagated crop such as grapevines can conveniently be divided into processes that prevent international virus spread (quarantine and phytosanitary regulation), and those that prevent local spread (certification). In the former, plant material distributed from one country to another is either placed in quarantine on entry and tested for viruses which are not known to occur in the importing country, or accompanied by a certificate from the exporting country that declares the plant material tested and free of specific exotic viruses, or stating that the exporting country does not contain those viruses. Local control of virus spread in vegetatively propagated crops, including grapes, is primarily through certification schemes that ensure the production of planting material tested for trueness of type of the cultivar/clone and tested free of specific viruses (Figure 1).
FIGURE 1. Steps in certification schemes to produce Vitis plants free of specific viruses. 1) Selection of specific Vitis cultivar maintained at 30°C for three months in vector-free phytotrons (heat therapy). 2) Cutting of a 0.24 mm section of the meristem of the heat-treated vine. 3) Growth of the meristem tissue and regeneration of new plantlets. 4) Establishment of the vine in insect-free greenhouses, called nuclear blocks. 5) Tests to confirm the absence of specific viruses. 6) Establishment of so-called foundation blocks, isolated from other vineyards from which mother blocks and nursery material are propagated for use in industry.
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.
FIGURE 2. ELISA for the detection of viruses. 1) A virus-specific antibody is stuck to the sides and bottom of a small plastic well, and all unbound antibodies are washed away. 2) Sap from a vine is added to the well. 3) If virus is present (red route), the virus will stick to the antibody. All unbound plant material is then washed away. 4) Where the virus is stuck (red route), the addition of a second antibody to the virus will cause it to stick to the free parts of the virus. This antibody is chemically coupled with an enzyme and is called a conjugate. Unbound conjugate is washed away. Where no virus is found in the well (black route) the conjugate will not stick and is totally washed away. 5) By adding the substrate of the enzyme, a colour reaction takes place in the well where the conjugate is stuck to the virus (red route) (A), but where there is no virus no colour reaction takes place (B). The colour, which can be measured, therefore shows the presence of the virus.
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 detect variants of the virus) of each in reciprocal tests against their supplied positive controls (virus-infected material) and a limited number of South African GFkV sources. No differences were observed in the specificity of three of the four supplied ELISAs to GFkV and only minor differences in sensitivity were observed between the same three kits. All three of these could therefore be utilised within the VIA scheme. The remaining commercial ELISA kit yielded very poor results and is not recommended for routine use.
The most affordable GFkV ELISA was selected for further study. We demonstrated that GFkV was a stable virus, still reacting in ELISA seven days after storage at 4°C of a macerate in a phosphate-based buffer with various additives. This buffer proved to be more effective than a Tris-based buffer to extract and stabilise GFkV for ELISA and is recommended for general use in all the ELISA systems.
It was also determined that GFkV can be detected throughout the growing season from grapevine plants but that the highest ELISA readings, and thus optimal time for sampling, is from March to June (Figure 3).
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.
We confirmed that phloem-rich tissue, either petioles and leaf veins or phloem scrapings of cane material, must be sampled and tested. In instances where cane scrapings were utilised, we demonstrated they are best extracted directly in extraction buffer, rather than pre-pulverised with liquid nitrogen. We identified petioles from basal (older) leaves as the best tissue for sampling and testing in the ELISA tests. We have shown that the ELISA is sufficiently sensitive to allow detection of one infected sample amongst five pooled together (Figure 4).
FIGURE 4. Graph of ELISA results showing the effect of mixing one grapevine fleck virus-infected vine with increasing ratios of healthy vines in pools, to determine how many vines at a time can be tested and still detect the virus. Four GFkV-infected sources were tested using two different means of preparing the extracts.
To better determine the ability of the ELISA to detect South African isolates of GFkV, we identified 80 sources of GFkV among 229 sampled cultivars or rootstocks from the local Vitis germplasm collection and determined the genetic variation among these. We confirmed the RNA sequence of the whole virus of 11 representative variants. The selected GFkV ELISA was used to confirm the ability of the ELISA to detect GFkV of all variant categories. We have also demonstrated that the ELISA does not detect the related virus, grapevine rupestris vein feathering virus (Figure 5).
FIGURE 5. ELISA values obtained for 232 individual vines of Vitis accessions selected to reflect the genetic variability of GFkV. Blue bars = individual vines containing GFkV as determined by high-throughput sequencing (HTS), grey bars = did not contain GFkV reads as determined by HTS, and black bars = contained grapevine rupestris vein feathering virus (GRVFV) as determined by HTS.
To gain a measure of the incidence and possible evidence of spread of GFkV, we analysed a total of 449 samples collected from 19 vineyards by using the Agritest GFkV ELISA. None of the samples were infected and hence no evidence of GFkV spread was obtained within this limited survey.
We have established an entire protocol for the detection of GFkV by ELISA for immediate implementation, at least at a nuclear plant level, in the VIA’s certification scheme, and have recommended to the VIA and DALRDD that the most affordable kit be utilised using the protocol established through this study.
Control of grapevine pinot gris virus (GPGV) through science-based phytosanitary regulations
Grapevine pinot gris disease associated with grapevine pinot gris virus (GPGV) is a relatively new emerging disease of grapevines, first found in Italy in 2003. Strains of GPGV are associated with chlorotic mottling and deformations of grapevine leaves and result in reduced fruit set, uneven ripening of berries, and sometimes shoot stunting or necrosis (Figure 6). Some strains of the virus however do not cause any symptoms.
FIGURE 6. Symptoms in Vitis vinifera. Examples of stunted shoot growth and chlorotic leaf mottling of leaves. All images provided by Dr. Elisa Angelini, CREA, Italy.
The disease has not been observed in South Africa and the associated virus has not been found here. However, the virus is increasingly found in countries outside of Europe after active tests for the virus were conducted in those countries. Generally, the virus in these countries is found in vines recently derived from planting material imported from GPGV-affected countries. It is now reported from various European countries, South Korea, Eastern Europe, China, Chile, USA, Canada, Uruguay, Brazil, Pakistan, and Australia (Figure 7).
FIGURE 7. European and Mediterranean Plant Protection Organisation (EPPO) map of the worldwide distribution of grapevine pinot gris virus (updated 2022-09). Yellow dots represent reported occurrence within the country.
No tests have previously been conducted to determine whether GPGV occurs in South Africa but its vector, an eriophyid mite, is reported here. As it has not been reported from here, it is considered an exotic virus to South Africa. To add it to the list of phytosanitary requirements for the import of grape material into South Africa, it is important to confirm its absence in South Africa. We did this by analysing 229 accessions of Vitis housed at the Vitis germplasm vineyard in Nietvoorbij using the next-generation sequencing (NGS) technique. We also tested 50 accessions of Vitis planting material imported into South Africa during the last 10 years, provided by three plant improvement organisations (PIOs).
GPGV was not observed in any of the 229 long established Vitis accessions housed in the Vitis germplasm collection, but was confirmed to occur in four of the 50 accessions recently imported into South Africa. The presence of this virus allowed us to attempt to optimise the GPGV polymerase chain reaction (PCR) test for this virus after the commercially available ELISA kit was found to perform sub-optimally. All vines derived from the confirmed GPGV-infected accessions were destroyed by the relevant PIOs and we still consider the South African grape industries (wine, table and raisin) to be free of this virus. As it is not possible to test all the grapevine planting material imported into South Africa retroactively, we invite any grower who observes symptoms as illustrated by Figure 6, to inform us as quickly as possible of this, as speed would be of the essence to prevent further spread. The phytosanitary authorities at Department of Agriculture, Land Reform and Rural Development (DALRRD) have been requested to consider the addition of GPGV to the list of exotic viruses which imported Vitis planting material must test free of, or be accompanied by a phytosanitary certificate from the exporting country declaring the material free of GPGV.
Conclusion
These two projects, funded by Winetech, have provided material outcomes toward improved control of viruses of grapevines in South Africa. In the first instance it has provided a validated protocol for the detection, and hence control, of grapevine fleck virus within the Vine Improvement Association grape certification scheme. Furthermore, the absence of grapevine pinot gris virus, the cause of an important emerging disease of grapevines in well-established South African Vitis stocks was demonstrated. In addition, the virus was intercepted and this prevented introduction into the South African grape industry of the virus by four instances of newly imported Vitis material.
* These studies were conducted during Gerhard Pietersen’s employment at Stellenbosch University and formed the basis of the MSc. studies of Katie Usher and Mmapula Lesailane.
– For more information, contact Gerhard Pietersen at gerhard@pathsol.co.za.
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