The efficacy of detection methods used by the South African Vine Improvement Association certification scheme has increased. By Jorisna Bonthuys
The more effective testing for the grapevine fleck virus (GFkV) is thanks to a recent investigation into the available enzyme-linked immunosorbent assay kits used for this purpose in table- and wine-grape plant material.
The project was initiated in 2019 under the leadership of Gerhard Pietersen, former professor in the Department of Genetics at Stellenbosch University (SU) and current senior researcher at Patho Solutions, a company involved in research, diagnostics and control of plant pathogens.
Funding was provided by the South African Table Grape Industry (SATI) and Winetech, the equivalent funding body for the wine grape industry. Genetic research techniques used as part of this project served as the topic for a master's degree thesis by Katie Usher.
The study aimed to assess the usefulness of commercially available enzyme-linked immunosorbent assay (ELISA) test kits in detecting grapevine fleck virus (GFkV) during routine testing done as part of the South African Vine Improvement Association (VIA) certification process.
Although GFkV is prescribed as a virus of concern within this scheme, the only means of testing for the virus in the past was to inoculate it onto a susceptible plant – known as an "indicator plant" – and then wait for symptoms of the disease to manifest. This, however, constitutes a lengthy and very expensive method and is best replaced by ELISA testing.Read More
Pietersen says plant certification schemes are critical for controlling viruses in vegetatively propagated crops (that is, crops propagated using pieces of the plant itself rather than its seed). Such schemes rely on efficient large-scale detection of target viruses. An ELISA test is often used for this purpose due to its robustness and because researchers can run thousands of these tests at the same time. In addition, they have been proven to detect viruses effectively, sometimes even without plant disease symptoms being present (constituting cases of so-called "latent infections").
Importantly, though, only ELISA tests that have been evaluated for local use and approved by the Department of Agriculture, Land Reform and Rural Development (DALRRD) may be used in the VIA certification scheme.
An effective test for GFkV
In this project, the researchers determined which internationally and commercially available ELISA test kits are suitable for detecting GFkV in table grape plant material.
Furthermore, the team compiled the entire detection protocol for this virus. This includes guidance on which plant tissue to use for testing purposes, the best time of the year to sample plant material for these tests and the best extraction methods to use when preparing plant material for analyses.
Four ELISA tests for this virus were purchased and tested against the positive controls of each test, along with several local GFkV isolates identified during a previous SATI-funded project.
Pietersen says three of the ELISA tests performed well against the local sources of this virus, and any of these tests can be used in the certification scheme. The names of the manufacturers of these kits are not made public, but are available to the VIA.
One of the three acceptable kits, referred to here as "ELISA A", was selected for further study. It did not show differences in sensitivity compared to the other tests, but was the cheapest to procure. This test can now be used as part of plant improvement efforts in the VIA certification scheme.
Using the ELISA A kit, the researchers showed that GFkV could be detected throughout the growing season. However, the highest ELISA values were obtained from March to June, making this the ideal time to test for the virus.
In addition, the researchers confirmed that the best tissue to use is phloem-rich tissue (the tissue that transports food to parts of the plant where it is required). Ideally, this should be from young leaves' petioles. This is because the stalks that attach the leaf blades to the stem are easier to prepare for ELISA testing than the canes of the vines, which are also rich in phloem. A superior extraction fluid was also identified.
It was also important to determine the genetic variability of the virus to ascertain whether the ELISA A test kit can effectively detect all local isolates of the virus.
To achieve this, the researchers subjected 229 Vitis accessions (cultivars, species or hybrids) from Nietvoorbij's collection of Vitis germplasm – the tissue used for plant breeding – to a relatively new technique known as "next-generation sequencing".
This technique allows researchers to detect all viruses (not just GFkV) and virus variants in a given plant sample with a single test. It also provides information on the genetic code of each virus and/or variant.
The scientists identified the genetic code of GFkV among more than 11 million pieces of genetic code produced by next-generation sequencing for each sample. (The remaining fragments of genetic code are those of the plant itself, other viruses or viroids, bacteria, fungi, or even insects found on the Vitis samples.) By analysing the GFkV-specific pieces of genetic material, the researchers were able to determine the presence or absence of the virus and closely related viruses in these grape plants, as well as the specific strains of virus present.
Eighty of the grape accessions in the germplasm collection were found to have GFkV. And researchers also managed to identify genetic variations of the virus.
A total of 232 individual vines representing all the replicated vines of the 80 accessions were sampled and subjected to the chosen four ELISA tests, with ELISA A proving capable of detecting them all.
Another valuable result of the study was showing that the kit does not detect a closely related virus, namely the grapevine rupestris vein feathering virus.
"The kit is excellent for detecting local variants of the virus," Pietersen says. "It will not provide false positives if grapevine rupestris vein feathering virus is present."
Understanding the spread
Another aim of the study was to determine the prevalence of GFkV in table and wine grapes. This information is crucial to informing decisions around whether or not to require testing for this virus at the foundation-block level, as part of the VIA certification scheme.
The researchers tested for GFkV in 19 mother-block vineyards known to be under inoculum pressure from grapevine leafroll-associated virus 3. This virus is known for causing grapevine leafroll disease, considered one of the most crucial grapevine viral diseases affecting vines worldwide. In this study, it is regarded as an indicator of conditions that allow the reinfection of healthy mother blocks. None of the hundreds of samples collected from these vineyards tested positive for GFkV.
The ability of GFkV to spread from plant to plant is poorly understood. Some reports suggest that natural spread can occur and others indicate that it does not. In their study of GFkV in the germplasm collection, Pietersen and Usher could not find conclusive evidence of its spread. They suggest this be investigated in a separate study that uses experiments specifically designed to test this phenomenon.
A recent discovery of a grape nuclear plant with GFkV by another one of his students, Seamus Morgan, provides the ideal opportunity for such a project. Pietersen believes future studies are needed to track the potential spread of GFkV in vineyards surrounding the progeny of this nuclear plant.
"This kind of research is vital to strengthening the local certification schemes aimed at improving the competitiveness of producers," he says. "The better the tests, the better the plant material made available as clones to the industry."
Featured Image: ELISA results: the yellow wells contained samples that tested positive for grapevine fleck virus (GFkV), e.g. well A. The colourless well B represents the absence of grapevine fleck virus in the sample, which prevents formation of the complex. And the Y particles in A and B represent antibodies directed specifically at GFkV.