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A variety of virus diseases, for example leafroll, stem grooving, corky bark, fanleaf and fleck occur on vines. From an economic point of view, leafroll is probably the most important virus disease in South African vineyards due to its increasing occurrence and effect on production and quality. Propagation material can be effectively cleaned of viruses by means of somatic embryogenesis or heat treatment combined with meristem culture. Leafroll is transmitted by mealybug, Planococcus ficus, and virus free material can therefore be re-infested in the field. The majority of research in South Africa is currently aimed at the detection and control of leafroll. Other aspects being studied are the determination of the possible viral origin of Shiraz disease and the identification of insects that are potential vectors (carriers) of viruses.

A virus study group was formed in 1992 with the exclusive purpose of co-ordinating research on vine viruses and virus diseases so as to avoid duplication. This group consists of researchers from ARC-Fruit, Vine and Wine Research Institute at Nietvoorbij in Stellenbosch, ARC- Plant Protection Research Institute (PPRI), Pretoria, the University of Stellenbosch as well as persons from the wine industry. The group convenes annually to discuss the progress being made with the research projects. Six research projects, partially funded by Winetech, are currently in progress. The following is a brief overview of the progress per project as reported at the last feedback meeting.

Control of leafroll by means of cross protection
Ms. R. Carstens (Infruitec/Nietvoorbij)

At present there are no control measures for leafroll in vines. Cross protection, where a mild strain of the virus is placed in a virus free (clean) plant to protect the plant against the severe strain, was previously used with success in citrus, for viruses belonging to the same family as leafroll. The focus is on grape vine leafroll associated virus-3 because it is common in vineyards. When a variety of indicator cultivars showed mild leafroll symptoms with the same virus isolate, this isolate was identified as a mild strain. These mild strains are graft transmitted to virus free Cabernet franc and Shiraz. The cross protection ability of these mild strains are then tested by infecting them with the severe strain. Mealybugs are used for this purpose. Symptom development on these plants is currently begin monitored. Very promising results were obtained this past season, but symptoms will have to be monitored for at least two more seasons before final recommendations in this regard can be made.

The cloning and molecular characterisation of the coat protein gene of GLRaV-3
Dr. J. Burger (US)

It is a world-wide trend to adopt a molecular approach in addition to conventional methods of disease control. This approach is especially applicable to viral diseases which, unlike other diseases, cannot be controlled chemically. The aim of this project is to identify, clone and characterise on a molecular level important resistant genes, for example the coat protein gene of leafroll-3 virus. These genes will be transferred by means of genetic transformation to plants. These plants are eventually challenged with the virus to determine whether the plant offers resistance to the specific virus. The project is making good progress and the first transformation vectors have already been developed.

Insects can act as carriers (vectors) of viruses. It has already been established that leafroll-3 virus is carried by mealybugs in the field. The tempo at which leafroll is spreading, however, gives rise to strong suspicions that mealybugs are not the only vector of the virus.

Insect surveys were done in blocks where leafroll is spreading. There was a wide variety of insects, mostly seed-feeders and predators. Insects have to feed on the phloem of the vine, where the leafroll occurs, to be able to carry the virus. Leafhoppers were identified as phloem feeders and transfer studies were conducted. The leafhoppers were tested with ELISA (a quick virus detection method) to determine whether they did in fact absorb the virus. No virus was found. It is planned to test the various life stages of the leafhoppers for transmission and also to use more sensitive detection methods.

The purpose of this project is to prepare antiserums against the various viruses in order to develop ELISA systems for quick detection of viruses. It is difficult, however, to find a vine in which specific viruses occur individually. Such material then has to be propagated under insect free conditions until a sufficient quantity of material is available to purify enough virus for antiserum production. Many sources were tested, but pure sources could not be found. An existing technique, cross adsorption, is adapted to get rid of these undesirable viruses. This cross adsorption is targeted at a specific virus, seeing that each virus has its own characteristics. The plant material containing these single viruses is scarce and the initial goal of preparing an antiserum for each individual virus had to be abandoned. An antiserum mixture, which can detect seven different viruses simultaneously, was subsequently developed. It is a huge improvement on the previous antiserum which could only locate three viruses simultaneously.

The preparation of an antiserum for the grapevine fleck virus was also researched. The available plant material containing this virus was also infested with leafroll-3 and because the concentration of the virus is very low, it is difficult to produce a good antiserum.

PCR (Polymerase Chain Reaction) is a very sensitive and specific technique with which viruses can be detected. Techniques have been developed for the detection of grapevine virus A (GVA) and grapevine virus B (GVB) in herbaceous plants and grapevine material. The detection has to be optimised, however, and the repeatability of the technique determined. The technique for GVA is ten times more sensitive than ISEM, a technique which uses the electron microscope to detect viruses. PCR is also cheaper and less labour intensive than ISEM. A multiplex PCR, with which GVA and GVB can be detected simultaneously, has also been developed. This PCR can detect viruses in herbaceous plants (Nicotiana benthamiana), but the technique for the detection in vines still has to be developed. A PCR for the detection of grapevine leafroll associated virus (GLRaV)-1, -2 and -4 is currently being developed. Its sensitivity will also be compared with ISEM and ELISA. GLRaV-3 can already be detected with a certain kind of PCR. The repeatability of the system has to be confirmed, however.

Determination of possible viral origin of Shiraz disease
Dr. D. Goszczynski (PPRI)

Shiraz disease occurs in the wine grape cultivars Shiraz, Merlot and Gamay and has a destructive effect on the vine. Vines can die within two years as a result of infection. Budding is delayed and bunches on affected vines are small, few and poorly coloured. Canes do not lignify properly and are rubbery. Where these green, non-lignified canes are pruned, the eyes sometimes do not bud at all the following season. This project, which aims at determining whether the disease is caused by a virus, was recently initiated. Plant material with symptoms of Shiraz disease was collected and virus purification is currently undertaken.

This project involves the repeatable production of sufficient callus and embryo's for use in transformation experiments. Firstly, induction of callus takes place and then callus with embryogenic potential is selected. This callus then has to be maintained and propagated as synchronised embryogenic callus (tissue in the same phase). The next step involves the maintenance of synchronised embryo cell lines. Good progress has been made over the past year with the maintenance and propagation of embryogenic cell lines of Chardonnay and Merlot noir. Large quantities of material are currently available. This material is also used with great success in transformation experiments by the Institute for Wine Biotechnology. The induction and maintenance of embryogenic callus are also progressing well in cultivars such as Roobernet, Pinot noir, Pinotage, Cabernet Sauvignon and Richter 99. It has been found in various instances that anthers, compared to ovaries, are more successful in the induction of embryogenic callus. A workable protocol has been developed for various wine grape and rootstock cultivars.

A virus research group gathers annually at Nietvoorbij to discuss progress with regard to virus research for the vine and wine industry.

Back: Mr Rian Burger (KWV), Prof. Barbara von Wechmar (US), Mrr Gawie Kriel (KWV), Johan Wiid (Nederburg), Dr Dariusz Goszczynski (PPRI), Mr Nico Spreeth (KWV).

Middle: Michael-John Freeborough (US), Dr Gerhard Pietersen (PPRI), Dr Johan Burger (US), Dr Andr� de Klerk (Nietvoorbij), Mr Kassie Kasdorf (PPRI).

Front: Dr Melanie Viviers (US), Ms Tertia van Tonder (PPRI), Roleen Carstens (Nietvoorbij), Dr Ilse Traumann (Nietvoorbij), Ms Kathy Wilsen (US).

Inset: Prof. Piet Goussard (US) and Ms Elleunorah Marais (Nietvoorbij).