A Technical Guide for Wine Producers

RECENT ARTICLES   |   WYNBOER HOME GRAPEVINE LEAFROLL DISEASE: A NEW CONTROL STRATEGY? Johan Burger, Department of Genetics, University of Stellenbosch Recent advances in plant genetic engineering holds great promise for modern plant breeding; also for grapevines. Can this new technology provide mechanisms to introduce resistance against the crippling grapevine leafroll disease? Grapevine leafroll disease Grapevine leafroll is widely regarded as one of the most devastating viral diseases of grapevine in South Africa. Damage directly attributed to the disease is nearly impossible to determine, but probably runs into millions of rands annually. The etiology of leafroll disease is not fully understood, but it is believed to be caused by a complex of viruses. At least seven distinct viruses, all members of the closterovirus group, have been associated with the disease. The most prominent of these is grapevine leafroll associated virus III (GLRaV-III, Fig. 1), a long, flexuous virus that is restricted to the phloem tissues of the grapevine. Disease symptoms are fairly characteristic, especially in red cultivars, where a typical interveinal reddening and downward rolling of leaf lamina occur (Fig. 2). Economical losses, however, are incurred by substantial reduction in yield, while poor fruit quality, e.g. reduced sugar content, has also been linked to these symptoms. Existing control measures It is well known that existing control measures are largely inefficient, but what are these and why are they not working? Viruses, unlike many other plant pathogens, cannot effectively be killed by chemicals. The application of pesticides is therefore limited to the control of possible insect vectors, but with the uncertainty about the identity of these (only the mealy bug, Planococcus ficus, is generally accepted as a vector for GLRaV-III, while several other insects are suspected to play a role in virus transmission, Martelli, 1997) it is clear that this approach can, at best, inhibit the spread of the virus. The introduction of natural resistance by conventional breeding seems such an unattainable prospect that no such breeding programme exists in South Africa, nor, to our knowledge, anywhere else in the world. The practice of sanitation seems currently the most effective. Sanitation measures include the use of virus-free propagation material, insect control (to eliminate possible vectors) and weed control (to eliminate possible alternative virus hosts). However, these are all preventative measures that offer no real resistance to virus infection. When new vineyards are established in a leafroll-prevalent area, using virus-free propagation material, re-infection is likely to occur in 5-6 years (pers. comm. P. G. Goussard) Similar rapid spread of the disease - from 11% to 90% infection in 5 years in New Zealand (Jordan et al., 1993) - suggests that other vectors must be involved. It is clear that viruses pose a unique problem when it comes to disease control, and that existing strategies do not offer any long-term solution. But are there any viable alternatives? New approaches With the dawn of the biotechnology era came the tools and techniques to study organisms in a more detailed manner. Similarly, the interactions between host and pathogen could be investigated on a molecular level. Early molecular host-pathogen interaction studies led to a hypothesis which states that the normal host-pathogen interaction will be altered if a gene from the pathogen is expressed in the host. This altered interaction may take the form of disease resistance, hence the term pathogen-derived resistance (PDR), first proposed by Sanford & Johnson in 1985. Molecular characterisation of virus genomes revealed that these simple pathogens possess a few primitive "genes". One such gene encodes a protein that coats the viral RNA genome to give the virus its characteristic shape (Fig. 1). This coat protein (CP) gene was used in the first experiment to prove the viability of PDR, when Powell-Abel et al. (1986) transformed and expressed the tobacco mosaic virus CP gene in tobacco plants and obtained resistance against the virus. Subsequently, many other forms of PDR against plant viruses (and other pathogens) have been developed. However, this coat protein-mediated resistance (CP-MR) mode of introducing virus resistance in plants, has over the years proven to be the most effective. The exact molecular mechanism of CP-MR has not been fully elucidated, but the general opinion is that the expressed CP somehow interferes with early replication events of the virus in plant cells. Examples of CP-MR to more than 25 different viruses from 12 virus groups have been reported (Grumet, 1994). A few of these are already in the process of being commercialised - in the USA three transgenic virus resistant crops were approved for commercialisation at the end of 1997 (Hagedorn, 1999). A parallel technology that is essential for successful PDR, is the genetic transformation of a crop. In this regard, grapevine initially proved to be extremely recalcitrant, with limited successes reported in rootstock species (Martinelli & Mandolino, 1994). Today, reasonable successes are achieved with some of the important wine and table grape cultivars (Kikkert et al. 1996; Perl et al. 1996). Recently, scientists at the Institute for Wine Biotechnology (IWBT) at the University of Stellenbosch managed to transform the grape cultivars Sultana, Chardonnay and Merlot. In grapevine, CP-MR is largely unexplored territory; with only a few examples that have been reported. These are the coat proteins of grapevine fanleaf virus (Krastanova et al., 1995; Mauro et al., 1995) grape chrome mosaic virus (Bouquet, 1993) and GLRaV-III (Ling et al., 1997). Leafroll resistant grapevine? We, in collaboration with the grapevine transformation team at the IWBT, decided on a CP-MR approach for leafroll disease and we targeted GLRaV-III as the principal pathogen. Viral RNA was extracted from infected grapevine and the GLRaV-III CP gene cloned by reverse transcription polymerase chain reaction. The identity of the gene was confirmed by nucleotide sequencing before it was modified to enable its cloning into plant expression vectors. The first set of vectors are currently being transformed into tobacco plants in our laboratories. The gene will initially be tested in tobacco for proper expression, while a second vector will be introduced into grapevine cultivars. While we cannot at this stage give an affirmative reply to the above question, we are confident that we are well on our way to produce leafroll resistant grapevine cultivars. It is an open question as to who will be ready first - the scientists to produce the resistant cultivars, or the industry and public to accept the genetically modified plants! References Brault, V., Candresse, T., Le Gall, O., Delbos, R.P., Lanneau, M. & Dunez, J. 1993. Genetically engineered resistance against grapevine chrome mosaic nepovirus. Plant Mol. Biol. 1: 89-97. Grumet, R. 1994. Development of virus resistant plants via genetic engineering. Plant Breed. Rev. 12: 47-79. Hagedorn, C. 1999. Transgenic crops. "http://fbox.vt.edu:10021/cals/cses/chagedor/crops.html". Jordan, D., Petersen, C., Morgan, L. & Segaran, A. 1993. Spread of grapevine leafroll and its associated virus in New Zealand vineyards. Proceedings of the 11th Meeting of the International Council for the Study of Viruses and Virus-like Diseases of the Grapevine, Montreaux, Switzerland, pp113-114. Kikkert, J.R., Hébert-Soulé, D., Wallace, P.G., Striem, M.J. & Reisch, B.I. 1996. Transgenic plantlets of "Chancellor" grapevine (Vitis sp.) from biolistic transformation of embryogenic cell suspensions. Plant Cell Rep. 15: 311-316. Krastanova, S., Perrin, M., Barbier, P., Demangeat, G., Cornuet, P., Bardonnet, N., Otten, L., Pinck, L. & Walter, B. 1995. Transformation of grapevine rootstocks with the coat protein gene of grapevine fanleaf nepovirus. Plant Cell Rep. 14: 550-554. Ling, K.S., Zhu, H.Y., Alvizo, H., Hu, J.S., Drong, R.F., Slightom, J.L. & Gonsalves, D. 1997. The coat protein gene of grapevine leafroll associated closterovirus-3: cloning, nucleotide sequencing and expression in transgenic plants. Arch. Virol. 142:1101-1116. Martelli, G.P. 1997. Grapevine virology highlights. Proceedings of the 12th Meeting of the International Council for the Study of Viruses and Virus-like Diseases of the Grapevine, Lisbon, Portugal, pp 7-14. Martinelli, L. & Mandolino, G. 1994. Genetic transformation and regeneration of transgenic plants in grapevine (Vitis rupestris S.). Theor. Appl. Gen. 88: 621-628. Mauro, M.C., Toutain, S., Walter, B., Pinck, L., Otten, L., Coutos-Thevenot, P., Deloire, A. & Barbier, P. 1995. High efficiency regeneration of grapevine plants transformed with the GFLV coat protein gene. Plant Science 112: 97-106. Perl, A., Lotan, O., Abu-Abied, M. & Holland, D. 1996. Establishment of an Agrobacterium-mediated transformation system for grape (Vitis vinifera L.): the role of antioxidants during grape-Agrobacterium interactions. Nature Biotechnology 14:624-628. Powell-Abel, P., Nelson, R.S., De, B., Hoffmann, N., Roger, S.G., Fraley, R.T., & Beachy, R.N. 1986. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science: 232: 738-743. Sanford, J.C. & Johnson, S.A. 1985. The concept of parasite-derived resistance: deriving resistance genes from the parasites own genome. J. Theor. Biol. 115: 395-405.

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