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Grapevine decline - American nepoviruses
G.P. Martelli
CAUSAL AGENTS
Four distinct nepoviruses, tomato ringspot (ToRSV), tobacco ringspot (TRSV), peach rosette mosaic (PRMV) and, to a much lesser extent, blueberry leaf mottle (BBLMV), are implicated in the genesis of this complex disease. These viruses have isometric particles about 30 nm in diameter and the bipartite genome typical of the nepovirus group (Harrison and Murant, 1977; Stace-Smith and Ramsdell, 1987: Martelli and Taylor, 1989).
All of these viruses have natural serological variants, but only ToRSV has two serologically distinguishable strains (Piazzolla et al., 1985) that infect grapevines and induce different diseases. There are biological variants that elicit different symptoms in naturally and artificially inoculated hosts.
GEOGRAPHICAL DISTRIBUTION
Grapevine decline is typical in the northeastern United States (Michigan, New York, Maryland and Pennsylvania) and Canada ( Ontario). ToRSV also occurs in grapes in a restricted area of central California.
ALTERNATE HOSTS
All four viruses have a wide range of wild and cultivated alternate hosts. These are both annual (weeds, vegetable crops) and perennial (weeds, shrubs, ornamentals, fruit trees) plants that serve as natural virus reservoirs.
FIELD SYMPTOMS
Symptomatological responses of grapevines vary according to the species (i.e. Vitis vinifera, Vitis labrusca, interspecific hybrids), the infecting virus and the climatic conditions. ToRSV-induced decline affects European cultivars (especially if self-rooted) more severely in colder than in warmer climates. Newly infected vines have normal growth but show occasional chlorotic mottling and rings on the leaves of a few shoots (Figure 39). In the following years affected vines decline rapidly, showing small mottled and distorted leaves, short internodes, stunted growth, little fruit set with straggling and shelled clusters, or no yield at all (compare Figures 40 and 41). Death of the vines may ensue, favoured by winter injury.
In Maryland and California ToRSV affects the yield rather than the vine's growth, which is more vigorous than normal. Yield is affected either by formation of small clusters (e.g. little grape disease of cultivars Vidal blanc and Carignan) or by severe reduction in fruit setting, as in the case of yellow vein disease in California. Although no visible foliar symptoms are associated with little grape disease, yellow vein is characterized by chrome-yellow flecking along the veins extending to the interveinal tissues (Figure 42).
The symptoms of TRSV-induced decline, which only occurs in New York State and Pennsylvania, are the same as those of ToRSV in native cultivars, but in V vinifera they are similar to the symptoms induced by European nepoviruses (Figures 43 and 44).
In Michigan, V labrusca and French hybrids affected by PRMV show a progressive decline over several years, accompanied by leaf and cane deformations, straggling and shot-berried clusters (Figures 45 and 46).
NATURAL SPREAD
Except for BBLMV, whose means of natural spread is unknown, the causal agents of grapevine decline are transmitted through soils by longidorid nematodes. The vector situation of these viruses is not as clear-cut as that of GFLV and other European nepoviruses. It is summarized in Table 5 (reviewed by Martelli and Taylor, 1989).
The efficiency of transmission is high for all virus/vector combinations except for PRMV/Longidorus elongatus. Natural virus reservoirs are primarily perennial weeds in which the viruses are endemic prior to the establishment of the vineyards; these weeds are also hosts for the vectors. All viruses are transmitted through seeds of weeds and grapevines, whose seedlings perpetuate the inoculum. Long-distance spread may take place through budwood from mildly infected vines, or from plants grown in warmer climates where vegetation is not much affected.
DETECTION
Detection is based on observation of field symptoms. Inspections for the identification of newly infected vines, which may show mild and localized responses, need to be carried out with special care. Summer, when bunches have reached full size, is the best time for detecting little grape and yellow vein diseases. Yellow vein can be confused with syndromes induced by chromogenic GFLV strains and vein banding disease. Differential traits are that yellow vein symptoms develop earlier than vein banding and are usually shown by fewer leaves (Goheen and Hewitt, 1962).
TABLE 5 Vectors of the causal agents of grapevine decline
| Virus | Vector |
| Tomato ringspot virus | |
| Type strain (decline) | Xiphinema
americanum sensu stricto Xiphinema rivesi |
| Californian strain (yellow vein) | Xiphinema californicum |
| Tobacco ringspot virus | Xiphinema
americanum sensu lato |
| Peach rosette mosaic virus | Xiphinema
americanum sensu stricto, Longidorus diadecturus, Longidorus elongatus |
IDENTIFICATION
Indexing by graft transmission
Indicators for yellow vein are the European varieties Grenache and Carignan, which reproduce the field syndrome following chipbud grafting. Vitis rupestris St George, Kober 5BB and cv. Mission, which do not show symptoms when inoculated with yellow vein sources, react symptomatically when grafted with chromogenic GFLV and thus serve as differential indicators for the two viruses.
Baco and Kober SBB, when inoculated with ToRSV sources, react with shock symptoms (chlorotic blotching and rings) that appear a few weeks after grafting (Figures 47 and 48). These are followed by systemic chronic symptoms similar to those seen in the field. Chardonnay reproduces more or less the field symptomatology when inoculated with TRSV sources (Figure 49).
Transmission to herbaceous hosts
All American nepoviruses are readily transmitted to herbaceous hosts by inoculation of sap from young leaves or roots, expressed in the presence of 2.5 percent aqueous nicotine or phosphatebuffered solutions. Inoculation procedures and precautions are the same as outlined for European nepoviruses. In addition, the herbaceous hosts used for isolation and diagnosis are the same as those used for European nepoviruses. ToRSV and TRSV have similar experimental hosts, which react with comparable, if not identical, symptoms. Thus, for example, either virus may induce tip necrosis in Phaseolus vulgaris (Figure 50), chlorotic local lesions and systemic mottling in Cucumis sativus (Figure 51), chlorotic/necrotic local lesions in Chenopodium amaranticolor (Figure 52) and Chenopodium quinoa followed by systemic mottling (Figure 53), and necrotic local lesions in Vigna unguicolata (Figure 54). Both viruses also cause chlorotic/necrotic rings and line patterns in various Nicotiana species, which after four to six weeks enter a recovery phase in which newly produced leaves are symptomless but contain virus.
Diagnostic hosts for PRMV are:
Diagnostic hosts for BBLMV are:
Serology
ToRSV, TRSV, PRMV and BBLMV are separate viruses that can be reliably distinguished and identified by serology. Immunodilfusion tests can be performed with sap expressed from symptomatic leaves of mechanically inoculated herbaceous hosts, but identification from fieldgrown vines is best made by immunoenzymatic procedures.
ELISA has been used successfully to detect ToRSV and PRMV from leaves of rooted cuttings and field-grown vines, with standard procedures (Ramsdell et al., 1979; Gonsalves, 1980). The efficiency of detection is satisfactory, but extreme care must be taken in collecting the test samples because of the irregular distribution of the virus in the vines. Young leaves from different parts of the canopy and from suckers should be collected for assaying.
SANITATION
The same techniques used for GFLV and other European nepoviruses are applicable.
REFERENCES
Gilmer, R.M. & Uyemoto, J.K. 1972. Tomato ringspot virus in Baco noir grapevines in New York. Plant Dis. Rep., 56: 133- 135.
Gilmer, R.M, Uyemoto, J.K. & Kelts, J.L. 1970. A new grapevine disease induced by tobacco ringspot virus. Phytopathalogy, 60: 619-627.
Goheen, A.C. & Hewitt, W.B. 1962. Vein banding, a new virus disease of grapevines. Am. J. Enol. Vitic., 13: 73-77.
Gonsalves, D. 1980. Detection of tomato ringspot virus in grapevines: irregular distribution of virus. Proc. 7th Meet. ICVG. Niagara Falls, NY. USA, 1980, p. 95- 106.
Gooding, G.V. & Hewitt, W.B. 1962. Grape yellow vein: symptomatology, identification. and the association of a mechanically transmissible virus with the disease. Am. J. Enol. Vitic., 13: 196-203.
Harrison, B.D. & Murant, A.F. 1977. Nepovirus group. Descriptions of Plant Viruses. No. 185. Kew, UK, Commonw. Mycol. Inst./Assoc. Appl. Biol.
Martelli, G.P. & Taylor, C.E. 1989. Distribution of viruses and their nematode vectors. Adv. Dis. Vector Res., 6: 151-189.
Piazzolla, P., Savino, V., Castellano, M.A. & Musci, D. 1985. A comparison of grapevine yellow vein virus and a grapevine isolate of tomato ringspot virus. Phytopathol. Mediterr., 24: 44-50.
Ramsdell, D.C., Andrews, R.W., Gillet, J.M. & Morris, C.E. 1979. A comparison between enzymelinked immunosorbent assay (ELISA) and Chenopodium quinoa for detection of peach rosette mosaic virus in Concord grapevines. Plant. Dis. Rep., 63: 74-76.
Ramsdell, D.C. & Myers, R.L. 1974. Peach rosette mosaic virus, symptomatology and nematodes associated with grapevine degeneration in Michigan. Phytopathology, 64: 1174-1178.
Stace-Smith, R. & Ramsdell, D.C. 1987. Nepoviruses of the Americas. Curr. Top. Vector Res., S: 131-166.
Uyemoto, J.K., Taschenberg, E.F. & Hummer, D.K. 1977. Isolation and identification of a strain of grapevine Bulgarian latent virus in Concord grapevines in New York State. Plant Dis. Rep., 61: 949-953.
Summary: grapevine decline (American nepoviruses) detection
GRAFT TRANSMISSION
Indicators
Grenache and Carignan (ToRSV yellow vein strain), Baco and Kober
58B (ToRSV, typestrain), Chardonnay (TRSV)
No. plants/teat
3-5 rooted cuttings
Inoculum
Wood chips, single buds, bud sticks
Temperature
22-24°C
Symptoms
Chrome-yellow flecks along the veins (ToRSV, yellow vein);
chlorotic blotches, rings, lines, foliar deformations (ToRSV,
decline strain); leaf mottling, malformations, reduced growth
(TRSV)
TRANSMISSION TO HERBACEOUS HOSTS
Diagnostic hosts
Phaseolous vulgaris(ToRSV and TRSV), Cucumis sativus (ToRSV
and TRSV), Chenopodium guinoa (PRMV), Nicotiana
clevelandii(BBLMV)
Inoculum
Tissues from young symptomatic leaves
Extraction
Grind in 2.5 percent aqueous nicetine
Temperature
Below 25°C Symptoma In P. vulgaris (ToRSV, TRSV),
systemic necrosis of the topmost leaves in about 2 weeks;
In C. sativus (ToRSV, TRSV), chlorotic local lesions in
5-7 days followed by systemic mottling;
In C. quinoa (PRMV), faintchloroticlocal lesions in about
a week, systemic mottling and tip necrosis in 10-12 days;
In N. clevelandii(BBLM), local necrotic rings and systemic
necrotic spotting in 10-12 days
OTHER TESTS
Serology (ELISA, ISEM)
Molecular hybridization (when probes are available)
FIGURE 40 Healthy Concord vine
FIGURE 41 ToRSV-infected Concord vine. Note the markedly reduced crop
FIGURE 42 Typical yellow vein symptoms elicited by the Californian strain of ToRSV
FIGURE 43 Shoot of a field-grown Chardonnay vine infected by TRSV (Photo: M.K. Corbett)
FIGURE 46 Straggling clusters in a PRMV-infected vine
FIGURE 54 Necrotic lesions on leaves of cowpea inoculated with TRSV (Photo: M.K Corbett)
FIGURE 55 Necrotic local lesions induced by PRMV in C. quince (Photo: D.C. Ramsdell)
G.P. Martelli
CAUSAL AGENTS
Grapevine leafroll may be induced by a complex of viruses' the majority of which belong to the closterovirus group. So far, at least five different long closteroviruses (i.e. with particle length ranging between 1 400 and 2 200 nary), denoted as grapevine leafroll-associated closteroviruses (GLRaV) I, II, III, IV and V, have been detected with varying degrees of consistency in infected vines (Gugerli, Brugger and Bovey, 1984; Hu, Gonsalves and Teliz, 1989; Zimmermann et al., 1990). All have been characterized serologically and differ from one another. Two additional long closteroviruses which seem to be serologically unrelated to all of the above have also been detected recently (Zimmermann et al., 1990). A shorter closterovirus (particles 800 nm long) called grapevine virus A (OVA) has also been found associated, though inconsistently, with the disease (Conti and Milne, 1985; Agran et al., 1990).
All the above viruses are phloem-restricted and non-mechanically transmissible, except for GVA, some isolates of which can be transmitted with great difficulty by sap inoculation (Conti et al., 1980; Agran et al., 1990). There is mounting evidence that most if not all of these viruses, alone or in various combinations, are able to induce leafroll symptoms and therefore qualify as possible elicitors of the disease.
The aetiological role of a potyvirus serologically close to potato virus Y (Tanne et al., 1977) is still undefined.
GEOGRAPHICAL DISTRIBUTION
The disease has been recorded from all major grapevine-growing areas of the world. The level of infection is often very high, so that cultivars grown in certain viticultural districts are totally diseased.
ALTERNATE HOSTS
No alternate hosts are known. Leafrollassociated closteroviruses have not been identified in any wild or cultivated plant species other than Vitis species and are not serologically related to any of the other known members of their taxonomic group.
FIELD SYMPTOMS
Affected vines may be smaller than healthy ones. Major external symptoms are downward rolling of the leaves accompanied by reddishpurple or yellow discolorations of the blades, depending on whether vines are red or white berried (Figures 56 and 57). Discoloured areas appear in the interveinal spaces of the lower leaves in early summer (Figure 58), becoming progressively stronger and extended so as to cover, with time, the whole foliar surface (Figure 59). The main veins may or may not retain the green colour in the advanced stages of the disease (Figure 60), and there is a difference in the hue, intensity and distribution of the reddish pigmentation over the leaf surface. In cases where the discoloration is particularly heavy, necrotic areas may develop in the interveinal tissues. Ripening of the fruits is affected. At harvest time, bunches are smaller than normal and may remain greenish or whitish (Figure 61). Clusters of certain red-fruited cultivars (e.g. Cardinal, Emperor) may become unmarketable because of the pale colouring of the berries.
As yet, it is not clear how extensively the variation in kind and intensity of symptoms shown in the field depends on varietal reactions or reflects differences in the type of agent(s) that cause specific responses. There are indications, however, that the presence of different closteroviruses is linked with differences in symptom expression. For example, GLRaV 1 seems to be related to a marked rolling of the leaf margins and a light reddish discoloration of the blades, whereas GLRaV III is more consistently associated with rolling of medium intensity and intense reddening with a deep purplish hue (Zimmermann, 1990).
NATURAL SPREAD
Although reports of leafroll spread within a vineyard are few, there is experimental evidence that spread is associated with pseudococcid mealybugs (Engelbrecht and Kasdorf, 1985). GVA can be acquired and transmitted by Pseudococcus longispinus, Planococcus ficus and Planococcus citri (Rosciglione et al., 1983; Rosciglione and Castellano, 1985), whereas GLRaV III is transmitted from grape to grape by P. longispinus and P. ficus (Rosciglione and Gugerli, 1989; Tanne, Ben Dov and Raccah, 1989).
Long-distance spread is through infected propagating material of European grapes (budwood) and especially of American rootstocks (rooted cuttings), most of which are symptomless carriers of the disease. In certain cases, leafroll-affected vines of Vitis riparia Gloire de Montpellier show a clear-cut rolling and yellowing of the leaves, which becomes evident in autumn.
DETECTION
Field symptoms are clearly expressed by redand black-fruited European scions, although mild forms of the disease are known, which are less readily detected. The best time for field detection is in autumn when the foliar reddishpurple discolorations are at their peak (Figure 62). Detection in white-berried cultivars depends very much on the varietal reaction and possibly on the viral complex responsible for the infection. In these varieties the symptoms are rarely distinctive enough to be identified with certainty. Likewise, no field detection is possible in American rootstocks because of their lack of visible response to the disease, except for the above-mentioned instance of V riparia. In these cases, laboratory detection is mandatory.
IDENTIFICATION
Indexing by graft transmission
Indicators for indexing are many, all belonging to black-fruited European varieties such as Mission, Pinot noir, Cabernet franc, Cabernet sauvignon and Barbera. The choice of the indicator may vary according to the conditions under which indexing is carried out. Thus, before establishing an indexing programme, it is advisable to run comparative graft transmission tests to assess the relative efficiency and reliability of symptom expression by each indicator.
The symptoms shown by the indicators are like those seen in the field: reddish discolorations on the interveinal areas of the leaves and downward rolling of their margins (Figure 63). In greenhouse indexing (green grafting) at 22°C, symptoms begin to appear about six weeks after grafting and are fully expressed in about three months (Walter et al., 1990). Responses in field indexing (chip-budding, cleft or whip grafting) are slower, as they may take up to two years to show completely. However, severe forms of the disease induce clear-cut reactions in the first year of vegetation.
Baco 22A is recommended as a routine indicator for leafroll, since certain forms of the disease elicit in it severe stunting, yellowing and rolling of the leaves. These differential responses of woody indicators have not yet been correlated with the presence of specific single viruses or associations of viruses.
Transmission to herbaceous hosts
GVA was the first grapevine closterovirus transmitted by mechanical inoculation (Conti et al., 1980). The transmission rate from expressed sap is extremely low and erratic, but with certain virus isolates transmission occurs more readily and can be enhanced by using partially purified preparations from grape leaves as inoculum.
Positive transmission is more consistently obtained, although not equally well with all GVA isolates, by means of pseudococcid vectors. Groups of five to ten crawling instars that have been allowed to feed on infected grapevine leaves are transferred, with excised pieces of the leaves they were feeding on, on to the leaves of young Nicotiana clevelandii and/or Nicotiana benthamiana seedlings. They are allowed to colonize the seedlings for a couple of weeks. The mealybugs may then be killed by insecticide treatment.
The herbaceous host range of GVA is extremely narrow, limited to N. clevelandii and N. benthamiana, both of which react with clearing and light yellowing of the secondary veins 10 to 12 days after infection (Figure 64).
Recent reports indicate that closteroviruses other than GVA can be mechanically transmitted from diseased grapevines. These viruses have not yet been characterized, although it appears that their herbaceous host range is limited to Nicotiana species.
Micropurification
Micropurification is the procedure (see details in Part III) for obtaining virus preparations from grapevine tissues for multipurpose use (antiserum preparation, serology, immune electron microscopy, electrophoresis, etc.). The best sources of virus are petioles and main veins of old symptomatic leaves of European scions (8 to 10g of tissues). Micropurification from American rootstock leaves, especially those containing Vitis rupestris plasma, gives inconsistent if not negative results (Boscia et al., 1990). With American rootstocks, cortical tissue is a far better source of virus for micropurification.
Serology
Immunodiffusion in agar gel may not be used with closteroviruses, but media containing SDS (see Part III) for disrupting virus particles are suitable. In any case, the antigen must be a concentrated, partially purified virus preparation.
ELISA. Immunoenzymatic procedures, as described in Part III, can be used for detecting all known closteroviruses in field- or greenhousegrown vines (Gugerli, Bruggerand Bovey, 1984; Teliz et al., 1987; Hu, Gonsalves and Teliz, 1989). Sources of antigen are fresh leaves, flowers, fruit, tendrils, bark and root tissues or wood shavings from dormant cuttings. The reactants can be polyclonal or monoclonal antibodies. Kits of both types of antibodies are commercially available for some closteroviruses (e.g. GVA and GLRaV I, as of 1990). Although the amount of plant material needed is as low as 40 mg (Teliz et al.,1987), larger samples (up to 1 to 2 g) are commonly used. Tissues are ground in ordinary ELISA extraction buffer, with or without the addition of nicotine ( 1 to 2.5 percent), and are used thus or at a dilution of up to 1 :50. Early field detection of viral antigens before symptoms appear on the foliage is possible from flower clusters and roots. ELISA reactions from basal leaves become consistently positive around blooming time. Viral antigens are evenly distributed in mature canes, which represent good antigen sources throughout the year (Teliz et al., 1987). Negative and positive controls, chosen as indicated for GFLV, should always be included in the tests. For ELISA detection of leafroll-associated closteroviruses in American rootstocks, wood shavings are far superior to leaf tissues, which for V. rupestris and its hybrids give consistent negative results regardless of the time of sampling (Boscia et al., 1990).
Immune electron microscopy (ISEM). GVA and other GLRaVs can be detected by ISEM following the procedure outlined in Part III. Although the starting material can be the same used for ELISA, cortical tissues taken after scraping off the bark are probably the best source of virus. Tissues are ground in three volumes of cold 2 percent PVP (MW 25 000 to 30 000) in 0.1 M phosphate buffer, pH 7, and the extract is used for observation (Conti and Milne, 1985).
Polyacrylamide gel electrophoresis (PAGE) PAGE is used either for separation and characterization of double-stranded viral RNAs (dsRNA) or, in conjunction with Western blotting, for separation and identification of viral coat proteins. PAGE and Western blotting are applied as described in Part III to extracts from stem phloem tissues (Hu, Gonsalves and Teliz, 1989; Hu et al., 1990). Both techniques disclose the presence of viral infections, and Western blotting also leads to the identification of the viral agent.
Molecular hybridization
Cloned cDNA probes have been prepared to genomic RNA of GVA, GLRaV III and the potyvirus serologically related to PVY. These probes have been successfully used for the identification of both viruses in field-grown grapevine leaf extracts denatured with formaldehyde (concentrated, partially purified preparations) or with 50 mM NaOH and 2.5 mM EDTA (grapevine sap) (Gallitelli, Savino and Martelli, 1985; Tanne, Naveh and Sela, 1989; Minafra, Russo and Martelli, 1990) and processed as described in Part III.
SANITATION
With many European red-berried cultivars visual selection helps reduce disease incidence. However, leafroll-free plants are best obtained by the following methods: prolonged heat treatment (60 to 120 days or more at 38°C) of grafted buds, or of whole plants with removal and rooting of shoot tips under mist (Goheen, 1977); heat treatment in vitro according to Galzy's method (Valet and Mur, 1976); micrografting (Bass and Legin, 1981.) and in vitro meristem tip culture (Barlass et al., 1982).
REFERENCES
Agran, M.K., Di Terlizzi, B., Boscia, D., Minafra, A., Savino, V., Martelli, G.P. & Askri, F. 1990. Occurrence of grapevine virus A (OVA) and other closteroviruses in Tunisian grapevines affected by leafroll disease. Vitis, 29: 49-55.
Barlass, M., Skene, K.G.M., Woodham, R.C. & Krake, L.R. 1982. Regeneration of virusfree grapevines using in vitro apical culture. Ann. Appl. Biol., 101: 291-295.
Bass, P. & Legin, R. 1981. Thermotherapie et multiplication in vitro d'apex de vigne. Application a la separation ou a l'elimination de diverges maladies de type viral et a l'evaluation des dégâts. C. R. Seances Acad. Agric. Fr., 67: 922933.
Boscia D., Savino, V., Elicio, V., Jebahi, S.D. & Martelli, G.P. 1990. Detection of closteroviruses in grapevine tissues. Proc. 10th Meet. ICVG, Volos, Greece, 1990, p. 52-57.
Conti, M. & Milne, R.G. 1985. Closterovirus associated with leafroll and stem pitting in grapevine. Phytopathol. Mediter., 24: 110- 113.
Conti, M., Milne, R.G., Luisoni, E. & Boccardo, G. 1980. A closterovirus from a stem-pitting diseased grapevine. Phytopathology, 70: 394-399.
Engelbrecht, D.J. & Kasdorf, G.G.F. 1985. Association of a closterovirus with grapevines indexing positive for grapevine leafroll and evidence for its natural spread in grapevine. Phytopathol. Mediterr., 24: 101-105.
Gallitelli, D., Savino, V. & Martelli, G.P. 1985. The use of a spot hybridization method for the detection of grapevine virus A in the sap of grapevine. Phytopathol. Mediterr., 24: 221 -224.
Goheen, A.C. 1977. Virus and virus-like diseases of grapes. Hortscience, 12: 465-469.
Goheen, A.C., Harmon, F.N. & Weinberger, J.H. 1958. Leafroll (white emperor disease) of grapes in California. Phytoputhology, 48: 51 -54.
Gugerli, P., Brugger, J.J. & Bovey, R. 1984. L'enroulement de la vigne: mise en evidence de particules virales et développement d'une méthode immunoenzymatique pour le diagnostic rapide. Rev. Suisse Vitic. Arboric. Hortic., 16: 299-304.
Hu, J.S., Gonsalves, D., Boscia, D. & Namba, S. 1990. Use of monoclonal antibodies to characterize grapevine leafroll associated closteroviruses. Phytopathology, 80 (in press).
Hu, J.S., Gonsalves, D. & Teliz, D. 1989. Characterization of closterovirus-like particles associated with grapevine leafroll disease. J. Phytopathol., 128: 1-14.
Minafra, A., Russo, M. & Martelli, G.P. 1990. A cloned probe for the detection of grapevine virus A. Proc. 10th Meet. ICVG, Volos, Greece, 1990, p. 417424.
Rosciglione, B. & Castellano, M.A. 1985. Further evidence that mealybugs can transmit grapevine virus A (OVA) to herbaceous hosts. Phsytopathol. Mediterr., 24: 186- 188.
Rosciglione, B., Castellano, M.A., Martelli, G.P., Savino, V. & Cannizzaro, G. 1983. Mealybug transmission of grapevine virus A. Vitis, 22: 331 347.
Rosciglione, B. & Gugerli, P. 1989. Transmission of grapevine leafroll disease and an associated closterovirus to healthy grapevine by the mealybug Planococcus ficus. Phytoparasitica, 17: 63.
Tanne, E., Ben Dov, Y. & Raccah, B. 1989. Transmission of closterovirus-like particles by mealybugs (Pseudococcidae) in Israel. Proc. 9th Meet. ICVG, Kiryat Anavim, Israel, 1987, p. 71 73.
Tanne, E., Naveh, L. & Sela, 1989. Serological and molecular evidence for the complexity of the leafroll disease of grapevine. Plant Pathol., 38: 1831X9.
Tanne, E., Sela, I., Klein, M. & Harpaz, 1977. Purification and characterization of a virus associated with grapevine leafroll disease. Phytopathology, 67: 442-447.
Teliz, D., Tanne, E., Gonsalves, D. & Zee, F. 1987. Field serology of viral antigens associated with grapevine leafroll disease. Plant Dis., 71: 704-709.
Valat, C. & Mur, T.G. 1976. Thermothérapie du Cardinal Rouge. Prog. Agric. Vitic., 93: 200204.
Walter, B., Bass, P., Legin, R., Martin, C., Vernoy, R., Collas, A. & Veselle, G. l990. The use of a green-grafting technique for the detection of virus-like diseases of the grapevine. J. Phytopathol., 128: 137-145.
Zimmermann, D. 1990. La maladie de I'enroulement de la vigne: caractérisation de quatre particules virales de type closterovirus à 1 'aide d 'anticorps polyclonaux et monoclonaux. Ph.D. thesis. Univ. Louis Pasteur, Strasbourg. 256 pp.
Zimmermann, D., Bass, P., Legin, R. & Walter, B. 1990. Characterization and serological detection of four closterovirus-like particles associated with leafroll disease of grapevine. J. Phytopathol., 130: 277-288.
Summary: leafroll detection
GRAFT TRANSMISSION
Indicators
Several cultivars of red-fruited Vitis vinifera (Pinot noir,
Cabernet franc, Merlot, Barbera, Mission)
No. plants/test
3-5 rooted cuttings
Inoculum
Wood chips, single buds, bud sticks, shoot tips
Temperature
22°C (green grafting)
Symptoms
Rolling and reddening of the leaves in 4-6 weeks (green
grafting) or 6-8 months to 2 years (field indexing)
TRANSMISSION TO HERBACEOUS HOSTS (GRAPEVINE VIRUS A)
Diagnostic hosts
Nicotiana clevalandii or Nicotiana benthamiana
Inoculum
Virus preparations micropurified from grapevine leaves or
cortical tissues; viruliferous mealybugs; tissues from young
leaves (with some virus isolates only)
Extraction
Grind leaf tissues in 2.5 percent aqueous nicotine
Temperature
Below 25°C
Symptoms
Systemic vein clearing and yellowing in 10-12 days
OTHER TESTS
Serology (ELISA, ISEM) and Western blot for closteroviruses
for which antisera are available
Electrophoresis (PAGE) for dsRNA pattern
Molecular hybridization (OVA, GLRaV 111)
FIGURE 56 Severe leafroll symptoms shown in autumn by a red-fruited European grape cultivar
FIGURE 58 Incipient rolling and reddening of the leaves in a vine infected by leafroll in spring
FIGURE 59 Progressive reddish discolorations in leaves from a vine affected by leafroll
FIGURE 60 Discoloured leaves with main veins retaining the green colour
FIGURE 61 Pale-berried bunches from a leafroll-infected vine
FIGURE 62 Red-fruited vines affected by leafroll are readily identified in the field in autumn
FIGURE 63 Leafroll symptoms shown in autumn by an indicator vine two years after graft inoculation
FIGURE 64 Vein yellowing in Nicotiana benthamiana infected with GVA
