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G.P. Martelli
Although six viroids have been recovered so far from grapevines (see Table 3), (Sano et al., 1985; Flores et al., 1985; Semancik, Rivera-Bustamante and Goheen, 1987; Rezaian, Koltunow and Krake, 1988; Puchta, Ramm and Sanger, 1989; Semancik and Szychowski, 1990), four are not known to induce disease. These viroids, i.e. hop stunt viroid (HSVd), citrus exocortis viroid (CEVd), Australian grapevine viroid (AGVd) and grapevine viroid - cucumber (GVd-c), have been found in vines without symptoms or with symptoms of different virus and/or virus-like diseases. The only disease for which a viroidal aetiology has reasonably been established is yellow speckle disease.
CAUSAL AGENT
The causal agents of yellow speckle disease are grapevine yellow speckle viroids 1 and 2 (GYSVd-1 and GYSVd-2), viroids with chain lengths of 367 and 363 nucleotides, respectively (Koltunow and Rezaian, 1989).
GEOGRAPHICAL DISTRIBUTION
GYSVds may have a worldwide distribution. They have been found in vines originating from many different countries of all continents (Semancik, Rivera-Bustamante and Goheen, 1987; Szychowski, Goheen and Semancik, 1988; Minafra, Martelli and Savino, 1990).
ALTERNATE HOSTS
No natural hosts are known other than Vitis species.
FIELD SYMPTOMS
Yellow speckle is an elusive disease whose outward expression is conditioned by climatic and possibly varietal factors. Symptoms, when shown, consist of a few to many minute chrome yellow spots or flecks scattered over part or all of the leaf surface (Figures 104 and 105) or gathering along the main veins to give a vein banding pattern (Figure 106).
GYSVd-induced vein banding is very similar, if not identical, to the symptoms of a disease also known by the name of vein banding (Figure 107), which has long been regarded as part of the fanleaf degeneration complex (Goheen and Hewitt, 1962). Although vein banding may show in GFLV-free vines, it is more often associated with GFLV infections. In fact, it has been suggested that the presence of GFLV enhances the expression of GYSVd in the form of vein banding patterns (Krake and Woodham, 1983). Similar enhancement may occur in vines concurrently infected by grapevine chrome mosaic virus (Figure 108). Unlike GFLV-induced yellow discolorations, the symptoms of yellow fleck appear in the height of summer on a limited number of mature leaves, and they persist for the rest of the vegetating season.Yellow speckle symptoms may also show concurrently with symptoms of other diseases such as, for instance, leafroll (Figure 109).
NATURAL SPREAD
No vector is known. Natural dissemination takes place by mechanical inoculation through surfacecontaminated cutting tools during management operations (pruning and propagation); by graft transmission: and by distribution of infected propagating material. The absence of symptoms in most European scion varieties and all American rootstocks greatly facilitates inadvertent viroid dissemination, making viroid dispersal virtually impossible to prevent. None of the grapevine viroids is known to be seedtransmitted.
DETECTION
Detection is based on observation of field symptoms. A search for symptoms (especially vein banding) is best made in late summer. For disease detection in locations where symptoms do not develop under field conditions, a sell: indexing procedure may be used. Freshly collected or cold-stored grapevine cuttings are placed in a growth chamber at 32°C under continuous illumination (fluorescent tubes and incandescent bulbs giving 10 000 lux at the level of vegetation) and are left to root and grow for four weeks (Mink and Parsons, 1975). Translucent chlorotic spots develop along the major veins and interveinal tissues of GYSVd-infected vines 10 to 20 days after the growth starts.
IDENTIFICATION
Indexing by graft transmission
Indicators for yellow speckle are European cultivars Mataro and Mission. Symptoms are like those seen in naturally infected vines in the field but are equally erratic. Under most indexing conditions indicator responses are unreliable. If chip-bud-inoculated LN 33 cuttings are immediately placed in a growth chamber et 32°C for three weeks, then transferred to a cooler environment (10 to 20°C) under continuous illumination at 10 000 lux, they may develop yellow-green to chrome-yellow foliar spots on a few leaves, which sometimes give a vein banding pattern.
Transmission to herbaceous hosts
CYSVd- 1 was reported to be mechanically transmissible to
cucumber cv. Suyo, which it infects without symptoms (Semancik,
Rivera-Bustamante and Goheen, 1987). Other grapevine viroids are
pathogenic to tomato cv. Rutgers (HSVd, AGVd and CEVd), cucumber
(HSVd, AGVd, GVd-c) and Gynurnaur aurantiaca (CEVd), in which
they induce visible symptoms.
Polyacrylamide gel electrophoresis (PAGE) Bioassays are unreliable, so PAGE is used for detection and tentative identification of GYSVds and other grapevine viroids. The procedures for extraction, concentration and electrophoresis are as reported in Part 111. Identification is made on the basis of electrophoretic mobility compared with that of reference viroids of known size (e.g. HSVd and CEVd).
Molecular hybridization
Molecular probes to grapevine viroids have been made and have
proved useful for specific identification (Rezaian, Koltunow and
Krake, 1988; Semuncik and Szychowski, 1990).
SANITATION
Heat treatment does not free grapevine explants from yellow speckle. Sanitation is obtained by regenerating plantlets from fragmented shoot apices grown in vitro at temperatures between 20°C (night) and 27°C (day) (Barlass et al., 1982) or by culturing 0.1 to 0.2 mm long shoot tips at 25 to 27°C. The latter method also eliminates HSVd (Duran-Vila. Juarez and Arregui, 1 988).
REFERENCES
Barlass, M., Skene, K.G.M., Woodham, R.C. & Krake, R.L. 1982. Regeneration of virus free grapevines using in vitro apical culture. Ann. Appl. Biol., 101: 291-295.
Duran-Vila, N.,Juarez, J. & Arregui, J.M. 1988. Production of viroid-free grapevines by shoot tip culture. Am. J. Enol. Vitic., 39: 217-220.
Flores, R., Duran-Vila, N., Pallas, V. & Semancik, J.S. 1985. Detection of viroid and viroid-like RNA from grapevine. J. Gen. Virol., 66: 20952102.
Goheen, A.C. & Hewitt, W.B. 1962. Vein banding, a new virus disease of grapevines. Am. J. Enol. Vitic., 13: 73-77.
Koltunow, A.M. & Rezaian, M.A. 1989. Grapevine viroid 1 B. a new member of the apple scar skin viroid group, contains the left terminal region of tomato planta macho viroid. Virology, 170: 575-575.
Krake, R.L. & Woodham, R.C. 1983. Grapevine yellow speckle agent implicated in the aetiology of vein banding disease. Vitis, 22: 40-50.
Minafra, A., Martelli, G.P. & Savino, V. 1990. Viroids of grapevines in Italy. Vitis, 29: 173-182.
Mink, G.l. & Parsons, J.L. 1975. Rapid indexing procedures for detecting yellow speckle disease in grapevines. Plant Dis. Rep., 59: 869-872.
Puchta, H., Ramm, K. & Sanger, H.L. 1988. Nucleotide sequence of a hop stunt viroid isolate from the German grapevine cultivar "Riesling". Nucl. Acicts Res., 16: 2730.
Rezaian, M.A., Koltunow, A.M. & Krake, L.R. 1988. Isolation of three viroids and a circular RNA from grapevine. J. Gen. Virol., 69: 413422.
Sano, T., Uyeda, I., Shikata, E., Meshi, T., Ohno, T. & Okado, Y. 1985. A viroid-like RNA isolated from grapevine has high sequence homology with hop stunt viroid. J. Gen. Virol., 66: 333338.
Semancik, J.S., Rivera-Bustamante, R. & Goheen, A.C. 1987. Widespread occurrence of viroid-like RNA in grapevine. Am. J. Enol. Vitic., 38: 35-40.
Semancik, J.S. & Szychowski, J.A. 1990. Comparative properties of viroids of grapevine origin isolated from grapevines and alternate hosts. Proc. 10th Meet. ICVG, Volos, Creece, 1990, p. 270-278.
Szychowski, J., Goheen, A.C. & Semancik, J.S. 1988. Mechanical transmission and rootstock reservoirs as factors in the widespread distribution of viroids in grapevines. Am. J. Enol. Vitic., 39: 213-216.
Taylor, R.H. & Woodham, R.C. 1972. Grapevine yellow speckle: a newly recognized graft-transmissible disease of Vitis. Aust. J. Agric. Res., 23: 447-452.
Summary: yellow speckle detection
GRAFT TRANSMISSION
Indicators
Vitis vinifera cv. Mission or LN 33
No. plants/test
3-5 rooted cuttings
Inoculum
Wood chips, single buds
Temperature
32°C for 3 weeks, then 1 8-20°C (growth chamber). Above
25°C in the open field
Symptoms
In LN 33, yellow-green to chrome-yellow spots and/or vein
banding;
In Mission, yellow flecks and/or vein banding
OTHER TESTS
Sequential electrophoresis (sPAGE)
Molecular hybridization
FIGURE 104 Scattered yellow spots in a European grape leaf, typical of yellow speckle infection
FIGURE 105 Symptoms as in Figure 104, but with more intense yellow speckling
FIGURE 106 Strong yellow speckle symptoms with speckles tending to gather along the main veins
FIGURE 109 Symptoms of yellow speckle and leafroll appearing in autumn on the same vine
Enation
disease
Vein
necrosis
Vein mosaic and summer mottle
Asteroid
mosaic
G.P. Martelli
CAUSAL AGENT
The causal agent is unknown.
GEOGRAPHICAL DISTRIBUTION
Although known to occur in Italy since the beginning of the century (Graniti, Martelli and Lamberti, 1966), enation disease was recognized as a specific disorder by Hewitt (1954) in California. It occurs in most European countries, Israel, the United States, Venezuela, South Africa, New Zealand and Australia.
ALTERNATE HOSTS
No alternate host is known.
FIELD SYMPTOMS
Field symptoms include delayed bud breaking, slow bushy growth of the shoots in the initial stages of vegetation and presence of laminar or cup-shaped outgrowths (enations) on the underside of the eight to ten leaves at the base of the shoot (Figures 110 and 111). Leaves with enations are misshapen and deeply laciniated, and shoots are variously deformed and sometimes cracked in the basal internodes. Symptoms are erratic and do not recur every year on the same vines.
NATURAL SPREAD
No vector is known. Spread is through propagative material, which perpetuates the disease.
DETECTION
Strongly symptomatic vines are readily detected in the field, even at a distance, in the early stages of vegetation, because of the bushy compact growth. These vines usually bear outstanding foliar enations that make the disease conspicuous. A search for symptoms in summer is not advisable because the vines resume their normal growth and vigour, and enation-bearing leaves are prematurely shed.
IDENTIFICATION
The only method available is graft transmission to the indicator LN 33 (Martelli et al., 1966). Indexing, however, is highly unsatisfactory. Symptom appearance is slow (it may take up to three years) and occurs at a rate seldom exceeding 20 percent. Symptoms consist of the development of enations on the underside of a few leaves of the indicator (Figure 112). Symptoms should be read when shoots are 15 to 20 cm long, for enations are no longer produced in summer and tend to disappear from the leaves that bear them.
SANITATION
No information is available.
REFERENCES
Graniti, A., Martelli, G.P. & Lamberti, F. 1966. Enation disease of grapevine in Italy. Proc. Int. Conf: Virus Vectors Perennial Hosts and Vitis, 1965, p. 293-306. Div. Agric. Sci.. Univ. Calif., Davis.
Hewitt, W.B. 1954. Some virus and virus-like diseases of grapevine. Calif: Dept. Agric. Bull., 39: 47-64.
Martelli G.P., Graniti, A., Lamberti, F. & Quacquarelli, A. 1966. Trasmissione perinnesto della malattia delle enazioni. Phytopathol. Mediterr., 5: 122- 124.
Summary: enation detection
GRAFT TRANSMISSION
Indicators
LN 33 or Vitis vinifera cv.
Italia
No. plants/test
3-5 rooted cuttings
Inoculum
Single buds, bud sticks
Temperature
Fieid conditions
Symptoms
Enations and leaf deformation 1-3 years after grafting
FIGURE 110 Severely malformed basal leaves of a European grape cultivar with outstanding enations
FIGURE 111 Enations on the underside of a leaf, clustered along the main veins
FIGURE 112 Enations on indicator LN 33 Photo: U. Prota)
G.P. Martelli
CAUSAL AGENT
The causal agent of vein necrosis is unknown.
GEOGRAPHICAL DISTRIBUTION
The disease was first identified in France by Legin and Vuittenez (1973). It is now recorded from most European and Mediterranean countries, where it often has a high level of incidence (Credi, Babini and Canova, 1985; Savino, Boscia and Martelli, 1985), and it is likely to have a much wider distribution.
ALTERNATE HOSTS
No alternate hosts are known.
FIELD SYMPTOMS
There are no field symptoms. The disease is latent in all European grapevine cultivars and in most American rootstock species and hybrids.
NATURAL SPREAD
No vector is known. Spread is through infected propagating material.
DETECTION
No field detection is possible because of the lack of symptoms in affected vines.
IDENTIFICATION
Grafting is done to Vitis rupestris x Vitis berlandieri 110 R. Symptoms consist of necrosis of the veinlets on the underside of the leaf blade (Figure 113). The necrotic reactions develop first in the leaves at the base of the shoots and then, as the shoots grow, on the younger leaves. With time, necrotic spots also appear on the upper side of the leaf blade. Severe strains may induce necrosis of tendrils and dieback of green shoots. An almost complete cessation of growth ensues, and the indicator may die. In the field or greenhouse, symptoms appear six to eight weeks after inoculation, but mild strains may induce positive reactions the year after grafting. The symptoms are clearly shown, persist throughout the vegetative season and are readily recorded. Reading of symptoms should be made eight to ten weeks after inoculation.
SANITATION
Heat treatment at 38°C for no less than 60 days, removal and rooting under mist of shoot tips 0.5 cm long eliminates the disease from about 65 percent of the explants (Saving, Boscia and Martelli, 1985).
REFERENCES
Credi, R., Babini, A.R. & Canova, A. 1985. Occurrence of grapevine vein necrosis in the Emilia-Romagna region (northern Italy). Phytopathol. Mediterr., 24: 17-23.
Legin, R. & Vuittenez, A. 1973. Comparaison des symptômes et transmission par greffage d'une mosaïque nervaire de Vitis vinifera, de la marbrure de V. rupestris et d'une affection necrotique de l'hybride Rup-Ber 110 R. Rile. Patol. Veg., 9(suppl.): 57-63.
Savino, V., Boscia, D. & Martelli, G.P. 1985. Incidence of some graft-transmissible virus-like diseases of grapevine in visually selected and heattreated stocks from southern Italy. Phytopathol. Mediterr., 24: 204-207.
Summary: vein necrosis detection
GRAFT TRANSMISSION
Indicator
American Vitis hybrid 110 R
No. plants/test
3-5 rooted cuttings
Inoculum
Wood chips, single buds, bud sticks, shoot tips
Temperature
26°C (green grafting)
Symptoms
Necrosis of the veinlets, stunting and necrosis of the shoot
tips
FIGURE 113 Necrosis of the veinlets typically induced by vein necrosis in the indicator 110 R
G.P. Martelli
These diseases are caused by undetermined but possibly distinct aetiological agents (Woodham and Krake, 1983). They are treated together here for practical purposes because of the similarity of their symptoms.
CAUSAL AGENT
The causal agent(s) are unknown. A viroid aetiology was suggested for summer mottle because of the influence of high temperatures on symptom expression (Woodham and Krake, 1983).
GEOGRAPHICAL DISTRIBUTION
Vein mosaic, first identified in France (Legin and Vuittenez, 1973), is widespread throughout Europe and the Mediterranean. Summer mottle was recorded from Australia (Krake and Woodham, 1978), but it is not known whether or not it is restricted to that country.
ALTERNATE HOSTS
No alternate hosts are known.
FIELD SYMPTOMS
Vein mosaic is a semi-latent disease, for it does not show up consistently in affected vines, regardless of whether they are European scion varieties or American rootstocks. The characterizing symptoms consist of pale green discolorations of the tissues adjacent to the main veins, producing a feathering or banding effect (Figures 114 and 115). The size and vigour of the vines may be adversely affected. Leaf symptoms induced by summer mottle are very similar. The symptoms of both diseases appear in summer and persist through the autumn; sometimes they may be more obvious in greenhouse-grown vines than in the field.
NATURAL SPREAD
No vector is known. Spread is through infected propagating material.
DETECTION
Field detection of vein mosaic is not always possible because of the semi-latency of the disease. Also, when shown, symptoms may escape observation because of their mildness and irregular distribution in the vines.
IDENTIFICATION
The main indicator for vein mosaic is Vitis riparia Gloire de Montpellier, which reacts with chlorotic blotches and green mosaic along the veins (Figure 116), malformations of the leaf blade and occasional necrosis. Under greenhouse conditions, symptoms appear four to five weeks after inoculation, reaching full expression in a couple of months. Symptomatological responses may be slower in the field, but they usually show on the vegetation in the first year after grafting. LN 33 is another indicator for vein mosaic, reacting with pale green to yellowish vein banding.
Summer mottle does not induce symptoms in V. riparia or LN 33 (Woodham and Krake, 1983). Good indicators are European cultivars Sideritis, Cabernet franc and Mission, which exhibit a typical pale green to yellowish vein feathering and vein banding. Symptoms develop both in greenhouse and field in the first year's vegetation of inoculated vines.
SANITATION
Freedom from summer mottle has been obtained by regenerating vines from fragmented shoot apices grown in vitro at temperatures between 20°C (night) and 27°C (day) or at a continuous 35°C (Barlass et al., 1982).
REFERENCES
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.
Krake, L.R. & Woodham, R.C. 1978. Grapevine vein mottle, a new graft transmissible disease. Vitis, 17: 266-270.
Legin, R. & Vuittenez, A. 1973. Comparaison des symptômes et transmission par greffage d'une mosaïque nervaire de Vitis vinifera, de la marbrure de V. rupestris et d'une affection nécrotique des nervures de l'hybride Rup-ber 110 R. Riv. Patol. Veg., 9(suppl.): 57-63.
Woodham, R.C. & Krake, L.R. 1983. A comparison of grapevine summer mottle and vein mosaic diseases. Vitis, 22: 247-252.
Summary: vein mosaic and summer mottle detection
GRAFT TRANSMISSION
Indicators
Vitis riparia (vein mosaic), Vitis vinifera cv.
Sidentis,
Cabernet franc (summer mottle)
No. plants/test
3-5 rooted cuttings
Inoculum
Wood chips, single buds, bud sticks, shoot tips
Temperature
22°C (green grafting) or field conditions
Symptoms
Chlorotic blotches and green mosaic along the veins, leaf
deformation in 4-6 weeks
FIGURE 114 Chlorotic vein feathering induced by vein mosaic disease
FIGURE 115 Intense chlorotic vein banding induced by vein mosaic disease
FIGURE 116 Vein mosaic symptoms in the indicator v. riparia
G.P. Martelli
CAUSAL AGENT
The causal agent is unknown.
GEOGRAPHICAL DISTRIBUTION
Asteroid mosaic was first identified by Hewitt and Goheen ( 1959) in California and more recently in Greece (P.E. Kyriakopoulou, personal communication).
ALTERNATE HOSTS
No alternate host is known.
FIELD SYMPTOMS
Translucent spots with a star-like shape and fused lateral veins centred between primary veins in the blade are present on the leaves. Leaves are also asymmetrical and crinkled (Figure 117). Diseased vines are weak and bear little or no fruit.
NATURAL SPREAD
No vector is known. Spread is through infected propagating material.
DETECTION
Infected vines are symptomatic and can be identified in the field.
IDENTIFICATION
Four to eight weeks after graft inoculation to Vitis rupestris St George, narrow cream-yellow bands develop on the main veins of leaves (Figure 118). Leaves are distorted and small (Hewitt et al., 1962).
SANITATION
No information is available.
REFERENCES
Hewitt, W.B. & Goheen, A.C. 1959. Asteroid mosaic of grapevines in California. Phytopathology, 49: 541.
Hewitt, W.B., Goheen, A.C., Raski, D.J. & Gooding, G.V. 1962. Studies on virus and virus-like diseases of the grapevine in California. Vitis, 3: 57-83.
Summary: asteroid mosaic detection
GRAFT TRANSMISSION
Indicator
Vitis rupestris St George
No. plants/test
3-5 rooted cuttings
Inoculum
Wood chips, single buds, bud sticks
Temperature
Field conditions
Symptoms
Cream-yellow bands along the veins of the indicator and leaf
deformation in 6-8 weeks
FIGURE 117 Foliar symptoms of asteroid mosaic (Photo: U. Prota
FIGURE 118 Foliar reactions of V. rupestris to asteroid mosaic infection
Diseases induced by phloem- and xylem-limited prokaryotes
Flavescence
dorée
Grapevine
yellows
Pierce's
disease
A. Caudwell and G.P. Martelli
CAUSAL AGENT
Flavescence dorée (FD) is caused by a mycoplasma-like organism (MLO)
GEOGRAPHICAL DISTRIBUTION
The disease was first recorded in 1954 in the southwest of France (Armagnac), from where it spread to other southern viticultural districts of continental France, Corsica and northern Italy (Caudwell and Larrue, 1986) Flavescence dorée was apparently introduced into Europe from the Great Lakes area of the United States, the home of its vector, where a similar disease occurs in Vitis vinifera (Caudwell and Dalmasso, 1985; Pearson et al., 1985).
ALTERNATE HOSTS
No natural host other than the grapevine is known.
FIELD SYMPTOMS
Symptoms usually appear in late spring. Growth can be reduced and the internodes are shortened. The leaves have downward rolled margins, and the shoots may exhibit a drooping condition because of irregular maturation of the wood (Figure 119) and lack of phloem fibres. As the season progresses, the severity of symptoms increases: rolling of the leaves becomes more intense and the blades are discoloured, turning yellow in white-berried cultivars (Figures 120 and 121) or red in red-berried cultivars (Figures 122 and 123). Necrosis along the main veins may develop in autumn. The internodes may show black pustules (Figure 124) and sometimes longitudinal splitting of the bark. The crop is much reduced. Inflorescences may dessicate when symptoms first appear. If bunches are formed, the berries wither and dry up or drop at the slightest shaking of the vine. Some affected vines die, usually in the year following infection. The survivors recover naturally and do not show symptoms unless they are infected anew. Wild American Vitis species are infected without showing symptoms.
NATURAL SPREAD
The disease is transmitted in nature by Scaphoides titanus (= Scaphoideus littoralis), a strictly ampelophagous leafhopper species (Figures 125 and 126) that in Europe thrives in southern France, Corsica, southern Switzerland and northern Italy. This leafhopper lays eggs in the bark of two-year-old grape wood and has five larval stages and a single generation per year. Local spread is effectively accomplished by the vector, whereas long-distance dissemination is through infected propagative material.
DETECTION
Detection is based on observation of field symptoms in summer or autumn.
IDENTIFICATION
Indexing by graft transmission
The most sensitive indicator is the hybrid variety Baco 22A (Noah x Folle blanche). Chip-bud and cleft grafting are both suitable for inoculation. Grafted indicators show symptoms within two to three months. The symptoms are the same as those shown by naturally infected plants in the field, i.e. stunting and downward rolling and yellowing of the leaves. Aramon, Chardonnay, Sangiovese and Alicante Bouchet are other sensitive cultivars that can also be used as indicators.
Transmission by vectors
Adults and larvae of S. titanus readily transmit the disease agent to broad bean (Vicia faba) cv. Aria under greenhouse conditions. Colonies of S. titanus can be obtained by placing egg-bearing two-year-old grape pruning wood kept moist in a cage around vegetating vines (Caudwell and Larrue, 1977). Leafhoppers (fourth or fifth stage instars or adults) are placed on infected vine shoots for three to four weeks, then transferred for one to two weeks on to young (4 to 5 cm tall) broad bean seedlings. Inoculated broad beans are removed from the cage and grown in the greenhouse with 1 6-hour artificial illumination. Symptoms consisting of reduced growth and yellowing and upward rolling of the leaves accompanied by progressively severe flower abortion (Figure 127) develop at the top of the plant within two to three weeks.
Euscelidius variegatus, another leafhopper species, can be used to transmit the causal agent of the disease (FD-MLO) from broad bean to broad bean as well as to other plant species such as Vicia, Pisum, Chrysanthernum and Lupines species and Vinca rosea. Since E. variegatus does not thrive on Vitis species, its colonies are established and maintained on maize (Zea mais) or broad bean under controlled conditions.
Serology
To minimize non-specific reactions in serological identification of FD-MLO, antisera to E. variegatus FD can be used against extracts of V. faba FD. Conversely, antisera to V. faba FD can be used against extracts of leafhopper FD, i.e. extracts from infected E. variegatus reared artificially or viruliferous S. titanus collected from diseased vines in the field.
ISEM. Freshly carbonated grids are coated with a 10 g/ml solution of protein A in 0.1 M phosphate buffer, ph 7.2. After rinsing in the same buffer, the grids are sensitized by floating on a drop of undiluted antiserum to E. variegatus FD, are again rinsed in buffer and are floated for 15 minutes on a drop of infected broad bean leaf extract containing 0.05 percent Tween 20. After thorough rinsing with phosphate buffer containing 0.05 percent Tween 20, the grids are fixed with 1 percent glutaraldehyde, rinsed with distilled water and negatively stained with 2 percent ammonium molybdate. In infected samples FD-MLOs appear as vesicles. Similar results are obtained by using an antiserum to V.faba FD and extracts from infected leafhoppers.
ELISA. Individual leafhoppers are crushed in 0.5 ml of PBS buffer, and 0.1 ml of the extract is placed in a plate well. Anti-V faba FD rabbit immunoglobulins are added. The conjugate is an anti-rabbit goat IgG. If infected leaf extracts are employed for ELISA, the IgGs used are rabbit anti-leaflhopper FD. Both polyclonal and monoclonal antibodies can be used satisfactorily in ELISA (Boudon-Padieu and Larrue, 1986; Boudon-Padieu, Larrue and Caudwell, 1989; Schwartz et al., 1989).
SANITATION
Spreading of the disease occurs through infected propagation material and the vector. Symptomless grape budwood can host both the eggs of the leafllopper vector and the disease agent. Thus nurseries must be established in, and propagating material must be collected from, areas that are free from the disease or particularly well protected against the vector. The vector is controlled by: eliminating eggs by burning pruning wood, treating before bud burst with parathion-activated oils (I 500 ml of a suspension of 3 percent parathion and 78 percent paraffin oil per 100 litres of water); or one or two chemical applications against instars 30 and 45 days after the first hatching, followed by another treatment against adults in August.
FD-MLOs may be eliminated from infected wood by treating dormant canes with water at 45°C for 3 hours or at 50°C for 45 minutes (Caudwell et al., 1990).
REFERENCES
Boudon-Padieu, E. & Larrue, J. 1986. Diagnostic rapide de la flavescence dorée de la vigne par le test ELISA sur cicadelle vectrice. Application a des populations naturelles de Scaphoideus littoralis Ball. Confirmation de la presence de la flavescence dorée dans les Boûches-du-Rhône. Prog. Agric. Vitic., 103: 524-526.
Boudon-Padieu, E., Larrue, J. & Caudwell, A. 1989. ELISA and dot-blot detection of flavescence dorée-MLO in individual leafhopper vectors during latency and inoculative state. Curr. Microbiol., 19: 357-364.
Caudwell, A. 1964. Identification d'une nouvelle maladie a virus de la vigne, la flavescence dorée. Etude des phénomènes de localisation des symptômes et de rétablissement. Ann. Epiphytol. , 15(hors ser. l). 197 pp.
Caudwell, A. & Dalmasso, A. 1985. Epidemiology and vectors of grapevine viruses and yellows diseases. Phytopathol. Mediterr., 24: 170-176.
Caudwell, A. & Larrue, J. 1977. La production de cicadelles seines et infectieuses pour les épreuves d'infectivité chez les jaunisses a mollicutes des végétaux. L'élevage de Euscelis plebeja KBM et la ponte sur mousse de polyurethane. Ann. Zool. Ecol. Anim., 9: 443456.
Caudwell, A. & Larrue, J. 1986. La flavescence dorée dans le midi de la France et dans le Bas-Rhone. Prog. Agric. Vitic., 103: 517-523.
Caudwell, A., Larrue, J., Volos, C. & Grenan, S. 1990. Hot water treatment against flavescence dorée on dormant wood. Proc. 10th Meet. ICVG, Volos, Greece, 1990, p. 336-343.
Pearson, R.C., Pool, R.M., Gonsalves, D. & Goffinet, M.C. 1985. Occurrence of flavescence dorée-like symptoms on "White Riesling" grapevines in New York, USA. Phytopathol. Mediterr., 24: 8287.
Schwartz, Y., Boudon-Padieu, E., Grange, J., Meignoz, R. & Caudwell, A. 1989. Obtention d'anticorps monoclonaux spécifiques de ['agent pathogène de type mycoplasme (MLO) de la flavescence dorée de la vigne. Ann. Inst. Pasteur Res. Microbiol., 140: 311 -324.
Summary: flavescence dorée detection
GRAFT TRANSMISSION
Indicators
Hybrid Baco 22A; Vitis vinifera cvs Chardonnay, Aramon
No. plants/test
3-5 rooted cuttings
Inoculum
Wood chips, single buds, bud sticks
Temperature
Field conditions
Symptoms
Stunting, leaf yellowing and necrosis (white-berried
varieties) or leaf reddening and necrosis (red-berried varieties)
2-3 months or more after inoculation
OTHER TESTS
Serology (ELISA)
Molecular hybridization
FIGURE 119 Irregular wood ripening in a shoot of a vine affected by flavescence dorée
FIGURE 122 Intense reddening caused by flavescence dorée infection in cv. Pinot noir
FIGURE 123 Reduced vigour, stunting and leaf reddening of cv. Aramon caused by flavescence dorée
FIGURE 124 Black pustules on the canes of a vine infected by flavescence dorée Photo: G. Granatal
FIGURE 125 Instar of the flavescence dorée vector Scaphoideus titanus (Photo: A. Bruno
FIGURE 126 Adult of Scaphoideus titanus Photo: A. Brun)
G.P. Martelli and A. Caudwell
CAUSAL AGENT
The causal agent of grapevine yellows (bois noir, Vergilbungkrankheit, etc.) is unknown, but it is thought to be a mycoplasma-like organism (MLO). Whether one or more different MLOs are involved in the aetiology of the disease is not known.
GEOGRAPHICAL DISTRIBUTION
Syndromes comparable to bois noir, one of the grapevine yellows originally reported from France (Caudwell, 1961), have been recorded from many European and Mediterranean countries (Germany, Switzerland, southern Italy, Romania, Yugoslavia, Greece, Israel). Similar diseases occur also in Chile, Argentina and Australia (see Caudwell, 1988). Whether and to what extent these diseases are related to one another has not been established.
ALTERNATE HOSTS
No alternate hosts are known, although they are likely to exist in native flora (weeds and shrubs).
FIELD SYMPTOMS
Symptoms are practically the same as those of flavescence dorée, i.e. leaf rolling, yellowing or reddening of the leaves, necrosis along the veins, incomplete wood ripening, withering of berries and drying up of bunches (Figures 128 to 131).
NATURAL SPREAD
Visual evidence strongly indicates that the disease spreads naturally in the field. Vectors are not yet known but are likely to be leafhoppers.
DETECTION
Infected vines are readily identified because of the symptoms shown, especially in summer, which is the best time for field surveys.
IDENTIFICATION
Although cv. Chardonnay is a sensitive indicator for certain of these diseases (e.g. bois noir), it may not be totally dependable for others. Vines can be infected by grafting but not by Scaphoideus titanus. The symptomatological reactions of cv. Chardonnay tally with field symptoms. Serological tests are not available.
SANITATION
No information is available.
REFERENCES
Caudwell, A. 1961. Etude sur la maladie du bois noir de la vigne: ses rapports avec la flavescence dorée. Ann. Epiphytol., 12: 241262.
Caudwell, A. 1988. Bois noir and Vergilbungkrankheit. Other grapevine yellows. In R.C. Pearson and A.C. Coheen, eds, Compendium of grape diseases, p. 46-47. St Paul, MN, USA, Am. Phytopathol. Soc.
Summary: yellows detection
GRAFT TRANSMISSION
Indicator
Vitis vinifera cv. Chardonnay
No. plants/test
3-5 rooted cuttings
Inoculum
Wood chips, single buds, bud sticks
Temperature
Field conditions
Symptoms
Yellowing or reddening of the leaves and rolling of the lades
followed by necrosis of the veins, usually within the first year
after inoculation
FIGURE 128 View of a white-berried grapevine cultivar affected by bois noir (Photo: C. Granata)
