Ch07

Part I: Detection and identification of specific grapevine diseases or pathogens

True virus diseases
Rugose wood complex
Viroid diseases
Virus-like diseases
Diseases induced by phloem- and xylem-limited prokaryotes

True virus diseases

Grapevine degeneration - fanleaf
Grapevine degeneration - European nepoviruses
Grapevine decline - American nepoviruses
Leafroll

Grapevine degeneration - fanleaf

G.P. Martelli

CAUSAL AGENT

The causal agent of fanleaf is grapevine fanleaf nepovirus (GFLV), a mechanically transmissible virus with isometric particles 30 nm in diameter and a bipartite genome (Quacquarelli et al., 1976). GFLV populations are remarkably homogeneous serologically, so that natural serological variants are rarely found when screening is done by conventional serological techniques (Saving, Chérif and Martelli, 1985). Serological differences are detected, however, by monoclonal antibodies (Huss et al., 1987). Biological variants differing according to the symptoms induced in artificially inoculated herbaceous hosts or in naturally infected grapevines are many.

GEOGRAPHICAL DISTRIBUTION

The disease and the causal virus have 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 host is known. GFLV has never been found naturally in any wild or cultivated plant species other than Vitis vinifera and American Vitis species.

FIELD SYMPTOMS

Fanleaf degeneration is characterized by two distinct syndromes deriving from a differential reaction of the hosts to biologically distinct strains of the causal virus. Vein banding, another syndrome traditionally thought to be caused by chromogenic GFLV strains, may have a different aetiology, perhaps viroidal, as discussed under Viroid diseases.

Infectious malformations (fanleat proper)
Distorting virus strains may cause vines to be stunted or less vigorous than normal. Leaves are variously and severely distorted, asymmetrical, cupped and puckered and exhibit acute dentations (Figures 1 and 2). Chlorotic mottling may sometimes accompany foliar deformations (Figure 3). Canes are also malformed, showing abnormal branching, double nodes, different length or exceedingly short internodes, fasciations and zigzag growth (Figures 4 and 5). Bunches are reduced in number and size, ripen irregularly and have shot berries and poor berry setting (Figure 6). Foliar symptoms develop early in the spring and persist through the vegetative season, although some masking may occur in summer.

Yellow mosaic
Yellow mosaic is caused by chromogenic virus strains. Affected vines show chrome-yellow discolorations that develop early in the spring and may affect all vegetative parts of the vines (leaves, herbaceous shoot axes, tendrils and inflorescences) (Figure 7). Chromatic alterations of the leaves vary from a few scattered yellow spots, sometimes appearing as rings and lines, to variously extended mottling of the veinal and/or interveinal areas, to total yellowing (Figures 8 to 11). In spring, affected plants in a vineyard can readily be spotted from a distance (Figure 12). Malformations of leaves and canes are usually not prominent, but clusters may be smaller than normal and may have shot berries. In hot climates the newly produced summer vegetation has a normal green colour, while the yellowing of the old growth turns whitish and tends to fade away (Figure 13).

Symptoms induced by distorting virus strains show up equally well under field and greenhouse conditions, whereas chrome-yellow alterations evoked by chromogenic strains may not develop in greenhouses.

NATURAL SPREAD

Spread at a site (i.e. within a vineyard or between adjacent vineyards) is mediated by nematodes. Two longidorid nematode vectors of GFLV are known: Xiphinema index and Xiphinema italiae (Hewitt, Raski and Goheen, 1958; Cohn, Tanne and Nitzany, 1970). The former species is by far the more efficient vector under natural and experimental conditions. Although not all X. index populations are equally efficient in transmitting virus isolates (Catalano, Roca and Castellano, 1989), this nematode is to be regarded as the major, if not the only, natural and economically important GFLV vector. It has a limited range of alternate natural hosts (e.g. fig, mulberry, rose), but these hosts are immune to GFLV. No natural virus reservoirs are known other than grapevines. GFLV persists in volunteer plants and in the roots of lifted vines that remain viable in the soil, constituting an important source of inoculum. Transmission of GFLV through grapevine seeds has been reported (Lazar, Köber and Lehoczky, 1990), but it has negligible epidemiological significance.

Long-distance spread is passively but efficiently effected through dissemination of infected propagating material (budwood and rooted cuttings of rootstocks) (Martelli, 1978).

DETECTION

Field symptoms are often obvious enough to allow the detection of diseased vines. Difficulties may be encountered, however, even by keen and experienced observers, in the presence of tolerant varieties and/or infections by mild virus strains. Observations for symptoms should be carried out twice during the vegetating season: in midspring for growth abnormalities, deformation and chromatic alterations of the foliage; and at leaf shedding for abnormalities of the canes.

IDENTIFICATION
Indexing by graft transmission

Although none of the Vitis species used as indicators is immune to GFLV, the quickest and most typical responses are given by Vitis rupestris St George. In greenhouse indexing (chip-budding, green grafting) at 22 to 24°C shock symptoms appear as soon as three to four weeks after grafting. The symptoms consist of chlorotic spots, rings and lines (Figure 14), sometimes accompanied by malformations and localized necrosis of the tissues. These symptoms are transient. They are followed by chronic reactions that, for distorting virus strains, consist of reduced growth and severely deformed leaves with prominent teeth (Figure 15). Chronic symptoms develop within two to three months from inoculation and persist throughout the vegetating season (Hewitt et al., 1962; Martelli and Hewitt, 1 963).

Field indexing (cleft or whip grafting) reactions are slower. Symptoms appear during the first year of vegetation. They are of the chronic type for distorting virus strains but consist of various patterns of yellow discolorations, sometimes accompanied by leaf deformity (Figure 16) for chromogenic virus strains. With certain virus sources yellowing may appear in the second year after inoculation. Yellow discolorations do not usually develop in greenhouse indexing.

Indicators other than V. rupestris may react to GFLV infections with chronic-type responses. These appear in the first or second year after grafting and consist of deformations and/or bright yellowing of the foliage. The type and severity of the symptoms vary with the nature and virulence of the virus strain.

Transmission to herbaceous hosts
GFLV is readily transmitted to herbaceous hosts by inoculation of expressed sap, provided that some precautions are observed. The best inoculum sources are young, tender symptomatic leaves of the spring flush collected directly in the field or from greenhouse-grown cuttings. Young, succulent roots are equally good, if not superior, sources of inoculum. Suitable root material is readily obtained by forcing grape cuttings to root in sand in a tray or bench heated to 24 to 25°C. Old leaves collected in the field in summer or autumn. especially in hot climates, constitute a poor source of virus, even if they show symptoms. Their use may result in repeated failures to transmit the virus.

For inoculation, 3 to 5 g of tissues (either leaves or roots) are ground in a chilled mortar together with an equal volume of one of the extraction media described in Part II. Media containing nicotine are recommended when the inoculum consists of leaves, especially if they are aged, whereas ordinary phosphate buffer is quite suitable for young root tips.

The herbaceous host range of GFLV is fairly wide, comprising some 50 species in seven dicotyledonous families (Dies, 1963; Hewitt et al., 1962; Martelli and Hewitt, 1963 ; Taylor and Hewitt, 1964). Differential diagnostic hosts are:

Chenopodium amaranticolor and Chenopodium quinoa, both reacting with occasional chlorotic/necrotic local lesions seven to ten days after inoculation, followed by systemic mottling and deformation of the leaves (Figures 17 and 18);
Gomphrena globosa, which exhibits chlorotic local lesions about one week after inoculation, turning reddish with age, light green to yellow spots and twisting of systemically invaded opposite upper leaves (Figure 19);
Nicotiana benthamiana, which reacts with occasional faint yellowish lesions followed by systemic mottling and deformation of the leaves in 10 to 15 days.

Whereas the above hosts are infected symptomatically by most virus isolates, many additional herbaceous indicators, such as Phaseolus vulgaris, Cucumis sativus and Petunia hybrida, are more selective in their susceptibility, being infectible by a smaller number of strains.

Presence of trabeculae (endocellular cordons)
Trabeculae are intraxylematic ribbon-like inclusions that cross tracheary elements radially. These structures are readily observed in crosssectioned basal internodes of green or mature canes. Sections made by hand with a razorblade are mounted without staining in a 1:1 mixture of water and glycerol and observed at low power undera light microscope. Freshlycross-sectioned canes can also be observed directly with a good (10 to 12x) magnifying lens. Trabeculae are more frequently found in American rootstocks than in European scion varieties. Their presence can be taken as evidence of viral infection, but their absence does not guarantee freedom from GFLV.

Serology

Immunodiffusion in agar gel is not applicable to leaf extracts of GFLV-infected grapevines, regardless of the severity of symptoms shown and the conditions under which plants are grown (field or greenhouse). Positive reactions are more readily and consistently obtained using crude sap expressed directly, without addition of buffers, from leaves of systemically invaded herbaceous hosts. Young upper leaves with strong symptoms are the best source of antigen.

ELISA. Immunoenzymatic procedures (see details in Part III) are now routinely employed for detection of GFLV in field- or greenhousegrown vines (Bovey, Brugger and Gugerli, 1980; Engelbrecht, 1980; Walteretal., 1984).Sources of antigens can be buds, roots, leaves and wood shavings, the last two being the most commonly used. Reactants can be either polyclonal antibodies or virus-specific or strain-specific monoclonalantibodies(Hussetal.,1986,1987).

The amount of plant material needed can be as low as 100 to 200 ma. However, it is customary to use 1 g of leaf tissues and 0.5 g of wood shavings. These are obtained by removing the bark and scratching, with a scalpel or a razorblade, the cortex of mature canes freshly harvested or cold-stored at 4 to 6°C (Walter and Etienne, 1987). Tissue samples, regardless of whether taken from leaves or cortex, are ground in PBS-Tween containing 2 percent PVP and 1 to 2.5 percent nicotine. Addition of nicotine is considered of paramount importance if tests are to be made from leaf tissues. No nicotine is needed if the extraction medium is made up of 0.1 M Tris-HCI buffer, pH 8.2, containing 0.8 percent NaCI and 2 percent PVP (Walter and Etienne, 1987).

The advantage of wood shavings over leaves is twofold: they can be used throughout the year without the apparent loss of efficiency that arises because of seasonal variations of antigen titre in vegetating organs; and they give low and consistent background readings. If leaves are employed, it is important, if not mandatory, to use extracts of the same rootstock or European scion variety as negative and positive controls. There is, in fact, a marked difference in background readings between American rootstocks and European cultivars and, among V. vinifera varieties, between those with hairy and those with glabrous leaves.

Immune electron microscopy (IEM and lSEM). GFLV can be detected by ISEM following the procedure outlined in Part III. Consistent and satisfactory results are obtained in spring from upper symptomatic leaves (Bovey, Brugger and Gugerli, 1980; Russo, Martelli and Savino, 1980).

Molecular hybridization

Cloned cDNA probes have been prepared to genomic (RNA-1 and RNA-2) and satellite RNAs of GFLV. These probes were successfully used for identification of GFLV infections in field-grown grapevine leaf extracts denatured with formaldehyde (Fuchs et al., 1991) and processed as described in Part III.

SANITATION

Sanitary selection and heat therapy together are powerful tools for reducing the incidence of fanleaf in newly established vineyards. Virusfree material (Figure 20) is readily obtained through conventional (Goheen and Luhn, 1973) or modified (Stellmach, 1980) heat therapy applications, micrografting and in vitro meristem tip or shoot-tip culture (Barlass et al., 1982).

REFERENCES

Barlass, M., Skene, K.G.M., Woodham, R.C. & Krake, R.C. 1982. Regeneration of virus-free grapevines using in vitro apical culture. Ann. Appl. Biol., 101: 291-295.

Bovey, R., Brugger, J.J. & Gugerli, P. 1980. Detection of fanleaf virus in grapevine tissue extracts by enzyme-linked immunosorbent assay (ELISA) and immune electron microscopy (IEM). Proc. 7th Meet. ICVG, Niagara Falls, NY, USA, 1980, p. 259-275.

Catalano, L., Roca, F. & Castellano, M.A.1989. Efficiency of transmission of an isolate of grapevine fanleaf virus (GFV) by three populations of Xipizinema index (Nematode: Dorylaimidae). Nematol. Mediterr., 17: 13- 15.

Cohn, E., Tanne, E. & Nitzany, F. 1970. Xiphinema italiae: a new vector of grape fanleaf virus. Phytopathology, 60: 181 - 182.

Dias, H.F. 1963. Host range and properties of grapevine fanleaf and grapevine yellow mosaic viruses. Ann. Appl. Bial., 5 I: 85-95.

Engelbrecht, D.J. 1980. Indexing grapevines for grapevine fanleaf virus by enzymelinked immunosorbent assay. Proc. 7th Meet. ICVG, Niagara Falls, NY, USA, 1980, p. 277282.

Fuchs, M., Pinck, M., Etienne, L., Pinck, L. & Walter, B. l991. Characterization and detection of grapevine fanleaf virus using cDNA probes. Phytopathology, 81: 559-565.

Goheen, A.C. & Luhn, C.F. 1973. Heat inactivation of viruses of grapevines. Riv. Patol. Veg. Sci., 9: 287289.

Hewitt, W.B. 1954. Some virus and virus-like diseases of grapevines. Bull. Culif: Dept. Agric., 43: 47-64.

Hewitt, W.B., Goheen, A.C., Raski, D.J. & Gooding, G.V. Jr. 1962. Studies on virus diseases of the grapevine in California. Vitis, 3: 57-83.

Hewitt, W.B., Raski, D.J. & Goheen, A.C. 1958. Nematode vector of soil-borne fanleaf virus of grapevines. Phytopathology, 48: 586-595.

Huss, B., Muller, S., Sommermeyer, G., Walter, B. & Van Regenmortel, M.H.V. 1987. Grapevine fanleaf virus monoclonal antibodies: their use to distinguish different isolates. J. Phytopathol., 1 19: 358-370.

Huss, B., Walter, B., Etienne, L. & Van Regenmortel, M.H.V. 1986. Grapevine fanleaf virusdetection in various grapevineorgans using polyclonal and monoclonal antibodies. Vitis, 25: 178188.

Lazar, J. Kolber, M. & Lehoczky, J. 1990. Detection of some nepoviruses (GFV, GFVYM, GCMV, ArMV) in the seeds and seedlings of grapevine by ELISA. Kertgasdasag, 22(4): 58-72.

Martelli, G.P. 1978. Nematode-borne viruses of grapevine, their epidemiology and control. Nematol. Mediterr., 6: 1-27.

Martelli, G.P. & Hewitt, W.B. 1963. Comparative studies on some Italian and Californian virus diseases of grapevine. Phytopathol. Mediterr., 2: 275-284.

Quacquarelli, A., Gallitelli, D., Savino, V. & Martelli, G.P. 1976. Properties of grapevine fanleaf virus. J. Gen. Virol., 32: 349-360.

Russo, M., Martelli, G.P. & Savino, V. 1980. Immunosorbent electron microscopy for detecting saptransmissible viruses of grapevine. Proc. Phylapathol. 7th Meet. ICVG, Niagara Falls, NY, USA, 1980, p. 251-257.

Savino, V., Cherif C. & Martelli, G.P. 1985. A natural serological variant of grapevine fanleaf virus. Phylapathol. Mediter, 24: 29-34.

Stellmach, G. 1980. Moderate heat propagation of grapevines for eliminating graft transmissible disorders. Proc. 7th Meet. ICVG, Niagara Falls, NY, USA, 1980, p. 325-328.

Taylor, R.H. & Hewitt, W.B. 1964. Properties and serological relationships of Australian and Californian soil-borne viruses of the grapevine, end arabis mosaic virus. Aust. J.Agric.Res., 15: 571585.

Walter, B. & Etienne, L. 1987. Detection of the grapevine fanleaf virus away from the period of vegetation. J. Phytopathol., 120: 355-364.

Walter, B., Vuittenez, A., Kuszala, J., Stocky, G., Burckard, J. & Van Regenmortel, M.H.V. 1984. Detection sérologique du virus du courtnoué de la vigne par le test ELISA. Agronomie, 4:527534.

Summary: fanleaf detection

GRAFT TRANSMISSION
Indicator
Vitis rupestris St George
No. plants/test
3-5 rooted cuttings
Inoculum
Wood chips, single buds, bud sticks, shoot tips
Temperature
22-24°C
Symptoms
Acute phase (shock) symptoms. Chlorotic spots, rings and lines, localized necrosis 3-4 weeks after grafting (chip-bud or green grafting);
Chronic symptoms. Reduced growth, severely deformed leaves with prominent teeth (distorting strains), yellow discolorations and mild deformation of the leaves (chromogenic strains)

TRANSMISSION TO HERBACEOUS HOSTS
Diagnostic host
Gomphrena globosa
Inoculum
Tissues from young symptomatic leaves or succulent roots
Extraction
Grind in 2.5 percent aqueous nicotine
Temperature
Below 25°C
Symptoms
Chlorotic local lesions soon turning reddish in
7-8 days; twisting of the upper leaves in 10-12 days

OTHER TESTS
Serology (ELISA, ISEM)
Molecular hybridization

FIGURE 1 Severely malformed leaves and bushy vegetation in a vine infected by a distorting GFLV strain

FIGURE 2 Grape leaf with typical fanleaf symptoms

FIGURE 3 Chlorotic mottle and deformation caused by fanleaf infection

FIGURE 4 Abnormal branching in a shoot of a fanleaf-infected vine

FIGURE 5 Abnormal shoot in a fanleaf-infected vine

FlGURE 6 Bunches from a healthy (left) and a fanleaf-infected (right) vine. Note reduced size and extensive shot berry condition

FIGURE 7 Total yellowing of a spring shoot of a vine infected by chromogenic GFLV strain

FIGURE 8 Yellow rings and line patterns in a leaf infected by a chromogenic GFLV strain (Photo: B. Walter)

FIGURE 9 Yellow mosaic symptoms in a leaf of an American rootstock

FIGURE 10 Spring symptoms of yellow mosaic

FIGURE 11 Totally yellow and stunted vine affected by yellow mosaic next to a normally growing plant

FIGURE 12 A patch of vines with yellow mosaic seen from a distance

FIGURE 13 Yellow mosaic symptoms fading away in late summer

FIGURE 14 Shock symptoms (chlorotic rings and lines) in a V. rupestris indicator graftinoculated with a fanleaf source

FIGURE 15 Chronic symptoms (severely deformed leaves with prominent teeth) in a V. rupestris indicator graft-inoculated with a fanleaf source

FIGURE 16 Chrome yellow discolorations and deformations of leaves of a V. rupestris indicator graft-inoculated with a yellow mosaic source

FIGURE 17 Systemic mottling induced by GFLV in Chenopodium amaranticolor

FIGURE 18 Systemic mottling and deformation of the leaves of Chenopodium quinoa infected by GFLV

FIGURE 19 Twisting of the upper leaves typically induced by GFLV infections in Gomphrena globosa

FIGURE 20 Bunches from a fanleaf-infected vine before (left) and after (right) heat therapy

Grapevine degeneration - European nepoviruses

G.P. Martelli and B. Walter

CAUSAL AGENTS

In addition to GFLV there are seven distinct nepoviruses: tomato black ring (TBRV), arabis mosaic (ArMV), raspberry ringspot (RRV), strawberry latent ringspot (SLRV), grapevine chrome mosaic (GCMV), grapevine Bulgarian latent (GBLV) and artichoke Italian latent (AILV) viruses, involved to a varying extent in the aetiology of this disease. All these viruses have isometric particles about 30 nm in diameter and the bipartite genome typical of the nepovirus group (Harrison and Murant, 1977). All possess natural serological variants, but in most cases strains infecting grapevines belong to a single serotype. Some of these viruses are serologically distantly related to one another or to other nepoviruses, i.e. GCMV to TBRV, ArMV to GFLV, GBLV to blueberry leaf mottle virus (reviewed by Martelli and Taylor, 1989). There are biological variants that elicit different symptoms in naturally and artificially infected hosts.

GEOGRAPHICAL DISTRIBUTION

European nepoviruses prevail in central and eastern Europe, where they occur in many natural and agricultural environments together with their vectors. In grapevines, ArMV is common in certain areas of France (Charentes, Alsace) and Germany (Palatinate) and is more rarely found in Switzerland, Italy, Yugoslavia, Hungary and Bulgaria. TBRV, SLRV and RRV are largely restricted to Germany (Palatinate and Moselle), even though there are occasional records from other European (Italy, France, Portugal, Turkey) and non-European (Israel) countries. GCMV and GBLV prevail in eastern Europe (Yugoslavia, Czechoslovakia, former USSR, Hungary, Bulgaria).

ALTERNATE HOSTS

ArMV, TBRV, RRV, SLRV and AILV have a wide range of wild and cultivated alternate hosts that constitute their natural reservoirs. These hosts include annual and perennial weeds, vegetable crops, shrubs, ornamentals and fruit trees. No alternate hosts for GCMV and GBLV are known.

FIELD SYMPTOMS

Like GFLV, some European nepoviruses (e.g. ArMV and GCMV) have distorting and chromogenic strains that elicit different symptomatologies, characterized by chlorotic mottling, leaf and cane deformation or chromeyellow discolorations of the foliage (Figures 21 to 25). The symptoms shown in the field by vines affected by these viruses are very similar to, if not indistinguishable from, those of GFLV-induced degeneration.

A distinctive trait of the yellowing caused by GCMV is that, unlike GFLV yellowing, it shows up equally well in the field and greenhouse. Furthermore, vines infected by GCMV lack vigour, bear little or no crop and tend to decline and die within a few years after infection. Heavy yield losses (up to 80 percent of the crop) are associated with infections by other European nepoviruses such as SLRV, TBRV and RRV (Ruder, 1985). These viruses induce leaf deformity and yellowing (Figures 26 and 27). In the cultivars Gruner Sylvaner and Riesling, SLRV elicits a most peculiar reddish discoloration of the tip of the spring shoots (Figure 28). This discoloration disappears in summer, when the vines exhibit a more or less normal appearance but are virtually fruitless (Figure 29).

NATURAL SPREAD

Experimental evidence for nematode transmission from grape to grape has been obtained only for ArMV/Xiphinema diversicaudatum and TBRV/Longidorus attenuantus. The ecology of these viruses in vineyards follows the same pattern known for other crops, i.e. seed transmission in weeds and persistence of the pathogens in their seedlings, which constitute sources of inoculum and food for the vector (reviewed by Martelli, 1978).

Although there is plenty of visual evidence that RRV and GCMV spread naturally in vineyards in a manner suggestive of soil transmission, their vectors have not been identified. Long-distance spread is through infected propagative material (budwood, rootstock roofings, grafted vines).

Rooted plants are high-risk materials because they can carry viruses and vectors together and thus establish active infection foci in any new environment favourable to active multiplication of the nematode vectors.

DETECTION

The presence of the disease in vineyards is disclosed by its symptoms. Plants can be inspected for symptoms throughout the vegetating season, but special attention should be paid to the spring growth, which ordinarily expresses the strongest reactions. Infections by mild virus strains or tolerance in certain cultivars can result in attenuated forms of disease that may escape observation. In the field it is virtually impossible to establish whether any given symptomatology is induced by one virus or another, or by a mixed infection of two or more viruses.

Sorting and identification of viruses can only be done by biological assays and/or laboratory tests.

IDENTIFICATION
Indexing by graft transmission

Any of the current graft inoculation procedures described in Part 11 can be used.

Graft transmission of GCMV to Vitis species yields foliar distortions (Figure 30) and yellow discolorations (Figure 31 ) like those induced by other nepoviruses (GFLV in particular). Somewhat more specific reactions are given by the cultivars Pinot noir and Jubileum 75, both of which show extremely severe stunting and necrosis of the apex (Figure 32). No symptoms are produced in Vitis rupestris St George, which differentiates GCMV from GFLV. In field indexing, symptoms develop on the second year's growth (Lehoczky, 1985).

Siegfriedrebe (FS4 201-39) is regarded in Germany as the best indicator for ArMV, RRV and TBRV. Cane deformations and foliar discolorations appear a few weeks after inoculation, but these symptoms are not distinctive enough to separate viruses. The Pinot noir cultivar can also be used as an indicator for TBRV.

Transmission to herbaceous hosts
All European nepoviruses are readily transmitted to herbaceous hosts by sap inoculation. Sources and preparation of inoculum and inoculation procedures are the same as described for GFLV.

The separation of viruses based on differential host range responses is not easy and may pose problems even to experienced workers. Certain hosts, however, give reactions that provide useful hints for identification.

Symptoms in Chenopodium quinoa are generally milder for ArMV than for GFLV. They consist of systemic mottling and mild deformations of the leaves, with rare local lesions (Figure 33). ArMV can also induce striking chlorotic ringspotting in Nicotiana glutinosa (Figure 34).

TBRV is very severe in C. quinoa, producing distinct chlorotic/necrotic local lesions followed by systemic spread and necrosis of the plant tips (Figure 35). RRV induces local chlorotic or necrotic spots in Nicotiana rustica, followed by chlorotic/necrotic systemic rings, spots and line patterns.

SLRV may elicit chlorotic local lesions in inoculated Cucumis sativus cotyledons, followed by systemic interveinal chlorosis.

GCMV has fairly narrow host range (Martelli and Quacquarelli, 1972). Diagnostic hosts are:

Chenopodiurn quinoa. Small chlorotic local lesions, sometimes with necrotic centre, severe systemic chlorosis, necrotic speckling and apical necrosis (Figures 36 and 37);
Datura stramonium. No visible localized infections. Transient systemic, yellowish zonate spots;
Gomphrena globosa. Chlorotic or reddish local lesions. Transient systemic mosaic and vein clearing;
Phaseolus vulgaris. Systemic symptoms only; mosaic, chlorotic rings and specks, sometimes necrotic blotches.

GBLV also has a narrow host range (Martelli et al., 1977). Diagnostic hosts are:

Chenopodium quinoa. Chlorotic or necrotic local lesions in three to four days, followed by systemic leaf mottle and necrotic flecks (Figure 38);
Gomphrena globosa. Reddish local lesions and deformation but not twisting of the upper leaves;
Nicotiana clevelandii. Necrotic local lesions, systemic leaf mottling and stunting.

Serology

Immunodiffusion tests may be used successfully with extracts from GBLV-infected grapevine leaves, especially when collected from greenhouse-grown cuttings. The same does not apply to tissue extracts of vines infected by any other European nepovirus. However, with these other viruses positive reactions are obtained using crude sap expressed directly, without addition of buffered solutions, from leaves of systemically invaded herbaceous hosts. Young, upper symptomatic leaves are the best source of antigen.

ELISA. Immunoenzymatic procedures carried out as described in Part III can be employed for the detection and identification of different European nepoviruses in naturally infected vines (Tanne, 1980; Stellmach, 1985; Walter et al., 1984; Kölber et al., 1985). Sources of antigen can be buds, roots, leaves, bark and wood scrapings. The weight of tissues used varies from 200 mg to 1 g, and the extraction buffer, as discussed for GFLV, may or may not contain nicotine ( 1 to 2.5 percent). As with GFLV, the use of buds from dormant canes or wood shavings may bypass the problem posed by the seasonal variation of virus titre, which limits the utilization of ELISA to certain growth periods (Ruder, Alebrand and Altmayer, 1983; Kölber et al., 1985). Single as well as mixed infections can be picked up by ELISA, and the viruses can be identified. Precautions for positive and negative controls are the same as with GFLV.

Immune electron microscopy (ISEM). Detection and identification of European nepoviruses by ISEM can be carried out, as outlined in Part 111, using upper symptomatic leaves collected in spring (Russo, Martelli and Savino, 1980).

Molecular hybridization

Aprobe to ArMV has been prepared (Steinkellner et al., 1989). cDNA probes to GFLV RNA- 1 or RNA-2 can also detect some ArMV isolates.

SANITATION

The same techniques used for GFLV are applicable.

REFERENCES

Bercks, R. & Stellmach, G. 1966. Nachweis verschiedener Viren in Reisigkrankheiten Reben. Phytopathol. Z., 65: 288-296.

Harrison, B.D. & Murant, A.F. 1977. Nepovirus group. Descriptions of Plant Viruses, No. 185. Kew, UK, Commonw. Mycol. Inst./Assoc. Appl. Biol.

Kölber, M., Beczner, L., Pacsa, S. & Lehoczky, J. 1985. Detection of grapevine chrome mosaic virus in field-grown vines by ELISA. Phytopathol. Mediterr., 24: 135- 140.

Lehoczky, J. 1985. Detection of grapevine chrome mosaic virus in naturally infected vines by indexing. Phytopathol. Mediterr., 24: 129-134. Martelli, G.P. 197X. Nematode-borne viruses of grapevine, their epidemiology and control. Nematol. Mediterr., 6: 127.

Martelli, G.P., Gallitelli, A., Abracheva, P., Savino, V. & Quacquarelli, A. 1977. Some properties of grapevine Bulgarian latent virus. Ann. Appl. Biol., 85: 51-58.

Martelli, G.P., Lehoczky, J. & Quacquarelli, A. 1966. Host range and properties of a virus associated with Hungarian grapevines showing macroscopic symptoms of fanleaf and yellow mosaic. Proc. Int. Conf: Virus Vector Perennial Hosts and Vitis, 1965, p. 389-401. Div. Agric. Sci., Univ. Calif., Davis.

Martelli, G.P. & Quacquarelli, A. 1972. Grapevine chrome mosaic virus. Descriptions of Plant Viruses, No. 103. Kew, UK, Commonw. Mycol. Inst./Assoc. Appl. Biol.

Martelli, G.P. & Taylor, C.E. 1989. Distribution of viruses and their nematode vectors. Adv. Dis. VectorRes., 6: 151-189.

Rudel, M. 1985. Grapevine damage induced by particular virus-vector combinations. Phytopathol. Mediterr., 24: 183- 185.

Rudel, M., Alebrand, M. & Altmayer, B. 1983. Untersuchungen Ober den Einsatz des ELISA Test zum Nachweis verschiedener Rebenviren. Vein Wiss., 38: 177-185.

Russo, M., Martelli, G.P. & Savino, V. 1980. Immunosorbent electron microscopy for detecting saptransmissible viruses of grapevine. Proc. 7th Meet. /CGV, Niagara Falls, NY, USA, 1980, p. 251 -257.

Steinkellner, H., Himmler, G., Laimer, M., Mattenovich, D., Bisztroy, G. & Katinger, H. 1989. Konstruktion von cDNA von Arabis Mosaik Virus und deren Anwendung für Diagnose. Mitt. Klosterneuburg Rebe Wein Obstbau Frht.' 39: 242246.

Stellmach, G. 1985. ELISA testing of grapevine roofings reared from nepovirus-infected mother plants forced to rapid growth. Phytopathol. Mediterr., 24: 123- 124.

Tanne, E. 1980. The use of ELISA for the detection of some nepoviruses in grapevines. Proc. 7th Meet. ICVC;, Niagara Falls, NY, USA, 1980, p. 293-296.

Walter, B., Vuittenez, A., Kuszala, A., Stocky, G., Burckard, J. & Van Regenmortel, M.H.V. 1984. Detection sérologique du virus du courtnoué de la vigne par le test ELISA. Agronomie, 4: 527-534.

Summary: grapevine degeneration detection

GRAFT TRANSMISSION
Indicators
Several Vitis vinifera cultivars: Pinot noir, Jubileum 75 (GCMV); Siegfriedrebe (ArMV, RRV, TBRV)

No. plants/test
3-5 rooted cuttings Inoculum
Wood chips, single buds, bud sticks, shoot tips
Temperature
22-24°C
Symptoms
Severe stunting and necrosis of the apex of Pinot noir in the second year of vegetation (GCMV); foliar discolorations and cane deformations of Siegfriedrebe within the first year after inoculation

TRANSMISSION TO HERBACEOUS HOSTS
Diagnostic hosts
Datura stramonium (GCMV), Chenopodium quinoa (TBRV, GBLV), Nicotiana clevelandii (RRV), Cucumis sativus (SLRV), Nicotiana glutinosa (ArMV)
Inoculum
Tissue from young symptomatic leaves
Extraction
Grind in 2.5 percent aqueous nicotine
Temperature
Below 25°C
Symptoms
In D. stramonium (CGMV), transient, systemic, yellowish zonate spots;
In C. quinea (TBRV), necrotic local lesions in 6-8 days, followed by mosaic and necrosis of the plant tip in about 2 weeks;
In C. quinoa (GBLV), necrotic local lesions in 3-4 days, systemic chlorotic mottle and necrosis;
In M clevelandii (RRV), necrotic local spots and rings in 5-7 days and systemic veinal necrosis;
In C. sativus (SLRV), chlorotic local lesions in 5-7 days and systemic interveinal chlorosisor necrosis in 10-12 days;
In N. glutinosa (ArMV), chlorotic ringspots