Nematol. medit. (1978), 6: 1·27.

I stituto di Patologia vegetale, Universita degli Studi 70126 Bari, Italy

NEMATODE-BORNE VIRUSES OF GRAPEVINE, THEIR EPIDEMIOLOGY AND CONTROL (1)

by

GIOVANNI P. MARTELLI

INTRODUCTION

The discovery of a nematode vector for the fanleaf disease of grapevine (Hewitt et al., 1958) together with the demonstration that the viral agent was mechanically transmissible to herbaceous hosts (Cadman et aI., 1960) greatly stimulated investigations on viruses of cultivated Vi tis. Consequently, in little less than two decades many different viruses have been manually transmitted from diseased vines in several grape-growing countries. By 1976 there were 24 viruses recorded from grapevine (Martelli, 1978) half of which are either recognized or putative members of the nepovirus group (Table I). At present, nepoviruses are the only nematode-borne pathogens known to infect grapevine in which they may cause destructive diseases. Although occurring in the major viticultural areas of the world, not all the viruses are equally widespread. Thus, with the exception of grapevine fanleaf virus (GFV) which is ubiquitous (Hewitt, 1968) and uniquely specialized for having no natural host other than grapevine, other viruses like arabis mosaic (AMV), tomato black

(1) The present review is largely based on an invitation paper given at the Conference on «Plant Disease Epidemiology and Dispersal of Plant Para­ sites », London, 14-18 December, 1977. Grateful thanks are espressed to Drs. T. J. W. Alphey, H. F. Dias, R. I. B. Francki, F. Lamberti, A. F. Murant, D. C. Ramsdell, M. Rudel and J. K. Uyemoto for kindly providing unpublished data or manuscripts prior to publication. I am also much endebted to Dr. C. E. Taylor for reviewing the manuscript at a very short notice and for making many helpful suggestions.

-1- ring (TBRV), raspberry ringspot (RRV), strawberry latent ringspot (SLRV), grapevine chrome mosaic (GCMV), and artichoke Italian latent (AILV) are restricted to European countries (Table I). These will be referred to as European nepoviruses as opposed to American nepoviruses, i.e. tomato ringspot (TomRSV) tobacco rings pot (TRSV) and peach rosette mosaic (PRSV) which seem confined largely to North America. Besides GFV, grapevine Bulgarian latent (GBLV) is the only grapevine nepovirus recorded, so far, in both Europe and North America. Various aspects of the biology and ecology of nematode-transmit­ ted viruses have been reviewed in recent times in articles which also contain much information on those infecting grapevine (Murant, 1970; Taylor, 1971, 1972; Vuittenez et al., 1972; Weischer, 1973, 1975; Taylor and Robertson, 1975; Harrison, 1977; Lamberti, 1978).

THE DISEASES

Symptomatology

Most European nepoviruses induce diseases which are charac­ terized by a large and rather confusing array of symptoms. Field syndromes are often the same whether caused by anyone virus or by an association of different viruses, so that it is virtually impossible to distinguish disorders evoked by specific entities on the basis of symptomatology alone. Variability in symptom expression depends on the host (Vitis species or cultivar) and, to a greater extent, on the virus strain. In fact, severa) nepoviruses e.g. GFV (Hewitt et al., 1962), GCMV (Martelli et al., 1965), AMV (Stellmach, 1970a) and TomRSV (Gooding and Hewitt, 1962; Gilmer and Uyemoto, 1972) possess «distorting» and «yellow}} strains or isolates which mayor, more commonly, may not be serologically distinguishable but are liable to induce strikingly different syndromes. Thus for example, « distorting}} strains are associated with malformations of leaves and canes sometimes accompanied by chlorotic mottles, whereas «yellow}} strains induce bright chrome-yellow discolourations of the foliage without much deformation. Reduced vigour and low yield may be associated with infection by either type of strains.

-2- Among American nepoviruses, TRSV and TomRSV cause serious decline of Vitis vinifera L. cultivars and their hybrids, whereas PRMV is a major pathogen of Vitis labrusca. In all cases symptoms consist largely of delayed spring growth, mottling, malformation and rolling of the leaves, severe stunting and poor fruit set. Ultimately, affected vines may die (Gilmer et al., 1970; Ramsdell and Myers, 1974; Uye­ moto, 1975; Dias and Cation, 1976; Dias, 1977a). However, the serologically distinct yellow vein strain of TomRSV, the causal agent of grapevine decline in Northern U.S.A. and Canada, seriously affects the yield of Californian grape cultivars without hampering the growth of diseased plants which, surprisingly, appear more vigorous than healthy ones (Gooding and Hewitt, 1962). Whether or not symptoms like leaf enations and wood pitting may be associated with infection by nepoviruses is still controversial. As discussed in previous reviews (Hewitt, 1968, 1973; Martelli, 1975b; Goheen, 1977), both types of symptoms rather than characterizing specific diseases may represent host responses to more than one viral pathogen. It should be borne in mind, however, that GFV very often has been found associated with enations in Europe and California (Hewitt, 1968; Graniti and Martelli, 1970a), whereas GFV, TomRSV and TRSV have been recovered from stem-pitted vines in several countries (Hewitt et al., 1970; Graniti and Martelli, 1970b; Uyemoto, 1975). Interestingly, field spread of stem pitting is associated with the presence of Xiphinema index in Southern Italy (Graniti and Mar­ telli, 1970b) and France (Boubals, 1976a).

Economic importance

Nepoviruses are detrimental to the grape industry in many ways. They may cause: a) progressive decline and death of the vines; b) low quantity and quality of yield; c) shortening of the productive life of the vineyard; d) low proportion of graft take; e) reduced rooting ability of propagating material; f) decreased resistance to adverse climatic conditions (for details see: Brlickbauer, 1962; Hewitt, 1968; Stellmach, 1970b; Gilmer et al., 1970; Ramsdell and Myers, 1978). Information on crop losses due to nepoviruses is fragmentary for no systematic assessment has been carried out in any country on a sufficiently large scale. Besides, most of the available data for European nepoviruses refer only to GFV and its strains. Thus,

-3 Table I - Nematode-borne viruses of grapevine, their vectors and geographical distribution.

References Vir u s Vector Geographical distribution First First Sap - transmission Nematode transmission

A) Recognized nepoviruses 1. Grapevine fanleaf Distorting strains x. index Worldwide Cadman et al., 1960 Hewitt et al., 1958 X. italiae Cohn et al., 1970 Yellow mosaic strain X. index Worldwide Cadman et al., 1960 Hewitt et ai., 1962 Vein banding strain X. index Worldwide Hewitt et al., 1962 Alfaro and Goheen, 1974 2. Arabis mosaic Type strain X. diversicaudatum France, Germany, Bercks and Stellmach, Vuittenez et al., 1976 Hungary 1966 3. Tomato blackring English strain L. attenuatus Germany Stellmach and Bercks, Rudel, 1977 1963 4. Raspberry rings pot Bercks' isolate unknown Germany Bercks, 1968 Palatinate strain unknown Germany Vuittenez et al., 1970 5. Strawberry latent ring­ spot Type strain unknown Germany Vuittenez et al., 1970 6. Grapevine chrome mo- saic unknown Hungary Martelli et al., 1965 7. Artichoke Italian la- tent unknown Bulgaria Jankulova et al., 1978 8. Peach rosette mosaic X. americanum U.SA. (Michigan), Dias, 1968 Dias, 1975 Canada 9. Tomato ringspot Yellow vein strain x. americanum U.S.A. (California) Gooding and Hewitt, Teliz et al., 1966 Type strain unknown U.S.A. (New York) 1962 Canada, Yugoslavia (?) Gilmer and Uyemoto, 1972 10. Tobacco ringspot unknown U.S.A. (New York) Gilmer et al., 1970 B) Possible nepoviruses 11. Grapevine Bulgarian latent Type strain unknown Bulgaria Martelli et al., 1977 New York strain unknown U.S.A. (New York) Uyemoto et al., 1977 12. Grapevine CM-112 * unknown Portugal Ferreira and De Sequeira, 1972 * Recent findings indicate that this virus is serologically related to Grapevine Bulgarian latent virus (R. Hull, per­ sonal communication) Vuittenez (1970) reckons that a 50% reduction in weight is the average crop loss caused by GFV to susceptible V. vinifera cultivars in European countries. Such losses are aggravated by the poor quality of clusters which are often unmarketable [see also review by Bovey, (1970) and recent reports by Legin (1972) and Prota and Garau (1978)]. Some American nepoviruses are even more destructive to cultivars and interspecific hybrids of V. vinifera, possibly because of the greater susceptibility of these hosts. For instance, Californian vines infected by the yellow vein strain of TomRSV are reported to be virtually unfruitful (Gooding and Hewitt, 1962), and in Canada, Dias (1977a) has recently recorded crop losses ranging from 76 to 95% in a French hybrid infected by the type strain of TomRSV.

THE VIRUSES

Nepoviruses have isodiametric particles about 30 nm in diameter which sediment as several components when centrifuged, and possess two species of functional RNA, each contained in a specific particle. As a group, these viruses have been extensively reviewed (Martelli, 1975b; Harrison and Murant, 1977; Francki and Hatta, 1977). Recent studies have established that: (i) GCMV, GFV and PRMV have properties typical of members of the nepovirus group (Martelli and Quacquarelli, 1972; Martelli et al., 1977; Quacquarelli et al., 1976, 1978; H. F. Dias, personal communication); (ii) GFV isolates, whatever their geographical origin, have similar physico-chemical properties and hydrodynamic behaviour (Quacquarelli et al., 1976; Dias, 1978; Romero et al., 1978); (iii) grapevine isolates of AMV, TBRV, TomRSV and AIL V do not differ from the respective type cultures (Quacqua­ relli et al., 1978; Savino et al., 1977; Allen and Dias, 1977). Hence, on the basis of current physico-chemical determinations (Table II), grape isolates of nepoviruses are hardly distinguishable from each other and from strains of the same viruses infecting other plants. Strain differentiation based upon symptomatological responses of herbaceous hosts may also be problematic. Obvious differences between diverse isolates of the same virus do occur, but these are not necessarily correlated with specific hosts and/or syndromes. In fact, indications of host range studies may be misleading as exem­ plified by the recovery in France of viruses serologically identical

-5- Table II - Physico-chemical characteristics of grapevine isolates of some nepoviruses.

GFV • AMV • AILV + PRMV 0 TBRV + GCMV + I TomRSV §

Characteristics 1 I I I T ! B T ! M I M B I-T M I B T M T M I B ! B M B I~-B I I I I I i --- I I I ---

Sedimentation coefficient (Sw,20) 50 86 120 51 91 125 55 96 121 52 115 134 99 117 98 115

Percentage of RNA 0 30 41 0 28 41 0 34 41 0 36 42 35 42 35 42 0 40 41

Buoyant density (g/cm3) 1.31 1.41 1.49 1.43 1.50 + 1.47 1.51 1.44 1.50 1.44 1.50 1.49 1.50 1.51

Temperature of RNA release (OC) 66 60 61 68 64

M.wt. of RNA (x 106 ) 1.4 2.4 1.4 2.4 1.5 2.4 2.1 2.5 1.5 2.5 1.5 2.4 2.5 2.6

M.Wt. of protein subunits 54,000 54,000 54.000 55 - 58,000 54,000 54,000 58,000

* Quacquarelli et al., 1976. + Quacquarelli et al., 1978. o H. F. Dias, 1977 (personal communication). § Allen and Dias, 1977. with AMV but indistinguishable from GFV with respect to host range and symptoms (Vuittenez et al., 1968). Most grapevine nepoviruses show no serological specificity of their own and can be assimilated either to type strains or to recognized serotypes of the parent virus. For instance AMV isolates from Ger­ many and Hungary were indistinguishable from each other or from Harrison's (1958) AB 10 type culture (Martelli and Lehoczky, 1968); French AMV isolates behaved similarly (Vuittenez et aI., 1968). Like­ wise, grapevine SLRV and AILV were found to share most if not all antigenic determinants with their type strains (Vuittenez et al., 1970; Savino et a!., 1977), whereas TBRV from different German viticultural areas were all identified with the English strain of the same virus (Bercks, 1968; Vuittenez et al., 1970). In New York State two serotypes of TRSV are present, one of which was recovered from diseased grapes (Uyemoto, 1975) but it is not known whether it is specific for this host. Conversely, the yellow vein strain of TomRSV constitutes a noteworthy exception for it differs serologically from the more widely distributed type TomRSV and may represent, on the basis of present knowledge, a grapevine-specific strain of this virus (Gooding, 1963; Teliz et al., 1966; Allen and Dias, 1977). A similar situation may exist with RRV of Palatinate vines which is distantly related to typical RRV infecting raspberry and cherry in France, and is con­ sidered by Vuittenez et al. (1970) to be a virus in its own right. However, a thorough serological comparison of this virus with other German RRV grapevine isolates (Bercks, 1968) and other known RRV strains appears desirable before definite conclusions can be drawn. Large serological differences have also been detected between Euro­ pean and North American strains of GBLV (Uyemoto et al., 1977a; Martelli et al., 1978). A rather common feature of nepoviruses is their serological· var­ iability which allows for identification of different strains or serotypes within each viral «species ». As mentioned above, this applies also to many grape nepoviruses but certainly not to GFV, which is remarkable for its serological uniformity. Some GFV isolates may show a different degree of relatedness with AMV (Vuittenez, 1970; G. P. Martelli, unpublished information) but none of them has been found, so far, to differ antigenically from any other to the extent of forming spurs in gel-diffusion tests or leaving behind detectable amounts of antibodies after cross absorption with heterologous

-7- antisera (Cadman et al., 1960; Barabino, 1963; Dias and Harrison, 1963; Martelli and Hewitt, 1963; Taylor and Hewitt, 1964; Quacqua­ relli and Martelli, 1965; Feldman, 1966; Vuittenez, 1970; Savino et al., 1975; Garcia Gil de Bernabe, 1976; Saric and Hranueli, 1977; Romero et al., 1978). The reasons for this striking serological similarity among GFV populations, that is independent of differences in geographical provenance, host (species or cultivars of Vilis) and type of symptoms, are unknown but an explanation may lie in the comparatively low selection pressure to which GFV has been subjected in nature because of its strict adptation to a single host.

THE VECTORS

To date, four X iphinema species and a single Longidorus species have been experimentally identified as vectors of grapevine nepo­ viruses (Table I). A close association has been observed between x. vuittenezi and field spread of GFV in Hungary (Martelli and Sarospataki, 1969), between x. americanum and spread of both TomRSV (type strain) and TRSV in New York State and Canada (Gilmer et a!., 1970; Uyemoto, 1975; Dias, 1977a) and between X. di­ versicaudatum and spread of AMV in Germany (Bruckbauer and Rudel, 1976) and France (Vuittenez et al., 1978). Also, in Germany, L. macrosoma sometimes was found in vineyards where RRV was present (Weischer, 1968). Although in most of the above cases there is fair circumstantial evidence that the nematode species mentioned are responsible for the transmission of the viruses with which they are associated, definite proof of this is still lacking. In this connection it may be noted that a high proportion of cucumber seedlings became infected when grown in soil containing X. americanum collected from vineyards in New York State and Canada under TRSV-and TomRSV-infected vines (Gilmer et al., 1970; Dias, 1977a). However, as recently confirmed by Bercks et al. (1977), there is no experimental evidence that X. vuit­ tenezi may act as a possible vector of GFV in certain areas of Ger­ many (Rudel, 1971; Wei scher, 1973) and France (Vuittenez et al., 1972) where field spread of this virus takes place apparently in absence of the alleged vectors, X. index and X. italiae.

-8- Geographical distribution

Much information on the geographical distribution of longidorid nematodes is available from surveys carried out in the last decade in many grape-growing countries, particularly in Europa. It was reviewed in part by Weischer (1974). Regrettably, these field surveys seldom include sufficient information on soil types and environmental factors, which would provide a clearer insight into the still inadequately known ecology of virus vector species (for review see Weischer, 1975). According to published records, X. index is by far the most widespread vector of grapevine viruses. It occurs throughout Europe (Weischer, 1974) and the Mediterranean basin and is firmly estab­ lished in America (California, Chile and Argentina), South Africa and Australia (Siddiqi, 1974). The origin of this nematode is uncertain but the fact that its presence in a natural environment has been recorded only in Iran (D. Sturhan, in Weischer, 1975) may perhaps indicate its home in ancient Persia. If this were so, then both GFV (Hewitt, 1968, 1970) and its vector have a common area of origin from which, conceivably, they have spread together first in the Old World and, more recently, in other continents. This is in accord with the idea that X. index is not a native of California (Raski and Hewitt, 1963). X. index is generally insensitive to soil texture and pH (Prota, 1970; Weischer, 1975) - which accounts, at least in part, for its wide geographical distribution - but it thrives better in sands and light alluvial loams than in clays (Prota, 1970; Coiro and Lamberti, 1978). X. americanum probably does not occur in Western Europe (Martelli and Lamberti, 1967; Taylor, 1971) its closest record being from the Azores islands (Sturhan, 1973). However, it is widely dis­ tributed in the American continent and in the Far East where it is encountered in a great variety of environments and soils (Siddiqi, 1973; Wei scher, 1975). X. diversicaudatum, X. vuittenezi and L. macrosoma, although apparently rather less demanding in their environmental requirements, are more frequently found in Central Europe in heavy soils with high moisture content (Taylor, 1971, 1972; Weischer, 1975; Taylor and Brown, 1976). Conversely, X. italiae is restricted almost entirely to the sands and light soils of the Mediterranean coast-lines (Weischer, 1975). Temperature is another important factor in affecting nematode

-9- distribution. Thus, X. index and X. italiae are widespread only in areas with temperate climate and mild winters such as Italy (Roca and Lamberti, 1978), Mediterranean France (Dalmasso et al., 1972) and Spain (Arias, 1978). In colder regions like Alsace (Vuittenez et al., 1972), Hungary (Martelli and Sarospataki, 1969) and Germany (Rudel, 1971), X. italiae is not found and X. index, although present, is less frequent than X. vuittenezi. Moreover, temperatures around 20° C appear to be optimal for virus transmission by X. index (Das and Raski, 1968) and for the completion of life cycle [adult-to-adult in 3-4 months, the shortest time recorded with critical studies (Prota et al., 1977)]. L. attenuatus and X. diversicaudatum are more northerly species. They are inconsistently associated with grapevine in nature (Rudel, 1971; Roca and Lamberti, 1978), although X. diversicaudatum thrives very well on grapes under artificial conditions (Vuittenez et al., 1972). No record of any of these two species exists in vineyards outside Europe (Pitcher et al., 1974; Brown and Boag, 1975).

Vertical distribution in soil

The few available data on the vertical distribution in soils of nematode vectors of grapevine viruses, are consistent with the notion that such distribution is uneven and generally relates to the root system of the host plants. Thus, in Southern Italy it was found that the distribution pattern of X. index was not noticeably affected by soil temperatures and remained remarkably constant throughout the year (Lamberti and Martelli, 1965). In particular, in vineyards established on permeable soils the distribution curve of this nematode showed at first an increase of the populations with a peak in the 30-50 cm range, corresponding with the level of maximum root concentration, then a steep drop which continued steadily down to the deepest level sampled. A somewhat similar pattern, i.e. largest X. index populations established between 30 and 60 em in depth, was reported from France under the comparable soil conditions of Burgundy (Taylor, 1971), and from Spain (Weischer, 1973). In heavy clay and in irrigated vineyards the largest nematode numbers were found in the top 30 cm of soils again relating to the presence of abundant feeder roots (Lamberti and Martelli, 1965; C. E. Taylor, in Weischer, 1975).

-10- Conversely, in a fallowed vineyard soil in California, Raski et al. (1965) have recorded a more or less constant level of X. index popu­ lations from 60 cm down to 2 m over a period of 5 years. They also demonstrated that in deep soils X. index can reach considerable depths (down to 3.6 m) and persist there for many years in absence of actively growing host plants, but apparently surviving by feeding on root pieces remaining after the removal of the crop. In the shallower soils of Apulia (Southern Italy) X. index individuals were recovered from samples taken at a maximum depth of 2 m. Albeit few, these nematodes were viruliferous and readily transmitted GFV, as well as the more numerous individuals collected in upper soil levels (Lamberti and Martelli, 1965). Recent investigations on X. americanum in two Michigan vine­ yards affected by PRMV, have shown that this species has a vertical distribution comparable to that recorded for X. index and may reach 2 m in depth (Ramsdell and Myers, 1978).

VIRUS-VECTOR RELATIONSHIP

In the last few years no great advances have been made in the knowledge of virus-vector relationship inasmuch as grape nepoviruses are concerned. As repeatedly reviewed (Taylor, 1971, 1972; Taylor and Robertson, 1975) most of the work on this subject refers to GFV and X. index which seems to represent one of the most thoroughly studied virus-vector combinations.

Acquisition and retention

Acquisition access threshold, i.e. the minimum time required for a nematode to become viruliferous when feeding upon an infected root (Teliz et ai., 1966) was found to be 1 h with heavy inocula (800 individuals per test pot) of X. americanum and TomRSV (Teliz et al., 1966), 24 h with 600 individuals/pot of X. index and GFV (Raski and Hewitt, 1960) and only 15 min with extremely high inocula (3000 individuals/pot) of X. index and GFV (Das and Raski, 1968). Shorter acquisition times (5 min or less) were more recently established for «distorting» and «yellow» GFV strains by using 2500-3000 X. index individuals per pot (Alfaro and Goheen, 1974). If, as pointed

-11- out by Taylor (1971), shortening of acquisition time as a consequence of higher nematode populations used for transmission trials may merely reflect the increased possibility of nematodes finding the roots of bait plants rather than their efficiency as vectors, it therefore indicates that whenever given an opportunity, nematode vectors may acquire the virus quickly. The consensus is that a single brief feed on an infected plant is sufficient for making nematodes viruliferous (Taylor and Robertson. 1975). The rapidity of virus acquisition, however, seems of little biological significance in the economy of the transmission process because, by virtue of the limited mobility of virus sources (i.e. host roots) and nematodes, their interaction is long lasting once the contact is established. Obviously, the same applies to the inoculation access threshold, i.e. the minimum time required for a successful virus transmission to healthy plants (Teliz et al., 1966). The length of virus retention in the vector seems to have far more important biological implications in view of the possible sepa­ ration of nematode vectors from plants which act as hosts for them and for the virus (i.e. through removal of a crop at the end of the productive cycle). A prolonged virus persistence within the nematode would increase the chances of bridging the gap between the removal and replanting of a susceptible crop without depending necessarily on alternate hosts. In this respect X. index, like other Xiphinemas (Taylor and Robertson, 1975), seems to be very efficient as it is capable of re­ taining GFV for up to 8 months in the absence if host plants (Taylor and Raski, 1964) and up to 3 months when allowed to feed on fig, a virus-immune host (Das and Raski, 1968). In this latter work, occasional GFV transmissions were obtained using juveniles collected from X. index populations which had fed on fig for 12 weeks, a time sufficient for juveniles to moult and/or to reach the adult stage. This finding had re-proposed the idea of transovarial or transtadial passage of GFV in X. index (Das and Raski, 1968) but more recent evidence definitely seems to exclude such a possibility (Taylor and Robertson, 1975). In fact, the site of retention of GFV within the vector is the cuticular lining of the oesophageal lumen, from the anterior part of the odontophore to the end of the basal bulb (Taylor and Robertson, 1970; Raski et al., 1973), which is shed during moulting. Particles of both «distorting» and «yellow» strains of GFV

- 12- are equally well acquired by x. index and are arranged in mono layers at the sites of retention in the lumen of the oesophagus (Taylor and Robertson, 1970; Raski et al., 1973). Whether or not all virus particles acquired by the nematode are retained is difficult to assess but recent findings relative to AILV and Longidorus apulus (Taylor et al., 1976) provide evidence that only those particles which are free in the cytoplasm of host cells are specifically adsorbed at retention sites. Instead, virions contained within membranous tubules - which, incidentally, are a rather common intracellular feature of nepovirus infections (Martelli and Russo, 1977) - may not be adsorbed and pass into the intestine with other plant material (Taylor et al., 1976).

Efficiency of transmission

Virus transmissions by adult and juvenile stages have been experimentally obtained with X. index and GFV (Raski and Hewitt, 1960, 1963; Cohn et al., 1970), X. italiae and GFV (Cohn et al., 1970), X. americanum and TomRSV (Teliz et al., 1966), L. attenuatus and TBRV (Rudel, 1977). The available evidence strongly indicates that all stages of the aforementioned nematodes transmit the associated viruses with equal efficiency except perhaps for L. attenuatus, whose juveniles gave a lower number of positive TBRV transmissions to grapes as compared with adults (Rudel, 1977). These, in turn, ap­ peared less efficient than Xiphinemas. However, it is known that Longidorus species are generally more difficult to handle than Xiphinema. Thus, data on transmission are hardly comparable when experiments are performed under diverse conditions in different labo­ ratories (Taylor, 1971; Taylor and Robertson, 1975). The transmission efficiency of Xiphinema species improves con­ siderably with increased duration of acquisition time and of the number of individuals used in the tests. Thus, positive transmission of TomRSV by X. americanum was raised from 10 to 100% by extending acquisition period from 1 h to 4 days (Teliz et al., 1966), and again from 10 to 100% by increasing the inoculum from 1 to 100 nematodes (Teliz, 1967). Similarly, transmission of GFV and related strains by X. index increased from 10 to 80%, or from 30 to about 60%, by prolonging the acquisition time from 1 to 28 days (Raski and Hewitt, 1960) and from 5 min to 20 h (Alfaro and Goheen,

-13- 1974), respectively, and increased from 10 to 63% by increasing the inoculum from 5 to 60 individuals (De Sequeira and Dias, 1964). The duration of inoculation time may be also critical in determining transmission rate but the available data are scanty. Cohn et al. (1970) found that beyond 4 and up to 16 weeks there was no increase in transmission of GFV by X. index and only a slight increase was registered in the tests with X. italiae.

Specificity of transmission

So far, experiments carried out with grapevine nepoviruses have confirmed the established specificity of transmission reported in the literature (for review see Taylor and Robertson, 1975). For instance, greenhouse and field tests have recently re-affirmed that X. diversi­ caudatum does not transmit GFV whereas AMV is not moved from grape to grape by X. index (Vuittenez et ai., 1978). Previously, Taylor and Robertson (1970) had shown that no virus particles were present in the alimentary tract of X. diversicaudatum individuals which had fed on GFV-infected plants. Moreover, the TBRV isolate that infects German vines and is serologically assigned to the English serotype, was experimentally transmitted by L. attenuatus (Rudel, 1977), as expected (Harrison, 1964). However, differences may exist in the ability of different populations of a nematode species to transmit a given virus. This was shown by Vuittenez (1973, in Martelli, 1975a) who reported that AMV was moved from grape to grape by X. diver­ sicaudatum reared on grapevines, but not by populations of the same species reared on peach.

DISSEMINATION AND SURVIVAL

Long-range spread

Like other nematode-borne agents, grapevine nepoviruses cannot be disseminated over long distances by natural means because of the limited range of their vectors or of other biotic factors that may be involved in their dissemination. Therefore, long distance movement is achieved chiefly through infected propagating material. Such a situation is clearly exemplified by GFV, for there is little

- 14- doubt that this, more than any other virus, has been spread with diseased graftwood and rootstocks. Its disastrous dissemination on a world-wide basis started in the late 1800s in connection with the introduction into Europe of Phylloxera-resistant American rootstocks. It is believed that prior to that time GFV distribution in Europe was not as generalized as at present, the virus being probably confined, along with its vectors, within scattered enclaves. Evidence of this is provided by the rare occurrence of GFV in some areas of South Australia where no new grape introductions have been made since 1894 (Francki and Crowley, 1967) and by its absence in early California grape imports dating back to 1890 (Luhn and Goheen, 1970). Thus, not only the sanitary conditions of European vineyards were remarkably better at the turn of the century, but it is doubtful that, in those days, GFV represented much of an economic threat. In fact, self-rooted V. vinifera cultivars are very tolerant to this virus, but American rootstocks are not and they also render European grape scions grafted on them highly susceptible (Hewitt, 1968). It is not by chance that the first alarming outbreaks of GFV-induced « infectious degeneration}} in Italy were recorded in rootstock nurs­ eries (Baccarini, 1902; Pantanelli, 1910; Petri, 1910). With the two major American nepoviruses the reverse is true. Some Vitis species native of North America such as V. rupestris and V. labrusca, but not V. riparia, show a hypersensitive resistant response to TomRSV and TRSV, so that they are not invaded system­ ically (Uyemoto et al., 1977b). Also these species acquire neither of these viruses from soils containing viruliferous X. americanum, whilst self-rooted V. vinifera cultivars and French hybrids become severely diseased (Gilmer et al., 1970; Gilmer and Uyemoto, 1972; Dias, 1977). On the other hand, the hybrid rootstock V. berlandieri x V. riparia Kober SBB is reported to be resistant to the yellow vein strain of TomRSV (Gooding and Hewitt, 1962). Jt is therefore con­ ceivable that germplasm of American Vitis species introduced into Europe for producing Phylloxera-resistant rootstocks was essentially free from TomRSV and TRSV. If so, it explains why both these viruses, unlike GFV, have not travelled a long distance, remaining confined to the viticultural areas of North America, except for the single but still unconfirmed record of TomRSV in Yugoslavia (Saric and Vrdoljak, 1973). Other major European nepoviruses have never been found in vineyards outside Europe. Also, they are not as widespread in the

- 15------_.------

countries of occurrence as is GFV but have a more or less patchy distribution, suggesting that these viruses rather than being distri­ buted through infected propagating material, are likely to be picked-up in situ. In this connection it is interesting to note Stellmach's (1970) observation that the survival of vines grafted onto TBRV-infected rootstocks is exceedingly low, which minimizes the chances for TBRV dissemination through young grafts.

Short-range spread

Recent investigations (Uyemoto, 1975; Uyemoto et al., 1977b; Dias, 1977; Rudel, 1977; Ramsdell and Myers, 1978) have shown that most grapevine nepoviruses do not deviate from the classical ecol­ ogical behaviour which characterizes nematode-borne entities (for a review see Murant, 1970). For instance, their spread over short distances results from the combined action of viruliferous nematodes and infected seeds of primary host and weeds. Nematodes alone, because of their limited mobility, are not efficient agents of virus dispersal. In fact, the yellow mosaic strain of GFV was found to spread at rates not exceeding 1.3 to 1.5 m/year in California (Hewitt et al., 1962) and Italy (Lamberti and Martelli, 1965), the yellow vein strain of TomRSV moved quite slowly in Californian vineyards (Teliz et al., 1966) and PRMV also spread slowly in Michigan (Ramsdell and Myers, 1978). Conversely, in Northern U.S.A. vineyards TomRSV and TRSV appear to move much faster. Gilmer et al. (1970) reported that in a collection field in New York State, 22 % of European grape­ vines became infected with TRSV in a 2-year period. In the same State, in four vineyards planted in 1968-1969, 50% of the French hybrid vines were infected with TomRSV by 1973 (Uyemoto and Welsh, 1974). The discrepancy between the rates of natural spread of TomRSV recorded in California and New York is evident. It should be re­ membered, however, that all Californian vines are grafted, and thus virus dispersal is likely to be impaired by resistant rootstocks, whereas, in Northern U.S.A. V. vinifera and French hybrids grow on their own roots. This makes them very susceptible to TomRSV which is naturally established in many weeds, primarily plantain (Uye­ moto, 1975), that serve as hosts and sources of inoculum for X. ame­ ricanum. It is not surprising, therefore, that the favourable comb ina-

- 16 ,.- tion of three factors namely: (i) abundant and randomly scattered virus reservoirs; (ii) widespread occurrence of vector populations; (iii) extreme susceptibility of the hosts, results in an uncontrolled virus spread. The behaviour of grapevine nepoviruses with respect to seed­ transmission in grape is rather erratic. For example, GFV and the yellow vein strain of TomRSV, although pollen-borne, are not transmitted through seeds (Cory and Hewitt, 1968; Boubals, 1969; Hevin et al., 1973) nor do plants pollinated with GFV-carrying pollen become infected (Doazan, 1978). Instead, the type strain of TomRSV, PRMV and GBLV are all seed-borne although at a rate never exceeding 10% (Uyemoto, 1975; Uyemoto et al., 1977a; Ramsdell and Myers, 1978). Seed-transmission in grape is, however, of negligible ecological importance, owing to the low transmission rate and to the fact that grape seedlings have no practical use except for breeding work where, nevertheless, virus contamination of seeds could be troublesome. In herbaceous plants the situation is entirely different since virus transmission through seeds consistently occurs in both natu­ rally and artificially infected hosts. Several such examples have been recorded with TRSV (Gilmer et al., 1970), TomRSV (Uyemoto, 1975; Dias, 1977; Uyemoto et al., 1977), PRMV (Ramsdell and Myers, 1978) and TBRV (Rudel, 1977). There is little doubt that in all these cases weeds represent effective means of virus dissemination and survival as well as sources of inoculum and alternate hosts for the vectors. This is then a typical example of an aspect of the ecology of nema­ tode-borne viruses that involves several different plant species serving as hosts for the virus and the vector (Fig. 1). A remarkable exception to this pattern is provided by GFV which, although artificially transmissible to a fairly wide range of herbaceous plants, has no natural hosts other than the grapevine. Moreover, as already mentioned, this virus is not transmitted through grape seeds and it occurs only in a very low proportion of the seeds of artificially infected herbaceous hosts (Bruckbauer and Rudel, 1961; Dias, 1963). The latter notion, is of no ecological significance as none of the herbaceous hosts in which the virus is seed-borne is liable to be found infected in nature, nor, as is often the case with herbaceous plants and X iphinema, do they seem able to support active growth of vector populations (Taylor, 1971; Cohn, 1975). On the other hand, all the hosts of X. index so far known (Siddiqi, 1974) are non host

- 17- of the virus so that, in these circumstances, the spread and survival of GFV relies on a simpler ecological system whose basic terms are a single host (grapevine) and the vector (X. index and/or X. italiae) (Fig. 2). The only natural reservoir for GFV is therefore the grapevine itself, the roots of which remain viable for many years after removal of the mother plant. Any newly produced rootlet contains GFV, can support the growth of vector populations and constitutes a source of inoculum for X. index which lasts long enough to bridge the period between the uprooting of the preceding vineyard and the establishment of the next (Raski et al., 1965). For these reasons agronomic measures adopted for breaking up the ecological chain GFV-vector-grapevine, consist primarily in the careful removal of host roots and in the cultivation of crops like cereals or alfalfa which induce a rapid decrease of X. index populations below de­ tection level (Dalmasso et al., 1972 ;Lamberti et al., 1976).

GRAPEV I N E

NEMATODE/' ~ VIRUS VECTOR ~ S #f'i\ D S E I NG ) A L WI L D AND/OR NEMATODE C U L T I V ATE D VECTOR PLANTS

Fig. 1 - Ecological cycle of Tomato ringspot, Tobacco ringspot, Peach rosette mosaic and Tomato black ring viruses in nematode-infested vineyards.

-18- VIRUS V I R US

f RO 0 T S PROPAGATII NG MATERIAL NEMATODE

Fig. 2 - Ecological cycle of Grapevine fanleaf virus in nematode infested vineyards.

CONTROL

Resistant and healthy stocks

Based on the available knowledge, there is little hope of find­ ing, in American rootstocks, sources of resistance to European nepoviruses that can be readily utilized in a breeding programme. For instance, all American Vitis species and their hybrids which have been used so far as commercial or experimental rootstocks proved susceptible, although to a varying extent, to GFV and its strains. Also the widespread tolerance to GVF shown by V. vinifera cultivars (Hewitt, 1968) seems of little pratical importance until highly sus­ ceptible American rootstocks will be used, since no alternative means for controlling Philloxera are available. In fact, going back to self­ rooted European stocks seems presently unfeasible. Also, whether

-19------

or not V. vinifera is resistant to European nepoviruses other than GFV remains to be established. Resistant stocks may, however, represent an interesting ap­ proach to an efficient control of some American nepoviruses. As already mentioned, a high level of resistance to TomRSV and TRSV has been found in V. rupestris and V. labrusca. Moreover, the be­ haviour of hybrids like V. berlandieri x V. riparia Kober 5BB and V. riparia x V. rupestris Couderc 3309 is such as to suggest that they are both endowed with remarkable resistance to the same viruses (Gooding and Hewitt, 1962; Uyemoto et al., 1977b) despite the pres­ ence of susceptible V. riparia genes. Actually, C. 3309 is immune to TRSV but is systemically invaded by TomRSV (J. K. Uyemoto, personal communication). It is conceivable then, that in Nortehrn U.S.A. and Canada field contamination by some viruses could easily be controlled merely by grafting both ({ premium)} grapevines 0. e. French hybrids) and European scions on appropriate rootstocks. There is no need to emphasize the effectiveness of using healthy stocks as a measure for preventing spread of many virus-induced disorders, including those evoked by nepoviruses. Extensive investi­ gations carried out in many leading grape-growing countries [see recent reviews by Bovey et al. (1975); Goheen (1977); and reports of round table discussions at ICGV Meetings at Salice Terme, Italy (1973) and Cordova, Spain (1976)J have amply demonstrated that sanitary selection combined whith heat therapy are powerful tools for reduc­ ing the incidence of nepoviruses in newly established vineyards.

Weed control

As already mentioned, it has been shown that in some North American and German vineyards weeds may act as alternate hosts for TomRSV, TRSV, PRMV and TBRV, thus playing a major role in their short-range dissemination and perpetuation (Uyemoto, 1975; Dias, 1977; Rudel, 1977; Ramsdell and Myers, 1978). Therefore, in such cases weed control may usefully complement other sanitary measures by reducing the inoculum potential of the pathogens. However, the effectiveness of weed control in vineyards as an anti viral practice needs to be examined experimentally.

-20- Chemical control

Chemical control is obviously directed against virus vector ne­ matodes and can be carried out by application of soil fumigants or systemic nematicides. The latter chemicals, like oxime carbamates (e. g. oxamyl), have been used with some success againts Longidorus and X iphinema in annual crops but they may not be equally effective in long-lasting crops like grapevine (for a review see Alphey, 1978). Field trials with oxamyl and organophosphates againts virus-transmit­ ting X. americanum are in progress in North American vineyards but the results are not yet available (Uyemoto et al., 1977 b). The possibilities of controlling X. index by soil fumigation have been better explored. Most of the experimental work has been carried out in California and France. In the deep Californian soils, after a series of conflicting and, on the whole, rather unsatisfactory results (Hewitt et al., 1962) long­ lasting control of X. index and GFV has been achieved by dichloro­ propane-dichloropropene (D-D) treatments at rates of about 2500 l/ha. Split applications were made at a depth of about 75 em (2000 l/ha) and 20-25 em (500 l/ha) (Raski et al., 1973, 1976). In France, as recently summarized by Boubals (1976 b), a single D-D application at a rate of 1000 l/ha proved highly efficient in con­ trolling the GFV-X. index complex in the shallow soils of Fronti­ gnan. In these soils, the rate of virus recontamination was still extremely low 16 years after soil fumigation. However, the same chemical applied at similar or higher rates (1500 l/ha) did not give such a satisfactory control in deeper soils.

LITERATURE CITED

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- 23------

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Accepted for publication on 20 December 1977.

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