Summary Grapevine yellows are known worlwide and are dangerous diseases which disseminate by planting material. Flavescence dorée, Bois noir and Vergilbungskrankheit have been known in Europe for over 50 years. Over the past decade, methods to detect and characterize phytoplasma in grapevine tissues and in insect vectors have considerably improved knowledge on the etiology and epidemiology of GYs.

Present status of etiology and diagnosis of grapevine yellows

1. History of grapevine yellows
Grapevine Yellows (GY) are dangerous diseases of wild and cultivated vines that are associated to phytoplasmas and known worldwide. The first GY to be described was Flavescence dorée (FD) which appeared in south-western France (Caudwell, 1957). Because they have similar symptoms, other GYs were often called FD or FD-like diseases until more was known of their etiology.
It is only for the last 10 to 15 years that antibodies, then DNA-based methods, have allowed to distinguish between the various GY.
Once considered as a physiological disorder, FD was shown to be an infectious disease, based on graft transmission and insect vectoring by the ampelophagous leafhopper species Scaphoideus titanus Ball (Schvester et al., 1961). The role of phytoplasmas in FD etiology was demonstrated by Caudwell et al. In 1971. After the first outbreak in south-western France, FD appeared in Corsica in 1978 and in the North of Italy (Osler et al., 1975, Belli et al., 1985); then it extended progressively to all French southern vineyards and to the North of Spain, in Catalona (Batlle et al., 1997) in the 80's and 90's. At the moment, it is still extending northwards in France in Bordeaux and Cognac vineyards as well as in Veneto-Friuli-Giulia in Italy (Refatti, 1993; Belli, 1994).
Bois noir (BN) and the similar disease Vergilbungskrankheit (VK) have been known by vinegrowers for over 50 years. In the early 60ies, BN was described in Eastern France by Caudwell in 1961 and VK in Germany in the Rhine and Mosel valleys by Gärtel in 1965. From the beginning, both diseases have been distinguished from FD on the basis of their epidemiology (1), mainly because the vector of FD was not present in vineyards affected by BN.
GYs were also described in Romania (1968), USA (1977), Australia (1979), Greece (1979), Sicily (1982), Emilia-Romana (1984), Israel (1990), Toscana (1991) Switzerland (1992), Moldavia (1991), Hungary (1997), Slovenia and Croatia (1997). They are suspected to occur also in Southern America (Chile) and in South Africa.
The name of FD is restricted to the disease which is vectored by the leafhopper species S. titanus. Specific methods for the characterisation of phytoplasmas demonstrated that FD phytoplasma belong to the EY group (2). Conversely, GY similar to BN or VK are associated to phytoplasmas in the stolbur group. They were shown to occur in all viticultural countries of western Europe (2, 3, 4). The species Hyalesthes obsoletus (Hemiptera, Cixiidae), a vector of stolbur phytoplasma to solanaceous crops (5), was identified as a vector of VK and BN in Germany and in France (3, 6).
Other GY-associated phytoplasmas have been characterized in most countries. However their vectors are still unknown.

2. Symptoms and economical importance of Grapevine Yellows
Symptoms of Grapevine Yellows are detrimental to infected vines. Their vitality is affected, yields are reduced, and the quality of vines is decreased by high acid and low sugar contents of infected clusters.
Several cultivars which are used in famous vineyards for the production of high-quality products, such as Riesling, Chardonnay, Cabernet Sauvignon, Pinot noir, are highly susceptible to GY. Furthermore, the culture of many varieties of regional importance and essential for the production of unique specialities, as for example Cognac in France, Chianti in Italy, Rioja in Spain, is endangered.
Symptoms of GY are similar whichever the associated phytoplasma. Leaves show discoloration and down rolling of laminae. Flower withering and berries shrivelling result in reduction of quality and quantity of crop. Woody canes are poorly ripened, resulting in a strong weeping aspect of the affected branches. Stocks decline rapidly.
All varieties of Vitis vinifera are susceptible to GY, although in different grades. Some varieties recover from FD if they are protected against reinoculation. Irregular symptom expression is known for BN and VK, depending mostly on climatic conditions, but also on cultivars.
A few rootstock varieties have been shown to be tolerant to FD (7) , thus behaving as carriers of the disease. It is suspected that tolerant rootstock might counterbalance the process of recovery occuring in the scion.

3. Etiology of Grapevine Yellows

3.1. Detection of phytoplasmas in grapevine tissues and in insect vectors
Demonstration of phytoplasma etiology in diseased vines may be obtained by vector transmission, graft transmission and specific laboratory assays such as ELISA and PCR based methods. In France, ELISA and PCR have been routinely used since 1993 by the French Plant Protection Services for the survey of GY.

Indexing of infected cuttings
Indexing, i.e., grafting of infected cuttings with susceptible varieties has been widely used to explore the sanitary status of propagation material. The technique was largely used before the development of laboratory diagnostic methods. It is still valuable for sanitary control of large batches of propagation material, especially for rootstock material which do not express symptoms. In the latter case, scion indicators are grafted on top of the material to be screened for infection.
Index plants must be carefully watched for symptom expression, which can be early and severe such as rapid death of the scion, or alternatively be delayed until the second year after grafting.
Indexing is a biological amplification of phytoplasmas. It may thus be used before specific assays whenever direct detection is not enough sensitive and reliable.

ELISA
Specific antisera and monoclonal antibodies raised to FD phytoplasma and stolbur phytoplasma allowed detection of phytoplasmas in tissues of FD and BN/VK infected grapevine. Procedures for ELISA detection of other grapevine phytoplasmas have not been described.
FD-ELISA uses an indirect double sandwich assay with rabbit polyclonal antibodies (8) and a cocktail of mouse monoclonal antibodies specific for two or several epitopes of FD membrane proteins (9). The assay was readily applied to wild leafhoppers from vineyards (8). However, Caudwell and Kuszala showed that detergents must be added to the extraction buffer to obtain positive detection from FD-infected grapevine tissues (leaves and petioles). The latter conditions most probably reduce the sensitivity of the assays. They are nevertheless necessary to ensure access of antibodies to target membrane antigens in vine tissue extracts.
BN-ELISA uses a double sandwich assay with a single monoclonal antibody to a major antigen of stolbur phytoplasma protein (5). For detection in grapevine, an extraction buffer containing detergents is also necessary .
ELISA detection was also experimented with a monoclonal antibody raised to the GYU isolate, a GY-associated phytoplasma transmitted to periwinkle using dodder in Udine, Italy shown to belong to the WX group. This monoclonal antibody reacted specifically with GYU-infected periwinkle extracts, but not with grapevine infected with a phytoplasma in the WX group in Italy and New York, probably due to a too low titre of phytoplasma in a grapewine (10) and most probably because access to antigens was not possible in the conditions of extraction.

DNA-based methods
Non-ribosomal group-specific DNA probes were obtained by Daire et al. (1992) from randomly cloned phytoplasma DNA of FD and stolbur phytoplasmas, and by Chen et al. (10) for the GYU phytoplasma . Primers constructed from both ends of the latter fragments were used in PCR for specific diagnosis of FD, BN and GYU in grapevine (10, 11) and in insect vectors (6). PCR assays with these tools are especially suited to sanitary certification regarding a particular disease, such as FD which is a quarantine organism in the EC. Stolbur specific primers in the 16S rDNA have been similarly used for detection of VK-associated phytoplasma in grapevine, insect vector and alternative hosts (3).
Universal primers in the 16S rDNA and 16S-23S intergenic spacer have been widely used for wide range detection and characterization of grapevine phytoplasmas. Nested-PCR using two universal primer pairs followed by RFLP analysis (4) or nested-PCR of 16S rDNA using group-specific primers in the second amplification (12, 13, 14), have been used to further characterize the pathogenic agents of GY in different regions. Nested procedures may be necessary for sensitive detection in grapevine or in rootstock varieties where phytoplasma titre may be very low (2).


3.2. Diversity of grapevine phytoplasmas
Grapevine Yellows are associated to phytoplasmas which belong to different groups.
EY phytoplasmas
The main EY group phytoplasma of grapevine is FD phytoplasma. It is widely distributed in southern France, northern Italy (4, 13) and in Catalonia (Batlle et al., 1997) and Rioja in Northern Spain, where the leafhopper vector S. titanus is present. It is readily differentiated from elm phytoplasma by the specific amplification of non ribosomal with the primer pair FD2 (Daire, unpublished) or by RFLP analysis of the non ribosomal DNA fragment FD9 (11).
A second EY phytoplasma was detected in grapevine in Germany, in the Palatine area, where S. titanus does not live (Maixner et al.,1995) and in a few affected vine stocks in northern Italy (Bertaccini, submitted). This phytoplasma can be differentiated both from elm phytoplasmas and from FD sensu stricto by RFLP analysis of the FD9 fragment (11).
Stolbur phytoplasma
Phytoplasmas in the stolbur group (previously referred as a subgroup of AY) have been found associated to BN, VK and to GY in France, Germany, Switzerland, Italy and Sicily, Greece, Hungary and Israel (4, 12, 13 ).
WX phytoplasma
Phytoplasmas in the WX group have been identified in diseased grapevines in USA, Northern Italy and Israel (10, 12, 15, ).
AY phytoplasma
Phytoplasmas in the AY group have been detected in diseased vines in Italy, Slovenia and Croatia (16, Saric et al. 1997)).
Australian grapevine yellows
Phytoplasma associated to the Australian grapevine yellows (AGY), have been identified by Padovan et al. (14) and were given the name of Candidatus Phytoplasma australiense by Davis et al. (1997)



Epidemiology and control of Grapevine phytoplasmas

1. Geographic incidence of GY phytoplasmas related to the biology of insect vectors

1.1. FD phytoplasma.
S. titanus hatch on the middle of May. each of the five instars lasts for 7-10 days and evolve into the first winged adults on the beginning of July. Young instar larvae as well as adults may feed-acquire the FD phytoplasma in Spring at the moment of quick growth of young vine shoots. A latency of four weeks is required before feeding transmission occurs (8). Insects are infective until they die in September.
The area affected by FD is linked to the occurrence of S. titanus. The leafhopper of American origin, was probably introduced in Europe on the beginning of the century as eggs inserted in vine bark. It was identified as the vector of FD in France in 1963 by Schvester et al in 1965. It was reported from Italy in 1964, in Southern Switzerland in 1968 and in Croatia in 1987. The range of S. titanus is limited by climatic parameters because it requires cold winters to break the diapause of eggs, whereas warm summer temperatures are required to complete the life-cycle. However S. titanus is progressively extending the northern border of its range. It has settled in Burgundy and Western Switzerland along the past decade. Although the distribution of the vector exceeds that of FD, new outbreaks of FD are currently being reported on its northern and eastern areas of extension.
1.2. Grape EY-group phytoplasma of Palatine
Maixner and Reinert (17) evidenced that same profiles of EY-group phytoplasma were obtained from GY-infected grapevine in Palatine (11), from diseased alder (Alnus glutinosa) and from two alder-feeding Hemiptera, the leafhopper Oncopsis alni and the psyllid Psylla alni. Alder samples and insects had been collected in Palatine in the vicinity of infected vines. Transmission of the phytoplasma to alder was obtained only with O. alni. Transmission to grapevine with the leafhopper was not demonstrated. However it may be assumed that either or both alder insects are responsible for infection of grapevine in this region, as it is known that infective insects may inoculate plants during probing even if they do not feed.

1.3. BN and VK phytoplasma
Grapevine yellows of the stolbur-type like Bois Noir and Vergilbungskrankheit stretch out to the North and to other regions where S. titanus is not present. Caudwell et al. (1) and Carraro et al. (18) have shown that this vector is not able to transmit BN.
The planthopper Hyalesthes obsoletus was identified as a vector of VK (3) and of BN (6). The planthopper species occurs in a wide area around Mediterranean sea. The biology of H. obsoletus is completely different from that of S. titanus. The planthopper prefers herbaceous weeds instead of grapevine, from which it feeds only erroneously (6). H. obsoletus, like other members of the family Cixiidae, does not develop on leaves or shoots but deposits its eggs on the roots of weeds. The larvae hatching from the eggs in late summer are feeding in nests on the roots. Second and third instar larvae hibernate in the soil, deep enough to be protected from frost. The winged adults are the only stage which occurs above the soil, feeding on leaves and shoots of various plants. In Germany and France, they fly from mid of June until the end of July. Only during this time H. obsoletus feeds on grapevine occasionally and is thereby able to inoculate grapes with GY. Bindweed Convolvulus arvensis is an important reservoir of stolbur phytoplasma. Nymphs feeding on the roots of infected bindweed acquire the phytoplasma and the adults are already infective when they emerge from the soil.
It seems that the species is presently extending its range and abundance and this might account for the increase of stolbur phytoplasma associated GY in several countries. The species was found in Israel (Tanne, personal communication), however not in Spain where other Hemiptera have been found infected with a stolbur phytoplasma (Lavina et al, submitted). As stolbur phytoplasma is a ubiquitous pathogen agent that occurs on many plant species, other vectors might also account for the dissemination of stolbur phytoplasma-associated GYs. Pentastiridius beieri Wagner (1970), another Cixiid vector, has recently been identified in France (Gatineau et al., 1997), however it was not found in vineyards.

1.4. Other grapevine yellows
No other natural vectors of GY are known yet. Oliarus atkinsoni, a Cixiid planthopper, is a vector of Phormium yellow leaf, a phytoplasma that is closely related to AGY (14) . The species has also be found in Australian vineyards but its ability to transmit AGY has not been proven. Orosius argentatus, a vector of Solanum Big Bud (SBB) and Sweet Potato Little Leaf (SPLL) has also been found in Australian vineyards (14) and in Israel (Tanne, personal communication).
Although S. titanus is abundant in New York (USA) (Maixner et al., 1993), it has not yet been proven that this vector is able to transmit the a GY phytoplasma to American vines, neither that a FD phytoplasma is associated to a GY in New York.

2. Spread of grapevine yellows

2.1. Spread by vectors
Flavescence dorée
FD spreads epidemically and reaches a high incidence within a few years, because its vector, S. titanus, feeds only on grapes. Whereas it is widespread and highly abundant on wild Vitis riparia in its American area of origin (Maixner et al., 1993), cultivated grapes are the major hosts in Europe. S. titanus completes the whole life cycle on grapevine. Eggs which are deposited in the bark of canes are the overwintering stage of this univoltine species. Adults are extremely mobile and effective vectors. Therefore they are responsible for the epidemic spread that is typical for FD.
BN and VK
These diseases are less epidemic than FD. They usually spread slowly and their incidence is not as high as that of FD. However, in some regions this situation has changed, but the reasons for this change are not yet clear. Observations made from the epidemiological behaviour of BN (1) and VK, suggested that the diseases were transmitted from reservoirs to grapevine. This hypothesis was verified, when the planthopper Hyalesthes obsoletus could be identified as a vector of VK and BN, and wild plants like C. arvensis and other weeds in vineyards revealed to be frequently infected with the phytoplasmas causing these GY (3, 6). Infected grapevine is no significant source of inoculum, on the contrary it might be considered as a dead-end host. The feeding habit of H. obsoletus is also the reason for the less epidemic spread of BN/VK, since the probability of infection is relatively low for grapes, even in areas where high proportions of the vector populations are infected. However, the situation could dramatically change if new, more effective vectors were introduced to viticultural areas with important phytoplasma reservoirs.

2.2. Spread by propagation material
GY are disseminated by cutting and grafting of apparently normally ripened canes. However all the cuttings obtained from a single cane do not produce infected plants. This shows that phytoplasma incubate in dormant wood ant that it is unevenly distributed along the canes. It could nevertheless reflect a minimum level of contamination necessary for symptom expression. It could also be due to irrgular recovery of infected wood: acoording to cytological observations made by Meignoz et al. (1991) on FD-infected grapevine leaf tissues and by Credi (19) on BN-infected tissues, senescent phytoplasma bodies were observed inside disorganised phloem sieve tubes. This cellular defence mechanisms might be related to the recovery process described by Caudwell for a few V. vinifera varieties. They might also account for a rather low dissemination ratio observed with bench grafting of BN-affected vines in Italy as reported by Credi (19) and by Osler et al. (1997).
This irregular expression of symptoms will introduce difficulties in the methodology of sanitary control of propagation material.
Mother plants for scions
Infected mother-plants for scions may have been inoculated during the preceding summer. In this occurrence they will not exhibit symptoms at the moment of canes selection. Alternatively, normally ripened, however infected canes may develop on infected stocks beside symptomatic canes or shoots which may not be detected at the moment of canes selection. In both cases, scions will be made from infected material. FD has been shown to be transmitted in this way with a high frequency. Osler et al. (1997) indicated that transmission of BN by bench-grafting does occur, though with a low frequency.
Rootstocks
A few rootstock varieties have been shown to be responsible for FD dissemination (7). The latter varieties (3309C, SO4, Fercal, 110 R) are tolerant towards FD infection and act as healthy carriers. Canes taken from suspected mother-stocks were indexed to a sensitive V. vinifera indicator with transmission frequency by cuttings taken on infected mother-plants, ranging from 6 to 80 %.
Grafted plants
Grafted plants obtained with material from infected mother-plants for rootstocks or scions may express symptoms differently. Most of the grafts will either fail or provide letal plants. However a few healthy-looking plants may occur, that will show delayed incubation and symptom expression. This material is highly dangerous for the long distance dissemination of FD, as they may be planted in vineyards free of FD, where however S. titanus is present.

2.3. Strategies to reduce the importance and dissemination of GY diseases

Present knowledge on GY phytoplasmas, on their vectors and reservoirs, offer different ways to the control of these dansgerous diseases.

Control of vectors
Control of vectors is only feasable in the case of S. titanus, since it lives inside the vineyards in Europe and its life-cycle is well known. Insecticide control of the leafhopper is subjected to mandatory regulations in France. Spraying schedules have been established on the basis on the biology of transmission and are facilitated by the univoltine cycle of the vector. The first treatment occurs one month after beginning of hatching, to prevent feeding inoculation by individuals which would have acquired along with their very first meal after hatching. One or two additional monthly treatment may be recommended. Biological control with natural enemies is not available, as no parasitoids or predators of S. titanus have been identified in Europe. Search for natural enemies in its American region of origin is planned.
In the case of H. obsoletus, studies on its biology and ecology have shown that the planthopper is favoured by rocky soils that are sparsely covered by weeds but have no managed green over. Plowing of the soil in vineyards seems to be an effective measure to decrease the population density of the planthopper, probably due to the mechanical damage to the nymphs (3, 6).

Pruning of vines
Pruning of vines may have different incidence, depending on the disease. According to Maixner, field experiments indicated that it is possible to reduce the incidence of VK by cutting canes that carry symptomatic shoots. A considerable proportion of vines treated in that way stayed free of symptoms over several years. The measure is of no danger as infected vines are dead-end hosts. In the case of FD, pruning of infected canes may help in recovery, providing that an efficient control of S. titanus is insured. Otherwise the pruned vines would act as important phytoplasma reservoirs for the vector.

Prophylaxis
a. Reservoir plants
Reservoir plants should be identified and suppressed. In the case of FD, uprooting of infected stocks and of abandoned vine plots is mandatory. In the case of BN / VK, all measures that will eliminate herbaceaous reservoirs of stolbur phytoplasma are recommended, such as clean fallow lands and paths. Different hosts for stolbur phytoplasma and H. obsoletus have been identified according to the country and climate. C. arvensis and labiaceaes are however constant hosts in the different regions examined.
b. Sanitary status of planting material

As it is known that Grapevine phytoplasmas belong to very different groups, care should be taken whenever vine planting material is shipped to different regions, because phytoplasma vectors already present might act as new vectors for a particular GY if contaminated material was planted on the site.
Maixner and Reinert (17) emphasize that S. luteolus, the vector of American elm yellows, belongs in the subfamily of Deltocephalinae to the same genus as the vector of FD phytoplasma, which in its turn belongs to the EY group and is thought of American origin. Conversely, alder yellows phytoplasma (also detected in Palatine GY) and rubus stunt phytoplasma, are European EY phytoplasmas which are transmitted by leafhoppers in the subfamily Macropsinae , i.e. O. alni and Macrospis fuscula, respectively. Similarly, phytoplasmas in the stolbur group in Europe and the AGY/PpDB/PYL group in Australia are closely related phytoplasmas, for which Cixiid planthopper vectors are known both in Europe, Israel and Australia.
A method to cure infected vine material by soaking dormant canes or plants into 50°C hot water for 45 mn has been developed by Caudwell et al. in 1990. The procedure has proven to be safe and efficient and it has been recommended by the International Board for Plant Genetic Resource (FAO/IBPGR) for safe international movement of grapevine germplasm in 1991.


Discussion. Future research on GY phytoplasmas

Research in the past decade has provided important progress in typing and characterization of phytoplasmas. Etiology of GYs is now considered as established, even though the impossibility to cultivate phytoplasmas and the absence of known vectors have not allowed definite evidence of their role as unique pathogen agents responsible for GY. It must be emphasized that only in the case of FD and of BN/VK the demonstration of etiology has been obtained by transmission of the associated phytoplasma to healthy vine seedlings and characterization of the phytoplasma in vectoring insects and inoculated plants.
The methods already available for detection and methods to be developed for investigations of the interactions between phytoplasma and their hosts (20), should now focus research efforts at a better understanding of the mechanisms of specificity such as occur between phytoplasmas and their vector(s), but also at the study of plant reactions such as tolerance, sensitivity and recovery, which are obviously important phenomenon depending on vine varieties and the pathogen agent. A better understanding of these biological processes would certainly deliver important clues to the control and prevention of dissemination of GY diseases.
Other ways offered to research are the introduction of genes which would either restrain or limit phytoplasma multiplication in the host plant, or prevent feeding of the insect vector. Genes coding for antibodies directed to a membrane protein of the associated phytoplasma or for elicitins are good candidates. However, the genetic diversity of grape and rootstock cultivars must be preserved and such strategies are only long term objectives.

References

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