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Journal of Plant Pathology (2010), 92 (2), 303-326 Edizioni ETS Pisa, 2010 303

INVITED REVIEW GRAPEVINE YELLOWS IN ITALY: PAST, PRESENT AND FUTURE

G. Belli1, P.A. Bianco1 and M. Conti2

1 Dipartimento di Produzione Vegetale, Sezione Patologia Vegetale, Università degli Studi, Via Celoria 2, 20133 Milano, Italy 2 Istituto di Virologia Vegetale del CNR, Strada delle Cacce 73, 10135 Torino, Italy

SUMMARY for new plantations, and insecticide sprays against S. ti- tanus. Thanks to these measures, a sharp decrease of Following the discovery and description by the mid- FD incidence has been registered in the last few years in dle of the past century in France of an epidemic all affected areas of the country. Research on GY in grapevine disease called Flavescence dorèe (FD), com- Italy is now focusing mostly on epidemiological aspects parable disorders known with the general name of (e.g. new potential insect vectors), new control practices Grapevine Yellows (GY) were reported from all major such as genetic resistance to either FD or BN, or both, grape-growing countries of the world, where they con- use of thermotherapy on propagation material, and in- stitute a serious threat to viticulture. In Italy, FD and vestigation of the possible role of symbiotic micro-or- Bois noir (BN), the two most important diseases of the ganisms present in host plants and insect vectors as an- GY group, have been recorded since the 1970’s, both tagonists of the phytoplasma agents of GY. representing a major concern for grapevine growers. FD is caused by phytoplasmas of the ‘elm yellows’ or 16Sr- V taxonomic group and is transmitted by the leafhop- INTRODUCTION per Scaphoideus titanus in the persistent-propagative manner. Severe FD epidemics started about two Grapevine yellows (GY), a group of diseases that were decades ago and are still in progress in the main viticul- originally thought to be caused by viruses, are now known tural districts of northern Italy, i.e. Liguria (Italian Riv- to have a phytoplasma aetiology. The first such disorder to iera), Lombardy, Piedmont and Veneto. The disease in- be reported from Vitis vinifera, and the most widely cidence may exceed 50% and the economic losses can known of the GY group, is certainly Flavescence dorée be very high. BN is caused by phytoplasmas of the ‘Stol- (FD), which appeared in south-west France in the 1950’s, bur’ or 16S-XIIA group transmitted in the persistent- from where it spread to other viticultural districts of propagative manner by the planthopper Hyalesthes ob- France, northern Italy and neighbouring European coun- soletus. Other hopper species may also act as vectors tries. Bois noir (BN), whose symptoms are indistinguish- since the disease has been observed to spread actively in able from those of FD, was also first reported from France, geographic areas where H. obsoletus does not occur. then from the most important viticultural areas of Europe. Currently, the presence of FD seems limited to northern During the last forty years or so, other diseases re- Italy, i.e. the Po Valley and a few regions south to it, like sembling FD and BN have been observed and studied Marche, Tuscany and Umbria (central Italy). Its vector, in many countries all over the world. All these diseases however, has occasionally been found also in regions are associated with the presence of phytoplasmas, which further south. BN occurs throughout the country and are now regarded as their effective aetiological agents, its incidence, initially believed to be negligible by com- and closely resemble each other in symptomatology. In parison with that of FD, has recently attained economi- fact, affected vines show downward rolling of the leaves cally important levels in some districts. FD and BN are accompanied by yellow or bright red discoloration of symptomatologically undistinguishable from one anoth- veins and blades, berry withering and uneven or total er, molecular diagnosis is therefore necessary for the lack of cane lignification. GY, however, have different proper identification of the agent. Since 2000, compul- phytoplasma species as causal agent, as well as different sory control measures against FD are enforced in Italy insect vectors, which are either leafhoppers or plan- by a govermental decree. These consist mainly in the thoppers (Homoptera:Auchenorrhyncha) that feed ei- elimination of infected vines, the use of healthy material ther specifically or occasionally on the vines. It is worth noting that two or more different phytoplasma species may infect simultaneously individual grapevines, thus Corresponding author: G. Belli Fax: +39.02.50316781 causing mixed infections. The symptoms, however, do E-mail: [email protected] not differ from those outlined above. 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 304

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Although phytoplasmas are non culturable microor- be consequent to root damage, excluding any correla- ganisms and, in the case of GY, Koch’s postulates have tion with infectious agents. Finally, Caudwell (1957), af- not yet been fulfilled, when phytoplasmas of a specific ter two years of detailed observations and transmission group or subgroup are found consistently associated tests, determined that FD was an infectious disease, with a specific grape disease, they are regarded as being probably caused by a virus (phytoplasmas were un- its causal agents. In fact, the identification of phytoplas- known at that time and yellows diseases of different mas as the cause of GY was finalized in the last decades crops were thought to have a viral aetiology). A few when molecular detection methods became available, years later Schvester et al. (1961) provided experimental thus allowing to distinguish from one another the phy- evidence that FD was spread in the vineyards by the toplasma species involved in each single disease. leafhopper Scaphoideus titanus (originally named S. lit- The current information on GY vectors is still largely toralis), an insect accidentally introduced from North incomplete. Whereas it has long been known that FD is America some years before. Since FD was the first dis- transmitted in nature by the leafhopper Scaphoideus ti- ease of the GY group to be investigated in detail and tanus Ball and, more recently, that BN can be transmit- described, it is still universally considered as the typical ted by the planthopper Hyalesthes obsoletus Signoret, representative of the GY group. little is known on the vectors of other GY. Furthermore, Meanwhile, during the 1960’s, another different GY it seems clear that H. obsoletus may not be the only nat- disease was reported by Caudwell (1961) under the ural vector of BN, for this disease spreads actively also name of “bois noir” (BN). BN was primarily present in in areas where this planthopper does not occur, and a some vineyards of north-eastern France and was charac- few other hopper species were identified as potential terized by symptoms very similar to those of FD but, BN vectors, e.g. Pentastiridius beieri and Reptalus quin- because it was spreading more slowly, it was at first con- quecostatus (Gatineau et al., 2001; Holzinger et al., 2002; sidered a non epidemic form of FD. Ten years later, Trivellone et al., 2005). It seems, then, that the relation- Caudwell et al. (1971b) established that BN had to be ship between diseases and insect vectors is a subject that regarded as a disease distinct from FD, primarily on the needs to be more deeply investigated for a better under- basis of its non transmissibility by S. titanus. standing of GY epidemiology and for the hopeful devel- A few years earlier, similar symptoms had been ob- opment of new sustainable means for their control. served in Germany, in vineyards of the Mosel and the Damage caused by GY may in fact be extensive and Rhein Valley by Gaertel (1965), who considered them economically relevant, since most of the crop of diseased as FD expressions, albeit S. titanus did not occur in vines is lost. In the last decade of the past century, FD the affected stands. This disease was later studied in epidemics have affected the vineyards of all the northern the same area by Mendgen (1971), who called it regions of Italy causing dramatic yield losses, while BN “Vergilbungskrankheit” (VK), then by Maixner is expanding in both the northern and southern parts of (1994), who provided experimental evidence of its the country, with great concern of the growers. transmission by the planthopper Hyalesthes obsoletus. This review is intended to provide an historical view We now know that BN and VK are the same disease, of GY in Italy and to describe the present situation with i.e. the likely cause of the yellows-type symptoms de- a look at future prospects. scribed by Ravaz and Verge (1924), when S. titanus was not yet present in Europe. From the 1970’s onwards, diseases similar to FD and HISTORICAL BACKGROUND BN were recorded from countries other than France and Germany, such as Rumania (Rafaila and Costache, First reports of grapevine yellows in France and oth- 1970), Italy (Belli et al., 1973), Israel (Tanne and er countries. Symptoms resembling those of GY had Nitzany, 1973), Greece (Rumbos and Biris, 1979), Chile been observed in France since the beginning of the past (Caudwell, 1980), Australia (Magarey and Wachtel, century and described by Ravaz and Verge (1924) under 1982), USA (Pearson et al., 1985), and in many other the names of “flavescence” and “rougeau”. However, it grapevine-growing areas of the world (Martelli and was around 1950 that a new disease began to appear Boudon Padieu, 2006). and spread in the vineyards of south-west France (espe- cially in Gascogne and Armagnac, where the first epi- First reports of GY in Italy. Zelger (1964) was the demic outbreaks were recorded) thus attracting the at- first to observe the presence of GY symptoms in Italy, tention of local researchers. The new disorder was first concluding that they were caused by FD. This record briefly described in 1955 (Levadoux, 1955) under the came from vineyards of Alto Adige (northern Italy), name of “flavescence dorée” (FD) and was referred to a where the author did not find the recognized vector of physiological disorder. In the following year, Branas FD and where this disease is still not known to occur. It (1956) described in more detail the same disease under seems then likely that Zelger (1964) had recorded a case the name “maladie du Baco 22 A”, but considered it to of BN. 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 305

Journal of Plant Pathology (2010), 92 (2), 303-326 Belli et al. 305

In the same year Vidano (1964) reported the occur- lia (Granata, 1982), where the disease apparently spread rence of S. titanus in vineyards of the Italian Riviera with an epidemic FD-like behaviour, although S. titanus (Liguria), close to the French border. was not found in the area (Granata, 1985). More recent- The first reliable record of FD in Italy is by G. Belli ly, molecular tests have established that the Sicilian dis- and coworkers, who observed its occurrence on red- order is BN rather than FD (Albanese et al., 1996). berried grape cultivars (Barbera, Croatina, Uva rara) of At the beginning of the 1980’s, cases of GY, often in some vineyards of the Oltrepò pavese (Lombardy) (Belli epidemic form, were observed in various parts of Vene- et al., 1973), and then found S. titanus in the same area to (northern Italy) (Belli et al., 1983; Egger and Borgo, (Osler et al., 1975). The disease spread with a typically 1983), where S. titanus was also present (Belli et al., epidemic pattern and was successfully controlled with 1984, 1985). GY infections were later reported from three insecticide treatments during summer (Belli et al., several additional Italian regions, i.e. Emilia Romagna 1978). (Credi and Babini, 1984), Friuli Venezia Giulia (Carraro A few years later, GY symptoms were observed in et al., 1986), Trentino Alto Adige (Mescalchin et al., some viticultural districts of Sicily, especially on cv. Inzo- 1986), Tuscany (Egger and Grasselli, 1988), Piedmont

Lazio (1993)

Fig. 1. Map of Italy showing the progressive spreading of grapevine yellows (Flavescence dorée and Bois noir) in the different re- gions of the country, from the earliest record (Trentino-Alto Adige, 1964) to date. Regions marked by an asterisk are those where Flavescence dorée is currently widely spread. 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 306

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(Vidano et al., 1988; Conti, 2001), Apulia (Di Terlizzi et but can be limited to a sector or a branch, whereas the al., 1994), Campania (Marcone et al., 1996), Liguria rest of the plant looks normal. In most cases, this is the (Minucci et al., 1994; Conti et al., 1997), Umbria (Al- result of infections that took place during the previous banese et al., 1997), Sardinia (Garau et al., 2002), year. Some vines may also show symptoms during the Abruzzo (D’Ascenzo et al., 2003), Latium, Calabria and same year of infection, if inoculated early in the season. Molise (Prince et al., 1993; Pasquini et al., 2006, 2008). As already said, mixed infections can occur, which In most of these cases, BN was the prevailing, if not the means that two or more different phytoplasma species only disease present, especially in the central and south- can infect simultaneously the same vine. Mixed infec- ern areas of the country. In fact, the presence of FD has tions, however, do not show a symptomatology differing so far been experimentally ascertained only in northern from that of single infections, which makes visual evalua- Italy, except for occasional records in the Marche (Credi tion most difficult and complicates laboratory diagnosis. et al., 2002), Tuscany (Bertaccini et al., 2003a) and Um- Besides the typical symptoms outlined above, other bria (Natalini et al., 2005). The progressive spreading of minor symptoms may be observed, depending on the FD and BN in Italy is shown in Fig. 1. susceptibility of the cultivar, the physiological status of the infected vine and the environmental and growing conditions. So, it is also possible to observe delayed THE DISEASES sprouting, stunting and necrosis of the young shoots, shortening of the internodes and necrosis of inflores- Symptomatology. The different diseases grouped un- cences. In susceptible varieties or when environmental der the name of GY can be empirically distinguished on conditions are adverse, the vines may decline rapidly the basis of their aetiology and epidemiology but not and die within a few years, especially if re-inoculated symptomatology, for it is practically impossible to find several times by the insect vector. This situation, that any relevant aspect in symptom expression that may was observed quite frequently in Liguria under condi- help differentiating them from one another. In other tions little suitable for grape growing, determined a GY words, grapevine plants show basically the same type of disease locally called ‘Moria della vite’ (grapevine die- symptoms, regardless of the infecting phytoplasma sp- back) (Conti et al., 1997). cies. Some occasional differences can be observed only In conclusion, the economic damage caused by GY in specific cultivars or in some phases of the growing can be very severe, for the detrimental effects of the dis- season such as sprouting. Some cultivars are more or ease do not reflect only on the crop, which can be com- less tolerant and may therefore show symptoms milder pletely lost, but involves also the canes, which are un- than usual or no symptoms at all. These particular as- suitable as source material for vegetative propagation, pects of the symptomatological response have been in- and the whole plant, which may be killed. vestigated recently, the conclusion being that a few pe- Rootstocks may also be infected, albeit they rarely culiar symptoms may be associated more frequently show visible symptoms. Latently infected rootstocks with a particular GY disease, but such differences are represent a dangerous source of contamination to new too inconsistent to enable a reliable discrimination vineyards and new geographic areas. among them (Marzachì et al., 2001). Recovery (remission of symptoms) has been observed On the other hand, the syndrome currently induced first for FD in France (Caudwell, 1961) and Italy (Belli in grapevine by all GY, especially on sensitive cultivars, et al., 1978), later also for BN (Osler et al., 1993). Re- is quite typical, thus useful for the successful identifica- covery is particularly frequent in some varieties, if the tion of affected plants in the vineyards. In early spring, vines are not re-infected by the vectors every year. ‘Bar- infected vines may show irregular sprouting, then, at the bera’, is one of the varieties that frequently enjoys re- onset of summer, the leaves start to roll downwards pro- covery. During an experiment conducted for five years gressively and become chlorotic to yellow in white- in a cv. Barbera vineyard in Oltrepò pavese and regular- berried varieties (hence the name of “flavescence ly sprayed against S. titanus, more than 50% of the dis- dorée”) or purple-reddish in red-berried varieties. The eased vines recovered every year, and only two of the 37 abnormal discolouration may be limited only to the main initially diseased plants showed symptoms throughout veins or to some sectors of the leaf blade, but more fre- the five years of observation (Belli et al., 1978). In re- quently is extended to the whole blade, that later turns covered vines, remission of symptoms is often accompa- thicker and brittle. The berries start to wither and the nied by the disapperance of infection (Osler et al., 2006; bunches dry up. The canes mature irregularly or not at Zorloni et al., 2008). all so that, being supple, acquire a characteristically drooping habit. The canes retain the green colour and, Aetiology. As briefly reported above, the studies on when exposed to the low winter temperatures, tend to the complex aetiology of GY started about 50 years ago necrotize turning black (hence the name of “bois noir”). in France, even though leaf reddening (“rougeau”) and Symptoms do not always show on the whole vine, yellowing (“flavescence”) had been observed long before 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 307

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in the country (Ravaz and Verge, 1923). The experimen- were used for detecting MLOs associated with FD, and tal results obtained by the Dijion group (Caudwell, 1957; ELISA tests were developed for optimizing serological Schvester et al., 1961) provided convincing evidence that reactions (Schwartz et al., 1989). ELISA and other sero- FD, the major GY disease, had an infectious nature and logical assays were useful for detecting MLOs in herba- was transmitted by the leafhopper then known as S. lit- ceous host plants, but did not give positive results when toralis, so dismissing the hypothesis of a physiological applied to grapevines or other woody plants. As ascer- disturbance (Levadoux, 1955; Branas, 1956). For many tained subsequently, this was due to the fact that MLOs years, the causal agent was retained as being a virus, occur in low concentration and show an erratic distribu- which, however, was never isolated nor identified despite tion in the tissues of woody host plants. the efforts carried out in many laboratories. Since 1993, the MLOs associated with a variety of The turning point was in the early 1970’s, following a plant diseases have been called phytoplasmas by the In- study by Doi et al. (1967) who had observed the pres- ternational Committee on Systematic Bacteriology, Sub- ence of wall-less microorganisms resembling mycoplas- committee on the Taxonomy of Mollicutes. During the mas, then called mycoplasma-like organisms (MLOs), in 1990’s, the use of DNA-based molecular analyses al- the phloem of paulownia plants affected by lowed the gathering of precise data on the genetic diver- witches broom. Inspired by the Japanese report (Doi et sity of phytoplasmas associated with plant diseases. The al., 1967), Caudwell et al. (1971a) looked for and found use of specific cloned DNA probes and molecular char- MLOs in the tissues of FD-affected grapevines and acterization of phytoplasmas through strategies based on broad bean (Vicia faba) plants on which individuals of S. PCR amplification of the 16S rRNA gene, generated the titanus, previously reared on FD-affected vines, had fed first RFLP-based comprehensive scheme of taxonomic for some days. Further studies proved that MLOs were phytoplasma classification (Lee et al., 1998), recently ex- the etiological agents of a large group of plant diseases, panded through an automated RFLP approach (Wei et including all those known as GY. al., 2007). This new classification scheme, based on phy- Based on the diagnostic value of transmission tests, toplasmal 16S rDNA sequences available in GenBank Fortusini et al. (1989) and Carraro et al. (1994) showed database, arranged phytoplasmas in 28 groups compris- that the GY epidemics in vineyards of Lombardy and ing more than 50 subgroups. Furthermore, based on 16S Veneto were caused by FD. Later on, however, the rDNA sequence homology and on biological properties, availability of serological reagents confirmed the MLO- phytoplasmas were recently assigned to a Candidatus related aetiology of GY diseases, allowed to distinguish genus, called ‘Candidatus Phytoplasma’ (IRPCM, 2004), the different disorders from one other and to track both including both confirmed and tentative species. vectors and hosts involved in the GY epidemiology. In Although phytoplasmas of V. vinifera grown in differ- detail, polyclonal antisera and monoclonal antibodies ent countries appear indistinguishable by electron mi-

Table 1. Grapevine diseases caused by phytoplasmas and their insect vectors.

Disease Group and Disease agent Insect vector Geographical distribution subgroup Flavescence dorée (FD) 16SrV-C, 16SrV-D Ca. P. vitis1 Scaphoideus France, Italy, Spain, Portugal, titanus Ball Serbia, Slovenia, Switzerland

Bois noir (BN), 16SrXII-A Ca. P. solani1 Hyalesthes Vergilbungskrankheit 16SrXII-F obsoletus Europe, Israel, Chile (VK), Legno nero (LN) 16SrXII-G Signoret

Palatinate Grapevine 16SrV related Ca. P. ulmi Oncopsis alni Germany Yellows Schrank

Virginian grapevine 16SrIII-I Ca. P. pruni Unknown USA yellows (VGY)

Australian grapevine 16SrXII-B Ca. P. Unknown Australia yellows australiense

Australian grapevine 16SrII related Ca. P. Unknown Australia yellows australasia

Buckland valley grapevine 16SrI related Ca. P. asteris Unknown Australia yellows (BVGY) 1 Both Ca P. solani and Ca. P. vitis are incidental citations which do not constitute priority citations, according to rule 28b of the bacteriological code (Lapage et al., 1992). 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 308

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croscopy, molecular analyses have shown that several The variability of the 16S rRNA gene of the FD phy- genetically distinct species can infect grapevines either toplasmas is apparently insufficient to explain by itself in singe or mixed infections (Bianco et al., 1993; Prince these complex bio-ecological differences, therefore other et al., 1993; Marzachì et al., 2001). As shown in Table 1, studies were focused on the finer differentiation of the GY occurring in diverse viticultural areas of the world phytoplasma strains associated with FD. Molecular and showing similar symptoms are attributed to infec- characterization and phylogenetic analysis on the two tions by no less than seven distinct phytoplasma species genetic loci map and uvrB-degV, along with the secY (Daire et al., 1993; Prince et al., 1993; Davis et al., 1997; gene, showed the presence of three FD phytoplasma Botti and Bertaccini, 2007). strain clusters: FD1 (comprising strain FD70, from In Europe, FD is associated with infection by ‘Candi- France), FD2 (comprising strains FD92 and FD-D, datus Phytoplasma vitis’-related strains classified in the from France and Italy), and FD3 (comprising strains rDNA RFLP group 16SrV. Australian GY is attributed FD-C, from Italy) (Arnaud et al., 2007). Recently, enzy- to ‘Ca. Phytoplasma australiense’, a member of the stol- matic restriction analyses of rplV-rpsC and secY gene bur group 16SrXII, and to ‘Ca. Phytoplasma australa- fragments, PCR-amplified from symptomatic grapevines sia’, a member of group 16SrII. GY in Virginia (USA) is collected in Italian vineyards, showed that the genetic associated with ‘Ca. Pytoplasma asteris’ and ‘Ca. Phyto- heterogeneity among FD-C phytoplasma strains (identi- plasma pruni”- related strains. Palatinate grapevine yel- fication of five genetic variants) is larger than among lows (PGY) is associated with ‘Ca. Phytoplasma ulmi’, a FD-D strains (identification of one genetic variant) member of group 16SrV, and BN is attributed to ‘Ca. (Botti and Bertaccini, 2007). Interestingly, ‘Ca. Phyto- Phytoplasma solani’-related strains (Daire et al., 1993; plasma vitis’-related strains were detected primarily in Prince et al., 1993; Davis et al., 1997; IRPCM 2004; grapevine and in S. titanus individuals but also, though Contaldo et al., 2009; Quaglino et al., 2009b). more rarely, in plants of Clematis vitalba, Alnus gluti- This impressive inter-species diversity of putative GY nosa and Ailanthus altissima (Angelini et al., 2004; Bor- causal agents is further complicated by recent findings go et al., 2008). of substantial intra-species genetic diversity of GY Since the possible role of the three above wild plant agents (Constable et al., 2004; Botti and Bertaccini, species in the field spread of FD has not been deter- 2007; Quaglino et al., 2009b, 2009c). For example, Bot- mined, the biological cycle of ‘Ca. Phytoplasma vitis’ is ti and Bertaccini (2007) reported molecular genetic thought to involve a single host plant and a single insect markers of extensive diversity among ‘Ca. Phytoplasma vector, strictly dependent on the host plant for its sur- vitis’-related strains occurring in grapevines affected by vival. On one hand, this explains the typical epidemic FD disease in Italy and Constable et al. (2004), through spreading of FD disease in the vineyards and, on the heteroduplex mobility assay (HMA), showed the genet- other, has allowed the development of diverse control ic heterogeneity in ‘Ca. Phytoplasma australiense’-relat- strategies, based on insecticide treatments against S. ti- ed strains infecting grapevines in Australia. In the case tanus populations. of BN, strains classified in several subgroups (16SrXII- As already said, the occurrence of spontaneous re- A, XII-F and XII-G) within the stolbur phytoplasma mission of symptoms (recovery) in FD-affected vines group have been reported (Lee et al., 1998; Quaglino et has been repeatedly observed (Caudwell, 1961; Belli et al., 2009b). al., 1978), and this phenomenon has recently been ob- FD and BN are by far the two main diseases of the served also in apples infected with the apple prolifera- GY complex and deserve a more detailed information. tion phytoplasma (‘Ca. Phytoplasma mali’) (Carraro et They cannot be distinguished from one another through al., 2004; Musetti et al., 2004) and in apricots infected symptom observations in the field, but differ deeply in by European stone fruit yellows (ESFY) phytoplasma their aetiological agents and biological cycle (Conti and (‘Ca. Phytoplasma prunorum’). It has been suggested Alma, 2002). that SAR (systemic acquired resistance) might be in- volved in apple and apricot recovery (Osler et al., 1999; Flavescence dorèe. FD is caused by ‘Ca. Phytoplasma Musetti et al., 2005). Other studies have underlined the vitis’ (group 16SrV, subgroups rV-C and rV-D) (Martini importance of different types of stresses (elicitations dis- et al., 1999; Davis and Dally, 2001), transmitted by S. ti- played by endophytes, general forms of biotic or abiotic tanus (Caudwell et al., 1971), a leafhopper species that stress) in the induction of SAR or of induced systemic thrives exclusively on grapevines. Field observations in resistance (ISR) (Van Loon et al., 1998). So far, the in- vineyards of northern Italy have reported differences in formation available is still insufficient for a clear expla- symptom expression including remission and recovery nation of recovery, especially in grapevine, although it (Belli et al., 1978; Osler et al., 1993). There are also spe- seems reasonable to assume that interactions between cific varietal responses and different geographical distri- the pathogen, the host and the environment may play a bution of the “C” and “D” subgroups involved in the key role. disease (Belli et al., 2002; Martini et al., 2002). Recently, an increase of reactive oxygen species 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 309

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(ROS) and of pathogenesis-related (PR) proteins has markers to type BN phytoplasmas in plants and insects. been detected in grapevines displaying FD recovery Furthemore, sequence analysis of the tuf gene revealed (Musetti et al., 2007). Furthermore, the possible impli- that three tuf genetic lineages, denoted tuf-I, tuf-II and cation in this phenomenon of host and pathogen geno- tuf-III of ‘Ca. Phytoplasma solani’ [formerly called VK- type (possible presence of hypovirulent phytoplasma I, VK-II, and VK-III (Langer and Maixner, 2004)] are strains) and of the environmental conditions has been present in BN-diseased grapevines, as well as singly and advocated, as well as the involvement of grapevine bac- specifically in hosts alternative to V. vinifera (tuf-I in Ur- terial or fungal endophytes (Quaglino et al., 2005; tica dioica, tuf-II in Convolvulus arvensis, and tuf-III in Musetti et al., 2005, 2007; Bulgari et al., 2009; Martini Calystegia sepium). Intriguingly, tuf-I and tuf-II BN phy- et al., 2009). toplasma strains are differently distributed in distinct New molecular approaches have also disclosed that geographic regions of Italy (D’Ascenzo et al., 2008; Du- recovery is frequently associated with the apparent dis- rante et al., 2008; La Rosa et al., 2008; Romanazzi and appearance of the pathogen from the host so that recov- Murolo, 2008; Silletti et al., 2008), the presence of one ery can be regarded as a case of natural healing. Analy- or the other being clearly linked with the presence of its ses performed on cvs Barbera and Croatina, two impor- specific herbaceous host plant. tant grapevine cultivars of northern Italy, highlighted In comparison with ‘Ca. Phytoplasma vitis’ (FD), the that sanitation is stable if the plants are not infected biological cycle of ‘Ca. Phytoplasma solani’ (BN) looks again (P.A. Bianco, unpublished results). New advances much more complicated because of its presence in mul- on plant host–phytoplasma interaction will certainly tiple hosts (Batlle et al., 2000; Borgo et al., 2008; provide useful information for the comprehension of Maixner et al., 1995; Marcone et al., 1997; Skoric et al., the mechanisms involved in recovery and may help in 1998) and insects (Batlle et al., 2000; Boudon Padieu, the planning of strategies for the control of GY and 2000; Gatineau et al., 2001; Garau et al., 2004; Palermo similar diseases. et al., 2004; Bagnoli et al., 2008). Even if the role of the alternative hosts in the spreading of ‘Ca. Phytoplasma Bois noir. Based on of 16S rDNA sequence analysis, solani’ has not been ultimately elucidated, their active the aetiological agent of BN was identified as ‘Candida- involvement in the epidemiological cycle of BN is more tus Phytoplasma solani’, a phytoplasma species belong- than likely. ing to the stolbur group (subgroups 16SrXII-A, XII-F Due to the complexity of the ‘Ca. Phytoplasma and XII-G) (IRPCM 2004; Quaglino et al., 2009b). In solani’ epidemiology, efficient control strategies have the vineyards, ‘Ca. Phytoplasma solani’-related strains not been developed. Recovery of BN-affected grapevine are transmitted by the planthopper H. obsoletus (Ho- plants has also been reported (Osler et al., 1993), al- moptera:Cixiide) (Maixner, 1994; Sforza et al., 1998; Al- though less frequently than in FD-affected plants (Zor- ma et al., 2002), which feeds on a very wide range of loni et al., 2008), and this further makes it difficult to plants. This suggests that this phytoplasma thrives in evaluate the efficiency of various control strategies. various ecosystems and on different plant species, so In the last years, the genome of four genetically di- that selection of diverse strains is likely. On the other verse phytoplasma species (Oshima et al., 2004; Bai et hand, the presence and, particularly, the natural active al., 2006; Kube et al., 2008; Tran-Nguyen et al., 2008) spreading of stolbur phytoplasma in grape-growing ar- has been totally sequenced, allowing a better under- eas where H. obsoletus is absent, strongly suggests the standing of the biology of these mollicutes. Projects of existence of alternative vectors (Conti, 2001; Palermo et ‘Ca. Phytoplasma vitis’ and ‘Ca. Phytoplasma solani’ al., 2004; Bagnoli et al., 2008). genome sequencing should extend the knowledge on The biological complexity of BN disease has stimu- the aetiology of FD and BN, giving the opportunity for lated studies on molecular markers that characterize a in-depth study of the molecular mechanisms involved stolbur phytoplasma genetic diversity. Recently, exten- in the phytoplasma-host interaction and in the determi- sive intra-species divergence among ‘Ca. Phytoplasma nation of the different epidemiological patterns of GY solani’-related strains in Italian vineyards has been as- diseases. certained through automated RFLP analysis of the 16S rRNA gene, suggesting that this phytoplasma species Epidemiology. Phytoplasma diseases are transmitted has a complex population structure (Quaglino et al., by vegetative propagation of infected plants and by in- 2009c). sect vectors of the families Cicadellidae and Cixiidae Seruga Music et al. (2008) reported genetic diversity (leafhoppers:Auchenorrhyncha), and Psyllidae (psyl- in grapevine-associated strains of the 16SrXII-A sub- lids:Sternorrhyncha) belonging to the order Hemiptera. group on the basis of SSCP analysis of 16S rRNA, tuf Transmission by root anastomosis and through the seeds (elongation factor tu), and dnaB genes, and the molecu- of some host plants has also been reported, but not in lar characterization of the genes vmp1 (Cimerman et al., the case of FD or BN. 2009) and secY (Filippin et al., 2009) identified useful Transmission by vegetative multiplication of infected 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 310

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plant material is most important for the spread of phy- ed by insects feeding in the phloem. The ability to ac- toplasma diseases over long distances and the establish- quire phytoplasmas by feeding is higher for the imma- ment of initial foci of infection in new grapevine-grow- ture forms than the adult insects, and the retention of ing areas. This is particularly facilitated by the fact that infectivity is not affected by moulting, but can last for American rootstocks do not show symptoms of infec- the whole insect life (Conti and Alma, 2002). tion. The presence of FD is limited to Europe, where it Transmission efficiency of FD by grafting changes ac- was first reported some fifty years ago following the in- cording to the grapevine cultivar and the level of infec- troduction of S. titanus from the nearctic region (Caud- tion of propagation material. For instance, transmission well, 1957; Schwester et al., 1961). This insect feeds and rate is about 0.4% when ‘standard’ grapevine material multiplies only on grapevines, develops one generation of undetermined sanitary status is used for grafting, but per year, and is the only known natural vector of FD. raises to 16% if allegedly infected plants are used as The disease agent, however, has been transmitted exper- source material (Osler et al., 2002). The transmissibility imentally to broadbean and to other herbaceous hosts of BN has been tested by grafting healthy grapevines of also by the leafhoppers Euscelidius variegatus and Eu- cv. Chardonnay with buds from either healthy, infected scelis incisus (previously known as E. obsoletus). Be- or symptomless vines chosen at random in a vineyard sides, another phytoplasma of the same taxonomic with a low disease incidence. Five year observations al- group, which differs from the FD agent and infects lowed to conclude that: (i) BN is graft-transmissible at a grapevine causing the ‘Palatinate grapevine yellows’ rate of less than 3%; (ii) grafting is by far more success- (PGY) in an area where S. titanus does not occur, has ful when healthy rather than infected plants are used as been transmitted to grapevine by the leafhopper Oncop- donors; (iii) the incubation of BN in graft-inoculated sis alni (Maixner et al., 2000) (see Table 1). Further- grapevines may range from five months to as long as more, recent studies have shown that the planthopper two years (Osler et al., 1997). Dictyophara europaea is able to transmit ‘Ca. Phytoplas- Leafhopper and psyllid vectors of yellows diseases ma vitis’ from C. vitalba to V. vinifera (Filippin et al., are plant suckers that feed in the host phloem, being 2009), opening new avenues for in-depth investigating provided with piercing mouthparts. The nutritional re- the epidemiology of FD. lations with plants to which infective insects may trans- The FD phytoplasma is characterized by genomic mit phytoplasmas are of three types, namely: (i) ‘obli- variability and occurs as many variants or ‘strains’ which gate’, when the survival of the vector insect is totally de- can be differentiated by RFLP analyses carried out with pending on the concerned plant species (e.g. S. titanus the TaqI restriction enzyme on the 16S ribosomal gene. and grapevine); (ii) ‘facultative’, when the plant species Such variants may have different geographic distribution is important, but not strictly necessary, for the insect and pathogenic ability (Martini et al., 1999). The most vector to survive and multiply under field conditions important of them are the FD-C and FD-D variants (e.g. H. obsoletus and bindweed); (iii) ‘occasional’, when which were found consistently associated with the severe the plant considered is neither an obligate or facultative FD outbreaks of the last two decades in both France and host of the insect vector, which can feed just occasional- Italy. In the latter country, FD-C has been identified in ly on it. The vectors of the last type, whose adults feed Piedmont, Lombardy and in north-eastern Tuscany, on various herbaceous, shrubby and woody plants, may while the more virulent FD-D has been found associated sometimes behave in unpredictable ways, causing infec- with the severe epidemics in Veneto since their early ap- tion to plants previously unknown as phytoplasma pearance, in the 1990’s and, more recently, also in some hosts. This is one of the mechanisms accounting for the severely affected areas of Piedmont, Lombardy and outbreak of ‘new’ phytoplasma diseases which had been Emilia-Romagna (Botti and Bertaccini, 2007). hypothesized by Caudwell (1957) since his early studies S. titanus transmits all FD variants and isolates with an on GY. efficiency of over 30% (Boudon Padieu et al., 1989). Al- Phytoplasmas are transmitted by insects in a persist- though the transmission process has not been studied in ent propagative manner, which means that the infectivi- detail, it is generally accepted that the latent period of FD ty is retained by vectors for long periods of time (per- in its vector is about one month in field conditions, as sistence) and that phytoplasmas multiply in their bodies originally determined by Caudwell and Larrue (1986). (propagation). The transmission process consists of This means that, if the pathogen is acquired by the three steps, acquisition, latent period, and inoculation. leafhoppers in their earliest life stage, as is the case with Vectors acquire phytoplasmas by feeding on infected individuals hatched from eggs on infected grapevines, plants for some hours/days and become inoculative af- they become able to transmit from their 5th pre-imaginal ter a latent period of two or more weeks during which stage onwards. As a consequence, the risk for the the microorganisms multiply in their organs and he- grapevine plants to be exposed to FD inoculation be- molymph. After reaching the salivary glands, phytoplas- comes concrete when S. titanus populations are ap- mas can be inoculated into plants with the saliva excret- proaching the above developmental stage in the vineyard. 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 311

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Being able to fly, the adult forms of S. titanus can the areas involved ex novo are typically scattered and far move to longer distances than the immature ones and away from one another and from the north of the coun- are then more efficient in spreading FD. The leafhopper try, where the insect is established since long. The pro- itself has, however, a scarce tendency to migrate and gressive spreading of S. titanus in Italy is shown in Fig. 2. moves actively only from one plant to the nearest ones. As mentioned, phytoplasmas of the FD type have Its spreading to short distances and across Europe, after been detected in nature in a few plants other than V. the introduction which occurred probably at the begin- vinifera, such as C. vitalba, A. glutinosa and A. altissima ning of the past century, can be reasonably attributed to (Borgo et al., 2008). In the case of C. vitalba and A. the exchange of grapevine propagative material (canes glutinosa, the simultaneous presence of FD-type phyto- and grafted plants) carrying eggs that the insect lays un- plasmas in both these hosts, that were growing close to der the bark to overwinter (Bertin et al., 2007). This vineyards, has so far been reported only in one occa- trend concerning the natural spread of S. titanus is con- sion. The possible role of these plants in the epidemiol- firmed by its recent outbreaks in central and southern ogy of FD has to be investigated in more detail, for the Italy (Latium, Campania, Abruzzo and Basilicata) where natural cycle of the FD pathogen is still believed to take

Fig. 2. Map of Italy showing the progressive spreading of Scaphoideus titanus, the vector of Flavescence dorée, in the different re- gions of the country, from the earliest record (Liguria, 1964) to date. In the regions marked by an asterisk the leafhopper has been introduced recently, likely with grapevine propagative material, and has a restricted distribution. 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 312

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place between the grapevine and S. titanus. Holzinger et al., 2002; Trivellone et al., 2005). BN, a disease endemic in many grape-growing areas Phytoplasmas of the Stolbur group associated with of the world, was first recorded in France (Caudwell, BN have been detected in several herbaceous species, 1961) and, a decade later, in Germany (Mendgen, 1971). among which bindweed, nettle, Amaranthus retroflexus, Another grapevine disease reported from the French Cirsium arvense, Calystegia sepium, Plantago media, Champagne under the name of Vein yellowing leafroll Rubus spp., celery and tomato (Bertaccini, 2008; Borgo (Caudwell et al., 1983) could have been an outbreak of et al., 2008). These plant species are food hosts of H. BN characterized by unusually bright leaf vein symp- obsoletus and may act as inoculum source for BN infec- toms. tion to grapevine. As mentioned, the BN phytoplasma belongs to the Like the FD phytoplasma, the BN agent has different Stolbur taxonomic group 16SrXII (‘Candidatus Phyto- molecular variants, two of which, denoted tuf-I and tuf- plasma solani’). The Bulgarian name “stolbur” refers to II, prevail in the areas most severely affected by the dis- the etiological agent of diseases typical of solanaceous ease. As mentioned, these variants have diverse patho- plants (tomato, pepper and eggplant) characterized by genic characteristics and geographic distribution, which stunting, virescence, sterility and yellowing or purple-red- seems strictly linked with the weed host species occur- dening. of the leaves. Phytoplasmas of the Stolbur group ring in a particular area, i.e. tuf-I /nettle and tuf- occur worldwide. Likewise, BN, its grapevine counter- II/bindweed. This situation has recently been con- part, has been recorded in many European countries firmed in Apulia, where the molecular characterization (France, Switzerland, Germany, Hungary, Italy, Croatia, of Stolbur phytoplasma isolates disclosed the presence Greece), Israel and Australia (Bertaccini et al., 1995; of only tuf-II infections, in agreement with the facts Tanne et al., 1995). The European isolates of the BN that, contrary to bindweed, nettle is uncommon in the pathogen belong to the ribosomic subgroups 16SrXII-A, local vineyards (Silletti et al., 2008). rXII-F, and rXII-G, while the Australian isolates (Aus- It can be concluded that the epidemiological cycle of tralian GY) belong to the 16SrXII-B subgroup. FD is quite different from that of BN, as the former in- The most efficient vector of BN in Europe is the cixi- volves grapevine as its only natural host plant (although id leafhopper H. obsoletus which has one generation per other species are now known as occasional field hosts of year, overwinters underground as young nymph (mainly FD-type phytoplasmas) and one insect vector, while the of the 1st instar) on dicotyledonous plants among which latter involves several host plant species beside nettle (Urtica dioica) and bindweed (Convolvulus arven- grapevine and different leafhopper vectors. In other sis) are the favourite ones. It feeds on different plant words, the FD phytoplasma can be regarded as a species, occasionally on grapevines (Maixner, 1994; pathogen ‘specific’ to grapevine while the BN agent is Sforza et al., 1998). The adult forms emerge gradually not. This seems to be reasonably tenable until the puta- by the end of June/beginning of July and remain active tive epidemiological role of some wild hosts of FD-type until mid September. During this period, the plants, phytoplasmas will ultimately be shown. grapevine included, are exposed to the inoculation of According to present knowledge, it seem plausible to the phytoplasmas they transmit, Beside nettle, preferred maintain that: (i) in the case of FD, the natural infection food host of H. obsoletus are the already mentioned pressure is determined by the fact that grapevine is a bindeweed, Ambrosia artemisifolia, Artemisia vulgaris, host specific to both the phytoplasma and its vector; (ii) Calystegia sepium, Daucus carota, Galeopsis angustifolia, in the case of BN, the infection pressure is determined Ranunculus bulbosus, Solanum nigrum and Senecio eru- by the wide range of natural host plants, the potential cifolius. H. obsoletus tends to migrate more effectively infection sources, and on the polyphagy and mobility of than S. titanus from one plant to another to feed and more than one leafhopper vector. laying eggs, either in an active (flying) or passive (trans- ported by the wind) way. There is circumstantial evidence that H. obsoletus EVOLUTION OF DIAGNOSIS may not be the only natural vector of BN. As already mentioned, the main reason for this rests on disease The paramount importance of a reliable distinction spreading in areas where this leafhopper does not occur. of GY from similar but aetiologically diverse grapevine Thus, leafhoppers that can be regarded as potential vec- diseases and for discriminating different GY from one tors are: H. mlokosiewiczi, widespread in the Caucasian another, has attracted the attention of a number of re- area; Pentastiridius beieri, which transmits Stolbur-type search teams and fostered investigations aimed at at- phytoplasmas to sugarbeet in France; Oliarius atkinsoni, taining this goal. This activity, carried out on a world- which transmits a yellows-like disease to flax in New wide basis, has reflected in the development of a series Zealand, and Reptalus quinquecostatus, whose adults of detection techniques which, over time, have evolved collected in heavily infected vineyards were found to from biological diagnostic approaches to molecular host the BN phytoplasma (Gatineau et al., 2001; protocols. 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 313

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Early diagnostic approaches: symptomatology, in- synthesized on phytoplasma genome sequences and its dexing and insect transmission. The main GY symp- successful use for phytoplasma detection in insects was toms, such as leaf discoloration, bunch drying and irreg- reported (Kirkpatrick et al., 1987), recombinant DNA- ular wood ripening, are quite typical and oustanding in based techniques were rapidly developed by several lab- late summer, allowing to recognize GY and, by and oratories. However, while the DNA hybridization is af- large, to distinguish them from other grapevine disor- fordable for phytoplasma detection in herbaceous ders that may show similar alterations (e.g. leafroll or di- plants, including periwinkle (Catharanthus roseus), this rect damage due to leafhopper feeding). As previously assay is not completely reliable when applied to woody stated, however, symptom expression is quite uniform plants, grapevine in particular, mainly because of the among different GY, thus symptomatology is not useful low concentration of the pathogen and its erratic distri- for distinguishing one GY from another. Even indexing bution in these hosts. on the hybrid Baco 22A, used in the past, did not help The major improvement in the diagnosis of GY dis- much because phytoplasmas are poorly transmitted by eases and the specific identification of their etiological grafting on woody plants and because the symptomatic agents was achieved using PCR-based techniques response induced by different GY agents in Baco 22A is (Schaff et al., 1992). The availability in NBCI database the same. of the 16S rRNA gene sequences of an Aster yellows Transmission to Baco 22A or to Vicia faba by S. ti- phytoplasma (Lim and Sears, 1989), and of the FD- and tanus has been used in early days to distinguish FD BN-related phytoplasmas (Davis et al., 1993; Daire et from BN. Successful transmission was reagarded as pos- al., 1993), allowed the development of universal PCR itive response for FD. Since this type of test is laborious, assays for the detection of all known phytoplasmas and slow, and time-consuming, it was dismissed as soon as of phytoplasma-specific PCR protocols for the targeted serological and molecular assays became available. identification of the pathogens associated with different diseases (Deng and Hiruki, 1991; Prince et al., 1993; Serology. As from 1982, monoclonal antibodies and Lee et al., 1994), including those of the GY complex. polyclonal antisera were produced for the detection of The pressing need to dispose of sensitive and depend- FD phytoplasmas through immunization of rabbits with able tools for assessing the absence of FD and other partially purified extracts from FD-infected V. faba phytoplasma diseases from certified propagating materi- plants or from the experimental vector E. variegatus al of grapevine, strongly stimulated the studies for de- (Caudwell et al., 1982; Boudon Padieu et al., 1989; veloping these tools. Various protocols were therefore Schwarz et al., 1989; Seddas et al., 1996). These antisera devised for the reliable identification of grapevine phy- were also used for observing phytoplasmas using im- toplasma based on nested PCR for the amplification of munosorbent electron microscopy (ISEM) and fluores- universal or group-specific phytoplasma 16S rRNA gene cence light microscopy (Lherminier et al., 1989). Poly- (Lee et al., 1994, 1998; Daire et al., 1997; Martini et al., clonal antisera were successfully employed by 1999; Boudon Padieu et al., 2003) and on RFLP analy- Boudon Padieu et al. (1989) for dot blot- and ELISA- sis of the amplicons using appropriate restriction en- based diagnosis of the FD phytoplasma in infected zymes for determining subgroup affiliation (Bianco et broad bean plants and in single S. titanus individuals al., 1996; Lee et al., 1998, 2004). collected in GY-affected vineyards. However, these anti- The more recent multilocus sequence analyses of ribo- sera did not yield good results for FD agent detection in somal (rplV and rpsC) and extra ribosomal genes (secY, grapevines. Better responses were obtained with proto- map, uvrB, degV, and tuf) disclosed how wide is the ge- cols based on the use of monoclonal antibodies netic heterogeneity of FD and BN phytoplasmas (Angeli- (Schwarz et al., 1989; Caudwell and Kuszala, 1992). ni et al., 2001; Lee et al., 2004; Langer and Maixner, Seddas et al. (1993, 1995, 1996) reported the produc- 2004). In some cases, significant differences among phy- tion of antisera for the specific detection of the FD phy- toplasmas causing different grapevine diseases were asso- toplasma that were also capable of distinguishing this ciated with single nucleotide mutations (insertion, dele- agent from other phytoplamas of the same taxonomic tion and substitution), a condition called SNPs (single group (16SrV). No data, however, were provided on the nucleotide polymorphisms). The need then became evi- sensitivity of this test when performed on infected dent of suitable diagnostic tests for a faster and specific grapevines. detection of grapevine phytoplasmas. The innovative mo- The serology-based assays has been used in France lecular approaches developed so far to this aim are: (i) re- for excluding the presence of the FD agent in propagat- al time RT-PCR for of FD and BN phytoplasma detection ing material. However, the lack of suitable commercial (Bianco et al., 2004; Galetto et al., 2005; Angelini et al., ELISA kits strongly limited its application on a large 2007; Margaria et al., 2009; Pellettier et al., 2009); (ii) scale. nanobiotransducer for FD phytoplasma detection (Firrao et al., 2005); (iii) multiplex nested PCR for the simultane- Molecular assays. When the first DNA probe was ous identification of FD and BN agents (Clair et al., 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 314

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2003); (iv) ligase detection reaction (LDR) DNA microar- provinces of Vicenza and Verona and in the province of ray for detecting and and distinguishing FD and BN phy- Treviso (Belli et al., 1997; Pavan et al., 1997; Sancassani toplasmas (Frosini et al., 2002). et al., 1997). In these cases, the association of FD phyto- plasmas with diseased vines was unambiguously deter- mined thanks to the new nucleic acid-based diagnostic FLAVESCENCE DORÉE EPIDEMICS IN NORTHERN tools (Bianco et al., 1993; Bertaccini et al., 1996), and ITALY transmission by S. titanus was experimentally ascer- tained (Carraro et al., 1994). When FD was first reported from Italy (Belli et al., In the western part of Veneto (areas of Soave and of 1973), it showed the typical behaviour of an epidemic Colli Berici), cvs Garganega, Trebbiano di Soave, Tocai disease, spreading very rapidly in the vineyards of the rosso and Chardonnay were the most damaged. In many Oltrepò pavese, thanks to the activity of the vector vineyards more than 50% of the vines showed severe which was already in the area (Osler et al., 1975). The symptoms and strong yield reduction (Belli et al., 1997). initial outbreak was successfully controlled with repeat- In the north-eastern part of the region (Valdobbiadene), ed insecticide sprays (Belli et al., 1978). However, chemi- the disease was particularly severe on cvs Prosecco and cal treatments were soon dismissed at the site of the first Pereira. In several vineyards infection rates of up to outbreak and were not applied in most of the vineyards 90% were registered and many vines (cv. Pereira in par- of other viticultural districts of northern Italy, so that S. ticular) declined and died within 2-3 years (Pavan et al., titanus populations had the opportunity to increase and 1997). As to the FD strain, nested PCR and RFLP spread almost everywhere in this part of the country. At analyses (Martini et al., 1999; Borgo et al., 2001) the same time, many vineyards of cv. Chardonnay, showed that phytoplasmas of subgroups 16SrV-C and which had become a very fashionable variety, were 16SrV-D were both represented in diseased vineyards. planted in northern Italy with propagating material im- Of these, 16SrV-D prevailed and was widely distributed ported primarily from France, where GY were very ac- throughout the most important viticultural areas of tively spreading at that time. Thus, it seems plausible Veneto, while 16SrV-C was mostly confined to the east- that plenty of new inoculum was inadvertedly intro- ern part of the region (province of Treviso). Over time, duced from abroad throughout northern Italy where the regular monitoring and control of the vector, together new cv. Chardonnay vineyards were established. with a more extensive use of certified propagation mate- The most damaging FD epidemics exploded in Vene- rial, gradually reduced FD incidence to a low and en- to, Lombardy, Piedmont and in the eastern part of Lig- demic level (Sancassani et al., 2007). uria as reported hereafter. FD appeared later, in a less epidemic form, also in Friuli Venezia Giulia (extreme Relevant damage in Piedmont and Liguria. Interest north-eastern part of Italy) and Emilia Romagna, where it in FD and other GY in north-west Italy increased by was quickly controlled taking advantage of the experience the middle of the 1960’s, when S. titanus was first de- gained elsewhere. In these latter regions, BN was the real tected in Liguria (Vidano, 1964), then in Piedmont (Vi- matter of concern (Frausin, 2000; Credi et al., 2001). dano et al., 1988). Previous work carried out in France, where S. titanus had been recorded earlier (Bonfils and First occurrence and subsequent epidemics in Vene- Schwester, 1960), had already established that it could to. Veneto is the largest north-eastern Italian region most probably be acting as vector of FD (Schwester et where grape-growing and wine-making are economic al., 1961). In these early papers and in several others activities of paramount importance. It was also the first that followed, C. Vidano and co workers stated that typ- Italian region to suffer severe FD epidemics. The first ical symptoms of GY were present in many localities of oubreak occurred in 1982 in the area of Colli Berici, at Piemont, mostly in the provinces of Asti and Cuneo, on the border between the provinces of Vicenza and Pado- diverse grapevine cultivars among which Chardonnay va (Belli et al., 1983). ‘Chardonnay’ (often confused, at was that most frequently affected. He also observed that that time in Italy, with the cv. Pinot blanc) was the most vineyards affected by GY frequently hosted herbaceous affected variety. Several vineyards, in which only a few weeds with witches’ broom symptoms, and that GY oc- vines with symptoms had been observed during the pre- curred also in areas where S. titanus was not present. In vious year, showed infection rates that went suddenly some of these localities C. Vidano identified also other up to 25%. Huge populations of S. titanus were consis- leafhopper species, today known as phytoplasma vec- tently found in the same area (Belli et al., 1984, 1985). tors, such as H. obsoletus and E. incisus (Vidano et al., This epidemic was controlled satisfactorily with 1988; Conti and Vidano, 1988; Vidano et al., 1989). prompt insecticide treatments However, a few years lat- Serious outbreaks of GY in north-west Italy started er (1990-1994), new and much more severe FD out- to appear during 1990/91 in the provinces of Genova breaks occurred in other important viticultural districts and La Spezia, in the eastern coast (‘Riviera’) of Liguria of the same region located at the border between the (Boselli, 1999), but their phytoplasma aetiology was as- 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 315

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certained only some years later (Minucci et al., 1994; tory and grown until symptom appearance. The highest Conti et al., 1997). Infections moved progressively west, infection risk (8.7%) was detected in September (Conti being first recorded in 2004 in the province of Savona et al., 1997). Diagnostic tests conducted on sympto- (western Riviera). matic grapevines showed that both FD and BN phyto- The grape-growing area of Liguria is about 2.300 ha, plasmas were present in all surveyed areas at a rate of running from the sea borders to the Apennine foot hills. 28% and 19%, respectively. The AY phytoplasma was The yield is almost totally devoted to wine production. also detected in some restricted areas. Its incidence was The environmental conditions are not especially con- generally negligible although an occasional peak of 20- genial to grape growing due to a climate characterized 25% was registered. Each of the three phytoplasmas oc- by continuous wind, air salinity and pronounced daily curred in the vines either alone or in various combina- thermal excursion. Besides, the organic and mineral tions but the symptoms were always indistinguishable content of the soil is so poor that it just allows grapevine regardless of the type of infection (Minucci et al., 1994; survival. Notwithstanding these constraints, viticulture Bertaccini et al., 1995). is important in Liguria because of the output of fine In western Riviera the spatial distribution of GY and white wines (e.g. Pigato, Vermentino, Bianchetta Geno- relative agents was the same, but the infection incidence vese), and because the vineyards, frequently grown on was much lower, ranging from 0 to less than 10%. One terraces supported by hand-made stone walls, protect possibile explanation for this is that grapevines in west- the soil from erosion and confer quite an attractive, ern Liguria are grown in large, well tended vineyards, unique and pictoresque aspect to the landscape. while small family-type stands surrounded by wild flora Due to the adverse growing conditions, the results of and infested by weeds are not as frequent as in the east- GY disease in Liguria were more devastating than else- ern Riviera. where, causing generally the death of plants one to two Viticulture is of primary economic importance in years after infection. For this reason, since its early out- Piedmont and involves an area of about 53,500 ha, break the disease was called locally ‘Moria della vite’ (= practically all devoted to wine production (cultivation grapevine lethal disease). Plant mortality takes place es- of table grape is limited to less than 500 ha). The cli- pecially during summer in the front row of vineyards on mate and soil conditions are nearly everywhere congen- terraces which are most exposed to the drastic daily ial to grape-gowing with the only exception of some en- changes of temperature. Symptoms were severe on all claves in the mountains, where vineyards can be found the grapevine cultivar examined, namely: Bianchetta at an altitude of 1,000 metres (e.g. the Susa Valley) that Genovese, Bosco, Pigato, Trebbiano and Vermentino sets the extreme limit of grapevine cultivation. The most (white berry), and Barbera, Bonarda, Ciliegiolo, Dolcet- important areas for wine production are the provinces to ormeasco, Lumassina, Rossese and Sangiovese (red of Asti, Alessandria (south-east), Cuneo (south-west), berry). The only exception was the white berry cv. Al- and Novara (north-east). The widely known red wines barola which showed consistently a mild downward (Barolo, Barbaresco, Barbera, Dolcetto, Nebbiolo) are rolling and discoloration of the leaf lamina, and limited by far the most important, while white vines (Arneis, yield losses. Cortese) are less important and are produced in rela- Field surveys started in eastern Riviera (Sestri Lev- tively low amounts. ante, Cinque Terre) in 1993 and were extended to west- Early cases of GY infection were observed in the ern Riviera since 2004. Their aim was to record GY 1970’s in the provinces of Alessandria, Asti and Cuneo presence and distribution in the vineyards, check the (Belli et al., 1978, 1985), although only in 1998 the dis- seasonal infection risk, and collect samples for nucleic- ease turned serious, causing severe damage in the Colli based diagnostic tests (PCR, DNA probes and RFLP) Tortonesi (Tortona’s hills, Alessandria province), an area for detection of phytoplasmas of the taxonomic groups close to the eastern part of Liguria where GY epidemics 16SrI-B (Aster yellows), 16SrV (Elm yellows, FD), and had started in 1990/91. When it was first noticed, the 16SrXII-A (Stolbur, BN). disease was already widespread, the number of affected GY incidence ranged from 12 to about 40% being plants was quite high (18-50% in single vineyards), and highest in cvs Vermentino, Sangiovese and Trebbiano in the symptoms were very strong. Considering the ad- old vineyards that were heavily infested by weeds. In- vanced stage of damage, it was generally agreed that the fected plants occurred most frequently in the rows at epidemics had started years before, possibly at the time the periphery of crops, but were also scattered or in they had been observed in Liguria. The occurrence of groups of 5-10 within crops. The ‘infection pressure’ epidemics had been reported to the Plant Protection determining the seasonal risk of infection was assessed Service much later (1998) because their pathogenic na- by exposing periwinkle (Catharanthus roseus) bait plants ture had been misunderstood for years, and the inci- to insect feeding in GY-affected vineyards. Potted peri- dence had taken some years to reach a dramatic level. winkles were placed in the field, from June to October, The most severely affected cultivars were Barbera, removed after one month exposure, taken to the labora- Dolcetto and Cortese grown on the ‘Colli Tortonesi’. In 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 316

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and around the infected vineyards, symptoms of phyto- the disorder and did not take action. This second and plasma infection were observed commonly on wild severe FD outbreak was probably consequent to a simi- (bindweed) and cultivated plants (alfalfa). The former lar epidemic registered in the previous years in the vine- species showed yellowing, severe dwarfing and witches’ yards around Tortona (province of Alessandria, Pied- broom symptoms, while alfalfa showed a bright mont), at the borders of Oltrepò pavese. Migration of purple reddening and malformation of leaves and infected S. titanus populations from the Tortona area to growth reduction (Conti, 2001). Oltrepò Pavese could have brought enough inoculum to Surveys carried out in southern Piedmont in 1999 cause a rapid disease spreading. showed that the area of GY occurrence was about 1,800 Taking advantage of a prompt and specific project fi- ha in size and included the provinces of Alessandria and nanced by the regional administration, field surveys Asti, where the total acreage of vineyards with more were initiated soon after the first observations made by than 30% infection was estimated to be in excess of of the regional extension service and were conducted in 1,000 ha. GY was also present, although sporadically, in all the most important viticultural areas of Lombardy. the province of Cuneo (south-west Piedmont). Testing From the end of June to the middle of October 1999, a of infected vines showed that both FD and BN were total of 74 vineyards with suspected FD infection were present, FD being more widespread that BN, that monitored. In each of these vineyards, the number and showed a more irregular distribution and relatively high relative position of diseased vines were registered and incidences only locally. Phytoplasmas of the AY type samples with different type of symptoms were taken for were occasionally detected in Piedmont with infection molecular assays. The highest GY incidence was found rates always lower than 5%. in Oltrepò pavese, where in some vineyards it reached In the years that followed, GY spread progressively peaks of up to 90%, with dramatic economic losses. In to the provinces of Novara and Biella (north-east Pied- this area, infection was caused almost always by FD mont) and the Aosta Valley, where FD prevailed. In any phytoplasmas; BN was much les represented and gener- case, GY incidence in northern Piedmont was not so ally occured in mixed infection with FD. high as in the southern part of the region, where A different situation was encontered in other areas of grapevine crops in the provinces of Alessandria and, Lombardy, where FD was present (except in Valtellina) more so, Asti, are still the most affected. but BN was prevalent and the economic damage was The economic damage caused by GY in Piedmont can generally much lower. Barbera, Chardonnay, Pinot gris be well represented by the expenses met by the regional and Sangiovese proved to be the most sensitive culti- administration to control the disease, i.e. a financial con- vars, followed by Pinot noir, Croatina, Riesling and tribution of 1.5 million € in 1999-2000 to launch the Trebbiano. Very mild symptoms were observed on vines control project; about 1.375 million € in 2001-2003 for of cvs Cortese and Moscato (Belli et al., 2000). A situa- monitoring occurence and distribution of the disease; tion similar to that of Oltrepò pavese was found in about 34 million € subsidy to growers in 2005 from the Valtenesi, which is located on the south-western side of Italian Government or the European Community to re- the Garda lake, bordering with Veneto. In in this area, fund yield losses and encourage replanting. FD prevailed, with a very high incidence (up to 90%) and cv. Sangiovese appeared to be by far the most dam- Epidemics and damage in Lombardy. Lombardy, lo- aged (Scattini et al., 2000). The 1999 FD epidemic re- cated between Piedmont and Veneto, includes viticul- tained its severity in Oltrepò pavese in the following tural areas where premium wines are produced, such as years (2000-2003), and spread to the other viticultural the Oltrepò pavese (province of Pavia), Franciacorta districts of the region. (province of Brescia) and Valtellina (province of Son- Molecular analyses showed that FD phytoplasmas of drio). While in Oltrepò pavese and Valtellina the pro- subgroups 16SrV-C and 16SrV-D occurred in Lom- duction of red wines is prevalent, Franciacorta is fa- bardy. Whereas subgroup 16SrV-C was found only in mous for its excellent white sparkling wines. Other Oltrepò pavese, subgroup 16SrV-D was present in all wine-producing areas are in the provinces of Bergamo viticultural districts of the region (Bianco et al., 2003). (Valcalepio) and Mantova (Colli Mantovani). Since 2004 a progressive decrease of FD incidence As mentioned, the first FD epidemics in Italy oc- has been recorded in all the most important viticultural curred in Oltrepò pavese (Belli et al., 1973). A success- areas of Lombardy, especially in Oltrepò pavese; while ful strategy for controlling disease spreading, based on BN has gained momentum (Quaglino et al., 2006). This insecticide treatments against the leafhopper vector, was trend continued in 2007 and 2008, for a further de- implemented for a few years, to be abandoned when the crease of FD incidence was registered in most of the growers believed that the disease was no longer danger- monitored vineyards of the region, due to the increased ous. In early summer 1999, there was a relapse in the cases of recovery (Zorloni et al., 2008), beneficial effect same area which caused much concern to the growers of vector control and use of healthy grapevines for the who, at first, did not recognize the “yellow” nature of new plantations. 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 317

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INCREASE OF BOIS NOIR IN ITALY positive samples (47 of 48) were shown to be infected with the FD phytoplasma and only one with BN. In the As mentioned above, many of the early GY reports following years, there was a gradual increase of BN and in Italy must be referred to BN, especially those not as- decrease of FD. Finally, in 2005, out of a total of 90 pos- sociated with the presence of S. titanus as in the records itive samples, 33 were infected with FD, 47 with BN from Trentino Alto Adige (Zelger, 1964; Mescalchin et and 10 had a mixed FD-BN infection. Concerning BN al., 1986), Piedmont (Belli et al., 1978), Sicily (Granata, phytoplasmas, the VK-I type prevailed in the samples 1985), some areas of Veneto (Egger and Borgo, 1983), collected in Franciacorta, while VK-I and VK-II types Emilia Romagna (Belli et al., 1978; Credi and Babini, were equally distributed in the samples from the other 1984) and Friuli Venezia Giulia (Carraro et al., 1986), areas (Quaglino et al., 2006). Tuscany (Egger and Grasselli, 1988), Liguria (Minucci A region-wide survey was done for nine years (1998- et al., 1994), Apulia (Di Terlizzi et al., 1994). The pres- 2006) in Veneto by the local Plant Protection Service. ence of BN in Italy was confirmed only when new diag- Molecular testing of a large number of symptomatic nostic methods (especially, PCR and RFLP) became samples showed a slight decrease of FD from 2003, ac- available for laboratory identification of the agents pres- companied by a general and gradual increase of BN that ent in field samples showing GY symptoms (Bianco et became very pronounced during 2005 and 2006, reach- al., 1993; Daire et al., 1993; Davis et al., 1993; Bertacci- ing an incidence of 20% (Sancassani et al., 2007). Both ni et al., 1995). Initially, the BN phytoplasmas were con- BN strains were detected, with a prevalence of the VK-I sidered to belong to a subgroup of the AY group type (Angelini et al., 2008; Scopel and Causin, 2008). A (16SrI-G) and only later were included in a new group more recent investigation, carried out in 12 vineyards of (16SrXII). Since then, reports on BN occurrence in di- three different provinces (Verona, Treviso and Venezia), verse Italian regions and studies on its biological and disclosed a BN infection rate of 64% (Quaglino et al., epidemiological features became fashionable. 2009a). In the other two north-eastern Italian regions (Trenti- Northern Italy. In Piedmont, the first molecular diag- no-Alto Adige and Venezia Giulia), most of the regis- nostic tests for the presence of BN were carried out in tered GY infections were assigned to BN, whereas FD 1999, revealing that this phytoplasma occurred in 33% was found only in areas bordering with Veneto (Frausin, of the infected grapevine samples tested, either in single 2000; Osler and Refatti, 2002). This situation has not or in mixed infection with FD. In 2000, the frequency changed during the last years. As to Alto Adige, molec- of BN infection was about 27% (Marzachì et al., 2001). ular analyses of infected grape samples from several These data came from a total of 219 vines tested (123 in vineyards showed the presence of both VK-I and VK-II 1999 and 96 in 2000) belonging to 17 different cultivars strains of BN. VK-II, which was not detected up to grown in the province of Alessandria. The number of 2003, has recently become quite frequent (Baric and BN-positive samples was 49%, 21%, and 47% in the Dalla Via, 2007). years 2004, 2005 and 2006, respectively (Marzachì and Surveys carried out in the main viticultural districts Pacifico, 2006). The frequency of BN infections was of Emilia Romagna showed the FD was present mostly highest in the areas characterized by low FD incidence in the northern part of the region (provinces of Piacen- such as the provinces of Torino, Novara and Vercelli, za, Parma and Reggio Emilia), while BN was wide- while it was lower in the areas most severely affected by spread with a high incidence especially in the south- FD. The FD distribution in the territory appeared less eastern part of the region, where consistent populations influenced by the growing conditions than that of BN, of H. obsoletus were also found (Credi et al., 2001; in the sense that the spread of BN was limited in areas Bertaccini et al., 2003; Vicchi et al., 2008). of industrial viticulture, but not in areas of familiar viti- culture, where vineyards were heavily infested by weeds. Central and southern Italy. The surveys conducted in Starting from 2004, the BN isolates collected in Pied- the most important viticultural districts of central and mont were characterized at the molecular level, follow- southern Italy have shown that BN occurs in all moni- ing two different procedures (Langer and Maixner, tored areas of Tuscany, Latium, Molise, Apulia, Campa- 2004; Pacifico et al., 2005). This confirmed that both nia and Calabria, with a high incidence in Tuscany and the VK-I (tuf-I) and VK II (tuf-II) variants of BN de- Latium. The presence of VK-II type was ascertained in scribed in Germany existed in Piedmont, and that the all the investigated areas except for Latium, where only range of variability was higher in phytoplasma isolates the VK-I type was found. This variant, however, was not infecting bindweed (Marzachì and Pacifico, 2006). detected further south [Campania and Calabria In Lombardy, 24 vineyards from the most important (Carnevale et al., 2008; Pasquini et al., 2008)]. viticultural districts were investigated for six years An increase of BN infections has been registered in (2000-2005) in order to identify the phytoplasmas asso- the last years in Marche, Abruzzo and Apulia, where ciated with GY symptoms. In the first year, almost all many cultivars are severely damaged. Variants, VK-I 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 318

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and VK-II were identified in Marche and Abruzzo which established officially the measures that had to be (D’Ascenzo et al., 2008; Romanazzi and Murolo, 2008) enforced to contain FD infections throughout the coun- while only VK-II was detected in Apulia (Silletti et al., try. This decree categorized the affected areas according 2008), where, as previously stated, bindweed is much to the extant epidemiological situation, namely: common than nettle. In Sicily, of a total of 159 symptomatic samples from ‘Zona focolaio’ (= area with FD foci), corresponding different areas of the island 57 were infected by the BN to an area where the presence of FD has officially been variant of the VK-II type. However, the planthopper H. ascertained but where eradication seems still technically obsoletus has never been found in any of the the sur- possible. Compulsory control measures for these areas veyed vineyards (La Rosa et al., 2008). A similar situa- are: (i) immediate uprooting and destruction of any vine tion is present in Sardinia, where, even in absence of the with GY type symptoms, even before confirmation of known vector, BN is apparently spreading (Garau et al., FD infection by laboratory tests; (ii) insecticide treat- 2007). These and other recent observations (Tessitori et ment twice a year against S. titanus. The use of further al., 2009) support the hypothesis that still unidentified control measures can be imposed by the local Plant Pro- BN vectors other than H. obsoletus, may exist in the tection Service (PPS), according to each specific situa- main Italian islands. tion, including the complete eradication of vineyards with symptomatic plants.

CONTROL STRATEGIES ‘Zona insediamento’ (= area where FD is ultimately established), consisting of an area where the presence of Control measures against GY are being implemented both FD and S. titanus has been ascertained, in which in Italy, targeted mainly to FD because: (i) the economic the disease is so widespread that eradication seems no importance of BN infections has emerged only recently, longer possible. Such epidemiological situation must be following the extensive use of appropriate diagnostic as- recognized officially by the PPS concerned, which also says; (ii) the present knowledge of the epidemiology and decides on the control measures to be enforced in each control of BN and other GY is still inadequate. single case. As mentioned, after the first FD outbreaks in Oltrepò pavese (Belli et al., 1973) and Veneto (Belli et ‘Zona indenne’ (= FD-free area), referred to an area al., 1983), and the finding of S. titanus in the same areas where FD does not occur, in which control measures to (Osler et al., 1975; Belli et al., 1984), insecticide sprays be implemented for preventing FD introduction must against the FD vector were performed for some years in be issued by the PPS. most of the affected areas. However, these were mostly local initiatives, based on what had been done in In addition to the above, the following compulsory France. measures were issued for implementing FD control in A specific project for a better knowledge of FD and grapevine nurseries: (i) continuous surveys for symptom its control was initiated in the mid 1980’s in the detection; (ii) roguing and immediate laboratory testing province of Vicenza with the financial support of the lo- of plants showing suspected symptoms; (iii) monitoring cal Chamber of Commerce. In about ten years of work, the presence of S. titanus all the year round and spray- new data on FD epidemiology in that province and in- ing periodically with insecticides. dications for efficient disease control were gathered. Di- Since June 2000 the PPS of the different regions af- rections were then issued to grape growers suggesting: fected by FD started to organize and support control (i) use of healthy material for the new plantings; (ii) strategies in agreement with the national decree of com- roguing symptomatic vines in young vineyards to reduce pulsory control. inoculum potential; (iii) perform two insecticide sprays in summer, in mid June and twenty days later; (iv) elimi- Piedmont. Field surveys conducted since the early nation of incompletely lignified canes during winter appearance of GY had shown that this disease was pres- pruning. These measures led to a substantial reduction ent in the whole territory of the provinces of Asti and of FD in the Vicenza province (Belli et al., 1997). Alessandria, while its incidence was much lower in the Following the new and destructive FD outbreaks reg- provinces of Torino and Cuneo. In 2006, the areas clas- istered in the late 1990’s in Veneto, Piedmont and Lom- sified as ‘focolaio’ included all the municipalities of the bardy, and taking into accout that FD is included in the Asti and Alessandria provinces, about fifty municipali- EPPO quarantine list, the Italian Ministry of Agricul- ties in the province of Cuneo, sixteen in the province of ture enacted a decree for the compulsory control of the Torino, and five in the province of Novara, the last in disease (D.M. n. 32442 of May 31st, 2000: “Misure per la time to suffer FD outbreaks. lotta obbligatoria contro la Flavescenza Dorata della During the first 3 to 4 years of survey, GY infections vite”, published in the G.U. n.159 of July 10th, 2000) were found in some 400 nurseries of the about 2,000 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 319

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registered in Piedmont. The regional PPS decided to Lombardy. The ample field surveys and the diagnos- destroy the whole production of these nursery and to tic tests done during the years 1999 and 2000 (Belli et stop their activity for at least two years. After this period al., 2000) showed that FD was present in all the most of time, the situation of FD presence and incidence in important viticultural areas of the region, except in Val- each area was re-examined by the PPS to verify whether tellina (province of Sondrio), a valley located at the ex- nursery activity could be restored. This was authorized treme north of the region. The disease incidence was only in about 10% of the cases. particularly high in vineyards of Oltrepò pavese and Spraying vineyards with insecticides against S. Valtenesi (province of Brescia), but was increasing in titanus, appeared an efficient means to control leafhop- several other areas. per populations. In the environmental conditions of The regional PPS therefore enforced (with exception Piedmont the fisrt treatment was done in mid May and for Valtellina) a control strategy based on the following the second at the beginning of June. In some cases, measures: (i) two insecticide sprays against S. titanus in grapevine growers decided themselves to spray a third commercial vineyards, in mid June and during the sec- time 10 to 15 days after the last. The seasonal periods ond week of July (after about twenty days); (ii) more most appropriate for the first of the two insecticide frequent insecticide treatments, up to six in the nurs- sprays were established on the basis of the biological cy- eries: (iii) destruction of the small abandoned vineyards cle of S. titanus in the region. This is to say, that the first acting a possible niches for S. titanus; (iv) carfeul elimi- treatment had to be made before the insect juveniles nation of canes showing incomplete lignification during had turned into adults, for these can fly and move more pruning; (v) use of certified FD-free material for new actively, spreading FD most efficiently. plantings The use of insecticides for FD control was not de- These specifications were followed by the majority of prived controversies, raised by the “green people” who the growers and nurserymen, leading to a substantial complained about the contamination of the environ- improvement of the sanitary status of the regional vine- ment, and the beekeepers, complaining about the mas- yards with respect to FD. In fact, a progressive decrease sive killing of the bees. This latter problem was satisfac- of this disease has been recorded since 2004, especially torily solved by employing less toxic insecticides, conse- in Oltrepò pavese that was the most damaged area dur- quent to trials carried out by the regional PPS (Bosio, ing the late 1990’s. 2006), and by compensating beekeepers for the possible damages caused to their operations. Veneto. The monitoring programme initiated in 1998 An additional FD control measure was adopted in established that FD was present in all the viticultural ar- 2005 in Piedmont, first in Italy, which consisted in sub- eas of the region, primarily in an endemic form. There- mitting grapevine propagation material (cuttings and fore, the whole regional territory was classified as “zona grafted nursery productions) to heat water treatment for insediamento”. It ensued that the control strategy de- eliminating phytoplasma infection (Bianco et al., 2000; vised by the regional PPS was based on the following: Tassart Subirats et al., 2003). Before resorting to the (i) insecticide sprays against S. titanus; (ii) use of healthy routine use of thermotherapy, experiments were con- material for the new plantings. ducted to find out the most appropriate conditions for As to issue (i), two treatments were recommended in the treatment, which were shown to be an exposure to commercial vineyards in June and July with the sugges- 50°C for 45 min (Mannini, 2007). Submitting grapevine tion of reducing them to one where the S. titanus popu- propagation material to such treatment not only con- lations were low. By contrast more frequent treatments tributed to reduce the spread of FD infections, but was had to be done in the nurseries to prevent infection of also useful to kill the eggs of S. titanus if present in the the young vines. As to issue (ii), collection of buds for bark of the treated grapevines. graftings was allowed only from mother plant blocks The GY situation in Piedemont has widely changed that had not shown GY symptoms for at least two years. today, because the whole grapevine-growing area of the This strategy afforded satisfactory results. FD has de- region has been recognized as ‘Zona insediamento’, and creased so as to be no longer the most serious problem. this has considerably reduced the use of insecticides On the contrary, BN has increased, becoming the real against FD vectors. On the other hand, although this new threat (Sancassani et al., 2007). means that the attempts to eradicate FD in the region have failed and that the presence of the disease has to be definitively accepted, it appears that the control FUTURE PROSPECTS measures implemented since the early FD outbreaks have actually reduced the disease incidence and thaught GY certainly constitutes a serious concern for Italian the growers to keep it under control, and to co-exist viticulture, considering the wide distribution of FD in with it. the northern part of the country and the alarmingly fre- quent presence of BN in the majority of the national ter- 002_JPPReview(Belli)_303 9-07-2010 11:56 Pagina 320

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ritory. This has stimulated during the last 20 years or so, croorganisms and GY vectors may open new avenues for much research activity in several Italian laboratories. the development of biological control strategies. Thanks to these efforts, supported by national and re- Finally, there is much to do in the field of genetic re- gional funds, and thanks to the productive collaboration sistance. Since the knowledge on plant genes inducing with some foreign research institutions (especially from phytoplasma resistance is very scarce, opportunities to France and the USA), we have gained a much better in- select resistant varieties by traditional or molecularly as- sight of GY diseases. sisted breeding are limited. Hence, the usefulness of Particularly beneficial has been the introduction of fostering studies for the identification of genetic traits molecular methods for the diagnosis and characteriza- for phytoplasma resistance tion of phytoplasmas involved in the aetiology of these diseases. Thanks to these avant-garde techniques, sub- stantial advances were made in the molecular knowl- REFERENCES edge of these agents, their biological and genetic charac- teristics, insect vectors and alternative host plants. Albanese G., Davis R.E., Granata G., Dally E.L., Santuccio T., However, there is no doubt that the knowledge on Tessitori M., 1996. Analisi del DNA per l’individuazione e FD is more advanced than that on BN, especially with l’identificazione di fitoplasmi in piante di vite affette da gial- lumi in Sicilia. Petria 6: 65-76. respect to epidemiology and control. We currently know that the main factor favouring the spreading of Albanese G., Granata G., Collodoro S., Egger E., Baioletti P., D’Arcangelo M., 1997. Individuazione e caratterizzazione FD in the vineyards of any given area, is the presence of molecolare di fitoplasmi in piante di vite con sintomi di gi- important populations of S. titanus. We also know that allume in Umbria. Rivista di Viticoltura e di Enologia 50: this insect thrives normally only on grapevines and ac- 3-9. quires the FD agent only by feeding on these hosts Alma A., Soldi G., Tedeschi R., Marzachì C., 2002. Ruolo di which makes relatively easy the chemical control of the Hyalesthes obsoletus Signoret (Homoptera, Cixiidae) nella vector and, therefore, of the disease it transmits. trasmissione del legno nero della vite in Italia. Petria 12: On the contrary, BN spreads even in areas where its 411-412. only known natural vector, H. obsoletus, seems to be ab- Angelini E., Clair D., Borgo M., Bertaccini A., Boudon-Padieu sent or very rare, e.g. in Sardinia and Sicily. This E., 2001. Flavescence dorée in France and Italy. Occurence leafhopper lives usually on herbaceous plants from of closely related phytoplasma isolates and their near rela- which it acquires the BN agent(s) and feeds occasionally tionships to Palatinate grapevine yellows and an alder yel- lows phytoplasma. Vitis 40: 79-86. on grapevines. Thus the use of insecticides to control Angelini E., Squizzato F., Lucchetta G., Borgo M., 2004. Detec- BN is useless. There is, therefore, much room and need tion of a phytoplasma associated with grapevine Flavescence for future research on the relationships between BN dorée in Clematis vitalba L. European Journal of Plant phytoplasmas and their vectors, aimed at identifying ef- Pathology 110: 193-201. ficient control measures for this disease. Angelini E., Bianchi G.L., Filippin L., Morassutti C., Borgo M., Results of recent studies can improve the control 2007. A new TaqMan method for the identification of phy- strategies currently enforced in some Italian regions. toplasmas associated with grapevine yellows by real-time For example, the frequent cases of recovery from FD PCR assay. Journal of Microbiological Methods 68: 613-622. and BN observed in several cultivars (Osler et al., 2003; Angelini E., Filippin L., Bellotto D., Stringher L., Borgo M., Zorloni et al., 2008), and the fact that vines infected 2008. Giallumi della vite in provincia di Treviso: caratteriz- with BN are not a source of infection, makes it ques- zazione dei fitoplasmi associati. Petria 18: 211-213. tionable the usefulness of rouging symptomatic plants. Arnaud G., Malembic-Maher S., Salar P., Bonnet P., Maixner Furthermore, a generalized application of hot water M., Marcone C., Boudon-Padieu E., Foissac X., 2007. Mul- tilocus sequence typing confirms the close genetic inter-re- treatment to propagation material, as already done in latedness between three distinct flavescence doree phyto- Piedmont (Mannini, 2007), could allow the collection of plasma strain clusters and group 16SrV phytoplasmas infect- cuttings and budsticks for graftings also from mother ing grapevine and alder in Europe. Applied and Environ- plant stands with a low FD incidence which is now for- mental Microbiology 73: 4001-4010. bidden according to the extant decree for the compuso- Bae S., Fleet G.H,. Heard G.M., 2006. Lactic acid bacteria as- ry control of this disease. sociated with wine grapes from several Australian vineyards. 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Received March 25, 2010 Accepted April 28, 2010