DETECTION, CHARACTERIZATION, EPIDEMIOLOGY AND ERADICATION OF PLUM POX VIRUS MARCUS TYPE IN SPAIN

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1 Journal of Plant Pathology (2010), 92 (3), Edizioni ETS Pisa, DETECTION, CHARACTERIZATION, EPIDEMIOLOGY AND ERADICATION OF PLUM POX VIRUS MARCUS TYPE IN SPAIN N. Capote* 1, M.A. Cambra 2, P. Botella 3, M.T. Gorris 1, M.C. Martínez 1, A. López-Quílez 4 and M. Cambra 1 1 Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, Valencia, Spain 2 Centro de Protección Vegetal, Diputación General de Aragón, Zaragoza, Spain 3 Universidad Cardenal Herrera-CEU, Moncada, Valencia, Spain 4 Universidad de Valencia, Valencia, Spain SUMMARY Sharka disease caused by Plum pox virus (PPV) is widespread in a number of Spanish stone fruit-growing areas; only the common Dideron type (PPV-D) is prevalent. Systematic surveys of field and packing houses were conducted in 2002 for detection of the aggressive Marcus type (PPV-M). The focus of PPV-M was an infected cv. Royal Gem peach (Prunus persica L.) orchard in Zaragoza province (Ebro Valley, Aragón). Over three years, the virus has spread from this orchard to distant plots by means of grafting of infected plant material and to neighbouring plots by aphid transmission. Serological and molecular analyses of symptomatic trees and fruits confirmed the presence of PPV-M in these plots and segregation of the initial viral population into three serogroups and seven different haplotypes as a consequence of host/individual change or vector transmission. Not only did peach cv. Royal Gem become infected, but also cvs Calante and Gladys, in 4 orchards with a total area of 19.5 ha. Spatial analysis of the spread of infection showed a compound contagion process with long-range (up to 150 meters) and short-range movements to adjacent trees in the same row. The main aphid vectors present in the area were Aphis spiraecola Pagenstecher, A. gossypii Glover and Myzus persicae Sulz.. Experimental aphid transmission assays showed that the introduced PPV-M isolate was more efficiently transmitted to other peach cultivars from GF305 peach seedlings, rather than to P. persica cv. Gladys young trees. All tree blocks infected with PPV-M were uprooted. Eight years later (2010) no more PPV-M has been detected in commercial Prunus orchards in the area suggesting successful eradication. Key words: PPV-M, aphid transmission, segregation of population, spatial spread. * Present address: Instituto Andaluz de Investigación y Formación Agraria, Pesquera, Alimentaria y de la Producción Ecológica (IFAPA), Alcalá del Río, Sevilla, Spain. Corresponding author: M. Cambra Fax: mcambra@ivia.es INTRODUCTION Plum pox virus (PPV), the causal agent of Sharka disease, produces severe damage and significant economic losses to stone fruit production (Cambra et al., 2006c). PPV was first detected in apricots in Bulgaria in 1917 (Atanasoff, 1932) and since then it has spread progressively to a large part of the European continent, the Mediterranean basin (which covers portions of Europe, Africa and Asia), and the Middle and Near East (Roy and Smith, 1994; García and Cambra, 2007). It has also been found in South and North America (Argentina, Canada, Chile and USA) and in Asia (China, India, Iran, Japan, Kazakhstan and Pakistan) (Capote et al., 2006a; Barba et al., 2010). PPV is transmitted by vegetative propagation; the use of infected plant material for propagation is the main cause of long-distance spread whereas, at a site, spread is mediated by a number of aphid species in a non-persistent manner (Labonne et al., 1995; Isac et al., 1998; Moreno et al., 2009). In the temperate Mediterranean area, the highest aphid populations are found in May. Aphis gossypii and A. spiraecola have been reported as the most abundant aphid species landing on Prunus trees in Spain, although other aphid species can also spread the virus with differing efficiency (Cambra et al., 2006b). To date, seven strains or types of PPV have been described, that differ in their pathogenicity, host range, serological and molecular characteristics and geographical distribution (Glasa and Candresse, 2005; García and Cambra, 2007): PPV-D or Dideron and PPV-M or Marcus are the most prevalent types; PPV-EA or El Amar is geographically restricted to Egypt (Wetzel et al., 1991); PPV-C or Cherry type is represented by sour and sweet cherry-infecting isolates (Nemchinov and Hadidi, 1996; Nemchinov et al., 1996; Crescenzi et al., 1997); PPV-W or Winona is the unusual and divergent W3174 isolate detected in Canada (James et al., 2003; James and Varga, 2005); recombinants between D and M types constitute the PPV-Rec type, showing D-type epidemiological behaviour (Glasa et al., 2004); and finally, a new group of recombinants named PPV-T, found in Turkey and characterized by a unique recombination in the HC-Pro gene (Glasa and Candresse, 2005; Serçe et al., 2009).

2 620 PPV-M in Spain Journal of Plant Pathology (2010), 92 (3), Each of the seven known strains or types can now be identified using a range of serological or molecular techniques some of which are recommended for routine analysis (EPPO, 2004; IPPC, 2009; Capote et al., 2009). PPV-D and PPV-M, the most common types, differ in epidemiological behaviour: PPV-D mainly affects apricots, causing severe losses in early cultivars but also to European and some Japanese plum cultivars and, to a minor extent, peaches. In Spain, where the peach industry is economically important, no temporal spread of PPV-D was observed in extensive plots of peach cultivars grown in the vicinity of PPV-infected Prunus orchards in different ecological areas (Cambra et al., 2006b). Conversely, PPV-M is more aggressive than PPV-D and affects all Prunus species, spreading very fast in peaches, apricots and plums. Numerous field surveys undertaken in Prunus-growing areas showed that Sharka spreads more rapidly in peach orchards infected with PPV-M than in apricot orchards infected with PPV-D (Adamolle et al., 1994; Gottwald et al., 1995), probably because the M type is more readily and efficiently transmitted by aphids and/or this type is more effective in overcoming peach resistance. PPV-M is currently present in several countries in eastern and central Europe, the Mediterranean basin, and the Middle and Near East including Albania, Bosnia and Herzegovina, Bulgaria, Croatia, Cyprus, Czech Republic, France, Germany, Greece, Hungary, Italy, Jordan, Macedonia, Montenegro, Poland, Rumania, Serbia and Montenegro, Slovakia, Slovenia and Turkey (Capote et al., 2006a). PPV-M however, has not been officially detected in other European and Mediterranean countries, North and South America or Asia, where the D type occurs. Only PPV-D is currently present in Spain where systematic surveys and analyses for detection and typing are performed every year. However, a focus of PPV-M of unknown origin, probably introduced three years before, was detected at the end of June 2002 by the Plant Protection Services of Aragón in four distant plots planted with P. persica cvs. Royal Gem, Calante and Gladys, located in the municipalities of Caspe, Maella and Fabara (Zaragoza/Aragón). Most of the infected trees showed typical PPV-M symptoms on leaves (obvious vein clearing and diffuse spots) and fruits (depressed chlorotic rings). Serological characterization confirmed the presence of PPV-M as revealed by molecular methods using specific RT-PCR primers and probes (Cambra et al., 2004). From this initial focus PPV-M has spread to neighbouring plots probably by aphid vectors and to more distant plots by confirmed grafting with infected plant material. The present work describes the methods used to detect and characterize PPV-M in Spain, the model of its spatial spread from the initial focus to neighbouring plots after three years of natural spread, and the differentiation of the initial PPV-M population into different serogroups and haplotypes. Aphid species in the infected area were identified, the transmission rate of the PPV-M isolates was determined, and the risk of new introductions and the success of the eradication were evaluated. MATERIAL AND METHODS Surveys for PPV-M detection and characteristics of infected field plots. Extensive surveys to detect PPV-M infection are routinely performed yearly by the Plant Protection Services in most important Spanish Prunusgrowing areas. Visual surveys for symptoms start with blossom and subsequently with leaves and fruits in the field and/or in packing houses. Plant material with suspicious symptoms is collected and analysed. Following this protocol, peach fruits showing PPV-like symptoms were collected from a packing house, stored at 4ºC and analysed following the European and Mediterranean Plant Protection Organization (EPPO) protocol for PPV diagnosis (EPPO, 2004). The origin of infected fruits was located in Caspe, Zaragoza province (plot No. 1; Fig. 1) where PPV-M-infected buds of cv. Royal Gem of unknown origin, illegally introduced into Spain were grafted. From this plot, infected buds were grafted to peach trees in two distant plots located in Maella (plot No. 2) and Caspe (plot No. 3). The Maella plot encompassed three blocks or parcels: block 1 where cv. Royal Gem infected buds were grafted; block 2 planted with cv. Calante, and block 3 planted with cv. Gladys. PPV-M from block 1 spread naturally to the neighbouring cvs Calante and Gladys by aphids, then buds from these infected Gladys trees were grafted to another distant Gladys plot located in Fabara (plot No. 4). The incidence of PPV-M in the area was determined by analysing 9,179 peach trees by 5B-IVIA DASI-ELISA (see below). Virus incidence was expressed as the number of infected trees on the total number of analysed trees. Inquiries in the area determined that the initial PPV-M infected material was probably introduced and grafted in 1999, three years before detection. The whole plots where PPV-M had been detected were uprooted once their infection was serologically confirmed. Virus isolates and plant extract preparation. Buds from 19 PPV-positive trees from different infected plots (cvs Royal Gem, Calante and Gladys) were selected for serological and molecular characterization and grafted onto GF305 peach seedlings for indexing and maintenance of viral isolates. PPV-M isolate MS89, maintained under quarantine facilities at IVIA (Instituto Valenciano de Investigaciones Agrarias) was used as positive control, and healthy GF305 was used as negative control.

3 Journal of Plant Pathology (2010), 92 (3), Capote et al. 621 The origin and most likely way of spreading of different PPV-M isolates are shown in Fig. 1. Crude extracts were prepared by grinding buds in individual plastic bags in the presence of 1/20 (w/v) PBS buffer, ph 7.2 supplemented with 2% (w/v) polyvinylpyrrolidone-10 (PVP-10) and 0.2% (w/v) sodium diethyldithiocarbamate (DIECA) (Cambra et al., 1994). The same crude extracts were used for both serological and molecular assays. Serological detection and typing of PPV-M isolates. Detection and further characterization of PPV isolates from infected cvs Royal Gem, Calante and Gladys were carried out by double antibody sandwich indirect- ELISA (DASI-ELISA) according to Cambra et al. (1994) and EPPO (2004) using 35 monoclonal antibodies (MAbs), including 5B-IVIA for universal detection of PPV (Cambra et al., 1994), AL for specific detection of PPV-M (Boscia et al., 1997), 4DG5 and 4DG11 for specific detection of PPV-D (Cambra et al., 1994), TUV and AC for specific detection of PPV-C (Myrta et al., 2000), EA24 specific for PPV-EA detection (Myrta et al., 1998), and 21 MAbs from different laboratories: EA2, EA4, EA5, EA7, EA8, EA9, EA11, EA12, EA13, EA14, EA15, EA16, EA18, EMA7, EMA88, 4CB1, 4DB12, 4BD7, 3C6,1EB6, 03, 04, 05, 06, 07, 08, 4B7C4 and 4-11 reported by Cambra et al. (2006a). Commercial kits were used for universal PPV detection, and for D and M serotyping according to manufacturer s instructions (AMR Lab and Real/Durviz, Spain). Fig. 1. Diagram of the plots in which Plum pox virus Marcus type (PPV-M) was detected in 2002, and had spread over the three previous years. The most probable means of transmission among different orchards are shown next to arrows. The percent of infection in each block is shown in brackets. The PPV-M isolates identified are shown in bold.

4 622 PPV-M in Spain Journal of Plant Pathology (2010), 92 (3), Molecular detection and typing of PPV-M isolates. Total RNA from plant material was extracted using RNeasy Plant Mini Kit (Qiagen, USA), following the manufacturers instructions. RT-PCRs were performed using P1/P2 primers for universal PPV detection (Wetzel et al., 1992) and P1/PD or P1/PM primers for specific detection of D or M isolates, respectively (Olmos et al., 1997). Hybridizations of the P1/P2-amplified-RT- PCR products were done using D and M-specific probes (Olmos et al., 2002). A 607 bp fragment corresponding to the 3 end of the RNA replicase (NIb) gene and the 5 end of the coat protein (CP) gene, the most variable region of the potyvirus genome (Aleman-Verdaguer et al., 1997), was amplified from the 19 selected PPV-M isolates by RT- PCR using primers 36 (5 -GAGGCAATTTGTGCWT- CAATGG-3 ) and 172 (5 -TGCAGGACTGTAATGT- GCCAA-3 ) (Capote and Cambra, 2005), and directly sequenced. The sequences obtained were compared with the same region of representative of PPV types Fig. 2. Neighbour Joining phylogenetic tree (NJ method of Tamura and Nei) of the 3 NIb-5 CP region of representative PPV type isolates and Spanish PPV-M isolates (in bold). The significance of the nodes in bootstrap analysis with 1,000 replicates is shown. B, isolate SK-68 (Accession No ); E, isolate PS (Accession No ); A, isolate CGG-M7 (Accession No. AY450597). from GenBank: PPV-D (isolate PENN2, accession No. AF401296), PPV-M (isolate SK 68, accession No. M92280; isolate PS, accession No. AJ243957; isolate CGG-M7, accession No. AY450597), PPV-Rec (isolate BOR3, accession No. AY028309), PPV-EA (accession No. X56258), PPV-W (isolate W, accession No. AY912055), and PPV-C (isolate PPV-SoC, accession No. X97398; isolate PPV-SwC 21, accession No. Y09851). Nucleotide sequences were aligned for Clustal-X analysis (Thompson et al., 1997) using the Align X program from the Vector NTi package. Phylogenetic tree was built using the neighbour joining method (NJ) (Saitou and Nei, 1987). Genetic analyses were done using the kimura-2 parameter algorithm from the MEGA 2.1 program. Aphid monitoring. Aphid species were monitored on ten adult and ten young peach trees in an orchard in the vicinity of plot No.1 (Caspe) by the sticky shoot method (Avinent et al., 1993; Cambra et al., 2000) in June Selected shoots (one shoot analysed per tree) were sprayed with glue (Souverode aerosol, Scotts, France) and collected after 10 days. Winged adult aphid species caught on the shoots were detached with turpentine, washed in water with soap, preserved in 70% alcohol, and counted and identified under a binocular microscope. Experimental transmission of PPV-M. Experimental transmission assays of PPV-M (isolate 22M) were done using Myzus persicae. Donor plants consisted of infected GF305 peach seedlings and young trees of cv. Gladys. Receptor plants were healthy GF305 seedlings and 15- day developed buds of cvs Calante, Mercil and Royal Glory grafted onto GF677 rootstock. After a 1 h pre-acquisition starving period, groups of 25 to 30 apterae young-adult aphids were released on the upper side of an infected GF305 or cv. Gladys detached leaf for virus acquisition. After a 10 min acquisition access period, aphids were transferred in groups of five onto GF305 seedlings and 15-day-old cvs Calante, Mercil and Royal Glory tree leaves for at least a 2 h inoculation period. Peach plants were finally sprayed with insecticide and transferred to an aphid-free quarantine greenhouse, where they were checked regularly for the appearance of PPV-M symptoms for six to eight weeks. Fifteen replicates of each donor/receptor test were used (2 donors x 4 receptors x 15 replicates = 120 tested plants). A 15-rack tray of GF305 seedlings was used as an uninoculated control in each transmission experiment. Leaf samples from all receptor plants were checked for PPV-M by DASI-ELISA using the universal 5B-IVIA and the M- specific AL MAbs eight weeks after inoculation. The transmission rates (number of infected plants divided by number of tested plants) were obtained and compared for each donor and receptor plant.

5 Journal of Plant Pathology (2010), 92 (3), Capote et al. 623 Spatial spread analysis of PPV-M. Spatial spread of PPV-M was studied in the isolated Maella plot (plot No. 2; Fig. 1) that consisted of three blocks: block 1 at the western side of the plot planted with cv. Royal Gem, and blocks 2 and 3 at the east side of the plot, planted with cvs Calante and Gladys, respectively. The location of infected trees was established by means of a GPS device (Fig. 3). The spread of infection was modelled by a spatial point process (Diggle, 2003; Møller, 2003); the plot was considered as a continuous region where an infected tree can appear anywhere. The intensity of the process was modelled by a mixture of two bivariate gaussian distributions as kernel function. Both processes generate two types of spread, over long and short distances. The long distance represents primary infections incurred directly from block 1 (Fig. 1). The short distance represents secondary infections from contiguous infected trees. Bayesian inference was performed in order to obtain posterior distributions of the parameters of the model. Markov Chain Monte Carlo methods (Chen et al., 2000) were employed to simulate from posterior distributions and to obtain characteristics of the two components of the contagion process (Møller and Waagepetersen, 2004). The statistical analysis was performed with R statistical programming language (R Development Core Team, 2000). RESULTS Detection and serological characterization of PPV- M isolates. PPV was detected in approximately 20% of the tested trees of cv. Royal Gem which was suspected to be the origin of PPV-M introduction from an unidentified country. The virus was spread naturally by aphids to other peach cultivars such as Calante and Gladys (incidence of 4.9% and 7.3%, respectively) grown near the originally infected plot and was also transmitted by grafting to a more distant cv. Gladys plot (incidence 1.2%) (Fig. 1). Infected plants of this cultivar showed typical PPV-M symptoms on flower petals but not on the leaves, while cv. Calante trees showed leaf vein clearing symptoms as observed in cv. Royal Gem trees. PPV-M isolates infecting peach cultivars in different plots were serologically classified as the M 1 subcluster described by Myrta et al. (2001). Three different serogroups were identified regardless of the cultivar they were isolated from. Serogroup 1 (including 1M, 3M, 4M, 5M, 6M, 12M, 13M, 14M, and 22M isolates) and serogroup 2 (including 8M, 10M, 11M, 18M, 19M, 20M, 23M and 24M isolates) were recognized by EA8, EA9 and 4-11 MAbs. EMA7 MAb reacted with serogroups 1 and 3 (including 2M and 9M isolates). MAbs 5B-IVIA, AL, EA5, EA12, 4DB12, 3C6, 05 and 4B7C4 reacted against all PPV-M isolates tested, whereas the rest of the MAbs used did not recognize any of the PPV-M isolates tested. Molecular analysis by RT- PCR and hybridization indicated the presence of PPV- M and the absence of PPV-D (data not shown). Sequence diversity analysis of PPV-M isolates. Sequence diversity analysis of the 19 PPV-M isolates showed 7 polymorphic sites in the 607 bp-long region analysed indicating seven different haplotypes in the population. The PPV haplotype present in cv. Royal Gem (haplotype H1) was also detected in cvs Calante and Gladys. In cv. Calante three haplotypes including Table 1. Distribution of the seven PPV haplotypes found in different peach cultivars infected with Plum pox virus Marcus type. The haplotype infecting Prunus persica cv. Royal Gem is underlined. Haplotypes infecting P. persica cv. Calante are in normal script. Haplotypes infecting P. persica cv. Gladys are in bold. No. of samples in each population Haplotype Royal Gem Calante Gladys Total Isolate number H M, 1M, 14M H M, 4M, 10M, 11M, 12M, 13M H M, 6M H M, 9M H M H M H M, 20M, 23M, 24M TOTAL

6 624 PPV-M in Spain Journal of Plant Pathology (2010), 92 (3), Table 2. Experimental transmission of PPV-M from GF305 peach seedlings and Prunus persica cv. Gladys to GF305 peach seedlings and P. persica cv. Mercil, P. persica cv. Calante and P. persica cv. Royal Glory. Transmission rates are expressed as a percent of PPV infected plants divided by total tested plants. Donor plant Receptor plant GF305 peach seedling P. persica cv. Gladys TOTAL Infected plants / tested plants Transmission rate (%) Infected plants / tested plants Transmission rate (%) Infected plants / tested plants Transmission rate (%) GF305 10/ / / Mercil 7/ / / Calante 4/ / / Royal Glory 4/ / / TOTAL 25/ / / H1and H2 (the most frequent haplotypes) and H3 were detected, whereas cv. Gladys trees carried five haplotypes (H1, H4, H5, H6 and H7) (Table 1). Only H1 was common to isolates from the three peach cultivars. The other haplotypes were unique to cv. Calante or cv. Gladys. The phylogenetic analysis of the nucleotide sequences obtained from different PPV-M isolates is shown in Fig. 2. The isolates grouped together with PPV-M isolate SK68 from eastern Europe (Hungary) sharing 98.3% to 99.7% sequence identity. Aphid species monitored. The main aphid species that landed on the leaves of infected peach trees in June 2003 were Aphis spiraecola (62.7%), A. gossypii (17.5%), M. persicae (9.1%), Hyalopterus pruni (8.5%), Brachycaudus helichrysi (1.1%) and A. craccivora (1.1%). Experimental transmission of PPV-M to different peach cultivars by M. persicae. Experimental transmission of the 22 PPV-M isolates from donor plants (GF305 peach seedling and cv. Gladys young trees) to receptor plants (GF305 seedlings and cvs Calante, Mercil and Royal Glory) by M. persicae led to high rates of transmission when GF305 was the donor (66.7% to GF305; and 46.7%, 26.7% and 26.7% to cvs Mercil, Calante and Royal Glory, respectively) and also if it was the receptor plant (overall transmission rate 43.3%). When the donor plant was cv. Gladys, transmission efficiency to other peach cultivars was lower (20.0% to GF305; and 13.3%, 26.7% and 0.0% to cvs Mercil, Calante and Royal Glory) (Table 2). Spatial spread of PPV-M. Fig. 3 shows spread of PPV-M from infected P. persica cv. Royal Gem (block 1 in plot No. 2) to cvs Calante and Gladys grown in the neighbouring blocks (blocks 2 and 3 in plot No. 2, respectively) by aphids The inference on the spatial spread model showed that about 43% of the trees were infected directly from block 1 (primary infection). Long-distance infection occurred within a radius of about 150 meters, with an average distance of 65 meters. Moreover the probability of infection decreased with the distance from the source, in the direction of the prevailing wind. In a secondary process, 57% of the trees were infected from other trees in the blocks. Short-distance transmission occurred as far as 14 meters away, in the direction of the rows of trees, showing an average distance of 6 meters. DISCUSSION A focus of PPV-M infection was detected and eradicated in peach orchards in north-east Spain. The quick action of the Plant Protection Services of the Aragón Govern was crucial for this early detection and successful eradication. Twenty ha of infected peaches were removed in July-August 2002 in the area, following a mandatory eradication program according to the Spanish Decree: Real Decreto 1190/98, BOE 141 de 13 Junio de 1998, and the European Union Directive 2000/29/UE, May 8 th, Surveys performed annually from early spring 2003 to date (8 years) have confirmed the complete eradication of PPV-M in this isolated area in which no other Prunus species are grown. We should note the agronomic and economic impacts that introduction and establishment of PPV-M could have caused in a Prunus growing area: PPV-M type is known to be more aggressive for all Prunus species than other types, spreading more readily by different aphid species, and can naturally be transmitted to and among peaches, apricots and plums. In addition, it has been shown that

7 Journal of Plant Pathology (2010), 92 (3), Capote et al. 625 Fig. 3. Aerial photograph of the Maella plot (Plot No.2) encompassing Prunus persica cv. Royal Gem (block 1) and P. persica cvs. Calante and Gladys (blocks 2 and 3, respectively) infected via aphids. Dark squares indicate individual PPV- infected trees. PPV-D, currently present in Spain, cannot cross-protect some Prunus trees against PPV-M infection. Thus PPV- M may replace a previous PPV-D population or, in other species, coexist with PPV-D in the same tree (Capote et al., 2006b). The initially detected PPV-M isolate was graft-transmitted from latently infected P. persica cv. Royal Gem material illegally introduced into Spain to four distant plots, and probably transmitted from this cultivar to cvs Calante and Gladys grown in the same plot by the predominant aphids (A. spiraecola, A. gossypii and M. persicae) feeding on Prunus in the area. All these aphid species are PPV vectors and could contribute to PPV-M spread within the same plot. During virus transmission, the isolate initially introduced (22M) segregated into three serogroups and seven different haplotypes randomly distributed among the affected peach cultivars. A field virus isolate may contain multiple genome variants (haplotypes), some of which can be separated by bottleneck processes such as aphid or graft transmission to different host species (Ali et al., 2006). Some of these variants can also differ from the source isolate by their serological reactivity. Population diversification is one of the strategies viruses use to evolve and adapt to new colonising hosts and ecological areas. PPV-M, the initial source of infection, could also be experimentally transmitted among peaches: from GF305 peach seedlings or cv. Gladys trees to GF305 and cvs Calante, Mercil and Royal Glory. The transmission was more efficient when using seedlings as donor or receptor plants than when using peach cultivars. These results indicate that transmission depends not only on the PPV type but also on the donor and receptor plant, and support the fact that peach seedlings used as rootstocks are more susceptible to PPV-M infection than trees of peach cultivars. Results on transmission rate of the PPV-M isolate to peach rootstocks and cultivars by M. persicae can be applied to any PPV-M isolate and to other aphid species vectors present in the area. Previous results on PPV transmissibility by A. spiraecola, A. gossypii and M. persicae showed that all these species transmitted two different isolates of the D and M types (Avinent et al., 1994). M. persicae was reported as the most efficient vector in experimental transmission of PPV-D isolates in North America (Gildow et al., 2004). However, the major occurrence of A. spiraecola in the area surveyed indicates this aphid species could be the most likely vector associated with PPV spread, as it has already been reported in other Mediterranean areas (Labonne et al.,

8 626 PPV-M in Spain Journal of Plant Pathology (2010), 92 (3), ; Cambra et al., 2006b). Analysis of the spatial spread of PPV in different countries shows differences in the behaviour of PPVviruliferous aphids. Analysis of peach orchards in Pennsylvania (USA) suggested a lack of movement of PPVviruliferous aphids to immediately adjacent trees and their preferential movement to trees located several tree spaces away (Gottwald, 2006). These results are consistent with those from apricot and peach orchards in Spain (Gottwald et al., 1995). These findings, however, do not seem consistent with those from PPV-M in France (Dallot et al., 2003; Labonne and Dallot, 2006) in which clustering of diseased trees were found, ellipsoidal in shape and with different intensity of aggregation, mainly depending on the incidence of the disease. Similarly, Varveri (2006) in Greece found clusters of PPV-M infected apricots around the initial infected ones. In the current investigation of the Spanish peach orchard, two types of movement are integrated in a compound model distinguishing long-range and shortrange spread. Long-range movement represents aerial dissemination that can occur from a few to more than one hundred meters, with variable direction depending on the wind. Meanwhile, short-range movement suggests secondary spread due to frequent aphid traffic to neighbouring trees with interlaced or more closed branches. The PPV-M isolate detected in Spain in 2002 is genetically close to a PPV-M isolate from Hungary (SK68), but its introduction could have come from any Mediterranean country during legal and illegal exchange of plant material with Spain. Probably, there was only one introduction but this was enough to spread the disease to different plots and distant blocks in a relatively short time. The existence of reliable and validated diagnostics for PPV (Capote et al., 2009) based on annual inspections and a combination of serological and molecular tests was crucial for the early detection and successful eradication of PPV-M infection from Spain. ACKNOWLEDGEMENTS This work was supported by grants from the Spanish Ministry of Science and Innovation (AGL , INIA-RTA03-099, INIA RTA and AGL ). The authors would like to thank Dr. María Cambra-López for critically reading the manuscript. They would also like to thank all personnel from the Plant Protection Services of Diputación General de Aragón for field surveys, Dr. Alfonso Hermoso de Mendoza (IVIA) for identification of aphid species and for providing individuals of Myzus persicae and Agromillora Iberia for providing plants for transmission experiments. REFERENCES Adamolle C., Boeglin M., Labonne G., Candresse T., Quiot J.B., A necrogenic strain of Plum pox potyvirus causing decline in some peach cultivars. 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