Journal of Plant Pathology (2013), 95 (3), 579-586 Caglayan et al. 579

EVALUATION OF THE SUSCEPTIBILITY OF DIFFERENT PRUNUS ROOTSTOCKS TO NATURAL INFECTION OF PLUM POX VIRUS-T

K. Caglayan1, C.U. Serce1*, M. Gazel1, K. Kaya1, F.C. Cengiz1, E. Vidal2 and M. Cambra2

1Mustafa Kemal University, Faculty of Agriculture, Plant Protection Department, 31034 Hatay, Turkey2Laboratorio de Virología e Inmunología, Centro de Protección Vegetal Biotecnología, Instituto Valenciano

de Investigaciones Agrarias (IVIA), Ctra. Moncada-Náquera km 5, 46113 Moncada, Valencia*Present address: Nigde University, Faculty of Agricultural Science and Technologies, Department of

Crop Production and Technologies 51240 Nigde, Turkey

SUMMARY

Plum pox virus (PPV) has been observed in Turkey since 1968, but was not widespread except in apricot and plum trees in home gardens and ornamental parks in restricted areas. Susceptibility of six different Prunus rootstocks to strain PPV-T was assessed under natural inoculum pressure in the Izmir-Aegean region during 2010-2011. Aphid populations were monitored from the first week of April to the middle of June by the sticky-plant method one year after the rootstock plantation was established. Aphids collected from different rootstocks were tested individually by squash real-time RT-PCR and all rootstocks were regularly tested by DASI-ELISA. The largest aphid populations were observed at the end of May and the most abundant aphid species as averages over the two years were Myzus persicae (20.15%), Hyalop-terus pruni (18.64%), Aphis craccivora (9.04%) and Aphis gossypii (8.36%). In 2011, the highest percentage of virulif-erous aphids was found in M. persicae (34.78%), followed by H. pruni (32.50%), Macrosiphum euphorbiae (25.00%), A. gossypii (23.80%), A. spiraecola (12.50%) and A. crac-civora (10.00%). Of the six Prunus rootstocks tested, only Nemaguard and Myrobalan 29C were infected by PPV-T, infection rate in 2010 being 6.0% (Nemaguard) and 4.0% (Myrobalan 29C). The infection rate increased to 16.0% for Nemaguard and 14.0% for Myrobalan 29C in 2011. However, the other rootstocks, Prunus marianna GF8.1, Docera6, GF677 and Garnem tested negative for PPV-T throughout 2011. PPV isolates obtained from naturally in-fected apricot trees (inoculum source) and from infected rootstocks in the experimental plot were characterized as PPV-T and had more than 99.5% nucleotide sequence identity.

Key words: Sharka disease, PPV Turkey strain, viru-liferous aphids, rootstock susceptibility, epidemiology, detection.

INTRODUCTION

In Turkey, Plum pox virus (PPV) was first recorded in the Edirne (Thrace) (Sahtiyancı, 1969) and subsequently in Ankara (Central Anatolia) (Kurçman, 1973) provinces. More recent surveys revealed new PPV outbreaks in the Aegean and Mediterranean regions (Gümüş et al., 2007; Gazel et al., 2010).

There is a wide genetic variability within PPV (Can-dresse and Cambra, 2006; Barba et al., 2011). Several strains or subgroups are recognized their classification being based on biology, serology and molecular proper-ties. The two most common strains are PPV-D (Dideron) and PPV-M (Marcus). Additionally, five other strains have been characterized, i.e.. PPV-Rec (Recombinant), PPV-EA (El Amar), PPV-W (Winona), PPV-C (Cherry) (Glasa et al., 2004b; James and Varga, 2005; Candresse and Cambra, 2006) and more recently, PPV-T (Turkey) (Ulubas Serce et al., 2009). Numerous PPV isolates were described as hav-ing different biological and epidemiological characteristics, such as those related to aggressiveness (Quiot et al., 1995), aphid transmissibility (Deborré et al., 1995) and symptom-atology (Jarausch et al., 2004; Palmisano et al., 2010). Se-rological characterization studies of Turkish PPV isolates using universal and strain-specific monoclonal antibodies (MAbs) showed that fifteen PPV-positive apricot samples from different regions of Turkey reacted with MAb4DG5 and MAbAL which made their serotyping difficult (Myrta et al., 1998).

A similarly ambiguous result (presence of both M- and D- specific epitopes in the same isolate) was reported by Candresse et al. (1998) with a Turkish PPV isolate. Partial sequencing of this isolate (Ab-Tk) revealed that it is a novel type of recombinant, characterized by a recombi-nation breakpoint in the HC-Pro gene, around position 1,566 of the genome (Glasa and Candresse, 2005). Fur-ther studies showed that Ab-Tk and several other Turk-ish PPV isolates should be considered as members of a new PPV strain for which the name PPV-T was proposed (Ulubas Serce et al., 2009). Three PPV strains (PPV-M, PPV-Rec and PPV-T) have been reported in Turkey each with a distinct distribution (Çağlayan et al., 2012). PPV-T,

Edizioni ETS Pisa, 2013

Corresponding author: K. Caglayan Fax: +90.326.2455832 E-mail: kcaglayano@yahoo.com

580 Susceptibility to infection of Plum pox virus-T Journal of Plant Pathology (2013), 95 (3), 579-586

originally detected and characterized in Turkey, prevails in central Anatolia (Ankara) and the Aegean regions (Izmir) where PPV has been endemic for years. PPV-M was reported primarily as new outbreaks in the eastern Mediterranean region and PPV-Rec was found only in Isparta, in the western Mediterranean region (Candresse et al., 2007). Although many studies are available on the epidemiological aspect of different PPV-M and D isolates from different countries, no such data are available for PPV-T. Therefore, in this study, the natural transmission status and rootstock reactions to PPV-T in the Aegean region of Turkey were investigated.

MATERIALS AND METHODS

Plant material and experimental nursery plots. An experimental nursery plot was established in a naturally PPV-infected apricot orchard in spring 2009 at İzmir (Ae-gean region). Spread of PPV and incidence of major aphid species were followed for two consecutive years, 2010 and 2011.

The susceptibility to natural PPV-T infection was evaluated of six different in vitro-propagated one-year-old PPV-free certified Prunus rootstocks (Agromillora Iberia, Spain), i.e. Myrobolan 29C (P. cerasifera), Garnem (P. dulcis × [(P. persica × Prunus davidiana) × P. persica]), P. marianna GF 8.1 (P. cerasifera × Prunus munsoniana), Nemaguard (P. persica × P. davidiana), Docera 6 (P. do-mestica x P. cerasifera) and GF677 (Prunus amygdalus × P. persica). Fifty plants for each rootstock were planted adja-cent to a PPV-infected apricot orchard but some of them died in the first year (Table 1). The plants were planted in two rows parallel to the inoculum source by using longi-tudinal design of 5 groups or replicates of 10 plants into each group (20 cm apart). The groups were randomly dis-tributed. The experimental field was managed according

to standard nursery practices without any phytosanitary treatment.

Monitoring of PPV spread. Serological tests were car-ried out one year after rootstocks planting, i.e. April of 2010 and 2011. Plants were regularly checked for PPV symptoms and individually sampled by collecting four fully expanded leaves from different parts of the canopy. Serological assay for PPV detection was by DAS-ELISA based on the 5B-IVIA (Cambra et al., 1994) monoclonal antibody kit (Plant Print Diagnostics, Spain), following the EPPO (2004) protocol for PPV detection.

Molecular characterization of PPV isolates. Samples from nine PPV-positive rootstocks (six Myrobalan 29C, three Nemaguard) in the experimental plot and six apri-cot samples randomly selected in the adjacent infected orchard were analyzed for PPV strain identification in the second year of evaluation. Leaves exhibiting PPV symp-toms were collected and RNA was extracted using a LiCl method (Spiegel et al., 1996). Four μl of RNA was used for cDNA synthesis using a reverse transcription kit (MBI Fermentas, Finland). PCR was performed using PPV uni-versal primer pairs that amplify 745 bp fragments: NCU-niFor 5’-GAGGCAATTTGTGCTTCAATGG-3’ and NCUniRev 5’-CGCTTAACTCCTTCATACCAAG-3’ (Predajna et al., 2012). Cycling parameters were: 95°C for 3 min, followed by 35 cycles of 94°C for 30 sec, 53°C for 1 min and 72°C for 1 min, followed by 72°C for 10 min. PCR amplicons were sequenced directly and analyzed using the basic local alignment search tool (BLAST). Multiple alignments of the nucleotide sequences of these isolates and reference isolates for each PPV strain ob-tained from GenBank were performed using BioEdit (Hall, 1999). Cluster analysis was done with the Mega 5.0 program (Tamura et al., 2011) using the neighbor-joining method with nucleotide identity distances. Bootstrap

Fig. 1. Vein clearing and chlorosis on the leaves of Nemaguard (left) and Myrobalan 29C (right) rootstocks in the experimental plot due to Plum pox virus (T strain) infection.

Journal of Plant Pathology (2013), 95 (3), 579-586 Caglayan et al. 581

analyses with 1000 replicates were performed to estimate the support for inferred phylogenies.

Monitoring of aphid species and detection of viru-liferous aphids. Adult winged aphid populations which were visiting the rootstock plants in the experimental plot were monitored by the sticky-plant method (Avinent et al., 1993; Marroquin et al., 2004) during spring 2010 and 2011. Three sticky shoots from each rootstock species were col-lected each week and new shoots were sprayed for next collections. Collected shoots were processed to identify aphid species and estimate their numbers (Capote et al., 2008). All identified abundant aphid species collected from complete sticky rootstock plants were used to esti-mate the number of PPV-viruliferous aphids visiting the experimental nurseries in 2011. Aphids were squashed in-dividually on nylon membranes using the round bottom of an Eppendorf tube to ensure complete disruption of each aphid. RNA was extracted from the individual squashed aphid specimens using 100 μl buffer [0.1M glycine, 0.05 M NaCl, 1 mM ethylenediaminetetraaceticacid (EDTA)] (Osman and Rowhani, 2006) and analyzed by squash real-time RT-PCR (Olmos et al., 2005).

RESULTS

Evaluation of the susceptibility of different Prunus rootstocks to natural PPV-T infection. Individual root-stocks of the experimental plot established in 2009 were tested by DAS-ELISA in April and September of 2010 and 2011. Infection rate in 2010 was 6.0% and 4.0% for Nemaguard and Myrobalan 29C, respectively. It increased to 16.0% for Nemaguard and 14.0% for Myrobalan 29C in 2011. The other four rootstocks were PPV-negative in these tests (Table 1). The most obvious symptoms on the two infected rootstocks were vein clearing and interveinal chlorosis (Fig. 1). Due to Turkish quarantine regulations, the PPV-infected orchard and all rootstocks were eradi-cated three years after the establishment of the experi-mental plots.

Characterization of PPV isolates. The sequences of PCR amplicons obtained from nine viral isolates recovered from the experimental plot were analyzed, showing 98% identity with the AbTk isolate of PPV-T, 95% identity with the BOR-3 and Niksic6 isolates of PPV-Rec, 94% identity with the N1, SK68 and PS of PPV-M strains (GenBank ac-cession Nos. EU734794, JQ794541, HQ452359, FJ361234, M92280 and AJ243957, respectively). The nucleotide se-quence identity of the nine above PPV isolates and of six additional isolates from the adjacent apricot orchard was greater than 99.5%. In a phylogenetic tree all these isolates

Table 1. Natural incidence of Plum pox virus-T in different Prunus rootstocks determined by DAS-ELISA in an experimental nursery plot in Izmir/ Turkey.

Name of the rootstocksNumber of infected / tested plants Natural infection rate (%)2010 2011 2010 2011

Nemaguard 3/50 8/50 6 16Myrobalan 29C 2/50 7/50 4 14Garnem 0/47 0/47 0 0P. marianna GF8.1 0/48 0/48 0 0GF-677 0/50 0/50 0 0Docera 6 0/40 0/40 0 0

Total plants 5/285 15/285 1.75 10.53

Fig. 2. Phylogenetic analysis of PPV isolates from Myrobolan 29C (TR-331, 332, 334, 336, 337, 338), Nemaguard (TR-329, 330, 333) in experimental plot and six apricot plants in a PPV-infected orchard (TR-157ap, TR-159ap, TR-179ap, TR-192ap, TR-218ap, TR-220ap). Reference sequences were retrieved from GenBank, the first capital letters are accession numbers followed by isolate names. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.

582 Susceptibility to infection of Plum pox virus-T Journal of Plant Pathology (2013), 95 (3), 579-586

clustered in the PPV-T group (Fig. 2). As it can be seen in Fig. 2, the isolate AbTk was slightly different from the other isolates obtained from the experimental nursery and the apricot orchard. Neverthless, the Mab 5B-IVIA reacted with all PPV-T isolates. The reactivity of Mab 5B-IVIA with all PPV strains was investigated in a PPV ring test performed in the frame of a COST-88 concerted ac-tion (Asensio, 1996). Its positive reaction was also proved for strains PPV-EA, PPV-SoC and PPV-W (Wetzel et al., 1991; Nemchinov and Hadidi, 1996; James and Varga, 2005). Mab 5B-IVIA reacts with the single protein mo-tif (DRDVDAG sequence) fully conserved in PPV-D and PPV-M, corresponding to positions 94 to 100 of the PPV

CP (Candresse et al., 2011). CP sequence alignments of PPV-T isolates with other reference isolates showed that this protein motif was also conserved in all sequenced iso-lates (Fig. 3).

Estimation of aphid species and number of virulifer-

ous aphids present in the experimental plot. The aphid species landing on the rootstocks in the experimental plot were identified during spring (from April to June) of 2010 and 2011. A total of 73 and 364 individual aphids were captured in 2010 and 2011, respectively. M. persicae and H. pruni were the most common aphids in both years, fol-lowed by A. craccivora and A. gossypii (Table 2).

In both years the total aphid number reached a peak at the end of May (26th of May in 2010, 31th of May in 2011). The most preferred rootstock by aphids was Nemaguard, followed by P. marianna GF 8.1, Garnem and Myrobalan 29C in both years (Fig. 4).

Among all aphid species collected in 2011, the high-est percentage of viruliferous individuals was detected in Myzus persicae (34.78%) by squash real-time RT-PCR, followed by H. pruni (32.50%), Macrosiphum euphorbiae (25.00%), A. gossypii (23.80%), A. spiraecola (12.50%) and A. craccivora (10.00%) (Table 3). An average of 24.19% (30 out of 124 analyzed individuals) of the aphid species that visited the experimental plot was viruliferous in the period studied.

The highest percentage of viruliferous aphids was found on the rootstocks they preferred the most, i.e. My-robalan 29C, P. marianna GF 8.1, Garnem and Nema-guard (Fig. 3, Table 3). The two most common aphid spe-cies, M. persicae and H. pruni, had the highest percentage of viruliferous individuals.

DISCUSSION

The PPV isolates found in the apricot orchard (inocu-lum source) and in the rootstocks of the experimental plot were of the PPV-T type, which is a common strain in old

Fig. 3. Local alignment of Plum pox virus coat protein amino acid sequences. The isolates beginning with TR- are PPV-T isolates sequenced in this study, others were retrieved from GenBank. The conserved protein motif is shaded.

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Fig. 4. Occurrence of PPV vector aphid species in different Prunus rootstocks. Number of alate individuals caught in spring of 2010 and 2011 (April–June) by the sticky plant method.

Journal of Plant Pathology (2013), 95 (3), 579-586 Caglayan et al. 583

stone fruit trees in Turkey. It is known that different PPV strains differ in terms of epidemiology and aggressiveness, PPV-M is more severe and more efficiently vectored than PPV-D (Wang et al., 2006), thus it can spread very rap-idly in peach orchards (Dallot et al., 1998). PPV-D isolates spread naturally in apricot and plum orchards but much more slowly from these hosts to peach trees (Quiot et al., 1995; Cambra et al., 2008). Limited information is available on the epidemic behavior and aggressiveness of PPV-Rec isolates and comparable data are lacking for PPV-T. The results of vector transmission studies (Glasa et al., 2004a) confirm that all the recombinant PPV isolates are aphid-transmitted, but transmission occurrs at different rates.

In this study, under severe PPV inoculum pressure the assayed rootstocks exhibited differences in their suscepti-bility to PPV-T infection by aphids (Table 1). Nemaguard and Myrobolan 29C proved highly susceptible, with in-fection rates of 16.0% and 14.0% in 2011, respectively, in agreement with results from similar studies (Vidal et al., 2010). The susceptibility observed in Nemaguard, an interspecific cross between peach and P. davidiana, agrees with the results reported by Pascal et al. (2002) and Ru-bio et al. (2003), indicating a certain level of susceptibility to a PPV-M and -D isolates, respectively. Although it is known that P. cerasifera is susceptible to PPV (James and Thompson, 2006), different clones have been reported as

Table 2. Occurrence of PPV-vector aphid species in the experimental plot in Izmir. Number of individuals caught and their per-centage among total aphid numbers in the seasons of 2010 and 2011 (April-June).

Aphid species2010 2011

Number of identified aphid Percentage (%) Number of

identified aphid Percentage (%)

Myzus persicae 20 27.39 47 12.91

Hyalopterus pruni 17 23.28 51 14.01Aphis craccivora 7 9.58 31 8.51Aphis gossypii 6 8.21 31 8.51Acyrthosiphon pisum 6 8.21 0 0Anoecia corni 5 6.84 0 0Aphis fabae 0 0 23 6.31Macrosiphum euphorbiae 4 5.47 21 5.76A. spiraecola 0 0 21 5.76Hyperomyzus lactucae 4 5.47 0 0Metopolophium dirhodum 3 4.10 0 0Capitophorus elaeagni 1 1.36 0 0Brachycaudus helichrysi 0 0 4 1.09Others 0 0 135 37.08

Total 73 364

Table 3. The number of viruliferous aphids/total number of aphids of that species collected from different Prunus rootstocks present in the experimental plot in 2011.

Garnem Myrobalan 29C Nemaguard GF677 P. marianna GF 8.1 Total

Myzus persicae 1/2 2/3 2/9 0/4 3/5 8/23 34.78%

Hyalopterus pruni 3/8 6/15 2/8 0/5 2/4 13/40 32.50%

Macrosiphum euphorbiae 0/0 0/0 1/4 0/0 0/0 1/4 25.00%

Aphis gossypii 2/6 2/4 1/7 0/3 0/1 5/21 23.80%

A. spiraecola 0/1 0/3 0/2 0/0 1/2 1/8 12.50%

A. craccivora 0/1 0/0 0/0 1/10 1/9 2/20 10.00%

A. fabae 0/2 0/0 0/1 0/4 0/1 0/8 0.00

Total 6/20 30.00%

10/25 40.00%

6/31 19.35%

1/26 3.84%

7/22 31.81%

30/124 24.19%

584 Susceptibility to infection of Plum pox virus-T Journal of Plant Pathology (2013), 95 (3), 579-586

resistant to PPV-D (Minoiu et al., 1998; Rubio et al., 2005). In our experiment Myrobalan 29C was found as sensitive as Nemaguard to PPV-T but the symptoms on this root-stock were always much milder than on Nemaguard (Fig. 1). Although some studies indicated the high susceptibility of different clones of “Marianna” plum to PPV-M (Dosba et al., 1994), PPV-C (Bodin et al., 2003) and PPV-D (Rubio et al., 2005), no infected Marianna 8.1 plants were detected in this study, either because of low inoculum pressure or presence of different PPV strains.

PPV isolates from nursery and adjacent orchard that were sequenced in this study were not 100% identical. The rapidly evolving ability of plant RNA viruses has a significant role in virus epidemiology, as it provides a se-lection of variants with increased pathogenicity (Moury et al., 2006). Jiridi et al. (2006) demonstrated that 15 years after inoculation of a peach seedling with PPV-M, the vi-rus could evolve into several distinct populations follow-ing the systemic invasion of the host. In a plum tree triply infected with PPV-M, PPV-D and PPV-Rec, after seven years only the more competitive isolate PPV-M was still detectable whereas the two other isolates (PPV-Rec and PPV-D) had been displaced (Predejna et al., 2012). On the other hand, nonpersistent transmission by aphids may have an important effect on the dynamics and evolution of virus populations, for example a major cause of virus strain dif-ferentiation could be genetic drift as a result of population bottlenecks during aphid transmission (Ali et al., 2006).

For epidemiological purposes, it is important to take into consideration the aphid vector species as well as the number of viruliferous aphids visiting the nursery plots. The most important PPV vectors reported from several countries are Brachycaudus cardui, B. helichrysi, M. persi-cae and Phorodon humuli (Sullivan, 2011) and A. spirae-cola (Cambra et al., 2006). Natural virus spread is low in summer but high in spring and autumn. Spring flights of B. helichrysi, M. persicae, and P. humuli are the most important for virus spread within and between orchards (Sullivan, 2011). In our conditions, the total aphid popula-tion reached a peak at the end of May in both years (26th of May in 2010, 31th of May in 2011).

Tables 2 and 3 show that the most abundant aphid species were M. persicae and Hyalopterus pruni, and that these two species had the highest percentage of virulifer-ous aphids. Although M. persicae is a well known PPV vector in many stone fruit-growing countries (Manachini et al., 2007), the role of H. pruni in the natural spread of PPV needs a better substantiated evidence (Gaborjanyi and Basky, 1995). Of the aphids that visited the experi-mental plot in Izmir, 24.19% (30 out of 124 analyzed in-dividuals) were viruliferous in the period studied, which explains the high incidence and rapid spread of PPV in the experimental plot in the studied area and the high risk of PPV dissemination in stone fruit orchards. Most of the aphid species that were previously reported as PPV vectors were also able to transmit PPV-T but more work needs to

be done to determine their role in the epidemiology of PPV-T in Turkey.

ACKNOWLEDGEMENTS

The research leading to these results received fund-ing from the European Community’s Seven Framework Programme (FP7/2007-2013) under Grant Agreement n°204429, SharCo project. The authors would like to thank R. R. Martin from USDA-ARS Horticulture Crops Research Unit for his kind help for English editing.

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Received March 15, 2013 Accepted May 16, 2013