Incidence of Plum Pox Virus and Its Strains in Kosovo

020_JPP718SC(Boscia)_725 16-11-2011 9:32 Pagina 725

Journal of Plant Pathology (2011), 93 (3), 725-728 Edizioni ETS Pisa, 2011 725

SHORT COMMUNICATION INCIDENCE OF PLUM POX VIRUS AND ITS STRAINS IN KOSOVO

A. Myrta1, F. Palmisano2, B. Pulaj3, L.R. Susuri4 and D. Boscia2

1 Certis Europe, Via Guaragna 3, 21047 Saronno (VA), Italy 2 Istituto di Virologia Vegetale del CNR, UOS Bari, Via Amendola 165/A, 70126 Bari, Italy 3 Intercooperation Horticultural Promotion in Kosovo, Mithat Frashëri Street. 6, 10000 Prishtina, Kosovo 4 Kosova Academy of Sciences and Arts, Agim Ramadani, 10000 Prishtina, Kosovo

SUMMARY fruit trees are currently infected and susceptible culti- vars can suffer heavy yield losses (Cambra et al., 2006). Sharka, caused by Plum pox virus (PPV), is the most Plum pox virus (PPV), the causal agent of the disease, destructive viral disease of plum, apricot and peach. Al- is currently grouped into seven main strains character- though PPV is widespread in all fruit-growing areas of ized by different serological, molecular and epidemio- eastern European countries, and causes serious yield logical properties. Whereas strains Marcus (PPV-M), losses, little is known about its occurrence and distribu- Dideron (PPV-D), El Amar (PPV-EA), and Cherry tion in Kosovo. Therefore, a survey was conducted in (PPV-C) (Pasquini and Barba, 1997; Candresse et al., orchards and nurseries at 18 sites located in seven dif- 1998) have been known for a number of years, Winona ferent districts to verify the presence of PPV and to de- (PPV-W) from Canada, Turkey (PPV-T) from Turkey, termine the virus strains occurring in the country by and recombinants between strains D and M (PPV-Rec) serological and molecular tools (ELISA and PCR). Field (Glasa et al., 2004; Serçe et al., 2009) have been de- observations and laboratory analysis disclosed a very scribed in the past decade. high incidence of PPV in nurseries and orchards at all Since no information is available on the occurrence locations. Characterization of 26 isolates representative and distribution of Sharka in Kosovo, a study was un- of all surveyed sites and Prunus species, revealed a pre- dertaken for assessing disease presence and characteriz- dominance of PPV-Rec (23 out of 26), and a low inci- ing local PPV strains. Surveys were conducted at 18 lo- dence of PPV-M and PPV-D. cations in seven districts (Fig. 1 and Table 1). Sites were visited in late autumn 2009 for collection of dormant Key words: Sharka, survey, diagnosis, monoclonal an- cuttings, and spring 2010 for symptom observation and tibodies, PCR. collection of leaves. All autumn samples were chip-bud grafted onto GF305 seedlings maintained in a green- house under controlled conditions and PPV testing was Fruit industry has traditionally been an important done using young leaves from the shoots that developed sector of Kosovo’s agriculture. European plum (Prunus after cutting back inoculated plants above the graft domestica), by far the leading stone fruit crop, is grown union. Autumn surveys involved orchards generally in most districts of this country in specialized orchards, more than 20-year-old, made up of non tended and un- a number of small private gardens, and as road-border- productive trees. By contrast, spring surveys were con- ing trees. The plum industry is based on imported culti- ducted in orchards generally not older than 5 years, and vars, the most important of which are Stanley, Presi- included also some nurseries. Samples consisted of dent, and the Cacanska group. leaves collected directly from the trees. Clear-cut symp- Since its first record by Atanassov (1932) on cv. Kjus- toms were observed in spring in orchards and nurseries, tendil in Bulgaria, plum pox disease (Sharka) has pro- allowing a visual evaluation of disease incidence. All gressively spread to most of the European continent and samples (Table 1) were tested for PPV by DASI-ELISA to some countries in North Africa, Near East, Asia and using the universal monoclonal antibody 5B-IVIA North and South America (Anonymous, 2006; Virsˇcek (Cambra et al., 1994) and by real-time RT-PCR accord- Marn et al., 2007; Maejima et al., 2010). Sharka is the ing to Olmos et al. (2005). most important virus disease of stone fruits in Europe, Virus strains were identified by serological and mo- especially in the western territories, causing reduced lecular tools. The following strain-specific monoclonal fruit quality and premature fruit drop. Millions of stone antibodies (Mabs) were used for DASI-ELISA: Mab4DG5 (PPV-D specific) (Cambra et al., 1994), MabAL (PPV-M specific) (Boscia et al., 1997), Corresponding author: D. Boscia Fax: +39.080.5443608 MabEA24 (PPV-EA specific) (Myrta et al., 1998) and E-mail: [email protected] MabAC (PPV-C specific) (Myrta et al., 2000). Molecu- 020_JPP718SC(Boscia)_725 16-11-2011 9:32 Pagina 726

726 Plum pox virus in Kosovo Journal of Plant Pathology (2011), 93 (3), 725-728

Table 1. Samples tested for PPV detection and strain identification.

Type of Samples Location District orchard (No.) Plum Apricot Peach Slivova Ferizaji Young 4 4 - - Sojeva Ferizaji Young 3 3 - - Lubishta Vitia Young 6 6 - - Koretini 1 Kamenica Young 3 3 - - Koretini 2 Kamenica Young 6 6 - - Caravrana 1 Malisheva Young 3 3 - - Caravrana 2 Malisheva Young 3 3 - - Klina Klina Young 3 3 - - Dolova Klina Young 3 3 - - Sojeva Ferizaji Old 6 6 - - Pozharani Vitia Old 2 2 - - Mogilla Vitia Old 3 3 - - Parteshi Gjilani Old 3 3 - - Cërnica Gjilani Old 4 - 2 2 Kravarica Gjilani Old 10 7 2 1 Gjilani Gjilani Old 4 2 2 - Llabjani Gjilani Old 3 3 - - Prishtina Prishtina Old 5 3 2 - Total 71 60 8 3

lar characterization was based on a two-step RT-PCR situation. The origin of such a high disease rate is plau- amplification of two different genomic regions (CP and sibly consequent to the widespread infection of nursery P3-6K1) followed by RFLP analysis. Amplification of stocks used for propagation (Table 2). the (Cter) CP was done by primers P1/P2 previously ELISA disclosed a high PPV incidence at all loca- described by Wetzel et al. (1992), while the region tions and orchards, i.e. 30 out of 31 samples from spring (Cter) P3-6K1-(Nter) CI was amplified using primers collection and 16 out of 46 samples from late autumn PCI and PP3 (Glasa et al., 2002). PCR products were collection, substantially confirming field observations, subjected to RFLP analysis using RsaI endonuclease for with a small discrepancy in virus detection (30 PPV- P1/P2 PCR products and DdeI endonuclease for the positive out of 31 symptomatic samples). This can be products amplified by PCI/PP3. explained by the fact that the single symptomatic but Sharka was found at all sites, including young or- ELISA-negative sample was in very poor conditions chards and nurseries. Symptoms were quite evident in when it arrived in the laboratory for testing. On the oth- spring, which allowed a visual assessment of disease in- er hand, three symptomless spring samples were also cidence. Only in one case it was below 10% whereas, ELISA-negative, confirming the correlation between more frequently, it was between 50 and nearly 100% in virus presence and field symptoms. There was also a dif- the orchards and close to 100% in 3 of 4 nurseries ference in PPV incidence between spring and autumn (Table 2), thus revealing a heavily compromised sanitary samples, most likely depending on the fact that spring

Table 2. Plum pox incidence based on symptoms shown by inspected trees.

Disease incidence (%) Locality District Nurseries Orchards Slivova Ferizaji 80-90 n.v. Sojeva Ferizaji n.v. 50-60 Lubishta Vitia n.v. <10 Koretini 1 Kamenica ~100 20-30 Koretini 2 Kamenica ~100 n.v. Caravrana 1 Malisheva n.v. ~100 Caravrana 2 Malisheva n.v. 50-60 Klina Klina ~100 n.v. Dolova Klina n.v. ~100 n.v.: no visit; 020_JPP718SC(Boscia)_725 16-11-2011 9:32 Pagina 727

Journal of Plant Pathology (2011), 93 (3), 725-728 Myrta et al. 727

Table 3. PPV strains identified in Kosovo.

Type of Virus isolates PPV strain identified District orchard (No.) M D Rec Ferizaji Young 3 1 (pl) 2 (pl) Vitia Young 1 1 (pl) Kamenica Young 2 2 (pl) Malisheva Young 2 2 (pl) Klina Young 2 2 (pl) Ferizaji Old 3 3 (pl) Vitia Old 3 3 (pl) 4 (pl) Gjilani Old 7 1 (pe) 2 (ap) Prishtina Old 3 1 (ap) 2 (pl) Total 26 2 1 23 pl: plum; pe: peach; ap: apricot

samples were symptomatic leaves whereas autumn sam- chards. Despite the high inoculum pressure of strain ples were dormant budsticks collected at random, thus PPV-Rec, the single peach tree analyzed for its presence reflecting the allegedly irregular virus distribution in the was infected with PPV-M, in accordance with the re- tree canopy. ported difficulty of PPV-Rec to infect peach naturally. Of the 26 representative PPV isolates selected and Results of this preliminary survey for PPV presence tested for strain typing, serological and molecular test- and distribution in Kosovo show a situation comparable ing confirmed the predominance of the PPV-Rec strain to that registered for the plum industry of neighboring (23 out of 26), and detected only one isolate of PPV-M countries, i.e. Albania (Myrta et al., 1994; Stamo and and two isolates of PPV-D (Table 3). PPV-Rec appeared Myrta, 2006), Serbia (Mandic, 2005; Dulic-Markovic to be endemic as it occurred in all areas in both old and and Jevremovic, 2006), Bosnia and Herzegovina (Matic young orchards. This agrees with previous findings et al., 2005), Croatia (Mikec et al., 2006), Bulgaria (Ka- from Serbia, where PPV-Rec prevails (Glasa et al., menova, 2003) and Romania (Isac and Zagrai, 2006; 2005), and may depend on the common origin of the Isac et al., 2008). propagating materials used for establishing Kosovo’s or- This is the first extensive report on the presence, incidence and identification of PPV strains in Kosovo. Hopefully, it can serve as a basis for additional investi- gations and for stimulating local institutions to devise and implement a strategy for the management for the disease it causes.

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Received March 13, 2011 Accepted June 30, 2011