Journal of Plant Pathology (2008), 90 (1, Supplement), S1.57-S1.61 Edizioni ETS Pisa, 2008 S1.57 ORNAMENTAL PRUNUS SPECIES AS NEW NATURAL HOSTS OF PLUM POX VIRUS AND THEIR IMPORTANCE IN THE SPREAD OF THE VIRUS IN HUNGARY D. Sebestyen1*, M. Nemeth2, R. Hangyal3, L. Krizbai1, I. Ember1*, K. Nyerges3, M. Kolber1*, E. Kiss4 and G. Bese4 1 Central Agricultural Office, Directorate for Plant Protection and Soil Conservation and Agri-environment, Budaörsi út 141-14, 1118 Budapest, Hungary 2 Hungarian Academy of Science, Plant Protection Committee, Budapest, Hungary 3 Agricultural Office, Plant Protection and Soil Conservation Directorate of county Fejér, Orszag ut 23, 2481 Velence, Hungary 4 Agricultural Office, Plant Protection and Soil Conservation Directorate of county Csongrád, Rárosi út 110, 6800 Hódmezővásárhely, Hungary SUMMARY To determine the natural incidence of Plum pox virus (PPV) in ornamental Prunus species, surveys of 120 species/cultivars were conducted in Hungary between 2002 and 2007. Fifty-nine PPV-infected trees were detected by ELISA and/or PCR. Twenty-five were P. cerasifera cv. Nigra with dark red leaves, which accounts for a significant proportion of street-grown Prunus trees in Hungary. Molecular assays showed a prevalence of strain PPV-D (22) over PPV-M (2) and mixed infection of strains M and D (1). Infection was identified in: *Persica x davidiopersica cv. Atropurpurea, P. cerasifera, *P. cerasifera (Blue-fruited), *P. cerasifera (Light-pink-fruited), *P. cerasifera cv. Nigra, *P. cerasifera cv. Pendula, *P.cerasifera cv. Pissardii, *P. cerasifera cv. Woodii, P. glandulosa, *P. glandulosa cv. Alba Plena, *P. japonica, *Prunus x blireana, *Prunus x blireana cv. Moseri. On cultivars with dark-red leaves, symptoms could not be recognized even if the virus concentration was high. It can be assumed that in Hungary the latently infected red-leaved cultivars can play an important role in the wide distribution of PPV. Therefore it is a must to implement the certification scheme for propagating materials also considering that a number of Prunus species and cultivars (marked by an asterisk above) have been identified as previously unreported natural hosts of PPV in Hungary. Key words: Prunus, PPV, natural hosts, new PPV records, ELISA, RT-PCR, indexing. INTRODUCTION Plum pox virus (PPV) is widespread on stone fruit species in Hungary, where it causes heavy yield losses (Nemeth, 1986). In the last decades natural occurrence * Present address:FITOLAB Plant Pest Diagnostic and Advisory Ltd, Istenhegyi ut 29, 1125 Budapest Hungary. Corresponding author: M. Kolber Fax: +36.1.2019682 E-mail: kolber.maria@fitolab.hu of this virus has been recorded also in some ornamental and wild Prunus species in Hungary and in other countries (Nemeth, 1986; James and Thompson, 2006). To provide scientific data to support the requirements of the certification scheme for the production of virus-free planting material, the present paper reports the results of a six-year survey conducted by the Plant Protection and the Soil Conservation Service in order to determine the rate of PPV infection of ornamental Prunus species in Hungary. MATERIALS AND METHODS Survey. Surveys and sampling in propagation sites, such as nuclear stocks, propagation blocks, nurseries, arboretums, public areas including parks and streetgrown trees, were carried out by plant pathologists and inspectors of the county Services in at least one location of each county. ELISA. Samples from the south-eastern part of Hungary were tested by ELISA at the Virus Laboratory of the Plant Protection and Soil Conservation Service (PPSCS) of county Csongrád using broad spectrum PPV kits from Loewe Biochemica (Germany). ELISA on samples collected in other parts of Hungary in 20022006 and molecular testing were done in the Central Laboratory for Pest Diagnosis of the Central Service for Plant Protection and Soil Conservation (PPSCS), Budapest. In 2007 ELISA testing of all samples was done in the PPSCS Virus Laboratory of county Fejér, Velence. Samples were always tested in duplicate. In the latter two laboratories universal (5B) and specific monoclonal antibodies raised either to PPV-D (Cambra et al., 1994) (Durviz, Spain), or PPV-M (Boscia et al., 1997) (Agritest, Italy) were used. PPV-infected trees were repeatedly tested in ELISA with the universal monoclonal antibody 5B throughout the duration of the survey. RT-PCR assays. ELISA-positive samples were further assayed by RT-PCR. RNeasy Plant Mini Kits (Qiagen, Germany) were used for RNA extraction. General PPV detection was conducted with P1/P2 primers (Wetzel et S1.58 Ornamental Prunus as hosts of PPV Journal of Plant Pathology (2008), 90 (1, Supplement), S1.57-S1.61 al., 1991). For strain-specific identification, P1/PD and P1/PM primers were used, according to Olmos et al. (1997). whitish or light yellow rings of different size, speckles, line pattern or light green vein banding. On P. glandulosa cv. Alba Plena the symptoms were pale and blurred, and similarly light were the green discoloured areas along the midribs and the sparse blotches on the leaves of P. japonica. Round blotches associated with the veins or long light green discoloured areas were present on P. cerasifera cv. Pendula. P. cerasifera cv. Pissardii leaves turned dark-red or purple, mainly along the main and secondary veins, with a higher intensity on the lower surface. The lower surface of Prunus x blireana leaves exhibited similar symptoms along the main, secondary and tertiary veins. On dark-red-leaved trees such as P. cerasifera cv. Nigra and Prunus x davidopersica cv. Atropurpurea symptoms were not visible. Woody indexing. Indexing of trees, selected among the ELISA-positive ones in 2002 and 2003, was done by chip budding onto GF 305 peach seedlings in 5 replicates in the experimental nursery of PPSCS of county Fejér. As controls, samples were collected from inoculated indicator plants and tested by RT-PCR. RESULTS AND DISCUSSION Field symptoms. Between 2002 and 2007, 120 ornamental Prunus species and cultivars in arboretums/ botanical gardens (288 samples), propagation sites (870 samples) and public areas including parks and streets (574 samples) were surveyed and sampled (Table 1). Symptoms affecting the leaf colour were observed only in a few trees of 10 ornamental Prunus species/cultivars. Among the green-leaved plants, the most conspicuous symptoms were shown by the leaves of P. glandulosa, i.e. ELISA. Fiftynine of 1732 samples collected from 93 locations of 15 counties were ELISA-positive. PPV-infected trees were repeatedly tested in successive years with consistent positive results. Reactions of the 5B monoclonal were convincing both in controls and positive samples throughout the six-year survey. Extinction values ranged Table 1. Results of surveys of ornamental Prunus species/cultivars for natural PPV infection in Hungary (2002-2007). Origin of samples Year From arboretums and botanical garden 2002 2/90 2003 (b) From propagation sites(a) From public areas Total 8/294 5/131 15/515 4/46 0/26 0/37 4/109 2004 0/67 2/191 11/245 13/503 2005 0/7 - 6/68 6/75 PPV-infected ornamental Prunus (number of samples) P. x blireana (6) P. x blireana cv. Moseri (1) P. cerasifera cv. Nigra (6) P. glandulosa (1) P. japonica (1) Persica x davidiopersica cv. Atropurpurea (1) P. cerasifera (1) cv. Nigra (1) cv. Pendula (1) P. cerasifera (Blue-fruited) (1) (Light-pink-fruited) (1) cv. Nigra (9) cv. Pissardii (2) P. cerasifera cv. Nigra (6) P. cerasifera (3) 2006 8/78 2/243 8/93 cv. Nigra (2) cv. Pissardii (4) cv. Woodii (4) 18/414 P. glandulosa (1) 2007 - 3/116 - 3/116 Total 14/288 15/870 30/574 59/1732 (a) (b) propagation sites: nuclear stocks, propagation blocks, nurseries. number of positive samples/Number of tested samples. cv. Alba Plena (2) P. x blireana (2) P. cerasifera cv. Woodii (3) Journal of Plant Pathology (2008), 90 (1, Supplement), S1.57-S1.61 between 0.400 and 3,000. Reactions of control PPV-M and PPV-D strains with the strain-specific monoclonals were generally slightly weaker but always convincing. With strain-specific ELISA testing, strain PPV-M was not detected at all, PPV-D was identified only in 4 of the 59 samples that had previously tested ELISA-positive using Sebestyen et al. S1.59 the 5B monoclonal (Table 3). Further PPV strain-specific serological investigations are planned. Woody indexing. PPV symptoms were observed on indicator plants of 10 of the 15 ELISA-positive trees selected for indexing in 2002 and 2003 (Table 3). Table 2. List of ornamental Prunus species, cultivars and botanical variety surveyed for natural PPV infection in Hungary (2002-2007). Persica x davidiopersica cv. Atropurpurea* Prunus cv. Accolade Prunus cv. Hally Jolivette Prunus cv. Okame Prunus cv. Pandora Prunus cv. Rubin Prunus cv. Shosar Prunus cv. Spire Prunus cv. Umineko P. americana P. armeniaca var. Ansu P. avium P. avium cv. Pendula, cv. Plena P. cerasifera* P. cerasifera (Blue-fruited)* P. cerasifera (Light-pink-fruited)* P. cerasifera cv. Hessei, Prunus cv.Trailblazer (= P. cerasifera cv. Hollywood) P. cerasifera cv. Nigra* P. cerasifera cv. Pendula* P. cerasifera cv. Pissardii* (= P. cerasifera cv. Atropurpurea) P. cerasifera cv. Woodii* P. cerasus cv. Pendula, cv. Semperflorens P. curdica P. davidiana P. dulcis (P. amygdalus) P. dulcis cv. Balaton, cv. Rosea Plena, cv. Spring Glow P. fruticosa P. glandulosa* P. glandulosa cv. Alba Plena* P. glandulosa cv. Rosea Plena P. grayana P. humilis P. japonica* P. kansuensis P. laurocerasus P. laurocerasus cv. Baumgartner, cv. Cherry Brandy P. laurocerasus cv. Grüner Teppich, cv. Herbergii, cv. Klári P. nipponica cv. Brillant P. padus P. padus cv. Coloratus, cv. Schloss Tiefurt Prunus pendula (= Prunus subhirtella cv. Pendula) P. pensylvanica P. persica P. persica cv. Bonanza P. pseudocerasus P. salicina P. sargentii P. serotina P. serrulata P. serrulata cv. Albo- plena, cv. Amanogawa P. serrulata cv. Edo-zakura, cv.Fukurokuju P. serrulata cv. Hisakura, cv. Hokusai P. serrulata cv. Kanzan, cv. Kiku-shidare-zakura P. serrulata cv. Mikuruma-gaeshi P. serrulata cv. Oyochin, cv. Pink Perfection P. serrulata cv. Royal Burgundy, cv. Shirotae P. serrulata cv. Taihaku, cv. Ukon P. serrulata cv. Yae-murasaki P. sibirica P. spinosa P. spinosa cv. Rosea, cv. Rubra P. sibirica P. subhirtella P. subhirtella cv. Autumnalis, cv. Autumnalis Rosea P. subhirtella cv. Fukubana, cv. Dahlem (= P. subhirtella cv. Plena) P. tenella P. tenella cv. Fire Hill, cv. Kati P. triloba P. triloba cv. Multiplex, cv. Rosenmund P. virginiana P. virginiana cv. Canada Red, cv. Schubert Prunus x blireana* Prunus x blireana cv. Moseri* Prunus x cistena S1.60 Ornamental Prunus as hosts of PPV Journal of Plant Pathology (2008), 90 (1, Supplement), S1.57-S1.61 P. laurocerasus cv. Magnoliifolia, cv. Marbled White P. laurocerasus cv. Mari, cv. Otto Luyken, cv. Piri, cv. Schipkaensis P. laurocerasus cv. Van Nes, cv. Zabeliana Prunus × eminens cv. Umbraculifera (= P. fruticosa cv. Globosa) Prunus × mohacsyana Prunus × persicoides (= Prunus x amygdalopersica) Prunus × persicoides cv. Spring Glow (=P. × amygdalopersica cv. Spring Glow) Prunus x schmittii Prunus x yedoënsis Prunus x yedoënsis cv. Moerheimii cv. Shidare-yoshino P. lusitanica P. maackii P. mahaleb P. mandshurica P. mume P. nipponica (= P. nipponica var. kurilensis) * Natural PPV host in Hungary Table 3. PPV-infected ornamental Prunus species and cultivars detected by ELISA, woody indexing and/or PCR in Hungary (2002-2007). Species/cultivar ELISA Indexing RT-PCR Strain-specific PCR 1/1 2/2 1/2 (1 PPV-D) 1/1 (PPV-D) 1/1 (PPV-D) 7/7 (PPV-D) 1/1 (PPV-D) 1/1 (PPV-D) 3/3 (PPV-D) 1/1 (PPV-M) 1/1 (PPV-D) 1/1 (PPV-D) 3/5 (3 PPV-D, 1 PPM-M, 1 PPV D+M) 1/1 (PPV-D) 1/1 (PPV-D) P. cerasifera (Blue-fruited) P. cerasifera (Light-pink-fruited)(a) P. cerasifera cv. Nigra P. cerasifera cv. Pendula(a) P. cerasifera cv. Pissardii(a) P. cerasifera cv. Woodii P. glandulosa(a) P. glandulosa cv. Alba Plena(a) P. japonica(a) 4/4(b) (1 PPV-D) 1/1 1/1 24/24 (1 PPV-D) 1/1 (1 PPV-D) 6/6 7/7 2/2 2/2 1/1 NT NT 2/3 1/1 NT 0 1/1 NT 1/0 1/1 1/1 7/8 1/1 1/2 3/3 1/1 1/1 1/1 Prunus x blireana(a) 8/8 4/6 5/6 Prunus x blireana cv. Moseri(a) Persica x davidiopersica cv. Atropurpurea 1/1 1/ 1 (PPV-D) 1/1 NT 1/1 1/1 P. cerasifera(a) (a) (a) PPV-infected; (c) (b) Number of positive samples/total number of tested sample; (c)not tested. RT-PCR. To verify the indexing results of the 15 ELISA-positive trees, molecular tests were positive with all the inoculated and symptomatic indicators of the 10 ELISA-positive trees. In the case of five ELISA positives, PPV was detected at least in one of the symptomless indicators. Twenty-five samples were identified as PPV-D, two as PPV-M and, in one case, a mixed infection (PPV-D and PPV-M) was found out of the 28 samples selected for strain-specific identification (Table 3). Further molecular studies of PPV isolates have been initiated and their characterization is in progress. Comparison is planned of the new findings with earlier results by Myrta et al. (2001), Salamon and Palkovics (2002) on other Hungarian PPV isolates, and with the classification of PPV strains reported by James and Glasa (2006). Based on the six-year study, consisting of surveys for new host species and laboratory analyses (serological and molecular assays), it can be concluded that natural PPV infection was found in 13 of the 120 ornamental Prunus species/cultivars tested. Although PPV was not detected during this survey in P. spinosa plants, infected blackthorn plants were often found in the course of woody indexing performed in the 60s’ in the frame of the Hungarian certification programme (M. Nemeth, unpublished information). Due to these indexing results, P. spinosa was included in the regulations for certification in the 70s. The presence of PPV in symptomatic and symptomless P. spinosa was confirmed by Salamon and Palkovics (2002) and identified as a new subgroup of M strain in Hungary. Journal of Plant Pathology (2008), 90 (1, Supplement), S1.57-S1.61 In the present study, 11 of 13 species/cultivars were identified as new natural PPV hosts for Hungary and also internationally. Only 10 of the 13 naturally PPV-infected species/cultivars/botanical varieties were symptomatic. On infected light red-leaved cultivars symptoms are clearly visible on the lower surface of the leaves. This is important for phytosanitary inspectors to bear in mind. On dark red-leaved ornamental Prunus cultivars (such as P. cerasifera cv. Nigra), PPV symptoms cannot be recognized even if the virus concentration is high. It can be assumed that in Hungary these latently infected dark red-leaved cultivars can play an important role in the wide distribution of PPV. Such trees in public areas are permanent virus sources. Symptomless infected trees in the vicinity of nuclear stocks and nurseries constitute a high risk. Therefore, it is a must for the future to enforce the requirements of the certification scheme for the production of propagating materials also to the natural PPV hosts newly identified in this study. Considering the high frequency of latent infections, the use of reliable laboratory methods in regular screening is essential. ACKNOWLEDGEMENTS The Authors wish to thank the plant pathologists and inspectors and of the county services for surveying and sampling, as well as Sütöri Diószegi Magdolna, (Corvinus University, Budapest) for her valuable assistance in the botanical identification of the collected plant material. Sebestyen et al. S1.61 REFERENCES Boscia D., Zeramdini H., Cambra M., Potere O., Gorris M.T., Myrta A., Di Terlizzi B., Savino V., 1997. Production and characterization of a monoclonal antibody specific to the M serotype of plum pox potyvirus. European Journal of Plant Pathology 103: 477-480. Cambra M., Asensio M., Gorris M.T., Pérez E., Camarasa E., Garcia J.A., Moya J.J., López-Abella D., Vela C., Sanz, A. 1994. Detection of plum pox potyvirus using monoclonal antibodies to structural and non-structural proteins. Bulletin OEPP/EPPO Bulletin 24: 569-577. 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