First Report of Prune dwarf virus and Prunus necrotic ringspot virus on Peach in Montenegro.

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In September and October 2011, samples were collected from mature peach trees (~17 years old) exhibiting symptoms of chlorotic rings and spots, vein clearing, mosaic, necrosis, leaf distortion, stunting, and rosette formation in a major commercial orchard (~80 ha) near Podgorica, Montenegro. Samples were collected from nine different peach varieties (cvs. Adriana, Caldesi, Gloria, Maria Marta, May Crest, Morsiani, Rita Star, Spring Belle, and Spring Crest). Samples (n = 58) were tested using DAS-ELISA for the presence of Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV). Commercial positive and negative controls were included in each ELISA (antisera and controls supplied by BIOREBA AG, Reinach, Switzerland). Only one symptomatic sample from cv. Gloria tested positive for PDV (sample reference: 399/11), a further 11 samples (cvs. Rita Star [six], May Crest [four] and Spring Crest [one]) were positive for PNRSV. Samples were also tested for Plum pox virus (PPV) by real-time RT-PCR (1). The PDV positive sample (399/11) showing mosaic was in mixed infection with PPV, as were 6 of the 11 PNRSV samples, including sample 373/11 with yellow mottling and leaf distortion symptoms. On single-infected PNRSV, sample 368/11 chlorotic line patterns and leaf deformations were observed. To confirm the presence of PDV and PNRSV, positive samples were also tested by RT-PCR. Total RNA was extracted using RNeasy Plant Mini kit (Qiagen, Hilden, Germany). RT-PCR was performed with primer pairs PDV2F/PDV1R (3) and MG1/MG2 (2) specific for PDV and PNRSV, respectively. Amplicons of the expected size, 173 bp for PDV and 675 bp for PNRSV, were obtained from corresponding ELISA-positive samples. Amplified products from three samples (PDV 399/11 and PNRSV 368/11 and 373/11) were cloned into pGEM-T Easy Vector (Promega, Madison, WI) then sent for sequence analysis (MWG-Biotech AG, Edersberg, Germany). Sequence data was compared to sequences published in GenBank. Analysis of sequence obtained from isolate 399/11 (cv. Gloria) corresponded to partial CP gene of PDV, with a high degree of similarity to isolates reported from other parts of the world ranging from 94.2 to 95.9%, showing highest similarity with isolate Ch 137 (L28145). Sequence analyses of CP gene from PNRSV isolates 368/11 (JX569825) and 373/11 (JX569826) proved to be 89.3 to 99.7% identical with corresponding sequences of isolates previously described. In particular, the Montenegrin PNRSV isolates were most closely related to Chilean NctCl.augl isolate from nectarine (EF565253). To demonstrate that the virus was infectious, seedlings of peach cv. GF305 were side grafted with bud-woods from PDV (sample 399/11) and PNRSV-positive samples (samples 368/11 and 373/11) and a healthy control sample. Grafted seedlings were kept in a greenhouse with a under 16-h light regime at 22 to 24°C and observed for symptom development. No symptoms were observed in grafted plants with the healthy control. All plants inoculated with virus-positive samples exhibited stunted vegetation and mild mottle with no difference in symptoms between the two viruses. Indicator plants of peach cv. GF305 inoculated with PPV dual-infected samples (399/11 and 373/11) were subsequently shown to be positive for PPV by real-time RT-PCR. Subsequent DAS-ELISA test on samples from experimentally inoculated trees using specific antisera as described above confirmed PDV and PNRSV infections as expected. These viruses have recently been reported from sour cherry (Prunus cerasus L.) in Serbia (4), ~600 km to the northeast. However, to our knowledge, this is the first report on the occurrence of PDV and PNRSV in Montenegro.