Molecular characterization of the coat protein gene of prunus necrotic ringspot virus infecting peach in Montenegro

In 2011 and 2012, two major peach-growing areas in Montenegro were surveyed for the occurrence of eleven peach viruses. Samples collected from 105 peach and nectarine trees were tested by Reverse transcription-polymerase chain reaction (RT-PCR) using sets of primers specific for the detection of Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV), Cherry mottle leaf virus (CMLV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Peach mosaic virus (PMV), Peach rosette mosaic virus (PRMV), Tobacco ringspot virus (TRSV) and Tomato ringspot virus (ToRSV). The occurrence of Strawberry latent ringspot virus (SLRSV) was examined using nested RT-PCR, while a Real-time quantitative RT-PCR method was applied for detection of Plum pox virus (PPV). PPV, PNRSV and PDV were detected in 42.9, 24.8 and 0.9 % of samples. Mixed viral infections were recorded in 6.7 % of samples, while results showed the absence of ACLSV, ApMV, PMV, CMLV, SLRSV, PRMV, TRSV and ToRSV from all assayed samples. In particular, the PCR products of complete coat protein (CP) gene was cloned and sequenced from fourteen PNRSV isolates. A high identity (87.5–100 %) was found between CP nucleotide sequences of Montenegrin isolates and other PNRSV isolates from different hosts and geographic regions retrieved from GenBank. CP nucleotide sequence comparison, as well as phylogenetic reconstruction confirmed the clustering of the isolates into three molecular groups (PV-96, PV-32 and PE-5), whereby Montenegrin isolates were separated into two phylogroups (PV-96 and PE-5). Comparative sequence and phylogenetic analysis showed that clustering of various isolates was not associated with geographic and host origin.