Discovery and characterization of Plum pox virus isolates in Montenegro

Abstract

In 2006, twenty extensive and intensive plum orchards, surrounding Nik s i C in Montenegro, were screened for the presence of symptoms of Plum pox virus (PPV) infection. Mild to severe symptoms were found in 15 orchards, usually only on some trees. In total 19 samples were collected and 17 proved to be positive by DAS-ELISA (Bioreba). Of these positives, 16 were selected for molecular identification. PPV isolates have been assigned to two major (PPV-D and PPV-M) and two minor (PPV-EA, PPVC) groups. Recently, a fifth group (PPV-REC) has been established, which corresponds to naturally-occurring recombinants between PPV-M and PPV-D isolates. All PPV-REC isolates share the same recombination breakpoint on the NIb polymerase gene and therefore conventional CP gene-based molecular and serological methods are not able to discriminate between PPV-M and PPV-REC isolates. Subgroup characterization was performed using two different PCR systems: one positioned downstream of the recombinant crossover (using the PPV-M and -D specific primer pairs P4/P3-M and P4/P3-D respectively; Dallot et al. , in press); and the other upstream of the recombination breakpoint (using the primer pairs CIf/CI-M (PPV-M specific) and CIf/CI-D (PPV-D specific); Glasa et al. , 2002). The results showed the presence of PPV-D type isolates in 4 samples. These samples were also negative for PPV-M when tested by TAS-ELISA (Agritest). The remaining 12 samples were PPV-M positive by TAS-ELISA. Of these, seven proved to be of PPV-REC type by RT-PCR. However, the isolate type could not be determined for the remaining five samples using the previously described method. Therefore they were further analyzed using primer pairs mM5/mM3, mD5/mD3 and mD5/ mM3 ( P ubr et al. , 2004), situated around the recombination breakpoint. All samples gave a positive result only with primer pair mD/mM3 and were therefore also identified as the PPV-REC type. Although plum production is important in Montenegro, data about PPV infection is scarce. Sharka symptoms were observed by Miju s kovi C (2002), but until now the presence of PPV was never confirmed. As a result, this is the first report confirming PPV as being present in Montenegro.