Serological and Molecular Typing of Plum pox Virus Isolates in the Transylvania, Romania
Keywords:Sharka, PPV strains, diagnosis, ELISA, RT-PCR, sequencing
Plum pox virus (PPV) is considered the most destructive viral pathogen of stone fruits. Although PPV is widespread in all plum growing areas from Romania and causes serious yield losses, little is known about the virus variability. For this reason we investigated 100 PPV isolates collected from 13 different plum orchards in the Transylvania - Romania. PPV detection was made by DAS-ELISA and by IC-RT-PCR. PPV strains were serologically determined by TAS-ELISA using PPV-D and PPV-M specific monoclonal antibodies. Molecular strain typing was done by RT-PCR targeting three different genomic segments corresponding to (Cter)CP, (Cter)NIb/(Nter)CP and CI. RFLP analysis was used to distinguish the two major strains, D and M based on a RsaI polymorphism located in (Cter)CP. All PCR products targeting (Cter)CP and 10 PCR products spanning the (Cter)NIb/(Nter)CP were sequenced. All PPV isolates typed as PPV-M by serological analysis and by molecular differentiation in the genomic region corresponding to (C-ter)CP proved to be PPV recombinant (PPV-Rec) when the molecular analysis were performed in the region corresponding to NIb/CP. Sequencing confirmed a high similarity with different sequences of PPV-Rec previously reported. All these recombinant isolates share the same recombination breakpoint and conserve the DAG motif, which is considered essential for aphid transmission. Overall results provided that in Transylvania the predominant strain is PPV-D, followed by PPV-Rec which shares the CP gene with M strain and, therefore, it is serologically detected as PPV-M with M-specific monoclonal antibodies. In this big plum growing area, mixed infections (PPV-D+PPV-Rec), which might generate additional variation by recombination, are also frequent.
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