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In the year 2004, a survey was carried out to determine the incidence and distribution of Plum pox virus (PPV) in Croatia. A number of consensus positives were obtained both by ELISA and PCR testing indicating PPV-M and PPV-D presence in various Prunus species all over the country. Additional investigations were conducted in the following years in order to search for a recombinant PPV (PPV-Rec) previously reported in the neighboring countries. Susceptible plum varieties, known as particularly endangered by recombinant PPV, were in the focus of our interest. The samples were collected in commercial orchards raised exclusively with imported plant material. A modified IC-RT-PCR approach was carried out combining Bioreba polyclonal antibodies and mD5mM3 primers in immunocapture and amplification step, respectively. Among 6 tested plum samples, representing three varieties from three different locations in the eastern part of Croatia, five were PPV positive. One sample (‘ Valjevka’ ) was proved to be positive for PPV-Rec. The PCR-based strain identification further revealed the presence of four PPV-M and three PPV-D strains. Thus, mixed infections were detected in three plum-samples (two M+D and possibly one M+Rec). Additionally, a plum growing spontaneously in a private garden in the urban Zagreb area dislocated from any commercial orchard and displaying severe leaf and fruit symptoms, was sampled. Unexpectedly, this tree was also proved to be PPV-Rec positive. Five sequences of (Cter)CP amplicons clustered in two groups, in accordance with the strain differentiation (PPV-M, D), sharing high sequence identity with some reference isolates. Even though (Cter)CP sequences of the two recombinant isolates (from ‘ Valjevka’ and wild plum) were identical, the phylogenetic analysis of (Cter)Nib/(Nter)CP regions (Cter)Nib/(Nter)CP-sequences showed some differences. The nucleotide identity between the two PPV-Rec isolates was 98.1%. The isolate ‘ Valjevka’ differed as little as 1.4% from SerbiaPO3 isolate, while wild plum isolate differed only 1.4% from the Slovakian isolate BRC8. These data raise intriguing questions about the PPV-Rec incidence and origin in Croatia. |
