Detection and Partial Molecular Characterization of Two Plum pox virus Isolates from Plum and Wild Apricot in Southeast Kazakhstan

Abstract

Plum pox virus (PPV) was detected in wild apricot and cultivated plum maintained in a germ plasm collection in Kazakhstan. Both isolates were typed as D strain, with no evidence of recombination. The virus was detected by triple-antibody sandwich enzyme-linked immunosorbent assay (ELISA) utilizing the universal PPV-specific monoclonal antibody (MAb) 5B as the secondary antibody, and by reverse-transcription polymerase chain reaction (RT-PCR) assay using primers that amplified a 243-bp fragment in the C-terminus of the coat protein (CP) coding region. Immunocapture (IC) RT-PCR was used to detect PPV in nine wild apricot accessions, including eight ELISA-negative and one ELISA-positive. The plum and apricot isolates reacted positively in Western blot assay with the universal MAb 5B, and negatively with the strain-M-specific MAb-AL. Restriction fragment length polymorphism analysis applied to the amplified 243-bp fragment showed that restriction sites for AluI and RsaI were present in the were present in the plum and apricot samples. An amplified 836-bp cDNA fragment derived from the P3-6K1 coding region of both isolates had restriction profiles typical for strain D. Nucleotide identities of 99 to 100% were observed for the 243-bp fragments of the Kazakhstan isolates when compared with the corresponding regions of strain D, and 94 to 95% identity with strain M. Nucleotide sequence analysis of the entire CP coding region of the plum and apricotisolates resulted in the identification of a unique deletion of six nucleotides (two deduced proline amino acid residues) in the N-terminal region in the plum isolate. This is the first deletion of this nature observed among PPV isolates. The DAG motif was present in both isolates. Several nucleotide substitutions in the CP coding region were common to the plum and apricot isolates and appear to be unique to the Kazakstan isolates. This suggests a close relationship between the isolates.

Sharka: The Past, The Present and The Future

“…PCR is widely used to amplify specific nucleic acid regions; thus, there is a necessity to have primers of known nucleotide sequence. In the past, different primers have been proposed and used, mainly on the HCPro [387], C-terminal part of NIb [265,388], C-terminal part of CP [249,265,389], N-terminal part of CP [390] or 3´-noncoding region [272]. The request to known nucleotide sequence is absolutely necessary also in hybridization techniques, which are popular due to relative simplicity.…”

Genetic variability of Plum pox virus isolates in the Czech Republic

“…One of them, Bosnian isolate BOS49Pl, was earlier characterized by Matić et al (2006). Recently, some other divergent isolates have been described from Kazakhstan (Spiegel et al 2004) and Pakistan (Kollerová et al 2006).…”

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