ANALYSIS OF PLUM POX VIRUS VARIABILITY AND DEVELOPMENT OF STRAIN-SPECIFIC PCR ASSAYS

A PCR-based detection technique named immunocapture-PCR (IC-PCR) which allows simple and very sensitive detection of plum pox virus (PPV) the causal agent of sharka disease of stone fruit trees has been developed. In its current version, this assay can detect about 2000 virus particles (10 femtograms of virus) diluted in 100 ul of crude plant sap. This is equivalent to a sensitivity about 2000 times better than that of a standard ELISA assay, the technique most commonly used to detect PPV.

IC-PCR was used, in conjunction with RFLP analysis or sequencing of the amplified material to study the molecular variability of PPV from unpassaged field samples, without the need to transfer the virus to herbaceous hosts or to purify it. In parallel, other techniques have also been used in order to characterise PPV isolates. The results obtained allow the discrimination of three groups of PPV isolates showing limited intra-group variability. Two of these groups correspond to the previously described D and M PPV serotypes, for which recent data indicate quite different epidemiological properties. Typing of PPV isolates belonging to the D or M serotypes can now easily be performed either using an RsaI polymorphism in the amplified cDNA fragment or, alternatively by employing serotype-specific primers developed from the sequence information obtained during this study. These serotype-specific primers should prove extremely useful for the rapid characterisation of the isolates responsible for new PPV outbreaks, a procedure recently rendered compulsory in France in an attempt to improve control of the recently introduced M serotype.