Genetic variability of apple proliferation phytoplasmas as determined by PCR-RFLP and sequencing of a non-ribosomal fragment

Abstract Apple proliferation phytoplasmas are considered as quarantine organisms in Europe and north America, but reliable polymerase chain reaction (PCR) primers for their identification in routine diagnosis were missing, because they show genetic variability. Therefore, 100 apple proliferation phytoplasma isolates, derived from most of the European countries where apple proliferation disease has been detected, were analysed for their genetic variability. A detailed restriction fragment length polymorphism (RFLP) analysis of a 1·5 kbp chromosomal DNA fragment amplified by PCR (PCR-RFLP) from various isolates of apple proliferation phytoplasma revealed three different subtypes named AP, AT-1 and AT-2. Sequence analysis of a 846 bp fragment of each subtype showed that the sequences differed only in the restriction sites responsible for the observed polymorphism. Thus, the apple proliferation phytoplasma subtypes are very closely related. The observed point mutations were responsible for specific amino acid changes in the putative protein PR3. No geographic prevalence of a given subtype could be observed. In a 5-year study a given subtype could be repeatedly amplified from the same tree indicating a stable maintenance of the subtype. The AP-specific primers used in this study enabled a one-step identification of all isolates suitable for routine diagnosis