Molecular characterization of ‘Candidatus Phytoplasma mali’ strains in outbreaks of apple proliferation in north eastern Italy, Hungary, and Serbia.

During 2005-2008 apple plants showing proliferation symptoms were observed in diverse cvs in different areas of north eastern Italy, Hungary and Serbia. Leaves and young shoots were collected from June to October in orchards with epidemic presence of apple proliferation, and in others where the symptomatic plants were present in a scattered way. PCR/RFLP analyses carried out on R16F2/R2 amplicons showed that all the samples were infected with ‘Candidatus Phytoplasma mali’. Further strain characterization was carried out using RFLP analyses with HpaII and FauI on almost full ribosomal DNA plus spacer region, AluI on rpl22-s3 genes, and RcaI and HincII on AP13/AP10 amplicons from representative samples collected in these geographic areas. Analyses of 16S plus spacer region distinguished two phytoplasma profiles (P-I and P-II). P-I was detected in reference strains AP, AT1, AT2, in samples from Serbia, and in the majority of samples from Trentino. The P-II profile was prevalent in samples from Veneto, and both profiles were identified in samples from Hungary, in some cases together. The analyses of rpl22-s3 genes allow to identify in all the samples showing P-I profile the presence of phytoplasmas belonging to rpX-A subgroup, while in samples showing P-II profile it was possible to distinguish the other three described rp subgroups. In the majority of samples from Veneto region phytoplasmas belonging to rpX-D subgroup were identified, while rpX-B and X-C subgroups were identified in a few samples from Trentino and Veneto regions respectively. Further RFLP characterization on AP13/AP10 amplicons differentiates among strains belonging to rpX-A subgroup: the Serbian samples show AP profiles, while those from Trentino show AT-2 profiles. In the samples from Hungary the presence of AT1, AT2, and AP profiles was identified. The combined use of these molecular markers allows differentiating ‘Ca. P. mali’ strains according with geographical and epidemic distribution