Differentiation among ‘Candidatus Phytoplasma mali’ strains by multiple genes analyses.

Samples from apple plants infected by apple proliferation (AP) phytoplasmas of different varieties and from diverse areas were examined by different molecular marker to verify strains differentiation. In the 16S plus spacer region two profiles (P-I and P-II) were distinguished. P-I profile was detected in reference strains, in samples from Serbia, and in the majority of samples from Trentino (Italy); the P-II profile was prevalent in samples from Veneto (Italy); both profiles were identified in samples from Hungary, in some cases both together in the same sample. The analyses of rpl22-s3 genes allow to identify in all the samples showing a P-I profile presence of phytoplasmas belonging to rpX-A subgroup, while in the samples showing a P-II profile it was possible to distinguish the other three reported rpX subgroups. In samples from Italy phytoplasmas belonging to rpX-D, rpX-B and rpX-C subgroups were identified with further local differences. RFLP analyses on AP13/AP10 amplicons differentiate among strains belonging to the rpX-A subgroup: the samples from Serbia show AP profiles, while those from Italy show AT-2 profiles. In the samples from Hungary the presence of AT1, AT2, and AP profiles was identified. The combined use of these three molecular markers allows differentiating ‘Candidatus Phytoplasma mali’ strains according with geographical and, in some cases, also with epidemic distribution. In several orchards of Veneto vector monitoring by yellow sticky traps was carried out and Cacopsylla melanoneura was consistently detected, while Fieberiella florii was erratically found, and only one specimen of Cacopsylla picta was captured. Work is in progress to further verify epidemiological application of these molecular markers for AP strain characterization in insect vector and in alternative host plants.