MOLECULAR DIFFERENTIATION OF 'CANDIDATUS PHYTOPLASMA MALI' AND ITS SPREADING IN FRIULI VENEZIA GIULIA REGION (NORTH-EAST ITALY)

A new PCR/RFLP method based on ribosomal protein (rp) gene sequences has been established for specific detection and differentiation of ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’). The primer pair rpAP15f/rpAP15r has been used in direct-PCR or in nested-PCR following direct-PCR with primer pair rpAP15f2/rp(I)R1A. This new method has been tested in an extensive survey carried out in Friuli Venezia Giulia (FVG) region, Italy, during years 2004–2005. Both symptomatic apple trees (89 samples) and psyllid vectors Cacopsylla picta and Cacopsylla melanoneura (457 individuals divided in 90 batches) collected in FVG were analysed for the presence of ‘Ca. P. mali’. RFLP analyses with AluI enzyme of rpAP15f/rpAP15r amplicons permitted to distinguish 4 different restriction patterns called rpX-A, rpX-B, rpX-C and rpX-D, respectively. In parallel, the PCR/RFLP method based on a non-ribosomal DNA fragment previously established was used in order to differentiate AP phytoplasma subtypes AP, AT-1 and AT-2. Serological detection (ELISA test) using monoclonal antibodies were also performed on the same apple samples. Combining the results obtained with the two PCR/RFLP-based systems, the following subtypes were found in apple trees: rpX-A/AP, rpX-A/AT-2, rpX-C/AT-1 and rpX-D/AT-1. In one sample rpX-A/AT-1 subtype was found in mixed infection with rpX-B/AT-1. Subtypes rpX-A/AP and rpX-A/AT-2 were the most common (54,9% and 30,5%, respectively) and reacted positively for the presence of ‘Ca. P. mali’ in ELISA; whereas rpX-C/AT-1 (1,2%) and rpX-D/AT-1 (12,2%) showed a restricted distribution and were negative in ELISA. The mixed infection rpX-A+rpX-B/AT-1 was not detected in ELISA; however, the serological characterization of these subtypes is under investigation. The periwinkle maintained strains AP15 (rpX-A/AP) and AT (rpX-B/AT-1) reacted positively in ELISA, even if AT reaction was much less intense. Results obtained from insects showed the presence of the same ‘Ca. P. mali’ subtypes found in apple trees from the same localities.