PCR/RFLP-based method for molecular characterization of ‘Candidatus Phytoplasma prunorum’ strains using the aceF gene.

New molecular typing tools for phytoplasmas belonging to the 16SrX phytoplasma group have recently been developed based on the non-ribosomal genes aceF, pnp, imp, and SecY. In the present work we chose to perform a PCR-RFLP method based on the aceF gene. This genetic marker had previously shown high variability among strains of the 16SrX group, moreover, it had allowed for the differentiation of French hypovirulent ‘Candidatus Phytoplasma prunorum’ strains from virulent ones.

Most of the stone fruit samples were collected in north-east Italy, although a few samples from Bosnia and Herzegovina, and Turkey were also included in the work to explore variability. French hypovirulent and virulent strains, one Azerbaijan strain and ‘Ca. P. prunorum’ strains maintained in periwinkles were used as reference strains. Some of the Italian samples were not collected in the field and they became infected by Cacopsylla pruni under controlled conditions.

Sequencing of the aceF gene was performed on some of the samples tested and based on the alignment, a few restriction enzymes were selected for ‘Ca. P. prunorum’ strain differentiation. Nested PCR was performed using previously developed primers on all samples and RFLP analyses were carried out with BpiI, HaeIII and Tsp509I enzymes. BpiI and HaeIII enzymes generated two different profiles, one profile was undigested and the second one constituted by two different fragments. The Tsp509I enzyme enabled three different pattern types to be distinguished. Combining the results obtained with the three restriction enzymes, it was possible to distinguish between the ‘Ca. P. prunorum’ strains investigated in this study: 6 different RFLP subgroups AceF-A, -B, -C, -D, -E and –F. We confirmed that strains belonging to 4 subgroups, AceF-A, -B, -C and -E were present in north-east Italy, where a large number of the samples were processed. The strains of AceF-A and -E subgroups were the predominant ones (21.6% and 17.0%, respectively) and mixed infections of AceF-A+E subgroups (17.0%), and AceF-B+E (14.8%) subgroups were quite common.