Molecular characterization of ‘Candidatus Phytoplasma mali’ strains from Bulgaria and Poland

During 2015, samples from 22 apple trees showing proliferation symptoms were collected in southwest Bulgaria and Central and South Poland and tested for phytoplasma presence. ‘Candidatus Phytoplasma mali’ was identified in 18 samples based on results of restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene amplified in nested PCR using primer pair P1/P7 followed by R16F2n/R16R2 and F1/B6 primer pairs. The nitroreductase and rhodonase-like genes and ribosomal protein genes rpl22 and rps3 were then analyzed using PCR-RFLP technique to study the genetic variability of the phytoplasma strains. Two restriction profiles, P-I or P-II, were obtained from fragments of 16S rDNA plus 16S-23S spacer region digested with HpaII enzyme. Restriction fragment length polymorphism analysis of nitroreductase and rhodonase-like genes using digestion with HincII endonuclease revealed that all ‘Ca. P. mali’ strains belonged to the subtype AP-15. Analysis of rpl22 and rps3 ribosomal protein genes digested with AluI enzyme resulted in classification of detected phytoplasma strains to rpX-A subgroup.