During 2010–2012, samples from 225 apple trees growing in six regions of Poland were tested for phytoplasmas. 16S?rRNA gene and 16S-23S spacer region sequences were amplified from total DNAs prepared from phloem tissue of apple shoots. According to the results of PCR-RFLP and sequence analyses, apple trees were infected by Candidatus Phytoplasma mali and Ca. P. asteris. Fragments of 16S rDNA plus 16S-23S spacer region of the Ca. P. mali isolates digested with HpaII enzyme showed two restriction profiles: P-I and P-II. Multiple alignments of 16S rRNA gene fragments revealed that the isolates of Ca. P. mali shared 100% sequence identity among themselves as well as with reference strains AT and AP-15 of apple proliferation phytoplasma. The nucleotide sequence of the same region of Ca. P. asteris isolates confirmed the phylogenetic relationship with reference strains OAY (MIAY) and AY1 of aster yellows phytoplasma PCR-RFLP analysis of ribosomal protein (rpl22 and rpS3), secY, and tuf genes did not show the sequence diversity of the isolates of aster yellows phytoplasma.
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