Genetic diversity of phytoplasmas infecting apple trees in the Czech Republic.

Apple trees are affected by the apple proliferation (AP) disease in several European countries. Samples from apple trees showing proliferation disease symptoms were collected during 2010 all over the Czech Republic. Phytoplasma strains were detected through polymerase chain reaction and restriction fragment length polymorphism analyses of 16S rDNA-spacer region-23rDNA, ribosomal protein gene sequences rpl22 and rps3 and non ribosomal DNA fragment (nitroreductase-like gene). All 74 apple trees examined were positive for phytoplasma presence, predominantly ‘Candidatus Phytoplasma mali’. ‘Ca. P. asteris’ alone and in mixed infection with ‘Ca. P. mali’ was also found. In the 16S plus spacer region of ‘Ca. P. mali’ two profiles were determined (P-I mainly, P-II rarely). Correspondingly, two genetic lineages were found in PCR amplified fragments covering 16S rDNA-spacer region-23rDNA (pattern ‘1a’ rarely, ‘2a’ mainly). ‘Ca. P. mali’ strains belonging to ribosomal protein rpX-A subgroup were identified in the majority of apple samples while phytoplasmas belonging to rpX-B subgroup were erratically found. Apple proliferation subtypes AP-15 and AT-2 revealed nearly equal occurrence. AT-1 subtype and a mixture of two or all three AP subtypes was not frequent. The PCR/RFLP results were confirmed by nucleotide sequence analyses of the 16S-23S ribosomal operon, ribosomal proteins L22, S3 and nitroreductase-like protein gene of five selected apple proliferation strains. This is the first study of molecular diversity among ‘Ca. P. mali’ strains in the Czech Republic.