Multilocus gene analyses of \u2018Candidatus Phytoplasma mali\u2019 confirms the genetic diversity of phytoplasma population in the Czech Republic

Apple trees are affected by the apple proliferation (AP) disease in several European countries. The AP is usually associated with the presence of \u2018Candidatus Phytoplasma mali\u2019 (\u2018Ca. P. mali\u2019), a quarantine pathogen in EU. Samples from several apple trees showing proliferation disease symptoms were collected during 2010 at 28 different locations 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 phytoplasmas, predominantly for \u2018Ca. P. mali\u2019 (60 trees). \u2018Ca. P. asteris\u2019 alone and in also mixed infection with \u2018Ca. P. mali\u2019 was detected sporadically. In the 16S plus spacer region of \u2018Ca. P. mali\u2019 two profiles P-I (39 trees) and P-II (5 trees) singly or together (8 trees) were identified. The presence of two genetic lineages designated in literature as pattern \u20181\u2019 (7 trees) and \u20182\u2019 (31 trees) singly and also mixed (7 apple trees) was detected while the only pattern named \u2018a\u2019 was identified in 45 apple trees. \u2018Ca. P. mali\u2019 strains belonging to ribosomal protein rpX-A subgroup were identified in the majority of apple samples (51 out of 55 plants positive in direct PCR amplification with rpAP15f/rpAP15r primers), while phytoplasmas belonging to rpX-B subgroup were detected sporadically (four trees). Nearly equal distribution of apple proliferation subtypes AP-15 and AT-2 (in 21 and 22 trees, respectively) was determined, while apple proliferation subtype AT-1 was detected in 7 trees. Nucleotide sequence analyses of the 16S-23S ribosomal operon, ribosomal proteins L22, S3 and nitroreductase-like protein gene of five selected apple proliferation phytoplasma strains confirmed the PCR/RFLP analyses results. This is the first study of molecular diversity among \u2018Ca. P. mali\u2019 strains in the Czech Republic