Detection and Molecular Characterization of ‘Candidatus Phytoplasma Trifolii’, a Member of the Clover Proliferation Grup, Infecting Tomato Plants from Iğdır Province in Turkey

Abstract

The tomato plant exhibiting leaf rolling, witches’ broom, distorted and elongated flower’s sepals in Iğdır province, Turkey, was observed. Total DNA extraction was performed from the symtomatic fresh tomato sample. All DNAs were subjected to Direct and Nested polymerase chain reaction (PCR) with universal primer sets that amplified the 16S rRNA of phytoplasmas. PCR products were purified from agarose gel and cloned into the pGEM T-Easy cloning vector. Recombinant plasmids were introduced into the prokaryotic cloning bacteria by electroporation. Plasmid isolation was performed by selecting one of the positive clones randomly and sequencing was performed by Next Generation Sequencing (NGS). Sequencing results revealed that the 16S rRNA gene associated with phytoplasma was 1251 nucleotides in size, and this sequence was denominated as ‘Iğdır 10’ isolates and recorded in the GenBank under the MT344968 accession number. The virtual restriction fragment length polymorphism (V-RFLP) and phylogenetic analysis of the 16S rDNA sequence confirmed that the cause of disease in infected tomato plants was 'Candidatus Phytoplasma trifolii' ('Ca. P. trifolii') (16SrVI-A, Clover proliferation group), with a 1.00 similarity coefficient. This present study is the first report of ‘Ca. P. trifolii’ and its nucleotide sequence analysis in naturally infected tomato in Iğdır province.