Authors: ŞEVKET ALP, MUSTAFA USTA, HİKMET MURAT SİPAHİOĞLU, ABDULLAH GÜLLER
Abstract: Marigold (Tagetes erecta L.) plants, also called Mexican or Aztec marigold, with symptoms of shoot proliferation, dwarfing, and reddening were observed in ornamental gardens of Van Province (Turkey). Five plants, two of them showing reddening and three symptomless plants, were sampled at the end of September 2014. Genomic DNA isolated from symptomatic and nonsymptomatic plant leaves was used to amplify 16S rDNA fragments by nested polymerase chain reaction (PCR). Of the 5 marigold samples tested by PCR, only the two showing reddening symptoms yielded the expected 1.2-kb DNA fragments. Amplified PCR fragments were cloned into a plasmid vector and transformed into competent Escherichia coli strain JM 109. Recombinant plasmid DNA was isolated and sequenced bidirectionally. The provided sequences were 1244 bp and 1245 bp in length and were designated as isolate 1 and isolate 2, respectively. BLAST analysis of the 16S rDNA sequence and virtual restriction fragment length polymorphism (RFLP) analysis confirmed the presence of the phytoplasma "Candidatus Phytoplasma solani". The in silico virtual RFLP pattern of isolate 1, based on the 16S rDNA F2n/R2 fragment, was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group XII, subgroup A (GenBank accession no.: AF248959). Isolate 1 was identified as a member of 16SrXII-A. Based on the same analyses, isolate 2 showed molecular characteristics different from reference patterns of all previously established 16Sr groups and subgroups. The most similar was the reference pattern of 16Sr group XII, subgroup A (GenBank accession no.: AF248959), with a similarity coefficient of 0.97. This is the first report of naturally occurring "Ca. P. Solani" affecting T. Erecta, which shows that this plant species is an alternate host of the agent.
Keywords: ?Candidatus Phytoplasma solani?, Tagetes erecta, nested PCR, molecular cloning
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