Molecular Detection and Characterization of ‘Candidatus Phytoplasma mali’ Associated With Phyllody on Narcissus tazetta in Türkiye

Abstract

In the winter of 2019, typical phyllody, flower sterility, and stunting symptoms were observed on daffodil plants (Narcissus tazetta) in the Mersin province of Türkiye. Molecular analyses were carried out on 16S rRNA genes of these plants to identify the cause of these symptoms and detect the presence of phytoplasma. DNA extracted from symptomatic plant samples was amplified via nested polymerase chain reaction using the universal primers P1/P7 followed by R16F2n/R16R2. The 16S rRNA gene amplicons from nine samples which were exhibiting severe phyllody symptoms were sequenced, nucleotide BLAST analysis revealed a 99.9% identity match among them and 99.91% with ‘Candidatus Phytoplasma mali’. Further analyses of the 16S rRNA gene-based phylogenetic tree, computer-simulated restriction fragment length polymorphism (virtual RFLP) and conventional RFLP analyses of 16S rRNA gene using MseI, TaqI, RsaI and SspI restriction endonucleases led to assign the phytoplasma to 16Sr group X and subgroup A. For finer characterization of ‘Ca. P. mali’ strain in daffodils, a PCR based multilocus sequence analysis (MLSA) was performed on three distinct genetic loci including nitroreductase and rhodanase-like genes, ribosomal protein genes rpl22 and rps3, and secY gene. The BLAST results showed 99.9% match to ‘Ca. P. mali’ AP-15 subtype (GenBank Accession Number: AJ542541). Electron microscopic examination of the infected daffodil plants revealed the presence of pleomorphic phytoplasma units in the sieve elements of the phloem tissues. Although 16SrX‑A subgroup phytoplasmas have frequently been reported from woody plants, to our knowledge this is the first report of ‘Ca. P. mali’ (16SrX-A) infecting daffodil plants worldwide.