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.

First Report of ‘Candidatus Phytoplasma australasia’ Strain Related to Witches’-Broom of Tomato in Türkiye

“…P. australasia' (16SrII-D subgroup) was identified in phytoplasma-symptomatic tomato plants in Turkey, which is the first record nationally (Fig 2). In Turkey, phytoplasmas belong to eight 16Sr groups which are I, IX, and X (Canik et al, 2011(Canik et al, , 2019, II (Ayvaci et al, 2020), V (Sertkaya et al, 2004), VI and XII (Usta and Güller, 2018), and XIV (Çağlar et al, 2013) have been identified in different hosts, including ornamental plants, orchards, weeds, and cultured crops.…”

“…PCR-targeting universal primers (R16mF2/R16mR1 for d-PCR and R16F2n/R16R2 for n-PCR) amplifying a segment of 1.8 kb and 1.25 kb, for both steps were adopted as specified by Lee et al, (1993) and Gundersen and Lee (1996), respectively. The reaction parameters and temperature cycles were applied as described by Usta et al, (2018). To ensure the reliability of the PCR assays, DNA of 'Ca.…”

“…P. trifolii' and 'Ca. P. solani' in Türkiye (Usta et al, 2018), 'Ca. P. ulmi' in central Italy (Del Serreno et al, 2001).…”

Pest categorisation of the non‐EU phytoplasmas of Cydonia Mill., Fragaria L., Malus Mill., Prunus L., Pyrus L., Ribes L., Rubus L. and Vitis L.

“…The sepals of the flowers acquired hypertrophied form, were fused, and created a bell‐shaped sterile bud: phyllody of green or anthocyanin color. The stems of the plants were lignified, and necrosis of the phloem was observed on stem cuts (Khalil et al., ); severe fruit deformation, flower sterility, aerial rooting, purplish leaves and leaf rolling (Usta et al., ); severe dwarfing, yellowing, and decreased flowering (Salas‐Munoz et al., ); stunting, yellowing or purplish leaves, proliferation of laterals buds, hypertrophic calyxes and virescent flowers (Choueiri et al., );…”