Molecular identification of ‘Candidatus Phytoplasma solani’ using SecY and Vmp1 Genes in Tomato Plants from Van province

Abstract

Tomato (Solanum lycopersicum) is one of the most financially important vegetable crops. It is a species belonging to the Solanaceae family and is cultivated in many countries, including Turkey. The natural presence of ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’) from the Stolbur group (16SrXII) in tomato plants is extensively characterized based on the 16S rRNA gene worldwide. Tomato plants displaying abnormality and sterility of flower, purpling, and bushy appearance were observed in the Van province, Turkey. DNA extraction from tomato leaves was performed, and the extracted DNA was used to amplify 16S rRNA sequences using universal primer pairs by double PCR assays. After confirming the presence of the probable agent (‘Ca. P. solani’), nested PCR testing was performed using an appropriate primer set amplifying the SecY and Vmp1 genes of the same DNAs. Amplified PCR yields were then cloned into a pGEM T-Easy vector and sequenced by New Generation System (NGS). Sequenced 1438 bp nucleotides for Vmp1 gene (MN104838) and 905 bp nucleotides for SecY gene (MN125054) revealed 97.38% and 100% maximum nucleotide similarity with formerly published Vmp1 and SecY gene sequences of ‘Ca. P. solani’ species belonged to the Stolbur group (16Sr-XII), respectively. According to the phylogenetic tree created using our gene sequences from ‘Ca. P. solani’, Vmp1, and SecY gene sequences showed a highly phylogenetic affinity with the same sequences of the same agent from Serbia and France, respectively. To the best of our knowledge, this study is the first report to reveal the phylogenetic relationships of ‘Ca. P. solani’ in stolbur group (16Sr XII-A) using SecY and Vmp1 genes in the tomato plant in Turkey based on the non-ribosomal genes.

Keywords

• ‘Ca. P. solani’,
• Vmp1
• SecY
• tomato
• Phylogeny
• Turkey

Van’dan Domates Bitkilerinde SecY ve Vmp1 Genlerini Kullanarak ‘Candidatus Phytoplasma solani’nin Moleküler Tanımlanması

Öz

Domates (Solanum lycopersicum) finansal olarak en öemli sebze bitkilerinden biridir. Solanaceae familyasına ait olan domates Türkiye’nin de içerisinde bulunduğu bir çok ülkede kültürü yapılmaktadır. Domates bitkilerinde Stolbur gruptan ‘Candidatus Phytoplasma solani’nin doğal varlığı dünya genelinde geniş ölçüde 16S rRNA geni üzerine dayalı olarak gerçekleştirilmiştir. Çiçekte anormallik ve kırıslık, morluk ve gür görünüm sergileyen domates bitkileri Türkiye’nin Van ilçesinde gözlendi. Domates yapraklarından DNA izolasyonu gerçekleştirildi. İzole edilen DNA’lar evrensel primer çiftleri çift PCR tekniğiyle 16S rRNA sekansını çoğaltmak için kalıp olarak kullanıldı. Muhtemel ajanın varlığının doğrulanmasının ardından, Nested PCR tekniği aynı DNA’ların SecY ve Vmp1 genlerini amplifiye eden uygun primer çiftleri kullanılarak gerçekleştirildi. Çoğaltılan PCR ürünleri daha sonra pGEM T-Easy vektöre klonlandı ve yeni nesil sekanlama teknolojisi ile sekanslandı. 1438 bp’lik Vmp1 geni ve 905 bp’lik SecY genine ait nükleotid dizileri Stolbur grupa ait ‘Ca. P solani’nin daha önceleri yayınlanan Vmp1 ve SecY geni ile sırası ile 97.38% and 100% maximum nükleotid benzerliği göstermiştir. ‘Ca. P solani’ye ait bizim gen sekanslarımız ile oluşturulan filogenetik ağaca göre Vmp1 ve SecY geni sekansları sırasıyla Sırbistan ve Fransa’dan aynı ajanın aynı genleri ile yüksek filogenetik ilişki göstermiştir. Bizim bilgimize göre bu çalışma ribosomal olmayan genlere dayalı Türkiye’de domates bitkilerinde SecY and Vmp1 genlerini kullanarak stolbur grupda (16Sr XII-A) ‘Ca. P. solani’nin filogenetik ilişkisini ortaya koyan ilk rapordur.

Anahtar Kelimeler

• Ca. P. solani’
• Vmp1
• SecY
• domates
• Filogeni
• Türkiye

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