Detection and Characterization of two Phytoplasma lineages on Cucumber (Cucumis sativus L.) with Same Symptomatology based on Virtual RFLP and Nucleotide Sequence Analysis of 16S rDNA

Abstract

Phytoplasma-like symptoms were observed in cucumbers (Cucumis sativus L.) in Van province of Turkey. The major symptoms observed were severe dwarfing, witches’ broom, rosetting, little leaf, and sterility of plants. Genomic DNA of 8 symptomatic and non-symptomatic plant leaves was isolated for the detection of pathogenic DNA. Of the 8 cucumber leaf samples tested by nested polymerase chain reaction (Nested-PCR), the four yielded the expected 1.25-kb DNA fragments when using universal primer pairs R16mF2/R16mR1 and R16F2n/R16R2. Randomly selected two DNA bands were further cloned into a proper plasmid vector. The recombinant plasmid DNA was sequenced bidirectionally. BLAST and virtual restriction fragment length polymorphism (RFLP) analyses of the 16S rDNA sequence revealed the presence of the “Candidatus Phytoplasma solani” (similarity coefficient 1.00) (GenBank accession no: KX977570) in one of the severely symptomatic cucumber samples and the “Candidatus Phytoplasma trifolii” (similarity coefficient 0.98) (GenBank accession no.: KR080212) in the other. The isolates were designated as Van-trifolii and Van-solani isolates, respectively. No significant differences were observed between the two different phytoplasmas’ symptomatology. To the authors’ knowledge, this is the first report of ‘Ca. P. trifolii’ and ‘Ca. P. solani’ in cucumbers in Turkey.

Keywords

• Candidatus Phytoplasma solani,Candidatus Phytoplasma trifolii,Characterization,Cucumis sativus,Detection

Hıyar (Cucumis sativus L.) Bitkisinde Aynı Simptomatolojiye Sahip İki Farklı Fitoplazma etmeninin 16S rDNA'sının RFLP ve Nükleotid Dizi Analizleri ile Teşhisi ve Karakterizasyonu

Öz

Van ilinde yetiştiriciliği yapılan hıyarlarda (Cucumis sativus L.) fitoplazma benzeri belirtiler görülmüştür. Gözlenen en önemli belirtiler arasında şiddetli cüceleşme, cadı süpürgesi görünümü, rozetleme ve küçük yapraklılık yer almaktadır. Toplanan 8 hıyar örneğine R16mF2/R16mR1 ve R16F2n/R16R2 primer çiftleri ile uygulanan Nested-PCR testi uygulanmıştır. Belirti gösteren ve göstermeyen hıyar yapraklarından izole edilen genomik DNA’ların kullanıldığı PCR reaksiyonunda fitoplazma etmenlerine ait 16S rDNA fragmentleri çoğaltılmış ve test edilen sekiz örneğin dördünde 1.25 kb uzunluğunda DNA fragmentleri elde edilmiştir. Beklenen band büyüklüğünü veren örneklerden rastgele seçilen iki örnek uygun bir plasmid vektörde klonlanmıştır. Saflaştırılan rekombinant plasmid DNA’sı çift yönlü olarak dizilenmiş ve 16S rDNA dizisinin BLAST ve RFLP analizleri yapılmıştır. Şiddetli fitoplazma belirtisi gösteren hıyar numunelerinin birinde ‘Candidatus Phytoplasma solani’ (benzerlik katsayısı 1.00) (GenBank erişim no: KX977570), diğerinde ise ‘Candidatus Phytoplasma trifolii’ (benzerlik katsayısı 0.98) (GenBank erişim no: KR080212) saptanmıştır. İzolatlardan birincisine‘Van-solani’ diğerine ise ‘Van-trifolii’ izolatı isimleri verilmiştir. İki farklı fitoplazma etmeninin oluşturduğu belirtiler arasında bir fark gözlenmemiştir. Yazarların bilgisine göre, Türkiye’de hıyarları doğal olarak enfekte eden "Ca. P. solani" ve "Ca. P. trifolii" fitoplazma etmenleri ilk defa bu çalışma ile rapor edilmiştir.

Anahtar Kelimeler

• Cucumis sativus,Candidatus Phytoplasma solani,Candidatus Phytoplasma trifolii,Karakterizasyon,Survey

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