Şanlıurfa ve Adıyaman illerinde yerel asma çeşitlerindeki fitoplazmaların 16s rDNA gen bölgesine dayalı filogenetik ve in silico RFLP analizleri ile tespiti ve moleküler karakterizasyonu

Türkiye'nin Güneydoğusundaki Şanlıurfa ve Adıyaman illerinde bağ (Vitis vinifera L.) üretim alanlarında 2016-2019 sonbahar sezonlarında, yapraklarda sararma, küçük yaprak oluşumu, kloroz, boğumlar arasında kısalma, şiddetli kızarmalar ve içe doğru kıvrılmalar gibi fitoplazma benzeri semptomlar sergileyen asma bitkilerindeki belirtilerin etmenini belirlemek ve karakterize etmek için çeşitli sürveyler yapılmıştır. Toplanan örneklerden elde edilen saflaştırılmış DNA, fitoplazmalara özgü üniversal primer setleri ile nested-PCR yöntemi ile yüksek oranda korunmuş fitoplazma 16S rDNA geni üzerinden incelenmiştir. Nested-PCR ile DNA amplifikasyonu ve RFLP analizleri ile örneklerdeki gözlenen simptomlar ve fitoplazma varlığı arasındaki beklenen korelasyon doğrulanmıştır. Örneklerden elde edilen fitoplazma türlerinin F2n / R2 amplikonları, birbirleriyle %99.99 homoloji göstermiş ve NCBI GeneBank veri tabanında bulunan bazı gruplara ait fitoplazmalar ile de %99 sekans homolojisi göstermiştir. DNA dizileme ve filogenetik analizler, toplanan izolatlardaki etmenlerin "Candidatus fitoplazma solani" (16SrXII-A) ve "Ca.P. asteris'in (16SrI-B) olduğunu ortaya koymuştur. Bildiğimiz kadarıyla bu, 16S rDNA genine dayalı farklı yerel asma çeşitlerini enfekte eden fitoplazmaların filogenetik ilişkisinin ilk raporudur. Bu tip enfeksiyonlara neden olan patojenin 16S rDNA dışındaki genlerin genetik çeşitliliği araştırma aşamasındadır.

Anahtar Kelimeler:

Asma sarılığı, Fitoplazma hastalığı, Şanlıurfa, Nested-PCR, Asma

Detection and molecular characterization of phytoplasmas based on 16s rDNA gene region by phylogenetic and in silico RFLP analysis of local grapevine cultivars in Şanlıurfa and Adıyaman

During the autumn seasons of 2016-2019, several surveys were carried out in grapevine (Vitis vinifera L.) cultivation areas in Şanlıurfa and Adıyaman provinces of South-eastern part of Turkey, to identify grapevine plants exhibiting phytoplasma-like symptoms including yellowing, small leaf formation, chlorosis, short internodes, severe redness and inward curling and to detect and characterize the causal agent responsible for these symptoms. Purified DNA obtained from collected samples was examined for the highly conserved phytoplasma 16S rDNA gene via nested-PCR with the universal phytoplasma-specific primer sets. DNA amplification via nested-PCR/RFLP analyses with some restriction enzymes confirmed the suspected correlations between the disease symptoms observed and phytoplasma presence in the samples. F2n/R2 amplicons of the phytoplasma strains obtained from the samples showed 99.99% homology with each other and 99% homology with phytoplasma DNA partial sequences belonging to some groups deposited in the GeneBank database. Sequencing and phylogenetic analysis revealed that collected isolates have 99% sequence identity with ‘Candidatus phytoplasma solani’ (16SrXII-A) and ‘Ca.P. asteris’ (16SrI-B). According to our knowledge, the results of this study is the first report of the phylogenetic relationship of phytoplasmas infecting different grapevine cultivars based on 16S rDNA gene. Genetic diversity of genes other than 16S rDNA of the pathogen causing these infections is under study.

Keywords:

Grapevine yellows, Phytoplasma disease, Şanlıurfa, Nested-PCR, Grapevine,

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