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

Yıl 2021, Cilt: 25 Sayı: 2, 204 - 213, 23.06.2021

Eray Şimşek , Mehmet Güldür

https://doi.org/10.29050/harranziraat.816630

Cited By: 4

Öz

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.

Anahtar Kelimeler

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

Destekleyen Kurum

HÜBAK

Proje Numarası

16045

Teşekkür

The authors also thank the Turkish Council of Higher Education Ogretim Uyesi Yetistirme Programı (OYP) program for partial funding of the study.

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

Öz

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

Proje Numarası

16045

Kaynakça

• Ahrens, U., & Seemüller, E. (1992). Detection of DNA of plant pathogenic mycoplasmalike organisms by a polymerase chain reaction that amplifies a sequence of the 16 S rRNA gene. Phytopathology, 82(8), 828-832.
• Babaei, G., Esmaeilzadeh-Hosseini, S. A., Eshaghi, R., & Nikbakht, V. (2019). Incidence and molecular characterization of a 16SrI-B phytoplasma strain associated with Vitis vinifera leaf yellowing and reddening in the west of Iran. Canadian Journal of Plant Pathology, 41(3), 468-474.
• Bekisli, M. İ., Bilgiç, C., & Gürsöz, S. (2015). Şanlıurfa İli Bağ Alanlarının Mevcut Durumu ve Sulama Sistemlerinin Değerlendirilmesi. Selcuk Journal of Agriculture and Food Sciences A, 27(1), 562-565.
• Bertaccini, A., & Lee, I.M. (2018). Phytoplasmas: an update. In Rao, G.P., Bertaccini, A., Fiore, N., Liefting, L. (eds.). Phytoplasmas: Plant Pathogenic Bacteria-I. Characterisation and Epidemiology of Phytoplasma-Associated Diseases (pp. 1-29). Springer Nature, Singapore.
• Bertaccini, A., Duduk, B., Paltrinieri, S., & Contaldo, N. (2014). Phytoplasmas and phytoplasma diseases: a severe threat to agriculture. American Journal of Plant Sciences, 5(12), 1-26.
• Canik, D., Ertunc, F., Paltrinieri, S., Contaldo, N., & Bertaccini, A. (2011). Identification of different phytoplasmas infecting grapevine in Turkey. Bulletin of Insectology, 64(Supplement), 225-226.
• Constable, F., & Bertaccini, A. (2017). Worldwide distribution and identification of grapevine yellows diseases. In Dermastia, M., Bertaccini, A., Constable F., Mehle, N. (eds.). Grapevine Yellows Diseases and Their Phytoplasma Agents (pp. 17-46). Springer, Cham.
• Dermastia, M., Bertaccini, A., Constable, F., & Mehle, N. (2017). Grapevine yellows diseases and their phytoplasma agents: biology and detection. Springer Nature Switzerland AG. Part of Springer Nature.
• Desjardins, P., & Conklin, D. (2010). NanoDrop microvolume quantitation of nucleic acids. JoVE (Journal of Visualized Experiments), (45), e2565.
• Duduk, B., Paltrinieri, S., Lee, M., & Bertaccini, A. (2013). Nested PCR and RFLP analysis based on the 16S rRNA gene. In M. Dickinson, J. Hodgetts, Phytoplasma (pp. 159-171). Totowa, NJ.: Humana Press.
• Ertunc, F., Orel, D. C., Bayram, S., Paltrinieri, S., Bertaccini, A., Topkaya, S., & Soylemezoglu, G. (2015). Occurrence and identification of grapevine phytoplasmas in main viticultural regions of Turkey. Phytoparasitica, 43(3), 303-310.
• Gibb, K. S., Padovan, A. C., & Mogen, B. D. (1995). Studies on sweet potato little-leaf phytoplasma detected in sweet potato and other plant species growing in northern Australia. Phytopathology, 85(2), 169-174.
• Gokbayrak, Z., & Soylemezoglu, G. (2010). Grapevine throughout the History of Anatolia. International Journal of Botany, 6(4), 465-472.
• Gorny, R. L. (2003). Viniculture and ancient Anatolia. In McGovern, P.E., Fleming, S.J., Katz, S.H. (eds). The Origins and Ancient History of Wine Food and Nutrition in History and Antropology (pp. 158-203). London, Routledge.
• Gundersen, D. E., & Lee, I. M. (1996). Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathologia mediterranea, 144-151.
• Gursoz, S., 1993. GAP Alanına Giren Güneydoğu Anadolu Bölgesi Bağcılığı ve Özellikle Şanlıurfa İlinde Yetiştirilen Üzüm Çeşitlerinin Ampelografik Nitelikleri İle Verim Ve Kalite Unsurlarının Belirlenmesi Üzerinde Bir Araştırma (Unpublished PhD Thesis). Çukurova University, Adana, Turkey.
• Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular biology and evolution, 33(7), 1870-1874.
• Lee, I. M., Bertaccini, A., Vibio, M., & Gundersen, D. E. (1995). Detection of multiple phytoplasmas in perennial fruit trees with decline symptoms in Italy. Phytopathology, 85(6), 728-735.
• Lee, I. M., Gundersen-Rindal, D. E., Davis, R. E., & Bartoszyk, I. M. (1998). Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. International Journal of Systematic and Evolutionary Microbiology, 48(4), 1153-1169.
• Lee, I. M., Gundersen-Rindal, D. E., Davis, R. E., Bottner, K. D., Marcone, C., & Seemüller, E. (2004). ‘Candidatus Phytoplasma asteris’, a novel phytoplasma taxon associated with aster yellows and related diseases. International journal of systematic and evolutionary microbiology, 54(4), 1037-1048.
• Pierro, R., Semeraro, T., Luvisi, A., Garg, H., Vergine, M., De Bellis, L., & Gill, H. K. (2019). The distribution of phytoplasmas in South and East Asia: an emerging threat to grapevine cultivation. Frontiers in plant science, 10, 1108.
• Quaglino, F., Zhao, Y., Casati, P., Bulgari, D., Bianco, P. A., Wei, W., & Davis, R. E. (2013). ‘Candidatus Phytoplasma solani’, a novel taxon associated with stolbur-and bois noir-related diseases of plants. International Journal of Systematic and Evolutionary Microbiology, 63(8), 2879-2894.
• Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic acids research, 25(24), 4876-4882.
• Zamharir, M. G., Paltrinieri, S., Hajivand, S., Taheri, M., & Bertaccini, A. (2017). Molecular identification of diverse ‘Candidatus Phytoplasma’species associated with grapevine decline in Iran. Journal of Phytopathology, 165(7-8), 407-413.
• Zhao, Y., Wei, W., Lee, M., Shao, J., Suo, X., & Davis, R. E. (2009). Construction of an interactive online phytoplasma classification tool, iPhyClassifier, and its application in analysis of the peach X-disease phytoplasma group (16SrIII). International Journal of Systematic and Evolutionary Microbiology, 59, 2582-2593.