Domates Bitkisinde Pospiviroid Cinsi Viroid Etmenlerinin Varlığının Araştırılması ve Moleküler Karakterizasyonu*

Year 2016, Volume: 31 Issue: 2, 27 - 34, 29.07.2016

Ruşen Kışlak Nüket Önelge

Abstract

Çalışma domates bitkisinde Pospiviroid cinsi viroidlerin varlığının araştırılması için yürütülmüştür. 2014-2015 arasında, 400 adet domates tohum, fide ile yaprak örneği toplanmış ve total nükleik asit ekstraktları elde edilmiştir. Pospi1-FW/RE ve Vid-FW/RE primerleri ile yürütülen RT-PCR çalışmalarında, 398 adet domates örneğinin bir viroidle bulaşık olmadığı görülmüştür. ‘’Çeri‘’ çeşidi iki domates örneğinin ise Citrus exocortis viroid (CEVd) etmeni ile bulaşık olduğu, CEVd-FW/RE primer çifti ile belirlenmiştir. Agaroz jel elektroforezi çalışmalarında bu örneklere ait 371 bp’lik DNA bantları görüntülenmiş ve CEVd ile enfekteli izolatlar moleküler açıdan da karakterize edilmiştir. Nükleotid dizilimi analizi sonuçları NCBI gen bankasına bildirilen domates, turunçgil ve diğer izolatlara %89-98 sekans benzerliği göstermiştir.

Keywords

Viroid, domates, Pospiviroid, RT-PCR, mekanik inokulasyon

Investigation and Molecular Characterization of Viroid Agents Existance at Pospiviroid Genus on Tomato Plant

Abstract

Study was conducted to investigate viroids’ existance at Pospiviroid genus on tomato plant. Between 2014-2015, 400 tomato seeds, seedlings and leaves samples were collected and total nucleic acid extracts of samples were obtained. In RT-PCR studies which were carried out by Pospi1-FW/RE and Vid-FW/RE primer sets, any viroid infection was detected in 398 tomato samples. Two tomato samples on ‘’Çeri‘’ cultivar, found infected with Citrus exocortis viroid (CEVd) by CEVd-FW/RE primer pair. 371 bp of DNA bands were shown belong to samples in agarose gel electrophoresis studies. CEVd infected isolates were also characterized molecularly. Nucleotid aligments analysis showed %89-98 sequence similiarity to some tomato, citrus and other isolates reported to NCBI Genbank database.

Keywords

Viroid, tomato, Pospiviroid, RT-PCR, mechanical inoculation

References

• Bora, T., Karaca, İ. (1970) Bitki hastalıklarının sörveyi: Kültür Bitkilerinde Hastalığın ve Zararın Ölçülmesi. 167:8-43, Ege Üni. Matbaası, İzmir.
• Diener, T. O. (1987) The Viroids. Plenum Publishing Corporation, USA.
• Fagoaga, C., Duran-Vila, N. (1996) Naturally occurring variants of Citrus exocortis viroid in vegetables. Plant Path. 45:45-53.
• FAO (2013) Faostat Agricultural Database. www.faostat.org. Accessed: 17.02.2015.
• Flores, R., Randles, J. W., Bar-Joseph, M., Diener, T. O. (2005) A proposed scheme for viroid classification and nomenclature. Archives of Virology 143(3):623-629.
• Flores, R., Grubb, D., Elleuch, A., Nohales, M. A., Delgado, S., Gago, S. (2011) Rolling-circle replication of viroids, viroid-like satellite RNAs and hepatitis delta virus: Variations on a Theme.
• Galindo, J., Smith, D. R., Diener, T. O. (1982) Etiology of planta macho, a viroid disease of tomato. Phytopatology 72:49-54.
• Hammond, R. W., Smith, D. R., Diener, T. O. (1989) Nucleotid sequence and proposed secondary structure of Columnea latent viroid (CLVd): A natural mosaic of viroid sequences. Nucleic Acids Research 17:10083-10094.
• Leontyeva, J. A. (1980) Tomatoes and Potato spindle tuber viroid. Zashc. Raste. 8: 22.
• Ling, K. S., Bledsoe, M. E. (2008) First report of Mexican papita viroid (MPVd) infecting greenhouse tomato in Canada. Plant Disease 93(8):839.
• Mishra, M. D., Hammond, R. W., Owens, R. A., Smith, D. R., Diener, T. O. (1991) Indian bunchy top disease of tomato plants is caused by a distinct strain of Citrus exocortis viroid. J. Gen. Virol. 72:1-5.
• Mongera, W., Tomlinsona, J., Boonhama, N., Marnb, M. V., Pleskob, I. M., Molinero-Demillyc, V., Tassus, X., Meekesd, E., Toonend, M., Papayiannise, L. (2010) Development and inter-laboratory evaluation of real-time PCR assays for the detection of pospiviroids. Journal of Virological Methods 169:207-210.
• Murray, M. G., Thompson, W. F. (1980) Rapid isolation of high molecular weight plant DNA. Nuc. Acids Res. 8(19):4321-4325.
• Önelge, N. (1997) Direct nucleotide sequencing of Citrus exocortis viroid (CEVd). Turk. J. Agric. For. 21:419-422.
• Puchta, H., Herold, T., Verhoeven, K., Roenhorst, A., Ramm, K., Schmidt-Puchta, W., Sanger, H. L. (1990) A new strain of Potato spindle tuber viroid (PSTVd) exhibits major sequence differences as compared to all other strains sequenced so far. Plant Molecular Biology 15:509-511.
• Reanwarakorn, K., Klinkong, S., Porsoongnurn, J. (2011) First report of natural infection of Pepper chat fruit viroid in tomato plants in Thailand. New Dis. Rep. 24:6.
• Singh, R. P., Boucher, A., Somerville, T. H. (1992) Detection of Potato spindle tuber viroid in the pollen and various parts of potato plant pollinated with viroid-infected pollen. Plant Dis. 76(9):951-953.
• Singh, R. P., Nie, X., Singh, M. (1999) Tomato chlorotic dwarf viroid (TCDVd): An evolutionary link in the origin of pospiviroids. J.Gen. Virol. 80:2823-2828.
• Şeniz, V. (1992) Domates, Biber ve Patlıcan Yetiştiriciliği. Tarımsal Araştırmaları Destekleme ve Geliştirme Vakfı, İstanbul.
• Verhoeven, J. TH. J., Jansen, C. C. C., Willemen, T. M., Kox, L. F. F., Owens, R. A., Roenhorst, J. W. (2004) Natural infections of tomato by Citrus exocortis viroid , Columnea latent viroid, Potato spindle tuber viroid and Tomato chlorotic dwarf viroid. Eur. J. Plant Pathol. 110:823-831.
• Walter, B., Thouvenal, J. C., Fauquet, C. (1980) Les viruses de la tomate en Cote d’Ivore. Annales de Phytopathologie 12:259-275.
• Walter, B. (1987) Tomato apical stunt: The Viroids. T. O. Diener (Ed.), 321-328, Plenum Publishing Corporation, USA.