Peach latent mosaic viroid (PLMVd) ve Hop stunt viroid (HSVd)’in farklı ekstraksiyon metotları kullanılarak RT-PCR ile teşhisi

Year 2023, , 40 - 44, 31.10.2023

Kamil Duman , Mustafa Gümüş

https://doi.org/10.16955/bitkorb.1239183

Abstract

Sert çekirdekli meyve ağaçlarında ekonomik zarara neden olan peach latent mosaic viroid (PLMVd) ve hop stunt viroid (HSVd)’leri RT-PCR ile tanılamak için 4 farklı nükleik asit ekstraksiyon yöntemi kullanılmıştır. Yaprak örnekleri İzmir ilinde farklı meyve bahçelerinden viroid benzeri belirti gösteren ağaçlardan alınmıştır. Silica capture, lithium chloride, citric buffer ve ames buffer yöntemleri kullanılarak total nükleik asit ekstraksiyonu gerçekleştirilmiştir. Dört farklı ekstraksiyon yöntemi erik, kayısı ve şeftali ağaçlarından farklı dönemlerde toplanan örnekler kullanılarak birbiri ile karşılaştırılmıştır. Altmış dört farklı sert çekirdekli bitki örneği RT-PCR test yöntemi ile değerlendirilmiş ve silica capture ekstraksiyon yöntemi, kullanılan yöntemler arasında en güvenilir sonucu vermiştir. Diğer 3 ekstraksiyon yöntemi ile yapılan ekstraksiyonlarda istenilen sonuç alınamamıştır.

Keywords

TNA ekstraksiyonu, RT-PCR, viroid, sert çekirdekli meyve, nükleik asit

Supporting Institution

Ege Üniversitesi Fen Bilimleri Enstitüsü

Thanks

Yüksek Lisans Tez çalışmam esnasında bilgi ve desteklerini esirgemeyen sayın tez danışmanım Prof. Dr. Mustafa GÜMÜŞ'e, saygıdeğer ağabeyim Doç. Dr. İsmail Can PAYLAN'a ve tezimin makaleye dönüşmesinde bilgi ve tecrübelerini esirgemeyen saygıdeğer büyüğüm Doç Dr. Birol AKBAŞ'a teşekkür ederim.

Diagnosis of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) by RT-PCR using different extraction protocols

Abstract

RT-PCR method was performed using four different nucleic acid extraction methods to identify viroids; peach latent mosaic viroid (PLMVd) and hop stunt viroid (HSVd) which causes serious damage to stone fruits. Leaf samples were collected from fruit orchards showing viroid-like symptoms in İzmir province. Silica capture, citric buffer, lithium chloride, and ames buffer methods were used to extract total nucleic acids. The four extraction methods were compared using samples collected during the vegetation period from naturally infected trees (plum, apricot, and peach). They were evaluated with RT-PCR tests. In 64 stone fruit tree samples, only the silica capture method gave reliable and accurate results in RT-PCR molecular tests for the detection of PLMVd and HSVd. The other wielded nucleic acid extraction methods were found to be ineffective for the isolation of the viroid RNAs.

Keywords

TNA extraction, RT-PCR, viroid, stone fruit, nucleic acid

References

• Akbas B., Degirmenci K., 2010. Simultaneous detection of Apple mosaic virus in cultivated hazelnuts by one-tube RT-PCR. African Journal of Biotechnology, 9 (12), 10.5897/AJB10.1749
• Astruc N., Marcos J.F., Macquaire G., Candresse T., Pallás V., 1996. Studies on the diagnosis of hop stunt viroid in fruit trees: Identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. European Journal of Plant Pathology, 102, 837-846.
• Candresse T., Lanneau T., Revers F., Grasseau N., Macquaire G., German S., Malinowsky T., Dunez J., 1995. An immunocapture PCR assay adapted to the detection and the analysis of the molecular variability of apple chlorotic leaf spot virus. Acta Horticulturae, 386, 136-147.
• Cieslinska M., 2004. Detection of strawberry mottle virus (SMoV) using RT-PCR: comparison of two RNA extraction methods. Journal of Fruit and Ornamental Plant Research, 12, 17-22.
• Demeke T., Adams R.P., 1992. The effects of plant polysaccharides and buffer additives on PCR. Biotechniques, 12 (3), 332-334.
• Diener T.O., Lawson R.H., 1973. Chrysanthemum stunt: a viroid disease. Virology, 51 (1), 94-101.
• Doyle J.J., Doyle J.L., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, 19, 11-15.
• Flores R., Delgado S., Gas M.E., Carbonell A., Molina D., Gago S., De La Pena M., 2004. Viroids: The minimal non-coding RNAs with autonomous replication. FEBS Letter, 567 (1), 42-48.
• Foissac X., Savalle-Dumas L., Gentit P., Dulucq M.J., Candresse T., 2000. Polyvalent detection of fruit tree Tricho, Capillo and Favea viruses by nested RT-PCR using degenerated and inosine-containing primers (PDORT-PCR). Acta Horticulturae, 357, 52–59.
• Góra-Sochacka A., 2004. Viroids; unusual small pathogenic RNAs. Acta Biochimica Polonica, 51 (3), 587-607.
• Hataya T., Nakahara K., Furuta K., Shikata E., 1999. Comparisons of gene diagnostic methods for the practical diagnosis of chrysanthemum stunt viroid in chrysanthemum plants. Archives of Phytopathology and Plant Protection, 32 (3), 179–192.
• Hataya T., Tsushima T., Sano T., 2017. Hop stunt viroid. In: Viroids and satellites. Hadidi, A., Flores, R., Randles J.W., Paukaitis, P. (Eds.). Oxford, UK: Academic Press, 199-210 pp.
• Hughes D.W., Galau G., 1988. Preparation of RNA from cotton leaves and pollen. Plant Molecular Biology Reporter, 6, 253-257.
• Korschineck I., Himmler G., Sagl R., Steinkellner H., Katinger H.W., 1991. A PCR membrane spot assay for the detection of plum pox virus RNA in bark of infected trees. Journal of Virological Methods, 31 (2-3), 139-145.
• Laulhere J-P., Rozier C., 1976. One-step extraction of plant nucleic acids, Plant Science Letters, 6 (4), 237-242.
• Loreti S., Faggioli F., Cardoni M., Mordenti G., Babini A.R., Poggi Pollini C., Barba M., 1999. Comparison of different diagnostic methods for detection of peach latent mosaic viroid. EPPO Bulletin, 29 (4), 433 – 438.
• Martelli G.P., Candresse T., Namba S., 1994. Trichovirus, a new genus of plant viruses. Archives of Virology, 134, 451-455.
• Navarro J.A, Vera A., Flores R., 2000. A chloroplastic RNA polymerase resistant to tagetitoxin is involved in replication of avocado sunblotch viroid. Virology, 268 (1), 218-225. doi 10.1006/viro.1999.0161.
• Podleckis E.V., Hammond R.W., Hurtt S.S., Hadidi A., 1993. Chemiluminescent detection of potato and pome fruit viroids by digoxigenin-labeled dot-blot and tissue blot hybridization. Journal of Virological Methods, 43, 147-158.
• Ragozzino E., Faggioli F., Alioto D., Barba M., 2003. Detection and differentiation of peach latent mosaic viroid and hop stunt viroid in stone fruit trees in Italy using multiplex RT-PCR. Phytopathologia Mediterranea, 42 (1), 79-84.
• Ragozzino E., Faggioli F., Barba M., 2004. Development of a one tube-one step RT-PCR protocol for the detection of seven viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid, and Pospiviroid. Journal of Virological Methods, 121 (1), 25-29.
• Rott M.E., Jelkmann W., 2001. Characterization and detection of several filamentous viruses of cherry: adaptation of an alternative cloning method (DOP-PCR), and modification of an RNA extraction protocol. European Journal of Plant Pathology, 107, 411-420.
• Schepetiuk S., Kok T., Martin L., Waddell R., Higgins G., 1997. Detection of Chlamydia trachomatis in urine samples by nucleic acid tests: comparison with culture and enzyme immunoassay of genital swab specimens. Journal of Clinical Microbiology, 35 (12), 3355-3357.
• Shamloul A.M., Faggioli F., Keith J.M., Hadidi A., 2002. A novel multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA) for simultaneous detection of six viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid, and Pospiviroid. Journal of Virological Methods, 105 (1), 115-121.
• Sipahioglu H.M., Usta M., Ocak M., 2006a. Use of dried high-phenolic laden host leaves for virus and viroid preservation and detection by PCR methods. Journal of Virological Methods, 137 (1), 120-124.
• Sipahioğlu H.M., Demir S., Myrta A., Al Rwahnih M., Polat B., Schena L., Usta M., Akkopru A., Selcuk M., Ippolito A., Minafra A., 2006b. Viroid, phytoplasma, and fungal diseases of stone fruit in eastern Anatolia, Turkey. New Zealand Journal of Crop and Horticultural Science, 34, 1, 1-6, doi: 10.1080/01140671.2006.9514380
• Sipahioglu H.M., Ocak M., Usta M., 2007. Comparison of three conventional extraction methods for the detection of plant virus/viroid RNAs from heat dried high-phenolic host leaves. Asian Journal of Plant Sciences, 6 (1), 102-107.
• TÜİK, 2019. Bitkisel Üretim İstatistikleri. Turkiye İstatistik Kurumu (TUİK), (http://www.tuik.gov.tr/). (accessed date: 09.09.2023).
• Wetzel T., Candresse T., Macquaire G., Ravelonandro M., Dunez J., 1992. A highly sensitive immunocapture polymerase chain reaction method for plum pox potyvirus detection. Journal of Virological Methods, 39 (1-2), 27-37.
• Wilde J., Eiden J., Yolken R., 1990. Removal of inhibitory substances from human fecal specimen for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions. Journal of Clinical Microbiology, 28, 1300-1307.
• Zacharzewska B., Przewodowska A., Treder K., 2014. The adaptation of silica capture RT-PCR for the detection of Potato Virus Y. American Journal of Potato Research, 91, 525–531. https://doi.org/10.1007/s12230-014-9383-y