Turunçgil Bitkilerinde Görülen Citlodavirus citri (CCDaV) ve Potexvirus citriflavivenae (CYVCV) Etmenlerinin Nitroselüloz ve Kaba Filtre Kağıtları Üzerindeki Kalıcılık Sürelerinin Araştırılması

Yıl 2025, Cilt: 40 Sayı: 2, 319 - 331, 30.12.2025

Nida Yalçın , Nuket Onelge

Öz

Bu araştırmada, turunçgil yetiştiriciliğinde ekonomik kayıplara yol açan önemli iki viral patojen olan Citlodavirus citri (CCDaV) ve Potexvirus citriflavivenae (CYVCV)'nin, nitroselüloz ve kaba filtre kağıtları üzerindeki canlılık ve tespit edilebilirlik süreleri karşılaştırmalı olarak incelenmiştir. Çalışmada virüsler, üç farklı yöntemle (CTAB protokolü ile ekstrakte edilmiş total nükleik asit/TNA, enfekteli bitki dokusunun direkt baskısı ve bitki öz suyunun damlatılması) membranlara aktarılmıştır. Beş hafta boyunca 7., 14., 21., 28. ve 35. günlerde RT-PCR/PCR ile yapılan analizlerde, CCDaV'nin TNA formunda her iki kağıt türünde de 35 gün boyunca stabil kaldığı tespit edilmiştir. CYVCV ise yalnızca TNA formunda benzer dayanıklılık göstermiş, diğer yöntemlerde özellikle kaba filtre kağıdında tespit süresi kısalmış veya hiç sonuç alınamamıştır. Mekanik inokulasyon testlerinde, CCDaV 21. güne kadar enfektivitesini korurken CYVCV hiçbir örnekte enfeksiyon oluşturamamıştır. Sonuçlar, özellikle nitroselüloz filtre kağıtlarının her iki virüsün de nükleik asit düzeyinde uzun süre saklanması için güvenilir bir ortam sağladığını göstermektedir. Bu bulgular filtre kağıtlarının, uzak mesafelere viral materyal gönderimi ve moleküler tanı laboratuvarları için sürekli kontrol materyali temininde pratik, ekonomik ve etkili bir araç olarak kullanılabileceğini ortaya koymaktadır.

Anahtar Kelimeler

CCDaV , CYVCV , PCR , Nitroselüloz filtre kağıdı , Kaba filtre kağıdı

Proje Numarası

FYL-2023-15726

Teşekkür

Çukurova Üniversitesi Bilimsel Araştırma Projeleri Birimine FYL-2023-15726 nolu projeye verdiği destekten dolayı teşekkür ederiz.

Investigation of the Persistence Periods of Citlodavirus citri (CCDaV) and Potexvirus citriflavivenae (CYVCV) Agents Present in Citrus Plants on Nitrocellulose and Coarse Filter Papers

Öz

This study comparatively investigated the stability and detection periods of two significant viral pathogens in citrus cultivation, Citlodavirus citri (CCDaV) and Potexvirus citriflavivenae (CYVCV), on nitrocellulose and coarse filter papers. The viruses were transferred to the membranes using three different methods: total nucleic acid (TNA) extracted via the CTAB protocol, direct tissue imprinting from infected plant material, and application of crude plant sap. Analysis via RT-PCR/PCR at 7, 14, 21, 28, and 35 days post-application revealed that CCDaV remained stable for 35 days on both paper types when applied as TNA. CYVCV showed similar durability only in TNA form, while its detection period shortened or yielded no results with other methods, particularly on coarse filter paper. In mechanical inoculation tests, CCDaV retained its infectivity up to 21 days, whereas CYVCV failed to induce infection in any sample. The results demonstrate that nitrocellulose filter papers provide a reliable medium for long-term preservation of both viruses at the nucleic acid level. These findings suggest that filter papers can serve as a practical, cost-effective, and efficient tool for shipping viral material over long distances and supplying consistent control materials for molecular diagnostics laboratories.

Anahtar Kelimeler

CCDaV , CYVCV , PCR , Nitrocellulose filter paper , Coarse filter paper

Proje Numarası

FYL-2023-15726

Teşekkür

We would like to thank the Cukurova University Scientific Research Projects Unit for their support of the project numbered FYL-2023-15726.

Kaynakça

• Ádám J., Stefanova B., Dragoyski K., Tóbiá I., Palkovics L., 2015. Use of FTA® (Whatman) membrane for collection and detection of Bulgarian PPV isolates. Journal of Mountain Agriculture on the Balkans, 18(2): 334-343.
• Afloukou F.B., Önelge N., 2020. Alternative hosts, vectors transmission, and molecular characterization of Citrus yellow vein clearing virus (CYVCV) isolates. PhD thesis, Çukurova University, Adana, 91 s.
• Ahlawat, Y.S., 1997. Virus, greening bacterium and viroids associated with citrus (Citrus species) decline in India. Indian J. Agric. Sci., 67: 51 – 57.
• Blab Market, 2023. Süzgeç kağıdı (filtre kağıdı) nedir? [online] Erişim adresi: https://www.blabmarket.com/blog/icerik/suzgec-kagidi-nedir-ve-nasil-kullanilir [Erişim tarihi: 14.10.2023]
• Catara A., Azzaro A., Davino M., Polizzi G., 1993. Yellow vein clearing of lemon in Pakistan. In: Proceedings of the 12th International Conference on IOCV, Riverside, CA, 364–367.
• Chandrashekara K.N., Prasannakumar M.K., Deepa M., Vani A., 2012. A rapid, sensitive and reliable method for detecting Ralstonia solanacearum using FTA (Whatman) card. Journal of Plant Pathology, 94(1): 219-221.
• Chen H., Zhou Y., Wang X., Zhou C., Yang X., Li Z., 2016. Detection of Citrus yellow vein clearing virus by real-time RT-PCR. Horticultural Plant Journal, 2(4): 188-192. https://doi.org/10.1016/j.hpj.2016.07.001 [Erişim tarihi: 27.05.2024]
• Correll J.C., Puhalla J.E., Schneider R.W., 1986. Vegetative compatibility groups among nonpathogenic root-colonizing strains of Fusarium oxysporum. Canadian Journal of Botany, 64: 2358-2361.
• Çınar A., Kersting U., Önelge N., Korkmaz S., Şaş G., 1993. Citrus virus and virus-like disease in the eastern Mediterranean region of Turkey. In: Moreno P., da Graça J.V., Timmer L.W. (Eds.), Proceedings of the 12th Conference of International Organization of Citrus Virologists (IOCV), Riverside, 397-400.
• Guo J., Lai X.P., Li J.X., Yue J.Q., Zhang S.Y., Li Y.Y., Gao J.Y., Wang Z.R., Duan H.F., Yang J.D., 2015. First report on Citrus chlorotic dwarf associated virus on lemon in Dehong Prefecture, Yunnan, China. Plant Disease, 99(9). https://doi.org/10.1094/PDIS-01-15-0011-PDN [Erişim tarihi: 11.07.2023]
• Interlab Laboratuvar Ürünleri., 2023. ISOLAB TR Catalog 2023-2025. [online] Erişim adresi: https://katalog.interlab.com.tr/#114 [Erişim tarihi: 01.10.2023]
• Korkmaz S., Çınar A., Bozan O., Kersting U., 1994. Distribution and natural transmission of a new whitefly-borne virus disease of citrus in the eastern Mediterranean region of Turkey. In: Proceedings of the 9th Congress of the Mediterranean Phytopathological Union, 437-439.
• Korkmaz S., 2002. Application of direct tissue blot immunoassay in comparison with DAS-ELISA for detection of Turkish isolates of Citrus tristeza closterovirus (CTV). Turkish Journal of Agriculture and Forestry, 26(4): 209-216. https://journals.tubitak.gov.tr/agriculture/vol26/iss4/5 [Erişim tarihi: 14.12.2023]
• Loconsole G., Saldarelli P., Doddapaneni H., Savino V., Martelli G.P., Saponari M., 2012a. Identification of a single-stranded DNA virus associated with citrus chlorotic dwarf disease, a new member in the family Geminiviridae. Virology, 432(1): 162-172. https://doi.org/10.1016/j.virol.2012.06.005 [Erişim tarihi: 28.01.2023]
• Loconsole G., Önelge N., Potere O., Giampetruzzi A., Bozan O., Satar S., Stradis A. DE, Savino V., Yokomi R.K., Saponari M., 2012b. Identification and characterization of Citrus yellow vein clearing virus, a putative new member of the genus Mandarivirus. Phytopathology, 102(12): 1168-1175. https://doi.org/10.1094/PHYTO-06-12-0140-R [Erişim tarihi: 28.01.2023]
• Mahrous R.M., Moustafa T.A., Farag A.Z., Sadik A.S., El-Afifi S.I., Gamal El-Din A.S., 2005. Isolation and identification of Spiroplasma citri using some sensitive methods. Arab University Journal of Agricultural Sciences, 13(3): 625-639. https://doi.org/10.21608/ajs.2005.15263 [Erişim tarihi: 03.03.2024]
• Michaud V., Gil P., Kwiatek O., Prome S., Dixon L., Romero L., Le Potier M.F., Arias M., Couacy-Hymann E., Roger F., Libeau G., Albina E., 2007. Long-term storage at tropical temperature of dried-blood filter papers for detection and genotyping of RNA and DNA viruses by direct PCR. Journal of Virological Methods, 146(1-2): 257-265. https://doi.org/10.1016/j.jviromet.2007.07.006 [Erişim tarihi: 02.02.2024]
• Murray M.G., Thompson W.F., 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 8: 4321-4325.
• Ndunguru J., Taylor N.J., Yadav J., Aly H., Legg J.P., Aveling T., Thompson G., Fauquet C.M., 2005. Application of FTA technology for sampling, recovery and molecular characterization of viral pathogens and virus-derived transgenes from plant tissues. Virology Journal, 2: 45. https://doi.org/10.1186/1743-422X-2-45 [Erişim tarihi: 02.02.2024]
• Önelge N., 1997. Direct nucleotide sequencing of Citrus exocortis viroid (CEV). Turkish Journal of Agriculture and Forestry, 21: 419-422.
• Önelge N., 2003. Türkiye’de limonlarda sarı damar açılması ile ilgili ilk rapor. Journal of Turkish Phytopathology, 32(1): 53-55.
• Önelge N., Satar S., Elibüyük O., Bozan O., Kamberoğlu M., 2011. Transmission studies on Citrus yellow vein clearing virus. In: 18th Conference of the International Organization of Citrus Virologists, Brazil, 11-14.
• Richert-Pöggeler K.R., Minarovits J., 2014. Diversity of latent plant-virus interactions and their impact on the virosphere. Plant Virus Host Interactions, 14: 263-275. https://doi.org/10.1016/B978-0-12-411584-2.00014-7 [Erişim tarihi: 19.01.2024]
• Roistacher C.N., 1995. A historical review of the major graft-transmissible diseases of citrus. FAO, Rome, 89 p.
• Roossinck M.J., 2005. Symbiosis versus competition in plant virus evolution. Nature Reviews Microbiology, 3: 917-924. https://doi.org/10.1038/nrmicro1285 [Erişim tarihi: 19.01.2024]
• Roossinck M.J., 2010. Lifestyles of plant viruses. Philosophical Transactions of the Royal Society B: Biological Sciences, 365: 1899-1905. https://doi.org/10.1098/rstb.2010.0057 [Erişim tarihi: 22.05.2024]
• Roy Y., Nassuth A., 2005. Detection of plant genes, gene expression and viral RNA from tissue prints on FTA cards. Plant Molecular Biology Reporter, 23(4): 383. https://doi.org/10.1007/BF02788886 [Erişim Tarihi: 14.11.2024]
• Smit P.W., Elliott I., Peeling R.W., Mabey D., Newton P.N., 2014. An overview of the clinical use of filter paper in the diagnosis of tropical diseases. American Journal of Tropical Medicine and Hygiene, 90(2): 195-210. https://doi.org/10.4269/ajtmh.13-0463 [Erişim tarihi: 22.12.2023]
• Tanaka S., Nishii H., Ito S., Kameya-Iwaki M., Sommartya V.E.P., 1997. Detection of Cymbidium mosaic potexvirus and Odontoglossum ringspot tobamovirus from Thai orchids by rapid immunofilter paper assay. Plant Disease, 81(2): 167-170. https://doi.org/10.1094/PDIS.1997.81.2.167 [Erişim tarihi: 30.03.2024]
• Tarım ve Orman Bakanlığı TAGEM., 2017. Turunçgil entegre mücadele teknik talimatı, Ankara. [online] Erişim adresi: https://www.tarimorman.gov.tr/TAGEM/Belgeler/yayin/Turun%C3%A7gil%20Entegre-29.08.2017.pdf [Erişim tarihi: 21.06.2023]
• Tarım ve Orman Bakanlığı TEPGE., 2024. Turunçgiller ürün raporu, Ankara. [online] Erişim adresi: https://arastirma.tarimorman.gov.tr/tepge/Belgeler/PDF%20%C3%9Cr%C3%BCn%20Raporlar%C4%B1/2024%20%C3%9Cr%C3%BCn%20Raporlar%C4%B1/Turun%C3%A7giller%20%C3%9Cr%C3%BCn%20Raporu%202024-408%20TEPGE.pdf [Erişim tarihi: 28.01.2025]
• Yang Z., Zhang L., Zhao J., Li T., Liu Q., Cao M., Zhou V., 2020. First report of Citrus chlorotic dwarf-associated virus on pomelo in Nakhon, Thailand. Plant Disease, 104(4). https://doi.org/10.1094/PDIS-10-19-2093-PDN [Erişim tarihi: 14.12.2023]