Türkiye'den Domates Halkalı Leke Virüsü İzolatlarının Kısmi RdRp Genlerinin Moleküler Karakterizasyonu

Öz

Domates halkalı leke virüsü (ToRSV); Nepovirüs grubundan Secoviridae familyasında yer alan, iki genomik RNA (RNA-1 ve RNA-2) kombinasyonu ile oluşan izometrik yapıda ve tek sarmal pozitif duyarlı bir virüs olup, çeşitli konukçularda ekonomik olarak yıkıcı etkilere neden olmaktadır. Bu çalışmada, Eylül 2014 - Temmuz 2015 tarihleri arasında Hakkari ilinin üç farklı ilçesinden virüs benzeri belirtiler gösteren domates (Lycopersicon esculentum), biber Capsicum annuum), hıyar (Cucumis sativus) ve asma (Vitis vinifera) bitkilerinden 300 yaprak örneği toplanmıştır. Örnekler, RNA'ya bağlı RNA polimeraz (RdRp) geni için spesifik primerler kullanılarak Ters Transkripsiyon Polimeraz Zincir Reaksiyonu (RT-PCR) ile 411 bp boyutundaki DNA fragmanlarında ToRSV enfeksiyonu için test edilmiştir. 300 yaprak örneğinden yedisinin (% 2.3) ToRSV ile enfekte olduğu tespit edilmiştir. ToRSV, domates, biber ve hıyar örneklerinde tespit edilirken asma örneklerinde tespit edilmemiştir. Bu çalışma sonucunda elde edilen DNA dizileri GenBank'a yüklenmiştir (Erişim numaraları; KT728407, KT728408, KT728409 ve KT728410). Ülkemiz ToRSV izolatları GenBank'ta bulunan ToRSV izolatları ile karşılaştırılmış ve dört ToRSV izolatı RdRp gen sekansının analizi ile nükleotid ve amino asit benzerliğinin sırasıyla% 83-100 ve% 94-100 olduğu tespit edilmiştir. Filogenetik analiz, ülkemiz ve GenBank'ta mevcut bulunan ToRSV izolatlarının üç farklı gruba ayrıldığını göstermiştir. Yapılan bu çalışma, bilgimiz dahilinde, Türkiye'deki ToRSV'nin kısmi RdRp dizisinin ilk raporudur.

Anahtar Kelimeler

• Domates halkalı leke virüsü
• Kısmi RNA bağımlı RNA polimeraz geni
• Ters Kripsiyon Polimeraz Zincir Reaksiyonu (RT-PCR)
• Dizi analizi

Molecular Characterization of Partial RdRp Genes of Tomato Ringspot Virus Isolates from Turkey

Abstract

Tomato ringspot virus (ToRSV) is a virus having isometric structure and single helix positive sense and is formed by a combination of two genomic RNA (RNA-1 and RNA-2) in the Secoviridae family from the Nepovirus group, and it causes economically devastating diseases in a wide range of hosts. In this study, 300 leaf samples from tomato (Solanum lycopersicum), pepper (Capsicum annuum), cucumber (Cucumis sativus) and grapevine (Vitis vinifera) plants exhibiting virus-like symptoms were collected from three different districts of Hakkari province between September 2014 and July 2015. The samples were tested for ToRSV infection on DNA fragments of 411 bp in size by Reverse Transcription Polymerase Chain Reaction (RT-PCR), using specific primers for the RNA dependent RNA polymerase (RdRp) gene. Seven out of 300 leaf samples (2.3%) were found to be infected with ToRSV. The virus was detected in tomato, pepper and cucumber samples, but its presence was not so evident in grapevine plants. The DNA sequences were submitted to GenBank (Accession numbers; KT728407, KT728408, KT728409 and KT728410). Our ToRSV isolates were compared with the ones available in GenBank, and the analysis of four ToRSV isolate RdRp gene sequences revealed that the nucleotide and amino acid homology were 83-100% and 94‐100%, respectively. Phylogenetic analysis suggested that Turkish ToRSV isolates and currently available ToRSV isolates in GenBank were divided into three distinct groups. This analysis provides, to the best of our knowledge, the first report on the partial RdRp sequence of ToRSV in Turkey.

Keywords

• Tomato ringspot virus
• Partial RNA dependent RNA polymerase gene
• Reverse Transcription Polymerase Chain Reaction (RT-PCR)
• Sequence analysis

References

1. Abou Ghanem-Sabanadzovic, N., Choueiri, E., El Zammar, S., & Jreijiri, F. (2003). Viruses of Stone fruit trees in Lebanon, Options Méditerranéennes, Série B. Etudes et Recherches, 45, 25- 27.
2. Al-Nsour, A., Mansour, A., Al-Banna, L., & Salem, N. (2010). Detection of Tomato Ringspot Virus on Stone Fruit Trees in Jordan. Jordan Journal of Agricultural Sciences, 6(2), 240-246.
3. Bitterlin, M. W., Gonsalves, D., & Scorza, R. (1987). Improved mechanical transmission of tomato ringspot virus to Prunus seedlings. Phytopathology, 77, 560-563.
4. Braun, A. J., & Keplinger, J. A. (1973). Seed transmission of tomato ringspot virus in raspberry. Plant Disease Reporter, 57, 431-432.
5. Brown, D. J. F., Halbrendt, J. M., Jones A.T., Vrain T. C., & Robbins, R. T. (1994). Transmission of three North American nepoviruses by populations of fourdistinct species of the Xiphinema americanum groups. Phytopathology, 84, 646-649.
6. Coneva, E., Murphy, J. F., Boozer, R., & Velasquez, N. (2010). Incidence and Distribution of Viruses in Blackberry (Rubus sp.) in Alabama. International Journal of Fruit Science, 10, 87–95.
7. Elekçioğlu, D.H., Ohnesorge, B., Lung, G., & Uygun, N. (1994). Plant parasitic nematodes in the East Mediterranean region of Turkey. Nematol. Medit., 22, 59-63.
8. Farmahini, M., Pourrahim, R., Elahinia A., Rouhibakhsh, A., & Farzadfar, S. H. (2014). An Investigation of Apple Chlorotic Leaf Spot (ACLSV) and Tomato Ring Spot (ToRSV) in Some Iranian Pear Gardens. Iranian Journal of Virology, 8(1), 25-32.

9. Fidan, Ü. (1995). Virus diseases of vegetables in greenhouses in İzmir and Muğla. Journal of Turkish Phytopathology, 24(1), 7-14.
10. Fuchs, M., Abawi, G. S., Marsella-Herrick, P., Cox, R., Cox, K. D., Carroll, J. E., & Martin, R. R. (2010). Occurrence of tomato ringspot virus and tobacco ringspot virus in highbush blueberry in Newyork state. Journal of Plant Pathology, 92(2), 451-459.
11. Golnaraghi, A. R., Shahraeen, N., Pourrahim, R., Farzadfar, Sh., & Ghasemi, A. (2004). Occurrence and relative incidence of viruses infecting soybeans in Iran. Plant Disease, 88, 1069-74.
12. Gospodaryk, A., Moroèko-Bièevska, I., Pûpola, N., & Kâle, A. (2013). Occurence of Stone fruit viruses in plum orchards in Latvia. Proceedings of the Latvian Academy of Sciences. Section B, 67, 2, 683, pp. 116-123.
13. Jovel, J., Walker, M., & Sanfaçon, H. (2011). Salicylic Acid-Dependent Restriction of Tomato ringspot virus Spread in Tobacco Is Accompanied by a Hypersensitive Response, Local RNA Silencing, and Moderate Systemic Resistance. Molecular Plant-Microbe Interactions, 24(6), 706-718.
14. Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Molecular Evolution, 16 (2), 111-120.
15. Li, R., Mock, R., Fuchs, M., Halbrendt, J., Howell, B., & Liu, Z. R. (2011). Characterization of the partial RNA1 and RNA2 3’ Untranslated region of Tomato ringspot virusisolates from North America. Canadian Journal of Plant Pathology, 33, 94-99.
16. Malhotra, B., Onyilagha, J. C., Bohm, B. A., Towers, G. H. N., James, D., Harborne, J. B., & French, C. J. (1996). Inhibition of tomato ring spot virus by flavonoids. Phytochemistry, 43, 1271-1276.
17. Massumi, H., Shaabanian, M., Hosseini Pour , A., Heydarnejad, J., & Rahimian, H. (2009). Incidence of viruses infecting tomato and their natural hosts in the southeast and central regions of Iran. Plant Disease, 93: 67-72.
18. Matthews, R. E. F. (1992). Fundamentals of plant virology. Academic press, Inc. California, USA, 403, pp. 44.
19. Mathews, D. M. (2010). Optimizing detection and management of virus diseases of plants (1) and Emerging tree diseases in Southern California (2). In: Proceedings of the Landscape Disease Symposium, Camarillo, CA, pp. 10-20.
20. Moini, A. A. (2010). Identification of Tomato ringspot virus (ToRSV) on apple in Iran. Australasian Plant Disease Notes, 5: 105-106.
21. Moini, A. A., Roumi, V., Masoumi, M., & Izadpanah, K. (2010). Widespread occurrence of Tomato ring spot virus in deciduous fruit trees in Iran. Julius-Kühn-Archiv.,427: 127-28.
22. Onyilagha, J. C., Malhotra, B., Elder, M., French, J. C., & Towers, G. H. N. (1997). Comparative studies of inhibitory activities of chalcones on tomato ringspot virus (ToRSV). Canadian Journal of Plant Pathology, 19(2), 133-137.
23. Pinkerton, J. N., Kraus, .J, Martin, R. R., & Schreiner, R. P. (2008). Epidemiology of Xiphinema americanum and Tomato ringspot virus on red raspberry, Rubus idaeus. Plant Disease, 92, 364-371.
24. Pourrahim, R., Rakhshandehro, F, Farzadfar, Sh., & Golnaraghi, A. R. (2004). Natural occurrence of Tomato ringspot virus on grapevines in Iran. Plant Pathology, 53, 237.
25. Rott, M. E., Gilchrist, A., Lee, L., & Rochon, D. (1995). Nucleotide sequence of tomato ringspot virus RNA-1. Journal of General Virology, 76, 465-473.
26. Safaizadeh, M., & Saidi, A. (2011). First report of Tomato ringspot virus on tomato in Iran. Journal of Plant Pathology, 93(4), 74-78.
27. Sanfaçon, H., Zhang, G., Chisholm, J., Jafarpour, B., & Jovel, J. (2006). Molecular Biology of Tomato Ringspot Nepovirus, a Pathogen of Ornamentals, Small Fruits and Fruit Trees. In: Teixcira da Silva, J. (Ed.), Global Science Books, London, pp.540-546.
28. Sanfaçon, H., Wellink, J., Le Gall, O., Karasev, A., van der Vlugt, R., & Wetzel, T. (2009). Secoviridae: a proposed family of plant viruses within the order Picornaviralesthat combines the families Sequiviridae and Comoviridae, the unassigned genera Cheravirus and Sadwavirus, and the proposed genus Torradovirus. Archives of Virology, 154, 899-907.
29. Sattary, M., Rakhshandehroo, F., & Mozaffari, J. (2011). Detection of Tomato ringspot virus in rose and almond in Fars Province of Iran. APS 6-10 agust, Hawai.
30. Sertkaya, G. (2010). Tomato ringspot nepovirus (ToRSV) in wild blackberry (Rubus fruticosus L.) in Hatay province of Turkey. 21st International Conference on Virus and other Graft Transmissible Diseases of Fruit Crops.
31. Sertkaya G., Yıldırım, A. E., Uremis, I., & Sertkaya, E. (2013). Investigation of Some Nepoviruses in the Vineyard Areas of Hatay Province. Journal of MKU Faculty of Agriculture, 18(2), 39-46.
32. Sokhansanj ,Y., & Rakhshandehroo, F. (2012). First Report of Tomato ringspot virus Infecting Pepper in Iran. Disease Notes, 96(12), 1828.
33. Sokhansanj, Y., Rakhshandehroo, F., Pourrahim, R. (2012). Fırst Report of Tomato Ringspot Virus on Eggplant in Iran. Journal of Plant Pathology, 94(4), S4.94.
34. Stace-Smith, R. (1996). Tomato ringspot nepovirus. Viruses of Plants, CAB International, Wallingford (GB), pp. 1309-1312.
35. Tamura, K,. Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution, 30, 2725-2729.
36. Turhan, P., & Korkmaz, S. (2006). Determination of Tomato Spotted Wilt Virus in Çanakkale by Serological and Biological Methods. Ankara University, Faculty of Agriculture. Journal of Agricultural Sciences, 12( 2), 130-136.
37. Walker, M., Chisholm, J., Wei, T., Ghoshal, B., Saeed, H., Rott, M., & Sanfacon, H. (2015) Complete genome sequence of three tomato ringspot virus isolates: evidence for reassortment and recombination. Archives of Virology, 160,543-547.
38. Wang, A., & Sanfaçon, H. (2000). Diversity in the coding regions for the coat protein, VPg, protease, and putative RNA-dependent RNA polymerase among tomato ringspot nepovirus isolates. Canadian Journal of Plant Pathology, 22: 145-149.
39. Wei, T., & Clover, G. (2008). Use of primers with 5 non-complementary sequences in RT-PCR for the detection of nepovirus subgroups A and B. Journal of Virological Methods, 153, 16-21.
40. Yeşilçöllü, S., Gümüş, M., & Paylan, I. C. (2011). Studies on the Detection of Viruses in Strawberry Growing Areas in Aegean Region. Journal of Turkish Phytopathology, 40(1-3), 13-20.