Marmara Bölgesi domates üretim alanlarında Cucumber mosaic virus izolatlarının yaygınlığı ve moleküler karakterizasyonu

Öz

Türkiye’de domates virüsleri ile ilgili olarak gerçekleştirilmiş çok sayıda çalışma vardır. Ancak domatesin en önemli virüs hastalıklarından bir tanesi olan Cucumber mosaic virus (CMV)’nin geniş alanlarda yaygınlığı ve genetik çeşitliliği üzerine gerçekleştirilmiş bir çalışma bulunmamaktadır. Bu bağlamda Marmara Bölgesi domates üretim alanlarında sürveyler düzenlenerek virüs ve virüs benzeri simptom gösteren 113 domates bitkisinden örnekler alınarak DAS-ELISA testi ile CMV enfeksiyonunun belirlenmesi amacı ile testlemeler gerçekleştirilmiştir. Gerçekleştirilen testlemeler sonucunda 34 bitkide CMV enfeksiyonu tespit edilmiştir. Enfekteli örnekler içerisinden elde edildikleri iller temel alınarak 10 CMV izolatı moleküler karakterizasyon çalışmaları için seçilmiştir. Seçilen bu izolatların kılıf protein (CP) ve hareket protein (MP) genleri RT-PCR ile amplifiye edilmiş ve sekanslanmıştır. Elde edilen sekanslar kullanılarak gerçekleştirilen çoklu dizi analiz çalışmaları sonucunda, Marmara Bölgesi CMV izolatlarının CP genine göre kendi içlerinde nükleotid ve amino asit düzeyinde %97-100, dünya izolatları ile nükleotid düzeyinde %77-100, amino asit düzeyinde ise %79-100 oranlarında benzerlikler gösterdiği tespit edilmiştir. MP gen bölgesine göre ise CMV izolatlarının kendi içlerinde nükleotid düzeyinde %97-100, amino asit düzeyinde ise %96-100 oranlarında benzerlikler gösterdiği belirlenmiştir, dünya izolatları ile gösterdikleri benzerlik oranları ise nükleotid düzeyinde %79-100, amino asit düzeyinde ise %81-100 olarak belirlenmiştir. Gerçekleştirilen filogenetik analizler sonucunda Marmara Bölgesi CMV izolatlarının hem CP hem de MP gen bölgesine göre birbirleri ile yakın ilişkili olduğu ve Ia altgrubunda olduğu belirlenmiştir.

Anahtar Kelimeler

• CMV
• DAS-ELISA
• RT-PCR
• sekanslama

Prevalence and molecular characterization of Cucumber mosaic virus isolates infecting tomato plants in Marmara region of Turkey

Abstract

There are many studies carried out on tomato viruses in Turkey. However, there is no study on the prevalence and genetic diversity of Cucumber mosaic virus (CMV), one of the most important viral diseases of tomatoes. In this context, surveys were conducted in the tomato production areas of the Marmara region, and samples were taken from 113 tomato plants showing virus and virus-like symptoms, and tests were carried out to determine CMV infection by DAS-ELISA. As a result of the tests, CMV infection was detected in 34 plants. And, 10 CMV isolates were selected for further studies. Coat protein (CP) and movement protein (MP) genes of selected isolates were amplified by RT-PCR and sequenced. As a result of multiple sequence analysis, CMV isolates from the Marmara region showed 97-100% similarity in nucleotide and amino acid levels within themselves, 77-100% in nucleotide level, and 79-100% in amino acid level in the world isolates according to the CP gene. According to the MP gene region, it was determined that the CMV isolates showed 97-100% and 96-100% similarities at the nucleotide and amino acid levels in each other, respectively. The similarity rates with world isolates were determined as 79-100% at the nucleotide level and 81-100% at the amino acid level. As a result of the phylogenetic analyses performed, tomato CMV isolates were closely related to each other according to both gene regions and were in the Ia subgroup.

Keywords

• CMV
• DAS-ELISA
• RT-PCR
• sequencing

Kaynakça

1. Balsak S. C., Kardeş H., Buzkan N., 2021. First report of cucumber mosaic virus in cyclamen (Cyclamen spp.) in Turkey. Journal of Plant Pathology, 103 (1), 361-362.
2. Clark M. F., Adams A. N., 1977. Characteristics of the microplate method of enzyme-linked immuno sorbent assay for the detection of plant viruses. Journal of General Virology, 34 (3), 475-483.
3. Çağlar B. K. 2006. Characterization of melon (CMV-K), tomato (CMV-D), pepper (CMV-B) isolates of cucumber mosaic virus (CMV) by biological, serological, molecular methods and effect of satellite-RNAs on virus. Çukurova University, PhD Thesis, 89p, Adana.
4. Çevik B., Erkıș, G. 2007. First report of tomato chlorosis virus in Turkey. Plant Pathology, 57 (4), 767.
5. Çulal-Kılıç H., Isparta L., Yardımcı N., Doğan K. 2017. Diagnosis of tomato spotted wilt virus (TSWV) in tomatoes grown areas in Isparta and Burdur Provinces, Turkey. The Journal of Graduate School of Natural and Applied Sciences of Mehmet Akif Ersoy University, 8 (1), 34-39.
6. Ergün M., Erkan S., Paylan İ.C. 2013. Cucumber mosaic virus in globe artichoke in Turkey. Canadian Journal of Plant Pathology, 35 (4), 514-517.
7. Fidan H., Sarikaya P., Calis O. (2019). First report of tomato brown rugose fruit virus on tomato in Turkey. New Disease Reports, 39, 18.
8. Güneş N., Gümüş M. 2019. Detection and characterization of tomato spotted wilt virus and cucumber mosaic virus on pepper growing areas in Antalya. Journal of Agricultural Sciences, 25 (3), 259-271.

9. Jacquemond M. 2012. Cucumber mosaic virus. Advances in Virus Research, 84, 439-504.
10. Karanfil A., Korkmaz S. 2017. Detection and molecular characterization based on coat protein gene of Cucumber mosaic virus (CMV) from cowpea production fields of Çanakkale province in Turkey. Plant Protection Bulletin, 57 (3), 293-304.
11. Karanfil A., Korkmaz S. 2021. Detection and phylogenetic analysis of coat protein gene region of cucumber mosaic virus Infecting cucurbit plants in South Marmara Region of Turkey. Journal of Agriculture Faculty of Ege University, 58 (2), 31-40.
12. Korkmaz S., Karanfil A. 2020. Genetic diversity of cucumber mosaic virus isolates infecting cucurbit plants from North-West Anatolia Region in Turkey. Project Result Report, COMU-FBA-2019-2873.
13. Kurtoğlu A., Korkmaz S. 2018. Determination and molecular characterization of cucumber mosaic virus (CMV) infection on spinach production fields of Çanakkale province, Turkey. The Journal of Turkish Phytopathology, 47 (2), 43-51.
14. Li R., Mock R., Huang Q., Abad J., Hartung J., Kinard G. 2008. A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens. Journal of Virological Methods, 154, 48–55.
15. Muhire B. M., Varsani A., Martin D. P. 2014. SDT: A virus classification tool based on pairwise sequence alignment and identity calculation. PLoS One, 9, 0108277.
16. Usta M., Güller A., Günay A. 2020. The molecular characterization of the coat protein sequence and differentiation of CMV-subgroup I on tobacco from native flora in Turkey. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 48 (2), 523-534.
17. Ohshima K., Matsumoto K., Yasaka R., Nishiyama M., Soejima K., Korkmaz S., Ho S. Y. W., Gibbs A. J., Takeshita M. 2016. Temporal analysis of reassortment and molecular evolution of cucumber mosaic virus: Extra clues from its segmented genome. Virology, 487, 188–197.
18. Özdemir S., Erilmez S. 2012. First report of Alfalfa mosaic virus and Cucumber mosaic virus in pepino in Turkey. Journal of Plant Pathology, 94 (4), 91.
19. Palukaitis P., Garcia-Arenal F. 2003. Cucumoviruses. Advances in Virus Research, 62, 41-323.
20. Palukaitis P., Roossinck M. J., Dietzgen R.G., Francki R. I. 1992. Cucumber mosaic virus. Advances in Virus Research, 41, 281-348.
21. Roossinck M. J., Zhang L., Hellwald K. H. 1999. Rearrangements in the 5' nontranslated region and phylogenetic analyses of Cucumber mosaic virus RNA 3 indicate radial evolution of three subgroups. Journal of Virology, 73, 6752-6758.
22. Sarı S. 2015. Characterization of Cucumber mosaic virus (CMV) isolates infecting summer-grown vegetable crops in Samsun province and investigations on host-symptom-satellite RNA interactions. Ondokuz Mayıs University, Master Thesis, 89p, Samsun.
23. Sertkaya G., Yılmaz M. 2017. Investigation on some begomoviruses, their natural transmission and other hosts in organic tomato production in greenhouses in Hatay province of Turkey. Journal of Agricultural Faculty of Mustafa Kemal University, 22(1): 1-15.
24. Uzunoğulları N., Gümüş M. 2015. Detection of cucumber mosaic virus (CMV) causing natural infection on some cultured plants in Marmara Region. Trakya University Journal of Natural Sciences, 16 (1), 9-15.
25. Yardimci N., Eryigit H. 2006. Identification of Cucumber mosaic virus in tomato (Lycopersicon esculentum) growing areas in the north‐west Mediterranean region of Turkey. New Zealand Journal of Crop and Horticultural Science, 34 (2), 173-175.
26. Yeşilyurt N., Çevik B. 2019. Genetic diversity and phylogenetic analyses of Tomato chlorosis virus isolates using the coat protein gene sequences. Journal of Plant Pathology, 101 (4), 1143-1150.
27. Yilmaz F., Sipahioğlu H. M. 2020. Detection of some important viruses in tomato production areas of Diyarbakır province by molecular methods and their molecular characterization. Plant Protection Bulletin, 60 (4), 49-58.
28. Zitter T. A., Murphy J. F. 2009. Cucumber mosaic. Plant Health Instructor, DOI: 10.1094. PHI-I-2009-0518-01.