Bingöl İlinde Kabak Bitkisini (Cucurbita pepo L.) İnfekteleyen Zucchini Yellow Mosaic Potyvirus (ZYMV) Virüs İzolatının Belirlenmesi ve Moleküler Karakterizasyonu

Kabak sarı mozaik potivirüsü (ZYMV), Potyviridae ailesine ait bir virüs olup dünya çapında birçok kabakgil bitkide ciddi ekonomik kayıplara neden olmaktadır. 2018 yılı Eylül ayında, Bingöl iline bağlı kabak yetiştiriciliği yapılan alanlarda kabarıklık ve buruşukluk gibi tipik viral hastalık belirtisi gösteren bitkiler ile sağlıklı bitkilerden örnekler alınarak ZYMV enfeksiyonu bakımından Reverse Transkripsiyon Polimeraz Zircir Reaksiyonu (RT-PCR) ile testlenmiştir. RT-PCR testi pozitif reaksiyon vererek ZYMV’nin kılıf proteinine karşılık gelen yaklaşık 840 bp DNA fragmenti oluşturmuştur. Ayrıca, çoğaltılan ZYMV'nin tam kılıf protein (coat protein, CP) geni, prokaryotik klonlama vektörü olan pGEM-T Easy vektörüne klonlanmış ve genin tam nükleotid ve amino asit dizisi tespit edilmiştir. Elde edilen ZYMV-CP gen dizisinin 279 amino asiti kodladığı (yaklaşık 31.2 kDa) ve 837 nükleotidi içerdiği ortaya çıkarılmış ve bu dizi ZYMV-Bingöl izolatı olarak isimlendirilerek MK689858 erişim numarası ile gen bankasına (NCBI) kaydedilmiştir. ZYMV-Bingol CP geninin dizisi, farklı coğrafi bölgelerden elde edilen 21 izolat ile analiz edilerek filogenetik ilişkileri belirlenmiştir. Nükleotid düzeyindeki moleküler analizlere göre ZYMV CP gen dizisisi, en yüksek % 100 oranında Türkiye izolatı (JF317296) ile ve en düşük % 91.64 oranında Kore izolatı (AF062518) ile benzerlik göstermiştir. Ayrıca, filogenetik analizler, ZYMV Bingöl izolatının Türkiye-Adana izolatı (JF317296) ve Pakistan izolatı (AB127936) ile gruplandığını belirlenmiştir. Bingöl ilinde kabak bitkisini enfekte eden ZYMV’nin bulunuşu ilk kez bu çalışmayla rapor edilmiştir.

Anahtar Kelimeler:

ZYMV (Zucchini yellow mosaic potyvirus), RT-PCR, klonlama

Occurrence and Characterization of Coat Protein Gene of Zucchini yellow mosaic potyvirus (ZYMV) Isolate Infecting Pumpkin (Cucurbita pepo L.) in Bingol Province (Turkey)

Zucchini yellow mosaic potyvirus (ZYMV) belongs to family Potyviridae, which causes serious economic losses in many cucurbits from worldwide. In 2018 (September), leaf samples of pumpkin exhibiting typical viral symptoms including mosaic, blistering and wrinkling and healthy pumpkin leaf samples were collected from Bingöl and screened by Reverse- Transcription Polymerase Chain Reaction (RT-PCR) against ZYMV infection. Tested leaf samples were reacted positive resulting in an expected about 840 bp DNA fragments of partial coat protein (CP) gene of ZYMV. ZYMV-CP gene was further inserted into a pGEM-T Easy prokaryotic cloning vector and their partial nucleotide sequences and deduced amino acid were ascertained. The provided ZYMV- CP gene sequence consisted of 837 nucleotides in length coded for 279 amino acid residues of approximately 31.2 kDa. The isolate was denominated as ZYMV-Bingol and registered with MK689858 accession number in the NCBI. The sequence of ZYMV- Bingol CP gene were aligned with 21 isolates deposited in GenBank from different geographical location and its phylogenetic relationships were determined. Molecular analysis of the ZYMV CP gene sequence indicated the highest similarity with 100% Turkish isolate (JF317296) and the lowest with 91.64% of Korea isolate (AF062518), at nucleotide level. Moreover, phylogenetic analyses revealed that ZYMV Bingöl isolate is clustered with the Turkish- Adana isolate (JF317296) and Pakistan isolate (AB127936). ZYMV has been reported for the first time in the pumpkin plant from Bingöl province of Turkey by this study.

Keywords:

ZYMV (Zucchini yellow mosaic potyvirus) RT-PCR, cloning, characterization,

PDF

___

Adams, M.J., Zerbini, F.M., French, R., Rabenstein, F., Stenger, D.C., Valkonen, J.P.T. 2012. Family Potyviridae. p. 1069-1089. In: King, A.M.Q.; Adams, M.J.; Carstens, E.B; Lefkowitz, E.J., eds. Virus Taxonomy: Classifcation and Nomenclature of Viruses; Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, San Diego, CA, USA.
Adlerz, W.C., Purcifull, D.E., Simone, G.W., Hiebert, E. 1983. Zucchini yellow mosaic virus: a pathogen of squash and other cucurbits in Florida. Proceedings of the Florida State Horticultural Society, 96: 72-44.
Al-Saleh, M.A., Amer, M.A., AL-Shahwan, I.M., Abdalla, O.A., Zakri, M.A. 2014. Characterization of different isolates of Zucchini yellow mosaic virus from cucurbits in Saudi Arabia. African journal of microbiology, 8(19): 1987-1994.
Balint, R., Plooy, I., Steele, C. 1990. The nucleotide sequence of zucchini yellow mosaic potyvirus. Abstract of the VIIIth International Congress of Virology, 8: 84-107.
Blua, M.J., Perrıng, T.M. 1992. Alatae Production and population ıncrease of aphid vectors on virus-infected host plants. Oecologia, 92: 65-70.
Bostan, H., Kaymak, H.Ç., Haliloğlu, K. 2002. Detection of Cucumber mosaic virus (CMV) and Zucchini yellow mosaic virus (ZYMV) in squash in Erzurum, Erzincan and Artvin provinces by serological and biological methods. Journal of Turkish Phytopathology, 31(1): 9-14.
Brandes, J. 1957. Eine elektronmikroscopishe Schnell-methode zum Nachweis faden-und sta¨bchenförmiger Viren, insbesondere in Kartoffeldunkelkeimen. Nachrichtenblatt des Deutschen Pflantzenschutz- Dienstesi, 9: 151-152.
Chinnaraja, C., Ramkissoon, A., Rajendran, R., Tony, S.T., Ramsubhag, A., Jayaraj, J. 2016. First report of Zucchini yellow mosaic virus and Squash mosaic virusiınfecting cucurbits in Trinidad. Plant Disease, 100(4): 866.
Coutts B.A., Kehoe, M.A., Webster, C.G., Wylie, S.J., Jones, R.A.C. 2011. Zucchini yellow mosaic virus: biological properties, detection procedures and comparison of coat protein gene sequences. Archives of Virology, 156: 2119-2131.
Coutts, B.A., Kehoe, M.A., Jones, R.A.C. 2013. Zucchini yellow mosaic virus: Contact transmission, stability on surfaces, and inactivation with disinfectants. Plant Disease, 97: 765-771.
Çıtır, A., Kutluk, N.D., Sağlam, N., İlbağı, H. 1998. Amasya, Çorum, Samsun ve Tokat illerinde hıyar ve kabak kültürlerinde görülen virüs hastalıklarının simptomolojik ve biyolojik yöntemlerle tanıları. Türkiye VIII. Fitopatoloji Kongresi, Ankara, s. 331-335.
Çulal Kılıç, H., Doğan, K., Yardımcı, N., Isparta, L. 2016. Detection of Zucchini Yellow mosaic virus from cucurbits in Burdur Province, Turkey. Journal of Multidisciplinary Engineering Science and Technology (JMEST), 3: 4.
Dağ, D.S. 2005. Gaziantep İl ve İlçelerinde Yetiştirilen Kabakgillere (Cucurbitaceae) Zarar Veren Virüslerin DAS-ELISA Yöntemiyle Saptanması. Yüksek Lisans Tezi. Gaziantep Üniversitesi.
Davis, R.F., Yılmaz, M.A. 1984. First report of Zucchini yellow mosaic virus on watermelon and squash in Turkey. Plant Disease, 68: 537.
Davis, R.F., Mizuki, M,.K. 1986. Seed transmission of Zucchini yellow mosaic virus in squash. Phytopathology, 76: 1073.
Davis, R.F. 1986. Partial characterization of Zucchini yellow mosaic virus isolated from squash in Turkey. Plant Disease, 70: 735-738.
Desbiez, C., Lecoq, H. 1997. Zucchini yellow mosaic virus. Plant Pathol., 46: 809-829.
Ertunc, F. 1992. Ankara İlinde Kabaklarda Enfeksiyon Olusturan Viral Etmenin Teşhisi Üzerinde Araştırmalar. Ankara Üniversitesi Ziraat Fakültesi Yayınları, No: 1252, 13s.
Foissac, L., Gentit, P., Svanetia-Dumas, L., Dulucq, M. J., Candresse, T. 2001. Polyvalent detection of fruit tree tricho, capillo, and foveaviruses by nested RT-PCR using degenerated and inosine-containing primers (PDO RT-PCR). Acta Horticulturae, 550: 37-43.
Fujisawa, I., Hanada, T., Anang, S.B. 1986. Virus diseases occurring on some vegetable crops in West Malaysia. Journal of Agricultural Research, 20: 78-84.
Ghanem, G.A.M., Noura-Hassan, M., Kheder, A.A., Mazyad, H.M., Abdel-Alim, A.I. 2016. Antiserum production, biological and serological detection of Cucurbit yellow stunting disorder crinivirus (CYSDV) in Egypt. International Journal of Advanced Research, 4(4): 1116-1128.
Harper, K., Creamer, R. 1995. Hybridization detection of insect-transmitted plant viruses with digoxigenin- labeled probes. Plant Disease, 79: 563-567.
Hosseini, S., Mosahebi, G.H., Koohi Habibi, M., Okhovvat, S.M. 2007. Characterization of the Zucchini yellow mosaic virus from squash in Tehran province. Journal of Agricultural Science and Technology, 9: 137-143.
Jossey, S., Babadoost, M. 2008. Occurrence and distribution of pumpkin and squash viruses in Illinois. Plant Dieases, 92: 61-68.
Kamberoğlu, M.A., Çalışkan, A.F., Desbiez, C. 2016. Current status of some cucurbit viruses in Cukurova Region (Adana and Mersin Provinces) of Turkey and molecular characterization of Zucchini Yellow mosaic virus isolates. Romanian Biotechnological Letters, 21(4): 11709-11719.
Karamanlı, A., Kamberoğlu, MA. 2010. Survey of Cucumber Mosaic Virus (CMV) and Zucchini Yellow Mosaic Virus (ZYMV) in Turkish Republic of Northern Cyprus in Cucurbits Growth Fields. Ç.Ü. Fen Bilimleri Enstitüsü, Cilt: 22-23.
Kaya, A., Erkan, S. 2011. Detection and incidence of viruses in cucurbits grown in Izmir, Aydın, Manisa and Balıkesir provinces. Bitki Koruma Bülteni, 51(4):387-405.
Khalifa, M.E., El-Fallal, A.A., El-Sayed, A.K., Diab, A., Sadik, A.S. 2015. Identification of an isolate of zucchini yellow mosaic potyvirus infecting squash in Egypt. Pakistan Journal of Biotechnology, 12(2): 173-180.
Kheder, A.A., Sulaiman, T.M., Ghanem, G.A.M., Tohamy, M.R. 2017. Biological, serological and molecular characterization of Egyptian Zucchini yellow mosaic virus isolate infecting squash plants in Fayoum Governorate. Egyptian Journal of Phytopathology, 45(2): 1-14.
Korkmaz, F., Topkaya, Ş., Yanar, Y. 2018. Tokat ili kabakgil üretim alanlarında enfeksiyon oluşturan virüslerin belirlenmesi. Gaziosmanpasa Journal of Scientific Research, 7(2): 46-56.
Köklü, G., Yilmaz, Ö. 2006. Occurrence of cucurbit viruses on field-grown melon and watermelon in the Thrace region of Turkey. Phytoprotection, 87(3), 123-130.
Lecoq, H., Wipf-Scheibel, C., Nozeran, K., Millot, P., Desbiez, C. 2014. Comparative molecular epidemiology provides new insights into Zucchini yellow mosaic virus occurrence in France. Virus Research, 186: 135-143.
Lisa, V., Boccardo, G., D’agostino, G., Delavalle, G., D’aquilio, M. 1981. Characterization of a Potyvirus that causes Zucchini yellow mosaic virus. Phytopathology, 71: 667-672.
Lisa, V., Lecoq, H. 1984. Zucchini Yellow Mosaic Virus. No: 282 in: Descriptions of Plant Viruses. Commonw. Mycol. Inst./Assoc. Appl. Biol., Kew, Surrey, England, 4 p.
Lovisolo, O. 1980. Virus and viroid diseases of cucurbits. Acta Horticulturae, 88: 33-82. Mahgoub, H.A., Desbiez, C., Wipf-Scheibel, C., Dafalla, G., Lecoq, H. 1997. Characterization and occurrence of zucchini yellow mosaic virus in Sudan. Plant Pathology, in press. Plant Pathology, 46(5): 800-805.
Mohammed, H.S., Zicca, S., Manglli, A., Mohamed, M.E., El Siddig, M.A., Tomassoli, L., El Hussein, A.A. 2014. Identification and phylogenetic analysis of common pumpkin viruses in Sudan. Journal of Plant Pathology, 96(1): 77-84.
Nagendran, K., Priyanka, R., Keerthana, U., Mohankumar, S., Karthikeyan, G. 2016. First report of Zucchini yellow mosaic virus on Cucurbita moschata in India. Journal of Plant Pathology, 98(1): 171-185.
Nasr-Eldin, M.A., Abdelkader, H.S., Abo-Senna, A.S., Othman, B.A. 2016. Characterization and phylogenetic analysis of Zucchini yellow mosaic virus infecting Cucurbita pepo in Egypt. Journal of American Science, 12(3): 93-104.
Ohtsu, Y., Sako, N. 1985. Zucchini yellow mosaic virus isolated from pumpkin in Milyako and Yaeyama Islands, Okinawa, Japan. Annals of the Phytopathological Society of Japan, 51: 234-237.
Ozaslan, M., Aytekin, T., Bas, B., Halil Kilic, I., Didem Afacan, I., Dag, D.S. 2006. Virus diseases of cucurbits in Gaziantep- Turkey. Plant Pathology Journal, 5(1): 24-27.
Özer, M., Sipahioğlu, H.M., Usta, M., Fidan, H. 2012. Cloning and sequencing of coat protein gene of Zucchini yellow mosaic virus isolated from squash and muskmelon in Turkey. Turkish Journal of Biology,. 36: 423-429.
Pospieszny, H., Cajza, M., Plewa, R. 2003. First report of Zucchini yellow mosaic virus in cucumber in Poland. Plant Disease, 87(11): 1399-1399.
Prieto, H., Bruna, A., Hinrichsen, P., Muñoz, C. 2001. Isolation and molecular characterization of a Chilean isolate of Zucchini yellow mosaic virus. Plant Disease, 85: 644-648.
Romay, G., Lecoq, H., Geraud-Pouey, F., Chirinos, D.T., Desbiez, C. 2014. Current status of cucurbit viruses in Venezuela and characterization of Venezuelan isolates of Zucchini yellow mosaic virus. Plant Pathology, 63: 78-87.
Sertkaya, G., Sertkaya, E., Yetişir, H., Kaya, K. 2004. Investigations on indicende and transmission of Zucchini yellow mosaic virus (ZYMV) in cucurbits Hatay province. Proceedings of the First Plant Protection Congress of Turkey, 8-10 September 2004, Samsun, Turkey, p. 217.
Sevik, M.A., Arli-Sokmen, M. 2003. Viruses infecting cucurbits in Samsun, Turkey. Plant Disease. 87: 341-344.
Shukla DD., Ward CW., Brunt AA. 1994. The Potyviridae. Wallingford, UK: CAB International. 516.
Simmons, H.E., Holmes E.C., Gildow F.E., Bothe-Goralczyk M. A., Stephenson A.G. 2011. Experimental verification of seed transmission of Zucchini yellow mosaic virus. Plant Disease, 95: 751-754.
Simmons, H.E., Dunham J.P., Zinn K.E., Munkvold G.P., Holmes E.C., Stephenson A.G. 2013. Zucchini yellow mosaic virus (ZYMV, Potyvirus): vertical transmission, seed infection and cryptic infections. Virus Research, 176(1-2): 259-64.
Spadotti, D.M.A., Wassano, D.T., Rezende, J.A.M., Camargo, L.E.A., Inoue-Nagata, A.K. 2015. Biological and molecular characterization of Brazilian isolates of Zucchini yellow mosaic virus. Scienta Agricola, 72: 187-191.
Svoboda, J., Polák, J. 2002. Distribution, variability and overwintering of Zucchini yellow mosaic virus in the Czech Republic. Plant Protection Science, 38: 125-130.
Şevik, M.A., Balkaya, A. 2015. Samsun, Sinop ve Bolu illerindeki bal kabağı (Cucurbita moschata Duch) popülasyonlarına ait tohum örneklerinde virüslerin tanılanması ve bulunma durumlarının belirlenmesi. Journal of Agricultural Faculty of Gaziosmanpasa University, 32(3): 70-77.
Thomson, K.G., Dietzgen, R.G., Gibbs, A.J., Tang, Y.C., Liesack, W., Teakle, D.S., Stackebrandt, E. 1995. Identification of Zucchini yellow mosaic potyvirus by RT-PCR and analysis of sequence variability. Journal of Virological Methods, 55: 83-96.
Tymchyshyn, O., Shevchenko, T., Shevchenko, O., Budzanivska I. 2017. Phylogenetic analysis of seed-transmitted isolate of Zucchini yellow mosaic virus. БІОЛОГІЯ, 2(74): 47-50.
Vucˇurovic´, A., Bulajic´, A., Stankovic´, I., Ristic´, D., Nikolic´, D., Berenji, J., Krstic´, B. 2012. First report of Zucchini yellow mosaic virus in watermelon in Serbia. Plant Disease, 96: 149.
Wang, H.L, Gonsalves, D., Provvidenti, R., Zitter, T.A. 1992. Comparative biological and serological properties of four strains of zucchini yellow mosaic virus. Plant Disease, 76: 530-535
Wang, D., Li, G. 2017. Biological and molecular characterization of Zucchini yellow mosaic virus isolates infecting melon in Xinjiang, China Can. Journal of Plant Pathology, 39(1): 48-59.
Yeşil, S. 2013. Konya, Karaman ve Aksaray İlleri Kabakgil Ekim Alanlarında Görülen Virüs Hastalıklarının Serolojik ve Moleküler Yöntemlerle Tespiti ve Enfeksiyon Kaynaklarının Belirlenmesi. Doktora Tezi. Selçuk Üniversitesi.
Yeşil, S. 2014. Virus Diseases of Edible Seed Squash (Cucurbita pepo L.) in Konya Province. In: Book of Proceedings Fifth International Scientific Agricultural Symposium (AGROSYM-2014), October, 23-26th, 2014, Jahorina, Sarajevo, Bosnia-Herzegovina, 226 p. ISBN: 978-99955-751-9-9.
Yeşil, S., Ertunç, F. 2012. Virus diseases of cucurbits in Konya province. Proceedings of the Xth EUCARPIA Meeting on Genetics and Breeding of Cucurbitaceae, pp. 791-796.
Yeşil, S., Ertunç, F. 2013. Virus diseases of Cucurbits in Karaman province. International Journal of Ecosystems and Ecology Sciences (IJEES), 3(2): 235-240.
Yılmaz, M.A., Lecoq, H., Abak, K., Baloglu, S., Sarı, N. 1992. Türkiye’de kabakgil sebze türlerinde zarar yapan virüsler. Türkiye I. Ulusal Bahçe Bitkileri Kongresi, İzmir, Bildiriler Kitabı, s. 439-442.
Yilmaz, M.A., Baloğlu S., Özaslan M., Güldür M.E. 1995. GAP Bölgesi kültür bitkilerinde belirlenen virüsler. GAP Bölgesi Bitki Koruma Sorunları ve Çözüm Önerileri Sempozyumu, Şanlıurfa, Turkey, s. 241-250.
Zhao, M.F., Chen, J., Zheng, H.Y., Adams, M.J., Chen, J.P. 2003. Molecular analysis of Zucchini yellow mosaic virus isolates from Hangzhou, China. Journal of Phytopathology, 151: 307-311.
Zheng, L., Rodoni, B.C., Gibbs, M.J., Gibbs, A.J. 2010. A novel pair of universal primers for the detection of Potyviruses. Plant Pathology, 59: 211-220.
Zitter, T.A., Hopkins, D.A., Thomas, C.F. 1996. Compendium of Cucurbit Diseases. St-Paul. MN: APS Press, 87 pp.