First Report of Natural Infection of Watermelon mosaic virus (WMV) Infecting Bottle Gourd and Snake Melon

Öz

Cucurbitaceous crops, one of the main crops of agriculture, are sensitive to many plant viruses. In August 2019, virus-like symptoms were observed on some cucurbit plants grown in private home gardens in Antalya and Denizli provinces (Turkey). A total of 53 leaf samples were sampled from plants with the most symptoms (melon (Cucumis melo L.), watermelon (Citrullus lanatus L.), bottle gourd (Lagenaria siceraria (Molina) Standl.), and snake melon (Cucumis melo var. flexuosus) and tested by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) against possible watermelon mosaic potyvirus (WMV) infection. The coat protein gene (CP) specific primer sets amplified a gene product of nearly 820 bp fragment from symptomatic plants. WMV infections were detected in 31 individual cucurbit plants, including 11 melons, 8 watermelons, 7 snake melons and 5 bottle gourds. The presence of viral infection was found only in ornamental squash plants in Antalya province and in all cucurbits sampled in Denizli province. To better comprehend the molecular characteristics of virus isolates, the amplified viral DNA fragments were cloned in a proper prokaryotic plasmid, sequenced by Next Generation Sequencing (NGS) and recorded to GenBank. Bioinformatic analyses using the Basic Local Alignment Search Tool (BLAST) showed that the identified CP gene sequences exhibited significant nucleotide homogeneity, supported by a high nucleotide similarity index with that of other isolates around the world. In addition, Turkish isolates isolated from Antalya and Denizli regions showed approximately 94% nucleotide similarity among themselves. For phylogenetic inference, WMV sequences were subjected to multiple alignments with isolates from different geographic origins of the same viruses. Molecular phylogeny showed that all WMV isolates are closely related to other world WMV isolates at variable rates. WMV is wide host range viruses in cucurbit crops, however, this work is the first scientific report of WMV isolates detected in bottle gourd and snake melon from the South and West Regions of Turkey all over the world.WMV are broad spectrum viruses in cucurbit crops. This work is the first scientific report of Watermelon mosaic potyvirus isolates detected in bottle gourd and snake melon from the South-West Region of Turkey in all over the world.

Anahtar Kelimeler

• WMV
• RT-PCR
• Molecular Phylogeny
• Bottle gourd
• Snake melon

Su Kabağı ve Acur’da Doğal Enfeksiyona Neden Olan Karpuz Mozaik Virüsü (WMV)’nün İlk Raporu

Öz

Tarımın ana ürünlerinden biri olan kabakgiller, birçok bitki virüsüne karşı hassastır. Ağustos 2019’da Antalya ve Denizli ilinde (Türkiye) müstakil ev bahçelerinde yetiştirilen bazı kabakgil bitkilerinde virüs tipi semptomlar gözlenmiştir. Çoğu belirtili olan bitkilerden (kavun (Cucumis melo L.), karpuz (Citrullus lanatus L.), su kabağı (Lagenaria siceraria (Molina) Standl.) ve acur (Cucumis melo var. flexuosus) toplam 53 yaprak örneği toplanmış ve olası watermelon mosaic virus (WMV) enfeksiyonuna karşı Revers-Transkriptaz Polimeraz Zincir Reaksiyonu (RT-PCR) ile test edilmiştir. Kılıf protein genine (CP) özel primer setleri, simptomatik bitkilerden yaklaşık 820 bp’lik bir gen ürününü amplifiye etmiştir. WMV enfeksiyonları, 11 kavun, 8 karpuz, 7 acur ve 5 su kabağı dahil olmak üzere 31 farklı kabakgil bitkisinde tespit edilmiştir. Antalya ilinde sadece su kabağı bitkilerinde, Denizli'den toplanan tüm kabakgil örneklerinde viral enfeksiyon varlığı saptanmıştır. Virüs izolatlarının moleküler özelliklerinin daha iyi anlaşılması için amplifiye edilmiş viral DNA fragmanları uygun bir prokaryotik plazmide klonlanmış, Yeni Nesil Dizileme (NGS) ile dizilenmiş ve gen bankasına kaydı yapılmıştır. Basic Local Alignment Search Tool (BLAST) ile gerçekleştirilen biyoinformatik analizler, belirlenen WMV-CP gen dizilerinin dünyadaki diğer izolatlarınkiyle yüksek bir nükleotid benzerlik indeksi ile desteklenerek önemli bir nükleotit homojenitesi sergilediğini göstermiştir. Ayrıca, Antalya ve Denizli illerinden izole edilen Türk izolatları kendi aralarında yaklaşık %94 oranında nükleotit benzerliği göstermiştir. Filogenetik çıkarımlar için, WMV dizileri, aynı virüslerin farklı coğrafik kökenlerinden gelen 30 izolatla çoklu hizalamaya tabi tutulmuştur. Moleküler filogeni ise tüm WMV izolatlarının değişen oranlarda diğer dünya WMV izolatları ile yakından ilişkili olduğunu göstermiştir. WMV, kabakgil bitkilerinde geniş konukçu dizisine sahip virüsler arasında yer almakta ve bununla birlikte bu çalışma, Türkiye’nin Güney ve Batı Bölgeleri’nde su kabağında ve acurda belirlenen WMV izolatlarının tüm dünyadaki ilk bilimsel raporudur.WMV, kabakgil bitkilerinde geniş spektrumlu virüslerdir. Bu çalışma, Türkiye'nin Güney-Batı Bölgesi’nden su kabağı ve acurda belirlenen Karpuz mozaik potyvirüs izolatlarının tüm dünyadaki ilk bilimsel raporudur.

Anahtar Kelimeler

• WMV
• RT-PCR
• Moleküler filogeni
• Süs kabağı
• Acur

Kaynakça

1. Akbar, A., Ahmad, Z., Begum, F. and Raees, N. (2015). Varietal Reaction of Cucumber against Cucumber mosaic virus. American Journal of Plant Sciences, 6: 833-838.
2. Al-Ani, R. A., Adhab, M. A., Ali, A. A. and Diwan S. N. H. (2011). Zucchini Yellow Mosaic Virus: Characterization and Management In Iraq. International Journal of Current Research, 3(11): 220-224.
3. Ali, A., Abdalla, O., Bruton, B., Fish, W., Sikora, E., Zhang, S. and Taylor, M. (2012). Occurrence of viruses infecting watermelon, other cucurbits, and weeds in the parts of southern United States. Plant Health Progress, 13(1): 9.
4. Ali, A., Natsuaki, T. and Okuda, S. (2006). The complete nucleotide sequence of a Pakistani isolate of Watermelon mosaic virus provides further insights into the taxonomic status in the Bean common mosaic virus subgroup. Virus Genes, 32: 307-311.
5. Amer, M. A. (2015). Biological and molecular characterization of cucumber green mottle mosaic virus affecting bottle gourd and watermelon plants in Saudi Arabia. International Journal of Agriculture and Biology, 17: 748-754.
6. Awala, F. O., Ndukwu B. C. and Agbagwa, I. O. (2019). Phytogeographical distribution and fruit diversity of Lagenaria siceraria species in Nigeria. American Journal of Plant Sciences, 10: 958–75.
7. Bisognin, D. A. (2002). Origin and evolution of cultivated cucurbits. Ciência Rural, 32(5): 715-723.
8. Brunt, A., Crdotree, K., Dallwitz, M., Gibbs, A. and Watson, L. (1996). Virus of Plants. Descriptions and lists from the VIDE database. S CAB International University Press. 477-478.

9. Chatzivassiliou, E. K., Papapanagiotou, A. P., Mpenardis, P. D., Perdikis, D. C. and Menexes, G. (2016). Transmission of Moroccan watermelon mosaic virus (MWMV) by Aphids in Greece. Plant Disease, 100(3): 601-606.
10. Coutts, B. A., Kehoe, M. A. and Jones, R. A. C. (2011) Minimising losses caused by Zucchini yellow mosaic virus in vegetable cucurbit crops in tropical, sub-tropical and Mediterranean environments through cultural methods and host resistance. Virus Research, 159: 141–160.
11. Delmiglio, C. and Pearson M. N. (2006). Effect sandincidence of Cucumber mosaic virus, Watermelon mosaic virus and Zucchini yellow mosaic virus in New Zealand’s only native cucurbit, Sicyos australis. Australasian Plant Pathology, 35: 29-35.
12. Dikici, S. and Tarla, G. (2020). Determination of Watermelon Mosaic Virus (WMV-2) By Serological Methods and Biological Index Studies in Some Cucurbits in Uşak. International Journal of Engineering Science Invention, 9(12): 12-15.
13. Fernandes, F. F. Valverde, R. A. and Black, L. L. (1991). Viruses infecting cucurbit crops in Louisiana. Plant Disease, 75: 431.
14. Fletcher, J. D., Wallace, A. R. and Rogers, B. T. (2000). Potyviruses in New Zealand buttercup squash (Cucurbita maxima Duch.): yield and quality effects of ZYMV and WMV 2 virus infections. New Zealand Journal of Horticultural Science and Crop Research, 28: 17–26.
15. Foissac, X., Svanella-Dumas, L., Gentit, P., Dulucq, M. J. and 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: 37-44.
16. Güller, A. and Usta, M. (2020). Occurrence of Cucumber mosaic cucumovirus and Watermelon mosaic potyvirus on Melon exhibiting viral symptoms in Bingöl province of Turkey and Their Phylogenetic Affinities. Turkish Journal of Agricultural and Natural Sciences, 7(4): 948–958.
17. Horuz, S. and Aysan, Y. (2018). Determination of appropriate method(s) to detect watermelon seedling blight and fruit blotch disease agent Acidovorax citrulli in cucurbit seeds. Journal of Tekirdag Agricultural Faculty, 15(3): 36-43.
18. Ilahy, R., Tlili, I, R’him, T., Chikh Rouhou., H., Mani, A., Hdider, C. and Lenucci M. S. (2019). A first insight on snake melon (Cucumis melo var. flexuosus): an under Tmated source of functional compounds. In: Mitra S., Banlk, A. K., Mani, A., Kuchi, V. S., and Meena. N.K. (eds) Trends & Prospects in Post-Harvest Management of Horticutural Crops. Today & Tomorrow's Printers and Publishers, New Delhi, India, pp. 529-542.
19. Jeger, M. J. (2020) The epidemiology of plant virus disease: Towards a new synthesis. Plants, 9: 1768. Kamberoglu, M. A., Desbiez, C. and Caliskan, A. F. (2015) Characterization of an emerging isolate of watermelon mosaic virus in Turkey. International Journal of Agriculture and Biology, 17: 211–215.
20. Kamberoğlu, M. A. and Keçe, M. A. (2016). Biological, Serological and Molecular Detection of Watermelon Mosaic Virus (WMV-2) in Watermelon Growing Fields in Eastern Mediterranean Region. Journal of Tekirdag Agricultural Faculty, 13(3): 156-164.
21. Karanfil, A. and Korkmaz, S. (2020). First Report of Cucumis Melo Endornavirus with a New Host, the Gherkin (Cucumis Anguria Linn.), in Turkey. Trakya University Journal of Natural Sciences, 21(1): 63-67.
22. Katis, N. I., Tsitsipis, J. A., Lykouressis, D. P., Papapanayotou, A., Margaritopoulos, J. T., Kokinis, G. M., Perdikis, D. C. and Manoussopoulos, I. N. (2006). Transmission of Zucchini yellow mosaic virus by colonizing and non-colonizing aphids in Greece and new aphid species vectors of the virus. Journal of Phytopathology, 154: 293–302.
23. Khalifa, M. E., El-Fallal, A. A., El –Sayed, A. K., Diab, A. and 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.
24. Kheder, A. A., Sulaiman, T. M., Ghanem, G. A. M. and 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.
25. Kızmaz, M. Z., Sagır, A. and Baloglu, S. (2016). Determination of ıncidence and agents of viral diseases in cucurbits growing areas in Diyarbakir and Mardin provinces. Journal of Agriculture Faculty of Ege University, 53 (4): 397-406.
26. Köklü, G. and Yilmaz, Ö. (2006). Occurrence of cucurbit viruses on field grown melon and watermelon in the Thrace region of Turkey. Phytoprotection, 87(3): 123-130.
27. Korkmaz, F., Karaca, K., Özaslan, C. and Yanar, Y. (2016). Sicyos angulatus: A natural host of Watermelon Mosaic Virus (WMV-2). Turkish Journal of Weed Science, 19(1): 1-3.
28. Korkmaz, F., Topkaya, Ş. and Yanar Y. (2018). Determination of viruses in cucurbits growing areas from Tokat. Gaziosmanpasa Journal of Scientific Research, 7(2): 46-56.
29. Korkmaz, G., Demirel, S., Usta, M. and Güller, A. (2021). Molecular Phylogeny of Watermelon mosaic virus (WMV) in Squash Plant Grown in Diyarbakir Province. ISPEC 6th International Conference on Agriculture, Animal Husbandry and Rural Development. May 16-18, Siirt, Turkey.
30. Krajcsi, P. and Wold S. M. (1998). Viral proteins that regulate cellular signalling. Journal of General Virology, 79: 1323-1335.
31. Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamura, K. (2018). MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35: 1547–1549.
32. Lecoq, H. and Desbiez, C. (2008). Watermelon Mosaic Virus and Zucchini Yellow Mosaic Virus. In “Encyclopedia of Virology, Third Edition” B.W.J. Mahy, & M.H.V. Van Regenmortel (Eds.), pp: 433-440. Elseiver, Oxford, UK, 5 Vols.
33. Mahgoub, H. A., Desbiez, C., Wipf-Scheibel, C., Dafalla, G. and Lecoq, H. (1997). Characterization and occurrence of zucchini yellow mosaic virus in Sudan. Plant Pathology, in press. Plant Pathology, 46(5): 800 – 805.
34. Martelli, G. P. and Russo M. (1985). Virus-Host Relationship. (R.I.B. Francki, Editor). The Plant Virus. Plenum Pres, 223 Spring Street, New York, N.Y. 10013. Vol. 1, 163- 205.
35. Masika, F. B., Kisekka, R., Alicai, T. and Tugume, A. K. (2017). Incidence of viruses and virus-like diseases of watermelons and pumpkins in Uganda, a hitherto none investigated pathosystem. African Journal of Agricultural Research, 12(3): 177-191.
36. McLeish, M. J., Fraile, A. and García-Arenal, F. (2020) Population genomics of plant viruses: The ecology and evolution of virus emergence. Phytopathology, 11: 32–39.
37. Niu, Y., Pang, X., Cheng, Y., Wang, D., Guo, S. and Liu, Y. (2017). Molecular detection of watermelon mosaic virus associated with a serious mosaic disease on Cucurbita pepo L. in Shanxi, China. Virus Disease, 28(3): 315–319.
38. Qiu, Y., Zhang, Y., Wang, C., Lei, R., Wu, Y., Li, X. and Zhu, S. (2018). Cucumber mosaic virus coat protein induces the development of chlorotic symptoms through interacting with the chloroplast ferredoxin I protein. Science Report, 8: 1205.
39. Randa‐Zelyüt, F., Karanfil, A., Sarı, M. and Korkmaz, S. (2022). First report of natural infection of wild carrot by Watermelon mosaic virus in Turkey. New Disease Reports, 46(1): e12107.
40. Saeed, M., Khan, M. S., Amir, K., Bi, J. B., Asif, M., Madni, A., Kamboh, A. A., Manzoor, Z., Younas, U. and Chao, S. (2022). Lagenaria siceraria fruit: A review of its phytochemistry, pharmacology, and promising traditional uses. Frontiers in Nutrition, 9: 927361. https://doi.org/10.3389/fnut.2022.927361
41. Santosa, A.I., Al-Shahwan, I.M., Abdalla, O.A., Al-Saleh, M.A. and Amer, M.A. (2018). Characterization of a Watermelon mosaic virus Isolate Inducing a Severe Disease in Watermelon in Saudi Arabia. Journal of Agricultural Science and Technology, 8: 220-229.
42. Sevik, M. A. and Arli-Sokmen, M. (2003). Viruses infecting cucurbits in Samsun, Turkey. Plant Disease, 87: 341-344.
43. Sharifi, M., Massumi, H., Heydarnejad, J. Pour A. H., Shaabanian, M. and Rahimian, H. (2008). Analysis of the biological and molecular variability of Watermelon mosaic virus isolates from Iran. Virus Genes, 37: 304-313.
44. Stephens, J. M. Gourd, Bottle– Lagenaria siceraria (Mol.) Standl. University of Florida Cooperative Extension Service, Institute of Food and Agriculture Sciences (EDIS) (1994). Available online at: http://edis.ifas.ufl.edu/pdffiles/mv/mv06900.pdf
45. Svoboda, J. and Hale, C. (2011). Pathogenic viruses on Cucurbitaceous vegetables and their spread in the Czech Republic. Acta Horticulturae, 917: 309-315.
46. Topkaya, S., Desbiez, C. and Ertunc, F. (2019). Presence of cucurbit viruses in Ankara and Antalya province and molecular characterization of coat protein gene of zucchini yellow mosaic virus Turkish isolates. Fresenius Environmental Bulletin, 28(4): 2442- 2449.
47. Usta, M., Güller, A. and Sipahioğlu, H.M. (2018). Molecular Characterization of Coat Protein Gene of Two Watermelon mosaic potyvirus Isolates Infecting Muskmelon at Van Province (Turkey). 1. International GAP Agriculture & Livestock Congress. 25-27 April 2018 – Şanlıurfa/Turkey.
48. Vural, H., Eşiyok, D. and Duman, İ. (2000). Cultivated Vegetables (Vegetable Cultivation). Ege University Faculty of Agriculture, Department of Horticulture, Ege University Press, Izmir.
49. Webb, R. E. and Scott H. A. (1965). Isolation and identification of Watermelon mosaic virus 1 and 2. Phytopathology, 55: 895–900.
50. Yeşil, S. (2013). Detection of virus diseases of cucurbits in Konya, Karaman and Aksaray provinces by serologic and molecular methods and determination of infection sources (Ph. D. Thesis) Selcuk University The Institute of Natural Sciences, Konya, Turkey.
51. Yeşil, S. (2018). Some virus diseases of edible seed squash (Cucurbita pepo L.) in Aksaray province, Turkey. Yuzuncu Yil University Journal of Agricultural Sciences, 29(Special Issue): 63-71.
52. Yeşil, S. (2019). Detection of viruses on edible seed squash (Cucurbita pepo L.) in Yozgat province, Turkey. Journal of the Institute of Science and Technology, 9(3): 1212- 1219. Yeşil, S. (2020). Detection of some virus diseases of edible seed squash (Cucurbita pepo L.) in Nevşehir Province, Turkey. Selcuk Journal of Agriculture and Food Sciences, 34(1): 49-56. Yeşil, S. and 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, Antalya, Türkiye. Yeşil, S. and, Ertunç, F. (2013). Virus diseases of Cucurbits in Karaman province. International Journal of Ecosystems and Ecology Sciences, 3(2): 235-240.