Detection and molecular characterization of some viruses on Cucurbitaceae plants growing in Hatay province by DAS-ELISA and RT-PCR methods

Abstract

Aims: In this study, it was aimed to investigate the occurrence of cucurbit yellow stunting disorder virus (CYSDV), cucurbit chlorotic yellows virus (CCYV) and beet pseudo-yellows virus (BPYV) in cucumber, zucchini and melon plants grown in Hatay province by Double Antibody Sandwich-Enzyme Linked Immuno Sorbent Assay (DAS-ELISA) and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) methods, to analyse the phylogenetic relationships of identified isolates.

Methods and Results: Totally 90 symptomatic and non-symptomatic cucumber, zucchini and melon samples were collected from Hatay province. DAS-ELISA and RT-PCR methods were used to investigate the presence of CCYV and RT-PCR method was used for CYSDV and BPYV. The most common symptoms observed are leaf shrinkage, deformation, leaf wrinkles, curling and vein clearing. According to the DAS-ELISA results, CCYC was not found in the samples tested. As a result of RT-PCR analysis, BPYV and CCYV could not found in the samples tested, but PCR amplicons of 364 bp amplifying the heat shock protein 70h (HSP70h) gene of CYSDV were obtained from 11 samples (9 zucchini, 2 melons). As a result of bidirectional sequence analysis of 10 CYSDV izolates, it was determined that the obtained nucleotide sequences were highly similar (99%) to that of the CYSDV isolates registered in the GeneBank. In this study, CYSDV was reported for the first time in the zucchini and melon plants growing in Hatay province.

Conclusions: CYSDV was detected for the first time in samples collected from zucchini and melon plants in Hatay province. BPYV and CCYV could not be detected in any of the samples tested. The fact that 12.22% of the tested samples were infected with CYSDV highlights the need of using healthy plant materials.

Significance and Impact of the Study: In this study, the presence of CYSDV was proven by RT-PCR analyses for the first time in zucchini and melon plants in Hatay province.

Keywords

• Boron content
• Olive orchard
• soil properties
• Gaziantep

Hatay ilinde yetişen Cucurbitaceae familyasına ait kültür bitkilerinde bazı virüslerin DAS-ELISA ve RT-PCR yöntemleriyle saptanması ve moleküler karakterizasyonu

Öz

Amaç: Bu çalışmada, Hatay ilinde yetiştirilen hıyar, kabak ve kavun bitkilerinde cucurbit yellow stunting disorder virus (CYSDV), cucurbit chlorotic yellows virus (CCYV) ve beet pseudo-yellows virus (BPYV)’lerinin Double Antibody Sandwich-Enzyme Linked Immuno Sorbent Assay (DAS-ELISA) ve Reverse Transcription-Polymerase Chain Reaction (RT-PCR) yöntemleriyle belirlenmesi, elde edilen virüs izolatlarının filogenetik ilişkilerinin ortaya konulması amaçlanmıştır.

Yöntem ve Bulgular: Hatay ilinden simptomlu ve simptomsuz 90 adet hıyar, kabak ve kavun örneği toplanmıştır. CCYV’nin varlığını araştırmak için DAS-ELISA ve RT-PCR yöntemleri, CYSDV ve BPYV’lerinin varlığını araştırmak için ise RT-PCR yöntemi kullanılmıştır. Gözlenen en yaygın simptomlar yapraklarda küçülme, deformasyon, yaprak kırışıklıkları, kıvrılmalar ve damar açılmalarıdır. DAS-ELISA testi sonuçlarına göre testlenen örneklerde CCYC bulunamamıştır. RT-PCR analizleri sonucunda testlenen örneklerde BPYV ve CCYV bulunamamış ancak CYSDV’nin heat shock protein 70h (HSP70h) genini çoğaltan 364 bp büyüklüğünde PCR amplikonları 11 örnekte (9 kabak, 2 kavun) elde edilmiştir. Bu ürünlerden 10 tanesinin doğrudan iki yönlü sekans analizi sonucunda, elde edilen nükleotit dizilerinin gen bankasında kayıtlı CYSDV izolatları ile yüksek oranda (%99) benzerlik gösterdiği saptanmıştır. Bu çalışma ile CYSDV ilk defa Hatay ilinde kabak ve kavun bitkilerinde rapor edilmiştir.

Genel Yorum: Hatay ili kabak ve kavun alanlarından toplanan örneklerde CYSDV ilk kez tespit edilmiştir. Testlenen örneklerinin hiçbirinde BPYV ve CCYV belirlenememiştir. Testlenen örneklerin %12.22 oranında CYSDV ile enfekteli bulunması, sağlıklı bitki materyallerinin kullanılmasının önemini göstermektedir.

Çalışmanın Önemi ve Etkisi: Hatay ilinde ilk kez bu çalışma ile kabak ve kavun bitkilerinde CYSDV’ünün varlığı, RT-PCR analizleri ile kanıtlanmıştır.

Anahtar Kelimeler

• Cucurbitaceae
• virüs
• RT-PCR
• DAS-ELISA
• sekans analizleri

Kaynakça

1. Abou-Jawdah Y, Sobh H, El-Zammar S, Fayyad A, Lecoq H (2000) Incidence and management of virus diseases of Cucurbits in Lebanon. J. Crop Prot. 19 (4): 217-224.
2. Abrahamian PE, Sobh H, Seblani R, Abou-Jawdah Y (2015) Co-infection of two criniviruses and a begomovirus enhances the disease severity in cucumber. Eur J Plant Pathol. 142(3): 521-530.
3. Abrahamian PE, Sobh H, Seblani R, Abou-Jawdah Y (2012) First report of Cucurbit chlorotic yellows virus on cucumber in Lebanon. Plant Dis. 96: 1704-1704.
4. Amer MA (2015) Serological and molecular characterization of cucurbit chlorotic yellows virus affecting cucumber plants in Egypt. Int. J. Virol. 11(1): 1-11.
5. Bananej K, Menzel W, Kianfar N, Vahdat A, Winter S (2013) First report of cucurbit chlorotic yellows virus infecting cucumber, melon and squash in Iran. Plant Dis. 97: 1005.
6. Berdiales B, Bernal JJ, Saez E, Woudt B, Beitia F, Rodriguez-Cerezo E (1999). Occurrence of cucurbit yellow stunting disorder virus (CYSDV) and beet pseudo-yellows virus in cucurbit crops in Spain and transmission of CYSDV by two biotypes of Bemisia tabaci. Eur. J. Plant Pathol. 105:, 211–215.
7. Bora T, Karaca G (1970) Bitki hastalıkları surveyi, kültür bitkilerinde hastalığın ve zararın ölçülmesi. Ege Üniversitesi Ziraat Fakültesi Yardımcı Ders Kitabı, Yayın No: 167, Ege Üniversitesi Matbaası, Bornova, 43 s.
8. Boubourakas IN, Avgelis AD, Kyriakopoulou PE, Katis NI (2006). Occurrence of yellowing viruses (beet pseudo-yellows virus, cucurbit yellow stunting disorder virus and cucurbit aphid-borne yellows virus) affecting cucurbits in Greece. Plant Pathol. 55: 276–283.

9. Celix A, Lopez-Sese A, Almarza N, Gomez-Guillamon ML, Rodriguez C (1996) Characterization of cucurbit yellow stunting disorder virus, a Bemisia tabaci transmitted closterovirus. Phytopathol. 86: 1370–1376.
10. Clark MF, Adams AN (1977) Charactristics of the microplate method of enzyme linked immunosorbent assay for the detection on of plant viruses. J. Gen. Virol. 34: 475-483.
11. Clark MF (1981) Immunosorbent assay plant pathology. Ann. Rev. Phytopathol. 19: 83-106.
12. Clover GRG, Elliott DR, Tang Z, Alexander BJR (2001) Occurrence of beet pseudo-yellows virus in cucumber in New Zealand. New Dis. Rep. 4, 8.
13. Desbiez C, Lecoq H (2000) First report of cucurbit yellow stunting disorder virus in Morocco. Plant Dis. 84: 5, 596.
14. Desbiez C, Lecoq H, Girard M (2003) First report of cucurbit yellow stunting disorder virus in commercial cucumber greenhouses in France Plant Dis. 87, 5, 600.
15. Duffus JE (1965) Beet pseudo-yellows virus, transmitted by the greenhouse whitefly (Trialeurodes vaporariorum). Phytopathol. 55: 450-453.
16. El Rahmany RG, El Attar AK, Zein HS, Abdallah NA, Mazyad HM (2014) Characterization of an Egyptian isolate of the cucurbit yellow stunting disorder virus. Arab J. Biotechnol. 17 (1): 29-42.
17. Fauquet MC, Mayo MA (1999) Abbreviations for plant virus names. Arch. Virol. 144: 1249-1273.
18. Fidan H, Unlu M, Unlu A, Yilmaz MA (2012) Cucurbit yellow stunting disorders (CYSDV) and Cucumber vein yellowing virus (CVYV) diseases on melon and cucumber in Turkey. Proceedings of the Xth EUCARPIA International Meeting on Genetics and Breeding of Cucurbitaceae October 15-18th, Antalya, TURKEY. pp 755-762.
19. Gazel M, Tunç B, Çağlayan K (2018) Hatay ve Tekirdağ illeri bağ alanlarında odun dokusunda deformasyona (rugose wood) neden olan virüslerin serolojik ve moleküler yöntemlerle saptanması ve karakterizasyonu. Mustafa Kemal Üniversitesi Ziraat Fakültesi Dergisi, 23:181-187.
20. Ghanem GAM, Noura-Hassan M, Kheder AA, Mazyad HM, Abdel-Alim AI (2016) Antiserum production, biological and serological detection of cucurbit yellow stunting disorder crinivirus(CYSDV) in Egypt. Int. J. Adv. Res. 4: 4, 1116-1128.
21. Gil‐Salas FM, Peters J, Boonham N, Cuadrado IM, Janssen D (2012) Co‐infection with cucumber vein yellowing virus and cucurbit yellow stunting disorder virus leading to synergism in cucumber Plant Pathol. 61: 468-478.
22. Gu QS, Liu YH, Wang YH, Huangfu WG, Gu HF, Xu L, Song FM, Brown JK (2011) First report of cucurbit chlorotic yellows virus in cucumber, melon, and watermelon in China. Plant Dis. 95:1, 73.
23. Gyoutoku Y, Hayashida S, Okazaki S, Okuda M (2008) The occurrence of melon yellowing disease caused by cucurbit chlorotic yellows virüs in Kumamoto prefecture. Ann. Phytopathol. Soc. Jpn. 74:219-219.
24. Gyoutoku Y, Okazaki S, Furuta A, Etoh T, Mizobe M, Kuno K, et al.(2009) Chlorotic yellows disease of melon caused by cucurbit chlorotic yellows virüs, a new crinivirus. Jpn. J. Phytopathol. 75,109-111.(In Japanase with Englısh abstract)
25. Hamed K, Menzel W, Dafalla G, Gadelseed AMA, Winter S (2011) First report of cucurbit chlorotic yellows virus infecting muskmelon and cucumber in Sudan. Plant Dis. 95: 1321. Hassan AA, Duffus JE (1991) A review of a yellowing and stunting disorder of cucurbits in The United Arab Emirates. Emirate J. Agric. Sci. 2: 1–16.
26. Huang L, Tseng HH, Li JT, Chen TC (2010) First report of cucurbit chlorotic yellows virus infecting cucurbits in Taiwan. Plant Dis. 94 (9): 1168.
27. Kao J, Jia L, Tian T, Rubio L, Falk BW (2000) First report of Cucurbit yellow stunting disorder virus (genus Crinivirus) in North America. Plant Dis. 84:101.
28. Keshavarz TM, Shams-Bakhsh K, Izadpanah S, Nassaj Hossini M (2013) Geographic distribution and phylogenetic analysis of cucurbit yellow stunting disorder virus in Iran Acta Virol. 57: 415–420.
29. Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 35:1547-1549.
30. Kuo YW, Rojas MR, Gilbertson RL, Wintermantel WM (2007) First Report of cucurbit yellow stunting disorder virus in California and Arizona, in association with cucurbit leaf crumple virus and squash leaf curl virus. Plant Dis. 91(3):330.
31. Louro D, Vicente M, Vaira AM, Accotto GP, Nolasco G (2000) Cucurbit yellow stunting disorder virus (genus Crinivirus) associated with the yellowing disease of cucurbit crops in Portugal. Plant Dis. 84: 1156.
32. Luis-Arteaga M, Alvarez JM, Alonso-Prados JL, Bernal JJ,Garcia-Arenal F, Lavin˜ a A, Batlle A, Moriones E (1998) Occurrence, distribution, and relative incidence of mosaic viruses infecting field-grown melon in Spain. Plant Dis. 82: 979–982.
33. Mackenzie DJ, Mclean MA, Mukerji S, Green M (1997) Improved RNA extraction for woody plants for the detection of viral pathogens by Reverse Transcription-Polymerase Chain Reaction. Plant Dis. 81: 222-226.
34. Okuda M, Okazaki S, Yamasaki S, Okuda S, Sugiyama M (2010) Host range and complete genome sequence of cucurbit chlorotic yellows virus, a new member of the genus Crinivirus. Phytopathology, 100, 560–566.
35. Orfanidou CG, Maliogka VI, Katis NI (2014) First report of Cucurbit chlorotic yellows virus in cucumber, melon, and watermelon in Greece. Plant Dis. 98: 1446.
36. Orfanidou CG, Maliogka VI, Katis NI, Kontosfyris G, Smith T, Caglayan K (2017) Fırst report of cucurbit chlorotic yellows virus in cucumber in Turkey J. Plant Pathol. 99 (2): 533-543.
37. Rubio L, Soong J, Kao J, Falk BW (1999) Geographic distribution and molecular variation of isolates of three whitefly-borne closteroviruses of cucurbits: Lettuce infectious yellows virus, cucurbit yellow stunting disorder virus, and beet pseudo-yellows virus. Phytopathology 89: 707-711.
38. Saitou N, Nei M (1987) The Neighbor-Joining Method-a new method for reconstructing phylogenetic trees. Mol Biol Evol. 4: 406–425.
39. Shakeel MT, Amer MA, Al-Saleh MA, Al-Shahwan IM, Kamran A, Orfanidou, CG, Katis NI (2018) Molecular characterization of Criniviruses involved in the etiology of cucumber yellowing disease in Riyadh region, Saudi Arabia. Eur J Plant Pathol. 150: 39-47.
40. Tomassoli L, Lumia V, Siddu GF, Barba M, (2003) Yellowing diseases of melon in Sardinia (Italy) Caused by beet pseudo-yellows virus. J. Phytopathol. 85: 59-61.
41. TÜİK (2021) Türkiye bitkisel üretim istatistikleri https:// biruni.tuik.gov.tr/medas/? kn=92& locale=tr. (erişim tarihi: 28/02/2022).
42. Wintermantel WM, Gilbertson RL, McCreight JD, Natwick, ET (2016) Host-specific relationship between virus titer and whitefly transmission of cucurbit yellow stunting disorder virus. Plant Dis., 100 (1): 92-98.
43. Wintermantel WM, Wisler GC (2006) Vector specificity, host range and genetic diversity of tomato chlorosis virus. Plant Dis. 90: 814-819.
44. Wisler GC, Duffus JE, Liu H.Y, Li RH (1998) Ecology and epidemiology of whitefly-transmitted closteroviruses. Plant Dis. 82: 270-280.
45. Zeng R, Dai FM, Chen WJ, Lu JP (2011) First Report of Cucurbit Chlorotic Yellows Virus Infecting Melon in China. Plant Dis. 95 (3): 354