Bu çalışmanın amacı, geriye doğru ölüm hastalığı belirtileri gösteren bağlardaki 2-10 yaşındaki asmalardan izole edilen Botryosphaericea ailesinin bir üyesi olan Diplodia seriata De Not.'nın, moleküler tanılamasını gerçekleştirmektir. Patojenin duyarlılığı, fosetil-Al+triadimenol, azoxystorbin+difecanozole, fludioxanil+cyprodinil, metrafenon, fluopyram+tebuconazole dâhil olmak üzere çeşitli fungisitlere karşı değerlendirilmiştir. Üreticiler tarafından getirilen asma örneklerinin kök ve kök boğazı kısımlarından elde edilen izolatlar, morfolojik ve moleküler yöntemlere göre D. seriata olarak tanımlanmıştır. Moleküler tanımlamada izolatların ITS (Internal Transcribed Spacer) ve TUB2 (β-tubulin) gen bölgeleri Real-Time PCR ile çoğaltılmış ve bu gen bölgelerinden nükleotid dizileri elde edilmiştir. Daha sonra MEGA 7 yazılımı kullanılarak ITS ve TUB2 dizileri hizalanmış ve kombine bir filogenetik ağaç çizilmiştir. Diplodia seriata izolatının filogenetik analizlere göre Diplodia türleri ile % 100 benzerlik indeksine sahip olduğu moleküler olarak doğrulanmıştır. Farklı konsantrasyonlarda (0, 1, 3, 10, 30, 50, 100 μL mL-1) kullanılan fungisitlerin ortalama etkili konsantrasyon (EC50) değerleri Probit analiziyle belirlenmiştir. Cyprodinil+fludioxonil 1 μL mL-1 konsantrasyonunda en yüksek etkinliği (%100) göstermiştir. EC50 değerlerine göre cyprodinil+fludioxonil (0.001 μL mL-1) en etkili fungisit olarak kaydedilmiş, ardından fluopyram+tebuconazole (0.520 μL mL-1) ve azoxystrobin+difenokonazol (2.958 μL mL-1) izlemiştir.

MOLECULAR IDENTIFICATION OF Diplodia seriata De Not. CAUSING DIEBACK EFFECT ON GRAPEVINES AND EVALUATION OF in vitro EFFICACY OF FIVE DIFFERENT SYNTHETIC FUNGICIDES AGAINST THIS DISEASE

The aim of this study was to realize the molecular identification of Diplodia seriata De Not., a member of the Botryosphaericea family, isolated from 2-10 years old vines in vineyards showing symptoms of dieback disease. The susceptibility of the pathogen against the fungicides with the fosetyl-Al+triadimenol, azoxystorbin+difecanozole, fludioxanil+cyprodinil, metrafenone, fluopyram+tebuconazole combinations were evaluated. The isolates obtained from the root and crown parts of the vine samples were identified as D. seriata according to the morphological and molecular methods. In molecular identification, the ITS (Internal Transcribed Spacer) and TUB2 (β-tubulin) gene regions of the isolates were amplified by Real-Time PCR and the nucleotide sequences were obtained in these gene regions. After using the MEGA 7 software, ITS and TUB2 sequences were aligned and a combined phylogenetic tree was made. It has been molecularly confirmed that the D. seriata isolate has a 100% similarity index with Diplodia species according to the phylogenetic analyses. The mean effective concentration (EC50) values of fungicides used with different concentrations (0, 1, 3, 10, 30, 50, 100 μL mL-1) were determined by Probit analysis. Cyprodinil + fludioxonil showed the highest efficacy (100%) at a concentration of 1 μL mL-1. According to EC50 values, cyprodinil + fludioxonil (0.001 μL mL-1) was recorded as the most effective fungicide followed by fluopyram + tebuconazole (0.520 μL mL-1) and, azoxystrobin + difenoconazole (2.958 μL mL-1), respectively.

Keywords:

Botryosphaericea, Dieback Diplodia seriata, Vitis vinifera,

PDF

___

1. Akgül, D.S., Savas, N.G. & Eskalen, A. 2014. First report of wood canker caused by Botryosphaeria dothidea, Diplodia seriata, Neofusicoccum parvum, and Lasiodiplodia theobromae on grapevine in Turkey. Plant Diseases, 98: 568.
2. Akgül, D.S., Savas, N.G., Teker, T., Keykubat, B., Mayorquin, J.S. & Eskalen, A. 2015. Fungal trunk pathogens of Sultana seedless vineyards in Aegean region of Turkey. Phytopathologia Mediterranea, 54(2): 380-393.
3. Akgül, D.S., Ozarslandan, M. & Erkılıç, A. 2020. Phylogenetic discrimination and pathogenicity of fungi causing Botryosphaeria dieback disease on grapevine in Southern Turkey. Plant Protection Bulletin, 60(2): 63-72.
4. Amponsah N.T., Jones E., Ridgway H.J. & Jaspers M.J. 2012.Evaluation of fungicides for the management of Botryosphaeria dieback diseases of grapevines. Pest Management Science, 68: 676-683.
5. Anonymous, 2019. Agricultural Products Markets GRAPE. https://arastirma.tarimorman.gov.tr/tepge/Belgeler/PDF%20Tar%C4%B1m%20%C3%9Cr%C3%BCnleri%20Piyasalar%C4%B1/2019Ocak%20Tar%C4%B1m%20%C3%9Cr%C3%BCnleri%20Raporu/2019-Ocak%20%C3%9Cz%C3%BCm.pdf. (Data Accessed: May 2020).
6. Billones‐Baaijens, R., Ridgway, H.J., Jones, E.E. & Jaspers, M.V. 2013. Prevalence and distribution of Botryosphaeriaceae species in New Zealand grapevine nurseries. European Journal of Plant Pathology, 135: 175-85.
7. Castillo-Pando, M., Somers, A., Green, C.D., Priest, M. & Sriskanthades, M. 2001. Fungi associated with dieback of Semillon grapevines in the Hunter Valley of New South Wales. Australasian Plant Pathology, 30: 59-63.
8. Chebil, S., Fersi, R., Bouzid, M., Quaglino, F., Chenenaoui, S., Melki, I., Durante, G., Zacchi, E., Bahri, B.A., Bianco, P.A. & Rhouma, A. 2017. Fungi from the Diaporthaceae and Botryosphaeriaceae families associated with grapevine decline in Tunisia. Ciencia e Investigación Agraria, 44(2): 127-138.
9. FAO. 2019. Food and Agricultural Organization, Statistics Division. http://www.fao.org. (Date Accessed: April 2020).
10. Glass, N. & Donalds, G.C. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology, 61(4): 1323-30.
11. Gramaje D., Úrbez-Torres, J.R. & Sosnowski, M.R. 2018. Managing Grapevine Trunk Diseases with respect to etiology and epidemiology: Current Strategies and Future Prospects. Plant Diseases, 102(1): 12-39.
12. Highet, A. & Wicks, T. 1998. The incidence of Eutypa dieback in South Australia vineyards. Annual Technical Issue. The Australian Grape Grower and Wine-maker, 414: 135-136.
13. Kühn A., Zappata, A., Gold, R.E., Zito, R. & Kortekamp, A. 2017. Susceptibility of grape pruning wounds to grapevine trunk diseases and effectiveness of a new BASF wound protectant. Phytopathologia Mediterranea, 56(3): 536 (abstract).
14. Larignon, P., Fulchıc, R., Cere, L. & Dubos, B. 2001. Observation on black dead arm in French vineyards. Phytopathologia Mediterranea, 40: 336-342.
15. Luque J., Martos, S., Aroca, A., Raposoand, R. & Garcia- Fihueres, F. 2009. Symptoms and fungi associated with declining mature grapevine plants in northeast Spain. Journal of Plant Pathology, 91: 381-390.
16. Ozben, S. 2011. Determination of Fungal Diseases and Their Prevalence in Vineyard in Ankara Provinces. Ankara University Graduate School of Natural and Applied Sciences Department of Plant Protection, Master Thesis, Ankara, 134pp.
17. Pascoe, I. & Cottral, E. 2000. Developments in grapevine trunk diseases research. Phytopathologia Mediterranea, 39: 68-75.
18. Phillips, A.J.L. 2002. Botryosphaeria species associated with diseases of grapevines in Portugal. Phytopathologia Mediterranea, 41: 3-18.
19. Phillips, A.J.L., Crous, P.W & Alves, A. 2007. Diplodia seriata, the anamorph of “Botryosphaeria” obtusa. Fungal Diversity, 25: 141-155.
20. Phillips, A.J.L., Alves, A., Pennycook, S.R., Johnston, P.R. & Ramaley, A. 2008. Resolving the phylogenetic and taxonomic status of dark-spored teleomorph genera in the Botryosphaeriaceae. Molecular Phylogeny and Evolution of Fungi, 21: 2955.
21. Phillips, A.J.L., Lopes, J., Abdollahzadeh, J., Bobev, S. & Alves, A. 2012. Resolving the Diplodia complex on apple and other Rosaceae hosts. Molecular Phylogeny and Evolution of Fungi, 29: 29-38.
22. Phillips, A.J.L., Alves, A., Abdollahzadeh, J., Slippers, B., Wingfield, M.J., Groenewald, J.Z. & Crous, P.W. 2013. The Botryosphaeriaceae: genera and species known from culture. Studies in Mycology, 76: 51-167.
23. Pitt, W.M., Sosnowski, M.R., Huang, R., Qiu, Y., Steel, C.C. & Savocchia, S. 2012. Evaluation of fungicides for the management of Botryosphaeria canker of grapevines. Plant Diseases, 96: 1303-1308.
24. Pitt, W.M., Huang, R. & Steel, C.C. 2010. Identification, distribution and current taxonomy of Botryosphaeriaceae species associated with grapevine decline in New South Wales and South Australia. Australian Journal of Grape and Wine Research, 16: 258-271.
25. Savocchia S., Steel C.C., Stodart, B.J. & Somers, A., 2007. Pathogenicity of Botryosphaeria species from declining grapevines in subtropical regions of Eastern Australia. Vitis, 46(1): 27-32.
26. Siebert, J.B. 2001. Eutypa: the economic toll on vineyards. Wines and Vines, 4: 50-56.
27. Slippers, B. & Wingfield, M.J. 2007. Botryosphaeriaceae as endophytes and latent pathogens of woody plants- diversity, ecology and impact. Fungal Biology Reviews, 21: 90-106.
28. Slippers, B., Boissin, E., Phillips, A.J.L., Groenewald, J.Z. & Wingfield, M.J. 2013. Phylogenetic lineages in the Botryosphaeriales: A systematic and evolutionary framework. Studies in Mycology, 76: 31-49.
29. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28: 2731-2739.
30. Úrbez-Torres, J.R. & Gubler, W.D. 2009. Pathogenicity of Botryosphaeriaceae species isolated from grapevine cankers in California. Plant Disease, 93: 584-592.
31. Úrbez-Torres, J.R. 2011. The status of Botryosphaeriaceae species infecting grapevines. Phytopathologia Mediterranea, 50: 5-45.
32. Uysal, A. & Kurt, S. 2019. In vitro sensitivity of anthracnose disease agent, Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., to some fungicides on lemon. Plant Protection Bulletin, 59(1): 53-62.
33. Yan, JY., Xie, Y., Zhang, W., Wang, Y., Liu, JK., Hyde, KD., Seem, RC., Zhang, GZ.,Wang, ZY, Yao, SW., Bai, XJ., Dissanayake, AJ., Peng, YL. & Li, XH. 2013. Species of Botryosphaeriaceae involved in grapevine dieback in China. Fungal Diversity, 61: 221-236.