Phylogenetic discrimination and pathogenicity of fungi causing Botryosphaeria dieback disease on grapevine in Southern Turkey

Abstract

Grapevine trunk diseases associated with Botryosphaeriaceae fungi are common and important diseases for vineyards in Turkey. Up to date, 24 species of this family have been reported to cause Botryosphaeria dieback disease all over the World. This study aimed to discriminate Botryosphaeriaceae isolates (obtained from symptomatic vines in Adana, Mersin, Kahramanmaraş) phylogenetically and to determine their pathogenicity. 22 Botryosphaeriaceae cultures were isolated by standard mycological isolation procedures obtained from representive 18 vineyards. In molecular identification of the isolates, ITS (Internal Transcribed Spacer) and TEF (Translation Elongation Factor 1-α) gene regions were amplified by PCR and nucleotide sequences were obtained from these regions. The gene sequences were compared with the sequences available in the NCBI GenBank database using the BLASTn pro¬gram. The isolates were pre-identified according to likelihood rates. The combined phylogenetic tree was generated after ITS and TEF sequences were aligned using MEGA7 software. Following BLASTn and phylogenetic analysis, 7 different Botryosphaeriaceae fungi (Botryosphaeria dothidea, Diplodia seriata, Lasiodiplodia theobromae, L. pseudoteobromae, Neofusicoccum parvum, N. vitifusiforme, Neoscytalidium novaehollandiae) were identified. Pathogenicity tests were performed on one-year-old grapevine plants under greenhouse conditions (for two months). According to lesion occurrence, by measuring lesion lengths, the pathogenicity of these isolates was confirmed. Variance analysis was performed for statistical separation of the isolates’ virulence. For the two-month incubation period, the most virulent species N. parvum produced 38.0-45.3 mm lesions, whereas D. seriata with the lowest virulence caused 17.6-20.0 mm lesions on vine seedlings.

Keywords

• Adana,Mersin,Kahramanmaras,Botryosphaeriaceae,trunk disease,vineyard

Türkiye’nin güneyinde asmada Botryosphaeria geriye ölüm hastalığına neden olan fungusların filogenetik ayrımı ve patojenisiteleri

Abstract

Türkiye’deki bağlarda Botryosphaeriaceae funguslarıyla ilişkili asma gövde hastalıkları oldukça yaygın ve önemli hastalıklardandır. Dünya genelinde şimdiye kadar bu familyadan 24 türün Botryosphaeria geriye ölüm hastalığına neden olduğu rapor edilmiştir. Bu çalışmanın amacı, 2015-2018 yılları arasında Adana, Mersin, Kahramanmaraş ve Gaziantep illerindeki simptomatik asmalarda Botryosphaeriaceae izolatlarını filogenetik olarak ayırt etmek ve izolatların patojenisitelerini belirlemektir. 18 bağa ait simptomatik asma örneklerinden standart mikolojik izolasyon prosedürleri ile 22 Botryosphaeriaceae kültürü izole edilmiştir. İzolatların moleküler tanısında ITS (Internal Transcribed Spacer) ve TEF (Translation Elongation Factor 1-α) gen bölgeleri PCR ile çoğaltılmış ve bu gen bölgelerindeki nükleotid dizileri elde edilmiştir. Gen dizileri BLASTn programı kullanarak NCBI Gen Bankası’nda bulunan dizilerle karşılaştırılmış ve benzerlik oranlarına göre ön tanı yapılmıştır. MEGA 7 yazılımı kullanarak ITS ve TEF dizileri hizalanmış ve kombine bir filogenetik ağaç çizilmiştir. BLASTn ve filogenetik analizlerden sonra 7 farklı Botryosphaeriaceae türü (Botryosphaeria dothidea, Diplodia seriata, Lasiodiplodia theobromae, L. pseudoteobromae, Neofusicoccum parvum, N. vitifusiforme, Neoscytalidium novaehollandiae) tanılanmış ve bunların patojenisiteleri sera koşullarında, 2 ay süreyle, 1 yaşındaki asma fidanları (Vitis vinifera L. Cardinal) üzerinde yürütülmüştür. Lezyonların oluşumuna göre, lezyon uzunluklarını ölçerek, bu izolatların patojenisiteleri doğrulanmış ve virülenslik seviyelerinin istatistiksel ayrımı için varyans analizi uygulanmıştır. Asma fidanları üzerinde 2 aylık sürede, en virülent tür N. parvum 38.0-45.3 mm’lik lezyonlar oluştururken, virülensliği en düşük bulunan D. seriata ise 17.6-20.0 mm’lik lezyonlar meydana getirmiştir.

Keywords

• Adana,Mersin,Kahramanmaraş,gövde hastalıkları,Botryosphaeriaceae,bağ

References

1. Akgül D. S., Güngör-Savaş N., Teker T., Keykubat B., Mayorquin J. S., Eskalen A., 2015. Fungal trunk pathogens of sultana seedless vineyards in the Aegean Region of Turkey. Phytopathologia Mediterranea, 54 (2): 380-393.
2. Akgül D. S., Awan Q. N., Güngör-Savaş N., 2016. Asmalarda bazı Botryosphaeriaceae funguslarının in vitro sıcak su uygulamalarına duyarlılıkları. Türkiye 6. Bitki Koruma Kongresi, 5-8 Eylül 2016, Konya, 609s. (özet).
3. Akgül D.S., Güngör Savaş N., Özarslandan M., 2019. First Report of Wood Canker Caused by Lasiodiplodia exigua and Neoscytalidium novaehollandiae on Grapevine in Turkey. Plant Disease, 103: 1036.
4. Alves A., Crous P.W., Correia A., Phillips A.J.L., 2008. Morphological and molecular data reveal cryptic speciation in Lasiodiplodia theobromae. Fungal Diversity, 28:1-13.
5. 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.
6. Auger J., Esterio M., Ricke G., Perez I., 2004. Black dead arm and basal canker on Vitis vinifera cv. Red Globe caused by Diplodia seriata in Chile. Plant Disease, 88: 1286.
7. Baumgartner K., Fujiyoshi P. T., Travadon R., Castlebury L.A., Wilcox W.F., Rolshausen P.E., 2013. Characterization of species of Diaporthe from wood cankers of grape in eastern North American vineyards. Plant Disease, 97:912-920.
8. Carbone I., Kohn L.M., 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia, 91(3): 553-556.

9. Carlucci A., Cibelli F., Lops F., Raimondo M., 2015. Characterization of Botryosphaeriaceae species as causal agents of trunk disease on grapevines. Plant Disease, 99, 1678-1688.
10. Cebeci E., Akın A., 2014. Mersin ili üzüm ihracatının Türkiye ekonomisi içindeki yeri ve öneminin değerlendirilmesi. ÇOMÜ Ziraat Fakültesi Dergisi, 2 (2): 119-129.
11. de Hoog G.S., Gerrits van den Ende A.H.G., 1988. Molecular diagnostics of clinical strains of filamentous Basidiomycetes. Mycoses, 41: 183-189.
12. Denman S., Crous P.W., Taylor J.E., Kang J.Ch., Pas¬coe I., Wingfield M.J., 2000. An overview of the taxo¬nomic history of Botryosphaeria, and a re-evaluation of its anamorphs based on morphology and ITS rDNA phylogeny. Studies in Mycology 45: 129–140.
13. Djoukeng J.D., Polli S., Larignon P., Abou-Mansour P., 2009. Identification of phytotoxins from Diplodia seriata, a pathogen of black dead arm disease of grapevine. European Journal of Plant Pathology, 124: 303–308.
14. Evidente A., Punzo B., Andolfi A., Cimmino A., Melck D., Luque J., 2010. Lipophilic phytotoxins produced by Neofusicoccum parvum, a grapevine canker agent. Phytopathologia Mediterranea, 49: 74–79.
15. Gramaje D., Cabral A., Mostert L., Lawrence D.P., Trouillas F.P., Urbez-Torres J.R., Alves A., 2019. The most informative loci for identifying grapevine trunk disease pathogens. 11. International workshop on grapevine trunk diseases Proceedings, 433-434 pp. 7th-12th July 2019 Pentincton, British Columbia, Canada.
16. Kumar S., Stecher G., Tamura K., 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33: 1870-1874.
17. Mohammadi H., Gramaje D., Banihashemi Z., Armengol J., 2013. Characterization of Diplodia seriata and Neofusicoccum parvum associated with grapevine decline in Iran. Journal of Agricultural Science and Technology, 15, 603-616.
18. Nejat N., Sijam K., Abdullah S.N.A., Vadamalai G., Dickinson M., 2009. Molecular characterization of a phytoplasma associated with coconut yellow decline (CYD) in Malaysia. American Journal of Applied Sciences, 6: 1331-1340.
19. Oksal E., Çelik Y., Özer G., 2019. Neoscytalidium dimidiatum causes canker and dieback on grapevine in Turkey. Australasian Disease Notes, https://doi.org/10.1007/s13314-019-0363-4
20. Özben S., 2011. Ankara ili bağ alanlarında görülen fungal hastalıkların ve yaygınlık oranlarının belirlenmesi. Ankara Üniversitesi Fen Bilimleri Enstitüsü, Yüksek Lisans Tezi, 134 s. Ankara.
21. 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.
22. Pitt W. M., Sosnowski M. R., Rujan H., Steel C. C., Savocchia S., 2012. Evaluation of fungicides for the management of Botryospaheria canker of grapevines. Plant Disease, 96, 1303-1308.
23. Savocchia S., Steel C.C., Stodart B.J., Somers A., 2007. Pathogenicity of Botryosphaeria species from de¬clining grapevines in sub tropical regions of Eastern Australia. Vitis, 46: 27–32.
24. Siebert J. B., 2001. Eutypa: the economic toll on vineyards. Wines & Vines 4, 50–56.
25. Slippers B., Boissin E., Phillips A. J. L., Groenewald J. Z., Lombard L., Wingfield M. J. Postma A., Burgess T., Crous P. W., 2013. Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary framework. Studies in Mycology 76, 31–49.
26. Smith D.R., Stanosz G.R., 2001. Molecular and mor¬phological differentiation of Botryosphaeria dothidea (anamorph Fusicoccum aesculi) from some other fungi with Fusicoccum anamorphs. Mycologia 93: 505–515.
27. Smith H., Wingfield M.J., Crous P.W. Coutinho T.A., 1996. Sphaeropsis sapinea and Botryosphaeria dothidea endophytic in Pinus spp. and Eucalyptus spp. in South Africa. South African Journal of Botany 62, 86–88.
28. Taylor A., Hardy G.E., Wood P., Burgess T., 2005. Identification and pathogenicity of Botryosphaeria species associated with grapevine decline in Western Australia. Australasian Plant Pathology, 34: 187–195.
29. Urbez-Torres J.R., Leavitt G.M., Guerrero J.C., Guevara J., Gubler W.D., 2008. Identification and pathogenic¬ity of Lasiodiplodia theobromae and Diplodia seriata, the causal agents of bot canker disease of grapevines in Mexico. Plant Disease, 92: 519–529.
30. Urbez-Torres J. R., Phillips A. J. L, Gubler W. D., 2015. Botryosphaeria dieback. In: Compendium of Grape Diseases, Second Edition. Wilcox W. F., Gubler W. D., Uyemoto J. K., (Eds.), APS Press. St. Paul Minnesota, USA, 33-39.
31. White T.J., Bruns T., Lee S., Taylor J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Snisky JJ, White TJ, eds. PCR protocols: a guide to methods and applications. San Diego, USA, Academic Press, 315–322.