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

Öz

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.

Anahtar Kelimeler

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

Kaynakça

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