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MOLECULAR IDENTIFICATION of Diplodia Seriata De Not. CAUSING

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MOLECULAR IDENTIFICATION of Diplodia Seriata De Not. CAUSING http://dergipark.org.tr/trkjnat Trakya University Journal of Natural Sciences, 22(1): 93-100, 2021 ISSN 2147-0294, e-ISSN 2528-9691 Research Article DOI: 10.23902/trkjnat.863506 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 Nurdan GÜNGÖR SAVAŞ*, Murat YILDIZ Manisa Viticulture Research Institute, Plant Protection Department, 45125 Yunusemre, Manisa, TURKEY Cite this article as: Güngör Savaş N. & Yıldız M. 2021. 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. Trakya Univ J Nat Sci, 22(1): 93-100, DOI: 10.23902/trkjnat.863506 Received: 18 January 2021, Accepted: 13 April 2021, Published: 15 April 2021 Abstract: 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 Edited by: isolate has a 100% similarity index with Diplodia species according to the phylogenetic Mehmet Bora Kaydan 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. *Corresponding Author: Cyprodinil + fludioxonil showed the highest efficacy (100%) at a concentration of 1 μL mL-1. -1 Nurdan Güngör Savaş According to EC50 values, cyprodinil + fludioxonil (0.001 μL mL ) was recorded as the most [email protected] effective fungicide followed by fluopyram + tebuconazole (0.520 μL mL-1) and, azoxystrobin + difenoconazole (2.958 μL mL-1), respectively. ORCID iDs of the authors: NGS. orcid.org/0000-0002-3450-4747 MY. orcid.org/0000-0002-0758-0429 Özet: Bu çalışmanın amacı, geriye doğru ölüm hastalığı belirtileri gösteren bağlardaki 2-10 yaşındaki asmalardan izole edilen Botryosphaericea ailesinin bir üyesi olan Diplodia seriata Key words: De Not.'nın, moleküler tanılamasını gerçekleştirmektir. Patojenin duyarlılığı, fosetil- Botryosphaeriaceae Al+triadimenol, azoxystorbin+difecanozole, fludioxanil+cyprodinil, metrafenon, Dieback fluopyram+tebuconazole dâhil olmak üzere çeşitli fungisitlere karşı değerlendirilmiştir. Diplodia seriata Üreticiler tarafından getirilen asma örneklerinin kök ve kök boğazı kısımlarından elde edilen Vitis vinifera L. 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) -1 göstermiştir. EC50 değerlerine göre cyprodinil+fludioxonil (0.001 μL mL ) en etkili fungisit olarak kaydedilmiş, ardından fluopyram+tebuconazole (0.520 μL mL-1) ve azoxystrobin+difenokonazol (2.958 μL mL-1) izlemiştir. Introduction Turkey is one of the countries including the most largest grape producer in the world with 4.1 million tons geographically favorable areas for viticulture. According per year (FAO 2019). In the country, the Aegean region to FAO 2019 statistics, Turkey has the 6th largest land area ranks first in terms of both area (1,392,082 da) and devoted to vineyards with 470,000 hectares and is the 5th production (1,952,356 tons per year) and the Manisa OPEN ACCESS © Copyright 2021 Güngör Savaş & Yıldız 94 N. Güngör Savaş & M. Yıldız province ranks first in Aegean region with 809,123 da and isolated the necrotic wood tissues showing symptoms of the 1,372,571 tons per year (Anonymous 2019). More than disease. Botryosphaeria colonies are similar to Alternaria 30% of the country's production is provided by this Nees colonies and are not noticed during the diagnostic region. Manisa province supplies 90% of Turkey's dried phase based on colony development (Pitt et al. 2010, grape production and is the leader in Sultani seedless Úrbez-Torres 2011). The asexual spores of Diplodia spp. grape production used for export (Anonymous 2019). with spores of anamorphic of Botryosphaeria species are very similar, and a classification based only on the sexual Botryosphaeria dieback, caused by members of the situation is not suitable, especially since it is known that fungi family Botryosphaeriaceae, is an important disease some species have only the asexual structures, and in some in vines seen all over the world and in Turkey. Members species, sexual development is extremely rare. Given these of Botryosphaeriaceae in the Dothideomycetes class are conditions, it has been determined that there are too many found largely as endophytes, parasites, and saprophytes in features in Botryosphaeriaceae that make it difficult to both annual and perennial plants under different classify species (Slippers et al. 2013). For this reason, ecological conditions in many regions of the world molecular diagnostics and even phylogenetic analysis are (Slippers & Wingfield 2007). The importance of these recommended. Ozben (2011) pointed to the presence of disease agents in vines was understood after it was first Botryosphaeria obtusa (Schwein.) Shoemaker and B. reported as a pathogen in the 2000s (Phillips 2002). The rhodina (Pat.) Griffon & Maubl. in the vineyard areas of Eutypa dieback disease caused by Eutypa lata (Pers.) Tul. Ankara, but the presence of these species has not been & C. Tul. has been thought to be responsible for cancers approved molecularly and phylogenetically. In a study and deaths seen on vines in Australia for many years conducted in the Aegean region, Akgül et al. (2015) (Highet & Wicks 1998, Pascoe & Cottral 2000, Castillo- morphologically and molecularly identified the presence of Pando et al. 2001, Siebert 2001). In Turkey, dieback, B. dothidea and D. seriata in Sultani seedless vineyards, symptoms on vine leaves, and the development of brown but phylogenetic analyses were not performed. Between color in wood tissue, which the shape of a “V”, have been 2015 and 2018, 22 Botryosphaeriaceae cultures were associated with Esca or Eutypa dieback disease as in isolated from the vineyard areas in the Mediterranean and Australia. Southern Anatolia regions and they were phylogenetically Identification of Botryosphaeria Ces. & De. Not. separated (Akgül et al. 2020). It is very difficult to combat diseases is problematic because symptoms occurring on wood trunk diseases contained in wood tissue, and the vines in the field are very similar to other diseases such as applications for accurate diagnostics and combating are Phomopsis dead arm disease caused by Phomopsis viticola extremely important. Therefore, the present study was (Sacc.) and Eutypa dieback caused by Eutypa lata performed in order to evaluate the molecular (Castillo-Pando et al. 2001). Species of Botryosphaeria characterization of Diplodia seriata isolated from the Ces. & De. Not., Diplodia Fr., Lasiodiplodia Ellis & vineyards in Aegean Region and its sensitivity against five Everh., Neofusicoccum Slippers & A.J.L. Phillips in the different fungicides. family Botryosphaeriaceae have been isolated and described for the first time from vines showing dieback Materials and Methods symptoms (Akgül et al. 2014). Diplodia seriata De Not. Isolation of the Disease Pathogen and Neofusicoccum luteum (Pennycook & Samuels 1985) Sultani seedless vines (N=23 samples) showing Crous, Slippers & A.J.L. Phillips 2006 were often isolated dieback disease symptoms were obtained from growers in from stems, branches and shoots that show symptoms 2019 and 2020 from the vineyard areas in Manisa and during surveys conducted in vineyards in the sub-tropical Denizli province in Aegean Region. In addition to the region of eastern Australia (Savocchia et al. 2007). symptoms the samples (including root regions) were those Members of the genera Diplodia and Botryosphaeria which dried in the vineyards. The information about the have often been isolated from the root and crown parts of rootstock of the vines and the area where the vineyards vine samples delivered to Manisa viticulture research were located were noted for those where D. seriate could institute plant health laboratory by producers in Aegean have been isolated (Table
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