Powered by TCPDF (www.tcpdf.org) New Disease Reports (2018) 38, 13. http://dx.doi.org/10.5197/j.2044-0588.2018.038.013 First report of Diplodia sapinea on Cedrus libani in Turkey F. Oskay 1, A. Lehtijärvi 2, H.T. Dogmuş-Lehtijärvi 3 and S. Woodward 4 1 Çankırı Karatekin University, Faculty of Forestry, 18200, Çankırı, Turkey;; 2 Bursa Technical University, Faculty of Forestry, 16310 Yıldırım-Bursa, Turkey;; 3 Süleyman Demirel University, Faculty of Forestry, TR 32260 Çünür, Isparta, Turkey;; 4 University of Aberdeen, School of Biological Sciences, Cruickshank Building, Aberdeen AB24 3UU, Scotland, UK *E-mail: fundaoskay@karatekin.edu.tr Received: 13 Aug 2018. Published: 29 Aug 2018. Cedar of Lebanon (Cedrus libani) is an ecologically and economically important conifer with a natural distribution in southern Turkey. Currently, no serious diseases affect C. libani in its native range, although it is susceptible to pathogens such as Heterobasidion annosum and Diplodia pinea in inoculation experiments (Lehtijärvi et al., 2011). Recent reports from the Balkans and Northern Europe, however, suggest that C. libani is susceptible to foliage and shoot diseases such as Diplodia pinea, Dothistroma septosporum and Sirococcus tsugae (Mullet et al., 2016; Zlatkovic et al., 2016). During disease surveys in July 2017 in the Atatürk Arboretum, İstanbul (41°10'30.53"N, 28°59'5.22"E), dieback was observed on branches of a young C. libani (Fig. 1). Abundant black pycnidia were observed on dead needles under a binocular microscope (Fig. 2). Examination under a compound microscope revealed the presence of non- or one-septate, light to dark brown, oblong-ellipsoid conidiospores, characteristic of the genus Diplodia (Figs. 3-4). Isolations were made by plating spore masses onto 2% malt extract agar in 90 mm diameter Petri dishes under aseptic conditions and incubating at room temperature for ten days in the dark. Blackish-dark grey colonies with grey aerial mycelia were subcultured on 2% malt extract agar covered with cellophane membrane and incubated as described above. DNA was extracted from mycelia using an E.Z.N.A. fungal DNA extraction kit (Omega Bio-tek, USA) and amplified by PCR using speciesspecific primers for both D. pinea and D. scrobiculata (Smith & Stanosz, 2006). The D. pinea primers amplified a product of 700 bp. The ITS region was also amplified and sequenced (White et al., 1990), and the sequence of a representative isolate was deposited in GenBank (Accession No. MH368280). Comparison of the ITS sequences obtained from C. libani showed 100% nucleotide identity with those of reference strains of D. sapinea (e.g. NR_152452.1). Inoculations were made on two-year-old C. libani plants to determine the pathogenicity of the fungus, with five replicate plants each for stem and shoot inoculations. A circular 4-mm wound was made on stems approximately 10 cm above the root collar, by removing the bark tissues with a surface-sterilised cork borer. For shoot inoculations, a small wound of c. 3 mm was made on the shoots using a scalpel to remove the bark, at c. 5 cm from the tip of the terminal shoot. Agar plugs (4 and 2 mm diameter for stem and shoots, respectively) taken from the margins of an actively growing D. sapinea colony on potato dextrose agar (PDA), were placed Figure 1 Figure 2 Figure 4 Figure 5 mycelium-side onto the wounds. All treatments were wrapped in plastic paraffin film. Controls (three replicates for both stem and shoot inoculations) were wounded, as described above, and treated with sterile PDA plugs. All plants were incubated at 24°C for eight weeks in a climate chamber. Following incubation, dieback symptoms were observed on all inoculated plants; foliage and stems were dead above the inoculation point (Fig. 5). Lesion extension towards the root collar was 24.2 ± 2.0 mm for stem and 26.0 ± 1.16 mm for the shoot inoculations. The pathogen was successfully re-isolated from symptomatic stem and shoot tissues, thus fulfilling Koch's postulates. To our knowledge, this is the first report of Diplodia sapinea causing dieback on C. libani in Turkey. Acknowledgements Financial support from the Süleyman Demirel University (Project No.4984-GÜP-17) is gratefully acknowledged. References Lehtijärvi A, Aday AG, Doğmuş‐Lehtijärvi HT, 2011. Cedrus libani: the most susceptible Turkish conifer species to local Heterobasidion isolates in spring inoculations. Forest Pathology 41, 1-6. http://dx.doi.org/10.1111/j.1439-0329.2009.00625.x Mullett MS, Fraser S, 2016. Infection of Cedrus species by Dothistroma septosporum. Forest Pathology 46, 551-554. http://dx.doi.org/10.1111/efp.12214 Zlatković M, Keča N, Wingfield MJ, Jami F, Slippers B, 2017. New and unexpected host associations for Diplodia sapinea in the Western Balkans. Forest Pathology 47, e12328. http://dx.doi.org/10.1111/efp.12328 Smith DR, Stanosz GR, 2006. A species-specific PCR assay for detection of Diplodia pinea and D. scrobiculata in dead red and jack pines with collar rot symptoms. Plant Disease 90, 307-313. http://dx.doi.org/10.1094/PD-90-0307 White TJ, Bruns T, Lee S, Taylor JW, 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR Protocols: A Guide to Methods and Applications. New York, USA: Academic Press Inc., 315-322. http://dx.doi.org/10.1016/B978-0-12-372180-8.50042-1 Figure 3 To cite this report: Oskay F, Lehtijärvi A, Dogmuş-Lehtijärvi HT, Woodward S, 2018. First report of Diplodia sapinea on Cedrus libani in Turkey. New Disease Reports 38, 13. http://dx.doi.org/10.5197/j.2044-0588.2018.038.013 This report was published on-line at www.ndrs.org.uk where high quality versions of the figures can be found. ©2018 The Authors New Disease Reports is a peer-reviewed on-line journal published by the British Society for Plant Pathology, for more information visit http://www.ndrs.org.uk/ Page 13