ABSTRACT
Diplodia pinea is a latent, opportunistic pathogen of Pinus and other coniferous species, including Pseudotsuga menziesii (3). The fungus causes twig blight, branch cankers, tree and seedling collar rot, root rot, and can also infect cones (1). D. pinea has often been reported causing tip and shoot blight on various Pinus spp. in different parts of Turkey. During disease surveys on Pinus spp. carried out in May 2012 in Izmit in the Marmara Region (37°36'54â³N, 31°20'00â³E), typical shoot blight symptoms of D. pinea infection were also observed on the neighboring P. menziesii trees. Shoots and cones of P. menziesii were investigated for the presence of D. pinea pycnidia. Pycnidia from cones and shoots were placed on potato dextrose agar (PDA) and incubated at 23°C. Three isolates were obtained from shoot and cone samples. Identification of the pathogen was based on morphological characteristics of the conidia and by PCR of the ITS region of nuclear rDNA. Colonies on PDA were woolly, whitish at first turning black, sometimes partly or entirely turning light gray. Micromorphological characteristics of the Diplodia isolates were similar to those described in (2): conidia width 18.4 µm (SD ± 2.8) (range 11 to 22 µm) × length 34.0 µm (SD ± 5.3) (range 20 to 41 µm) (n = 100). Conidia were at first hyaline, later becoming brown to dark brown, oblong ellipsoid, bicellular with a distinct septum. To confirm the identity of the isolates, genomic DNA was extracted and the internal transcribed spacer (ITS) region of the rDNA was amplified using primers ITS1 and ITS4 (4). Amplicons were 483 bp in length (GenBank Accession No. KF372874) and shared 98% nucleotide identity with HM100285.1 and 97% nucleotide identity with JX981458.1 of D. pinea. Inoculation tests were performed on 2-year-old P. menziesii seedlings by placing mycelial plugs of three isolates obtained from pycnidia on the main stem after wounding with a cork borer. Control seedlings were inoculated with PDA plugs without mycelium. All seedlings were incubated at 24°C for 3 weeks in a climate chamber. Following incubation, the seedlings displayed dark brown-to-black discoloration, measuring on average 10.7 ± 0.6 cm, of the bark and wood tissues around the inoculation points on the stems. The pathogen was successfully re-isolated from symptomatic stem tissues, thus fulfilling Koch's postulates. To our knowledge, this is the first report of P. menziesii as a host of D. pinea in Turkey. P. menziesii is not endemic to Turkey and to date has a limited distribution (approximately 140 ha), but it is an important fast growing tree species for new industrial plantations. References: (1) J. de Wet. PhD thesis, University of Pretoria, 2008. (2) J. de Wet et al. Plant Dis. 84:151, 2000. (3) G. Hausner et al. Can. J. Plant Pathol. 21:256, 1999. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.
ABSTRACT
Cedrus libani, commonly known as Lebanon cedar, is one of the most important coniferous tree species in Turkey. Its main distribution is in the Taurus Mountains in the Mediterranean Region. The total area of pure Taurus cedar forest covers 109,440 ha in Turkey, all located in the southwestern regions of the country. Due to its drought resistance, Taurus cedar has been commonly used for afforestations in these semi-arid areas (1). In September 2011, during surveys for Phytophthora spp. in forest nurseries in Adapazari and Izmir in eastern Turkey, initial symptoms such as death of fine roots, yellowing, and wilting of Taurus cedar seedlings were observed. Soil samples were collected from 10 symptomatic C. libani seedlings and isolation tests for Phytophthora species were carried out using leaflets from young Quercus suber, Azalea sp., and Rhodendron sp. saplings as baits floated over flooded soil. Necrotic baits were blotted dry, cut into small pieces, and placed on selective PARPNH carrot agar. Out growing colonies were subcultured on carrot agar and kept at 12°C for morphological and molecular identifications (2). In total, six Pythiaceous isolates were obtained from the C. libani soil samples. The isolates were investigated using a light microscope and grouped according to their morphological characteristics (3). DNA was extracted from two representative isolates using Qiagen DNeasy Plant Mini Kit following the manufacturer's instructions. PCR amplifications and sequencing of the internal transcribed spacer (ITS) region of rDNA and the ß-tubulin gene were performed using ITS1 and ITS4 and Tub1 and Tub2 primer sets (4). Sequencing of the PCR products in both directions was conducted by IonTek Inc. (Istanbul, Turkey) in an ABI PRISM automated sequencer. The obtained sequences were compared with those in the GenBank and Phytophthora database using BLAST search. On the basis of morphological features and molecular analyses, the two isolates were identified as Phytophthora syringae. Morphological characteristics on carrot agar were identical with the description of P. syringae (2). At 20°C, colonies reached 7 cm in diameter after 1 week. Sporangia were semipapillate to non-papillate, ovoid, with average length of 59 µm (SD ± 2.8) (range 58 to 70 µm). Oogonia were 38 µm (SD ± 5.4) in diameter (range 30 to 47 µm) with paragynous antheridia. The morphological identification was confirmed by sequence comparison at GenBank with 99% homology for both ITS and ß-tubulin. The ITS sequences of the two isolates were deposited in GenBank with the accession nos. KF430614 and KF944377. Under-bark inoculation tests with mycelia plugs were conducted with both isolates of P. syringae at 18°C in a growth chamber on a total of six 1-year-old shoots cut from two C. libani trees. Lesions with an average length of 19 mm (SD ± 6) developed after 10 days. P. syringae was consistently re-isolated from the margins of necrotic tissues. Control shoots remained symptomless. To our knowledge, this is the first report of damage caused by P. syringae on C. libani seedlings in forest nursery in Turkey. References: (1) T. Çaliskan. Pages 109-130 in: Proceedings of Workshop "Hizli gelisen türlerle ilgili rapor," Ankara, Turkey, 1998. (2) T. Jung et al. Eur. J. For. Pathol. 26:253, 1996. (3) T. Jung et al. Mycol. Res. 107:772, 2003. (4) L. P. N. M. Kroon et al. Fung. Genet. Biol. 41:766, 2004.
