First Report of Phytophthora chlamydospora Causing Root Rot on Walnut (Juglans regia ) Trees in Turkey

Publication Type:

Journal Article


Plant Disease, Volume 100, Issue 11, p.2336 (2016)



Common or English (Persian) walnut (Juglans regia L.) is an important nut crop in Turkey, which is the fourth largest producer of walnut in the world. In August 2015, we observed sudden wilt and subsequent death associated with root rot. Approximately 15% of 2,000 5- to 7-year-old J. regia cv. Chandler trees grafted on wild walnut (J. regia) in waterlogged areas of a commercial walnut orchard in Bingöl province of eastern Turkey were affected. Most fine roots were completely rotted and the inner bark of infected lateral and taproots showed a reddish brown discoloration. Tissue samples taken from the margins of root lesions of 10 symptomatic trees were placed on grated carrot corn meal agar (GCCMA) (Türkölmez et al. 2015) amended with 5 mg of pimaricin, 250 mg of ampicillin, 10 mg of rifampicin, 100 mg of pentachloronitrobenzene, and 50 mg of hymexazol per liter (P5ARPH). Plates were incubated for 5 days at 28°C in the dark. A Phytophthora species was consistently isolated from the tissues. On GCCMA, colonies had a distinct petaloid growth pattern and produced abundant spherical, thin-walled, intercalary and terminal chlamydospores (30 to 46 μm diameter) and hyphal chains of globose to subglobose swellings. After incubation of mycelial disks in nonsterile soil extracts at 25°C, all isolates produced hyaline, nonpapillate, noncaducous sporangia of ovoid to obpyriform shape, with internal proliferation, measuring 34.5 to 56.5 μm in length, 27.0 to 39.5 μm wide, with a length/breadth ratio of 1.4 to 1.7, formed on unbranched or sympodial sporangiophores. Growth rate on carrot agar (CA) at 25°C was 3.3 to 3.5 mm d−1; the optimum and maximum temperatures for mycelium growth on CA were 29 and 37°C, respectively. All these characteristics were similar to those described for Phytophthora chlamydospora Brasier and Hansen (Hansen et al. 2015), previously known as P. taxon Pgchlamydo. Genomic DNA was extracted from three representative isolates. The internal transcribed spacer (ITS) region of rDNA and cox2 gene regions were amplified using the ITS6/ITS4 and FMPhy10b/FMPhy8b primer pairs, respectively, and sequenced (GenBank accession nos. KU725882, KU725883, KU707216, KX446861, KX446862). BLAST searches of ITS region showed 100% identity to many P. chlamydospora isolates, including the ex-type culture P236 (AF541900) (Brasier et al. 2003), deposited in GenBank and of cox2 gene regions of two isolates had 100% identity with PD_01777_cox2 and PD_00174_cox2 accessions deposited at Phytophthora-ID databases, which confirmed the morphological identification. Pathogenicity of P. chlamydospora was evaluated using the soil infestation method on 10 1-year-old potted J. regia cv. Chandler seedlings by growing P. chlamydospora on sterilized millet seeds for 4 weeks at 29°C and adding infested millet seeds to potting soil at a rate of 3% (w/v). In 10 noninoculated control plants, sterilized millet seeds were added to the potting soil. Both inoculated and control plants were flooded for 24 h at 2 week intervals. All inoculated plants showed severe wilting within a month and necrosis on lateral and taproots and rot on fineroots after 2 months of incubation in a greenhouse where air temperatures ranged from 24 to 30°C, while control plants remained asymptomatic. Koch’s postulates were satisfied after reisolating P. chlamydospora, which was identified morphologically, from symptomatic roots of inoculated plants. P. chlamydospora has been previously recovered from several ornamental and woody species (Blomquist et al. 2012; Brasier et al. 2003; Ginetti et al. 2014), and to our knowledge, this is the first report of P. chlamydospora infection of walnut, which represents a new host for this pathogen.