Plasma copeptin levels in patients with restless legs syndrome.

OBJECTIVES: Copeptin, the C-terminal fragment of antidiuretic hormone, is a new biomarker that has been found to be elevated in several disorders and could be related with prognosis. This study aimed to compare plasma copeptin levels in patients with restless legs syndrome (RLS) with healthy individuals and to investigate whether plasma copeptin levels were associated with the severity of disease. MATERIAL AND METHODS: 41 patients with primary RLS, who were followed in Bakirkoy Psychiatry and Neurology Research and Training Hospital and 41 age- and sex-matched healthy individuals were included into the study. RLS patients were divided into subgroups as mild-moderate, severe, and very severe according to the severity of symptoms. Sleep quality and excessive daytime sleepiness were determined according to Pittsburgh Sleep Quality Index and Epworth Sleepiness Scale, respectively, and sleep quality scores were analyzed statistically among the groups divided according to disease severity. Copeptin levels in all the patients were compared to the controls. RLS subgroups were compared with each other to evaluate association between copeptin levels and disease severity. RESULTS: Plasma copeptin levels in RLS patients were significantly higher than controls (P < .001). However, there was no association between copeptin levels and disease severity. Excessive daytime sleepiness was found as 14.63% and low sleep quality as 68.29% in patients. CONCLUSIONS: Hypothalamic-pituitary-adrenal axis activation and sympathetic hyperactivity in RLS might be responsible for increased Antidiuretic hormone (ADH) and copeptin release. We think that copeptin might have a potential role in the pathogenesis of RLS and be a biomarker for this disease.

First Report of Campylocarpon fasciculare Causing Black Foot Disease of Grapevine in Turkey.

Soil-borne fungal diseases have become an important problem in grapevine nurseries of the Aegean region (western Turkey) in recent years. Reduced vigor, black vascular streaking in basal ends, blackish-sunken necrotic root lesions, and young vine death were observed in 15 grapevine nurseries of Manisa city in May 2011 and 2012. To determine the causal agents, symptomatic young grapevine (Vitis vinifera cv. Sultana 7) plants (grafted on 1103 Paulsen) were collected from nurseries (8 to 10 plants from each). Symptomatic basal end tissues were surface disinfested with 95% ethanol and flame sterilized. The internal tissues were plated onto potato dextrose agar amended with tetracycline (0.01%). Campylocarpon-like fungi were isolated (with 37.9% isolation frequency) from only one nursery (corresponding to 6.7% of all surveyed nurseries). Fungal colonies were incubated for 21 days in the dark to induce sporulation. Fungal colonies produced cottony aerial mycelium and turned chocolate-brown to dark brown on PDA. Abundant macroconidia were observed at branched conidiophores on long and cylindrical phialides. Microconidia were not observed. Macroconidia were generally 2 to 4 septate, cylindrical and slightly curved, with the following dimensions: 2 septate: 33.5 to 40.7 × 6.1 to 7.6 µm (mean: 35.9 × 6.8 µm), 3 septate: 36.2 to 43.4 × 6.6 to 8.3 µm (mean: 37.3 × 7.6 µm), and 4 septate: 48.9 to 53.5 × 7.6 to 8.3 µm (mean: 50.7 × 8.0 µm). Fifty macroconidia were measured. Morphologically, the isolates resembled the published description of Campylocarpon fasciculare Schroers, Halleen & Crous (2,4). For molecular identification, fungal DNA was extracted from mycelium and ribosomal DNA fragments (ITS1, 5.8S ITS2 rDNA), ß-tubulin, and histone H3 genes, amplified with ITS 4-5, Bt 2a-2b, and H3 1a-1b primers (3,5), and sequenced. Sequences were compared with those deposited in GenBank. The isolate (MBAi45CL) showed 99% similarity with Campylocarpon fasciculare isolates AY677303 (ITS), AY377225 (ß-tubulin), and JF735502 (histone H3). The DNA sequences were deposited into GenBank under accessions KJ573392, KJ573393, and KJ573394 for ITS, ß-tubulin, and Histone H3 genes, respectively. To fulfill Koch's postulates, pathogenicity tests were conducted under greenhouse conditions on own-rooted grapevines (Vitis vinifera) cv. Sultana 7. Plants were removed from the rooting bench and the roots were slightly trimmed and submerged in a 107 ml-1 conidial suspension of the isolate for 60 min (5). After inoculation, the rooted cuttings were planted in 1-liter bags containing a mixture of soil, peat, and sand (2:1:1, v/v/v), and maintained in the greenhouse (24°C. 16/8-h day/night, 75% RH). Ten plants were inoculated with the isolate and five plants were submerged in sterile distilled water (control). After 4 months, young vines were examined for vascular discoloration, reduced root biomass, blackish lesions, and recovery of fungal isolates. The experiment was repeated twice. Blackish-brown discoloration of xylem vessels and necrosis in the basal ends was visible in the inoculated plants but not in the control plants. The pathogen was successfully re-isolated from 69.1% of the inoculated plants. This report is important for the new studies aiming at black foot disease control in Turkey viticulture. References: (1) A. Cabral et al. Phytopathol. Mediterr. 51:340, 2012. (2) P. Chaverri et al. Stud. Mycol. 68:67, 2011. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (4) F. Halleen et al. Stud. Mycol. 50:431, 2004. (5) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

First Report of Phaeoacremonium scolyti Associated with Esca and Petri Diseases of Grapevine in Turkey.

Grapevine (Vitis vinifera L.) is a widely planted and economically important crop for production of raisin grapes, table grapes, and wine grapes in Turkey. Esca and petri diseases are two of the most important and destructive diseases of young and old vines worldwide (1). During the summers of 2009 and 2010, a survey was carried out in 63 vineyards in six locations of Ankara Province. Root and trunk samples were collected from 4- to 15-year-old grapevines showing esca and petri disease symptoms, including reduced trunk diameters, shortened internodes, stunted growth, chlorotic or necrotic leaves, and brown-to-black spots or streaks in the xylem vessels in cross or longitudinal sections of the rootstock trunk (1,3). Small pieces of internal tissues were surface disinfested in 1% sodium hypochlorite for 2 min, washed twice with sterile distilled water, and plated onto potato dextrose agar (PDA) amended with tetracycline hydrochloride (0.1 g liter-1). Plates were incubated at 25°C in the dark for 14 to 21 days. Phaeoacremonium spp. were consistently isolated from necrotic tissues. Single-conidial isolates of these Phaeoacremonium spp. were grown on PDA, malt extract agar (MEA), and oatmeal agar (OA) in the dark at 25°C for 2 to 3 weeks until colonies produced spores (3). Of these, Phaeoacremonium aleophilum was the most prevalent species, however, one isolate identified as P. scolyti was described by L. Mostert et al. (3). Conidiophores were mostly short and usually unbranched, subcylindrical to navicular, and often consisting of an elongate-ampuliform phialide. Phialides were terminal or lateral and pale brown to hyaline. Type II phialide were predominant. Type I phialide were 4 to 6 µm (average), type II phialide were 7 to 14 µm (average), and type III phialide were 14 to 20 µm (average). Conidia were hyaline, oblong-ellipsoidal, occasionally reniform or allantoid, 2.5 to 5 µm long (average), and 1 to 1.5 µm wide (average). Colony colors were reddish gray on PDA, pinkish white on MEA, and grayish pink on OA. Identity was confirmed by ß-tubulin sequence analysis using primers T1 and Bt2b (2). Additionally, the ß-tubulin gene fragment (primers T1 and Bt2b) of this isolate was sequenced (GenBank Accession No. JF909894). The sequence showed high similarity (99%) with the sequence of P. scolyti (GenBank Accession No. EU260415). Pathogenicity tests were completed using five, 3-month-old rooted cuttings of cv. Sultana. A hole approximately 3 mm in diameter was drilled on the crown 2 cm aboveground level from the bark to the pith and filled with a 30-µl spore suspension (106 spores/ml) harvested from 21-day-old cultures grown on PDA at 25°C in the dark. Five control plants were used. Controls were inoculated with sterile distilled water. Filled holes were sealed with a sheet of Parafilm. The plants were incubated for 3 months in a controlled environment facility at 25°C. After 3 months, the fungus was reisolated from black discoloration of vascular tissue and pith tissue of the crown area of all inoculated cuttings, completing Koch's postulates. The black discoloration was more compact near the point of inoculation. Control plants were asymptomatic and P. scolyti was not recovered from control plants. To our knowledge, this is the first report of the presence of P. scolyti causing esca and petri disease of grapevine in Turkey. References: (1) A. Eskalen et al. Plant Dis. 91:1100, 2007. (2) S. Essakhi et al. Persoonia 21:119, 2008. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006.

First Report of Cylindrocarpon macrodidymum Associated with Black Foot Diseases of Grapevine in Turkey.

Grape (Vitis vinifera) is widely planted and is an economically important crop in Turkey for domestic consumption and export. Black foot disease, caused by Cylindrocarpon macrodidymum Halleen, Schroers & Crous, is a recently identified but worsening problem in vineyards worldwide (3,4). Symptomatic grapevines show reduced vigor, shortened internodes, small leaves with interveinal chlorosis, and necrosis frequently leading to the death of the plants (1). Roots of symptomatic grapevines exhibit black, sunken, necrotic lesions with a reduction in root biomass. Pith of affected vines is discolored (4). During the summers of 2009 and 2010, a survey was carried out in 63 vineyards (4 to 15 years old) in six locations of Ankara Province. We collected 44 samples from roots and crowns of grapevines exhibiting black foot symptoms. In cross section, extensive necrosis at the base of the trunk and brown-black spots in xylem vessels were observed, resembling those previously reported for black foot disease (2,4). Isolations were made from roots, vascular elements, and pith tissue. In this study, 26 isolates were identified as C. macrodidymum on the basis of morphological characteristics. Isolates identified as C. macrodidymum had a dark orange-brown colony color and abundant aerial mycelia when grown on potato dextrose agar. Isolates produced ellipsoid or ovoid microconidia. The macroconidia were one to three septate, straight, and cylindrical. One-septate macroconidia were 24 to 32 × 5 to 7 µm; three-septate macroconidia were 26 to 40 × 5 to 6 µm. Chlamydospores developed in short, intercalary chains. Conidiophores were simple or complex and sporodochial. Isolate identities were confirmed by sequence analysis of the ribosomal DNA internal transcribed spacer (GenBank Accession No. HM245331) with primers ITS1 and ITS4 (4). Isolates had 99% genetic identity with other isolates of C. macrodidymum present in GenBank. In pathogenicity tests, one representative isolate was used to inoculate five grapevine plants. Tests were completed by drench inoculation onto 3-month-old rooted cuttings of cv. Sultana with 25 ml of a conidia suspension (106 conidia ml-1). Controls were inoculated with an equal volume of sterile distilled water. Plants were incubated for 4 months in a controlled environment facility at 25°C. After 3 to 4 months, inoculations resulted in reduction of root mass, and C. macrodidymum was reisolated from regions of brown streaking in wood and discolored vascular tissue in all inoculated plants, fulfilling Koch's postulates. Control plants were asymptomatic and C. macrodidymum was not recovered from control plants. To our knowledge, this is the first report of the presence of C. macrodidymum causing black foot disease on grapevine in Turkey. References: (1) S. Alaniz et al. Plant Dis. 93:821, 2009. (2) F. Hallen et al. Stud. Mycol. 50:431, 2004. (3) F. Halleen et al. Phytopathol. Mediterr. 45:S55, 2006. (4) E. Petit and W. D. Gubler. Plant Dis. 89:1051, 2005.