First Report of Leaf Spot Caused by Cladosporium herbarum on Centaurea solstitialis in Greece.

Centaurea solstitialis L. (yellow starthistle), family Asteraceae, an invasive weed in California and the western United States, is targeted for biological control. In the summer of 2003, an epidemic of unknown etiology on dying C. solstitialis plants was observed near Kozani, Greece (40°22'07″N, 21°52'35″E, elevation, 634 m). Plants had necrotic light brown leaf spots on the lower leaves and the decurrent leaf bases along the stems. Often, necrotic lesions extended along the stems to the capitula. Virtually all plants in a solid stand of C. solstitialis (approximately 0.5 ha) showed disease symptoms. Diseased plants were collected, air dried, and sent to the quarantine facility of the Foreign Disease-Weed Science Research Unit (FDWSRU), USDA/ARS, Fort Detrick, MD. On the basis of culture growth (45-cm diameter after 2 weeks at 25°C on malt extract agar), fungal morphology (1), and comparison with 21 internal transcribed spacer sequences in GenBank, the putative causal organism was identified as Cladosporium herbarum (Pers.:Fr.) Link. (teleomorph = Davidiella tassiana (De Not.) Crous & U. Braun). Sixteen C. solstitialis plants in the rosette stage and 16 plants in the bolted stage were inoculated with an aqueous suspension of spores (106 conidia ml-1) and placed in an environmentally controlled chamber at 25°C with 8 h of dew and 12 h of light daily. Plants in the rosette stage were resistant, but the fungus was very aggressive on bolted plants. Within 4 to 6 days of inoculation, necrosis developed on leaves and stems and then spread up the stems to the capitula, often resulting in plant death. The fungus also infected developing flowers. Cladosporium herbarum was reisolated from each of the 16 bolted C. solstitialis plants in two separate tests at the FDWSRU and from all bolted inoculated plants at the European Biological Control Laboratory (EBCL) in Greece. In the greenhouse at the EBCL, the pathogen readily spread to (and was isolated from) another 10 noninoculated C. solstitialis plants in close vicinity to the inoculated C. solstitialis plants. Results of host range tests will establish if this isolate of Cladosporium herbarum has the potential as a biological control agent of C. solstitialis in the United States and does not pose a threat to other Centaurea spp. used in horticulture. A voucher specimen has been deposited with the U.S. National Fungus Collections (BPI 863446). Live cultures are being maintained at the FDWSRU and EBCL, Greece. To our knowledge, this is the first report of a disease caused by Cladosporium herbarum on C. solstitialis. Reference: (1) M. H. M. Ho et al. Mycotaxon 72:115, 1999.

First Report of Anthracnose of Salsola tragus Caused by Colletotrichum gloeosporioides in Greece.

In early October of 2005, dying Salsola tragus L. (Russian thistle, tumbleweed), family Chenopodiaceae, plants were found along the Aegean Sea at Kryopigi Beach, Greece (40°02'29″N, 23°29'02″E, elevation 0 m). All of the 30 to 40 plants in the area were diseased and approximately 80% were dead or dying. All plants were relatively large (approximately 1 m tall × 0.5 m diameter), and living portions of diseased plants were flowering. Dying plants had irregular, necrotic lesions extending the length of the stems. Leaves of these plants were also necrotic. Lesions on stems and leaves were dark brown and usually coalesced. Diseased stem pieces were taken to the European Biological Control Laboratory, USDA, ARS at the American Farm School in Thessaloniki, Greece. There, diseased stem pieces were surface disinfested for 15 min with 0.5% NaOCl and placed on moist filter paper in petri dishes. Numerous, waxy subepidermal acervuli with black setae were observed in all lesions after 2 to 3 days. Conidiophores were simple, short, and erect. Conidia were one-celled, hyaline, ovoid to oblong, falcate to straight, 12.9 to 18.0 × 2.8 to 5.5 µm (mode 16.1 × 4.5 µm). These characters conformed to the description of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. in Penz. (2). Conidia were placed on modified potato carrot agar and axenic cultures from these isolations were sent to the quarantine facility of the Foreign Disease-Weed Science Research Unit, USDA, ARS, Fort Detrick, MD for testing. On the basis of DNA sequences, two variants within S. tragus have been described in California and named "Type A" and "Type B" (1). Conidia were harvested from 14-day-old cultures grown on 20% V8 juice agar, and healthy stems and leaves of 18 30-day-old plants of S. tragus Type A and 10 Type B plants were spray inoculated with an aqueous conidial suspension (1.0 × 106 conidia/ml plus 0.1% non-ionic surfactant). Three control plants of each type were sprayed with water and surfactant only. Plants were placed in an environmental chamber (18 h of dew in darkness at 25°C). After 1 day, all plants were transferred to a greenhouse (20 to 25°C, 30 to 50% relative humidity, and natural light augmented with 12-h light periods with 500-W sodium vapor lights). Lesions developed on stems of inoculated Type A plants after 5 days. After 14 days, all inoculated Type A plants were dead. Lesions on Type B plants were small and localized; all plants were diseased but no plants died. No symptoms occurred on control plants. C. gloeosporioides was reisolated 14 to 21 days after inoculation from stem pieces of all inoculated plants of both types of S. tragus. This isolate of C. gloeosporioides is a destructive pathogen on S. tragus Type A and is a potential candidate for biological control of this weed in the United States. To our knowledge, this is the first report of anthracnose caused by C. gloeosporioides on S. tragus in Greece. A voucher specimen has been deposited with the U.S. National Fungus Collections, Beltsville, MD (BPI 871126). Nucleotide sequences for the internal transcribed spacers (ITS 1 and 2) were deposited in GenBank (Accession No. DQ344621) and exactly matched sequences of the teleomorph, Glomerella cingulata. References: (1) F. Ryan and D. Ayres. Can. J. Bot. 78:59, 2000. (2) B. C. Sutton. Page 15 in: Colletotrichum Biology, Pathology and Control. J. A. Bailey and M. J. Jeger, eds. CAB International Mycological Institute, Wallingford, UK, 1992.

First Report of an Ovary Smut of Italian Thistle Caused by a Microbotryum sp. in Greece.

Italian thistle (Carduus pycnocephalus L.), family Asteraceae, is a common weed in Greece. It is also a problematic invasive weed in the western United States and a target of biological control efforts. In May 2005, smutted capitula of Italian thistle were found in an abandoned field in Halkiades, Greece. A total of 38 smutted plants, representing approximately 20% of those plants present, were found in a portion of the field that was lightly infested with Italian thistle. In most cases, capitula of all diseased flowers were smutted. In one or two cases, capitula on some branches of the plants were smutted, whereas capitula on other branches were healthy. Diseased capitula were noticeably more globose than healthy ovoid capitula, and diseased capitula did not open completely. When diseased capitula were split open, the ovaries in all florets within the capitula were filled with powdery masses of smut teliospores. Diseased capitula were collected, air dried, and sent to the quarantine facility of the Foreign Disease-Weed Science Research Unit (FDWSRU), USDA/ARS, Fort Detrick, MD. Teliospores within the capitula were extracted and observed microscopically. Teliospores of isolate DB05-014 were relatively uniform in shape and size, globose, 12.0 to 17.3 × 12.3 to 18.0 µm (mean 14.5 × 15.1 µm), violet tinted pale to medium yellowish-brown; wall reticulate appearing as coarse, radiate wings on the spore margin, 5 to 7 polyangular meshes per spore diameter, muri, 0.7 to 2.0 µm high in optical median view appearing as gradually narrowing blunt spines, 0.5 to 1 µm wide at their basis; in scanning electron microscopy (SEM), the meshes were subpolygonal, wall and interspaces were finely verruculose. Teliospores were more globose and slightly smaller than the description of Microbotryum cardui (A. A. Fischer Waldh.) Vánky (2), but the mean sizes were within the described range. When compared with teliospores of M. cardui on C. acanthoides, the numbers of polyangular meshes per spore diameter were within the range of the description using SEM, but the muri were about one-half of the height of those described. Nucleotide sequences for the internal transcribed spacers (ITS 1 and 2) and 5.8S ribosomal region (GenBank Accession No. AY280460) were aligned with sequences of other smut fungi using the BLAST algorithm of the National Center for Biotechnology Information. The closest alignment of DB05-014 was with M. scorzonerae (590 of 627 bp identities or 94% with 2% gaps). No sequences of M. cardui were available for comparison, but only M. cardui has been reported on Carduus spp. (1,2). Another smut reported on a Carduus sp. is Thecaphora trailii (1). DB05-014 is a likely variant of M. cardui from a previously unknown host. Italian thistle is an annual plant that reproduces solely by seeds (achenes). Because of the lack of seed production on smutted plants and the systemic nature of the disease, this fungus has great potential as a biological control agent for Italian thistle in the United States. A voucher specimen has been deposited with the U.S. National Fungus Collections (BPI 871812). To our knowledge this is the first report of a Microbotryum sp. parasitizing C. pycnocephalus. References: (1) K. Vánky. European Smut Fungi. Gustav Fischer Verlag, Stuttgart, Germany, 1994. (2) K. Vánky and D. Berner. Mycotaxon 85:307, 2003.

First Report of Leaf Spot Caused by a Cercosporella sp. on Acroptilon repens in the United States.

In August 2005, leaf spots were observed on a sample of Acroptilon repens (L.) DC. (Russian knapweed [RK]) collected at the Charles M. Russell National Wildlife Refuge in Montana. Symptoms included circular to subcircular brown spots, 1 to 18 mm in diameter, with indefinite margins that sometimes had a thin, purple-to-rose border. Leaves placed in moist chambers developed conidiophores and conidia within 48 h. Stroma were subcuticular, pale yellowish; conidiophores were hyaline, zero to two septate, thin walled, smooth, unbranched, 31 to 91 × 2.8 to 5.6 µm; and conidia were solitary or in secondary short chains, ovoid to subclavate, 13 to 52 × 3.2 to 6.8 µm, zero to three septate, hyaline, and thin walled. Conidial scars and hyla were umbronate, somewhat thickened, refractive, and not darkened. In three tests, 50 RK plants spray inoculated with 106 conidia/ml developed symptoms similar to those on the sample and the fungus was reisolated each time. Fungal identification, Cercosporella acroptili (Bremer) U. Braun, was based on morphology (1) and comparisons with the type specimen and a Turkish isolate (FDWSRU 98-001). ITS 1 and 2 sequences (GenBank Accession No. 779164) also were identical to a known isolate of C. acroptili. A specimen (BPI No. 871029) has been submitted to the USDA-ARS-SBML. RK is a major weed pest in the western United States and has been target of biological control research in recent years. Reference: (1) U. Braun. A Monograph of Cercosporella, Ramularia, and Allied Genera (Phytopathogenic Hyphomycetes) Vol. 1. IHW-Verlage, Eching-by-Munich, 1995.

First Report of Puccinia jaceae var. solstitialis Pycnia on Yellow Starthistle in the United States.

The rust fungus Puccinia jaceae (Otth) var. solstitialis field isolate FDWSRU 84-71 (formerly TR 84-96) was first released in California for the biological control of yellow starthistle (YST; Centaurea solstitialis) in July 2003. This isolate was collected by S. S. Rosenthal in 1984, east of Yarhisar and Hafik (SIVAS), Turkey. It is macrocyclic and autoecious (1), completing its entire life cycle on YST. Observations were made in field plots west of Woodland, CA that had been inoculated on a monthly regimen between January and June of 2005. On February 22 and March 2, 2006, pycnia were observed in and around one of the plots (38°42.767'N, 121°53.732'W at an altitude of 57 m). Pycnia were yellow, flask shaped, and small (less than 100 µm in diameter), occurring in clusters on abaxial leaf surfaces or on petioles. DNA was isolated from pycnial and uredinial samples collected from the site and used for polymerase chain reaction amplification of the ITS2 region with P. jaceae specific primers. Sequences of amplicons from both samples were identical to FDWSRU 84-71 (GenBank Accession No. AF047728). Further amplification of pycnial DNA with PCR primers specific to the released P. jaceae isolate (2) produced the expected 851-bp amplicon. To our knowledge, this is the first report of pycnia from Puccinia jaceae var. solstitialis in the United States, suggesting that the YST rust is fully functional and completes its life cycle in California. References: (1) D. B. O. Savile. Can. J. Bot. 48:1553, 1970. (2) L. F. Yourman and D. G. Luster. Biol. Control 29:73, 2004.

Cercosporella acroptili and Cercosporella centaureicola sp. nov.--potential biological control agents of Russian knapweed and yellow starthistle, respectively.

Russian knapweed (Acroptilon repens [L.] DC.) and yellow starthistle (Centaurea solstitialis L.) are invasive weeds in the western United States, and both weeds are targeted for biological control. Cercosporella acroptili (Bremer) U. Braun was identified as a possible biological control agent for A. repens, and a morphologically similar Cercosporella sp. recently was found damaging to C. solstitialis in the field. Because both fungi are potentially important for biological control of the respective weeds, studies were undertaken to ascertain whether the isolates were identical based on morphology, pathogenicity, growth and spore production, and genetics (molecular characterization of the internal transcribed spacer regions of the ribosomal RNA genes). Differences in these variables between the two isolates were sufficient to indicate that the isolate from C. solstitialis was distinct and justified a new description at the species level: Cercosporella centaureicola sp. nov.

Development of a policy for patients receiving neuromuscular blocking agents.

PURPOSE: To develop a policy to standardize the care of patients paralyzed with neuromuscular blocking agents (NMBAs). METHODS: Criteria were developed to assess adherence to the policy. Categorical and continuous data were collected and analyzed. RESULTS: Deficiencies were identified in frequency of train-of-four monitoring and in carrying out daily discontinuation of NMBAs to allow effects of paralysis to dissipate. CONCLUSIONS: The policy was useful as an educational tool. A NMBA order form should be developed to improve adherence to the policy.

Identification of two cytosolic ascorbate peroxidase cDNAs from soybean leaves and characterization of their products by functional expression in E. coli.

Screening of a cDNA library from soybean (Glycine max (L.) Merr. cv. Century) with probes based upon cytosolic ascorbate peroxidase (APx; EC 1.11.1.11) genes identified two full-length clones (SOYAPx1, SOYAPx2) apparently encoding for different soybean leaf cytosolic APxs. The deduced amino acid sequences of the two APx cDNA products differed in 13 of the 250 amino acids. The SOYAPx1 cDNA was identical to the cytosolic APx cDNA previously found in soybean root nodules. Escherichia coli expression systems were developed using both soybean APx cDNAs. Recombinant SOYAPx1 and SOYAPx2 were then utilized to characterize the enzymatic properties of the two APx cDNA products.