First Report of Leaf Necrosis on Microstegium vimineum Caused by Bipolaris microstegii in Maryland.

Japanese stiltgrass (JSG, Microstegium vimineum) is an invasive weed causing significant ecological changes in the United States. Severely diseased plants in a shaded location 2 × 4 m in size were discovered in August 2012 at a residence on Indian Springs Rd., Frederick, MD (39.46747° N, 77.46106° W). JSG in larger monoculture stands at sunny locations within 6 to 10 m of diseased plants had a few, small, necrotic spots. Diseased plants had leaves with brown, often large, elliptical, necrotic spots up to 0.5 × 1.5 cm. Lesions were surrounded by a diffuse chlorotic margin, and larger lesions had tan centers. Diseased plants were smaller in stature than neighboring, non-symptomatic plants. Symptoms were similar to those on JSG reported by Kleczewski and Flory (2). Field samples of diseased leaves in moist chambers at room temperature and lighting produced dematiaceous conidiophores and conidia typical of Bipolaris within 2 days. Subcultures of the isolate (FDWSRU 12-049) were made from the conidia. Cultures growing on modified potato carrot agar (broth from 140 g each of potatoes and carrots and 20 g agar in 1 L water), were gray, velutinous or tomentose, and had clumps of short aerial hyphae on the upper surface. Healthy plants were grown in potting soil from seeds collected at the disease site. A minimum of five 4-week-old plants were spray-inoculated in each of three replications by conidia from detached leaves in a suspension of 105 spores/ml, given a 16-h dew period at 25°C, and placed in a 25°C greenhouse for observation. Non-inoculated plants were included for comparison. Brown or dark tan necrotic, irregular, often linear, spots with entire margins developed on all inoculated individual plants and not on control plants. The Bipolaris species was recovered from inoculated plant samples incubated in moist chambers, thus fulfilling Koch's postulates. Conidia were produced sympodially on dematiaceous conidiophores, often in clusters of two to three spores at the terminus, were medium to dark brown, straight or slightly curved, nearly fusiform with obtuse apices, and typically had 8 to 10 distoseptate cells that were 74.8 ± 2.3 × 16.4 ± 0.3 µm, and Q = 4.6 ± 0.1 (mean ± ci, P = 0.05; n = 100). A sequence of the ITS1-5.8S-ITS2 region of DNA, extracted using a DNeasy Plant Mini Kit (QIAGEN), was 100% identical to that of the type specimen of B. microstegii from M. vimineum (BPI 883727; GenBank Accession No. JX089579), using BLAST. On the basis of fungal morphology and molecular characteristics (1), along with symptomatology and published information (2), the causal agent of this disease has been determined to be B. microstegii. Dried specimens of both the isolate and diseased leaves were deposited in the U.S. National Fungus Collections (BPI 892680) and sequence information was submitted to GenBank (KF150215). Bipolaris leaf spot was also found on JSG in Howard and Prince Georges counties, MD, in 2009, but the causal agents were not formally characterized (K. Rane, Univ. Maryland; personal communication). This is the first confirmed identification of B. microstegii on JSG in Maryland, a plant that occurs as extensive monocultures in natural areas. These results provide a basis for characterization of this disease in the mid-Atlantic region. References: (1) P. W. Crous et al. Fungal Planet description sheets: 128-153. Persoonia 29:146, 2012. (2) N. M. Kleczewski and S. L. Flory. Plant Dis. 94:807, 2010.

First Report of Pilidium concavum causing Leaf Necrosis on Fallopia japonica in the United States.

Fallopia japonica (Houtt.) Ronse Decr. (= Polygonum cuspidatum Siebold & Zucc.; Japanese knotweed, JKW) is an invasive perennial forb in the Polygonaceae. It has been identified as a target for biological control in many parts of the world, including the United States. Several potted JKW plants in an outdoor study at the Oregon Department of Agriculture, Salem (44.93° N, 122.99° W) developed leaf spots. Samples collected on August 20, 2007, were sent to the FDWSRU for identification of the disease. The necrotic leaf spots were brown and large, 1 to 3 cm in diameter, and in some cases occupying 30% of the leaf area. Both hemispherical and discoid conidiomata with gloeoid spore masses (3) developed in necrotic areas of all leaves placed in moist chambers. Discoid conidiomata had dark, pedicellate bases subtending a fimbriate disc on which pale brown to brown gloeoid conidial masses were produced. Hemispherical conidiomata were black, circular, sessile, and somewhat flattened, within which similar, gloeoid conidial masses were produced. Conidia from each type of conidioma were unicellular, cylindrical to fusiform, hyaline, and 4.5 to 7.2 × 0.9 to 1.8 µm (mean 5.7 × 1.33). Artificial inoculation of 15 plants was made on two occasions with a suspension of 106 conidia per ml, followed by two 16-hr dew periods at 25°C that were separated by an 8-hr "day;" a similar set of 15 non-inoculated plants served as controls each time. Symptoms similar to those in the original sample developed within 2 months after inoculation. The fungus was easily reisolated, and conidia from each type of conidioma produced similar growth on artificial media and similar disease after inoculation. The characteristics of conidial size and distinctly different conidiomata are diagnostic of Pilidium concavum (Desm.) Höhn (3,4). A sequence of the ITS1-5.8S-ITS2 region DNA, extracted using a DNeasy Plant Mini Kit (QIAGEN), was found identical to that of P. concavum from Rosa sp. (BPI 1107275; GenBank Accession No. AY487094), using BLAST. This isolate, FDWSRU 07-116, has been deposited in the US National Fungus Collection (BPI 883546) and at the Centraalbureau voor Schimmelcultures (CBS 132725). Sequence data have been deposited in GenBank (JQ790789). To our knowledge, this is the first report of P. concavum causing disease on a member of the Polygonaceae in North America (1), a disease clearly different from a Japanese Mycosphaerella sp. under consideration for biological control of JKW in the United Kingdom (2). References: (1) D. F. Farr, and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , May 15, 2012. (2) D. Kurose et al. MycoSci. 50:179, 2009. (3) M. E. Palm, Mycologia 83:787, 1991. (4) A. Y. Rossman, et al. Mycol. Progr. 3:275, 2004.

Field Damage to Yellow Starthistle Infected by Synchytrium solstitiale, and Greenhouse Maintenance and Host Range of the Fungus.

Yellow starthistle (YST, Centaurea solstitialis) is a major weed pest of the western United States. Synchytrium solstitiale, a pathogen of YST, caused significant damage to symptomatic (versus asymptomatic) plants in a field study in France. Before it was evaluated as a candidate for biological control of YST in the United States, protocols for pathogen maintenance under greenhouse conditions were developed. Maintenance, increase, and host range determination protocols involved incubation at 10/15°C (night/day) with an 8-h photoperiod either of potted or exhumed (i.e., roots of 4-week-old plants grown in flasks of water) plants inoculated with galled leaf tissue, or potted plants in which inoculum was wrapped within healthy leaves by a plastic wrap. The leaf-wrap protocol, used during the host range determination, always resulted in disease of YST. Several safflower (Carthamus tinctorius) cultivars and other plants related to YST became diseased following this protocol, thus raising concern about host specificity. Development of disease on nontarget species precludes proposal of S. solstitiale for biological control of YST at this time.

First Report of Crown Gall Caused by an Agrobacterium sp. on Diffuse Knapweed (Centaurea diffusa).

Diffuse knapweed (DK) plants were discovered in Mosier, Wasco County, OR (45.6842°N, 121.4021°W) with crown gall-like symptoms near the soil line. Specimens were collected on 27 July 2004 and sent to the USDA-ARS at Ft. Detrick, MD for identification of disease and pathogen. Pure culture of a bacterium was obtained on potato dextrose agar, and hyperplasia and hypertrophy developed on carrot disks and tomato stems after wound inoculation with a needle contaminated by the agar culture. The same bacterium was reisolated from the galls on DK, thus fulfilling Koch's postulates. Pathogenicity tests involving needle inoculations of stems and petioles resulted in gall formation on Acroptilon repens, Carthamus tinctorius, Centaurea solstitialis, C. maculosa, C. cyanus, Crupina vulgaris, Helianthus annuus, and Rubus armeniacus. In biochemical tests typically used for identification of Agrobacterium species (3), the DK strain grew on D1M agar but not on 2% NaCl medium, produced acid from erythritol but not from melezitose, converted malonic acid to base, and turned litmus milk alkaline. These results are characteristic of Agrobacterium rhizogenes (= Biovar 2), except for the litmus milk reaction. Using 16S rRNA cluster analysis by unweighted pair group method with arithmetic mean (UPGMA, 500 replicates) and basic local alignment search tool (BLAST), the DK strain clustered most closely with A. rubi (GenBank Accession Nos. D12787 and AM181759). The DK strain differed from A. larrymoorei (GenBank Accession No. Z30542), A. tumefaciens (GenBank Accession No. AJ389896), A. rhizogenes (GenBank Accession No. AB247607), and A. vitis (GenBank Accession No. AB247599) on the basis of 16S rRNA sequence cluster analysis. The DK strain differed from A. rubi on the basis of differential reactions with erythritol, litmus milk, and 2% NaCl medium (2,4); and the 16S rRNA sequence of the DK strain differed from that of A. rubi by 11 bp (99.2% similarity). Comparisons also were made between the DK strain and two strains (83A and 135A) of A. tumefaciens (= Biovar 1), described from New Mexico on A. repens (1), a plant species in the same tribe and subtribe of the Asteraceae as DK. Host range reported for the two A. repens strains after artificial greenhouse inoculations was similar to that of the DK strain and it included diffuse knapweed (1). However, 16S sequencing, which confirmed identification of both A. repens strains as A. tumefaciens, showed they differed from the DK strain. The DK strain belongs in the genus Agrobacterium, but it could not be assigned to any known species on the basis of data from phenotypic or 16S sequence comparisons. To our knowledge, this is the first report of crown gall on diffuse knapweed in the field. This strain has been deposited into the International Collection of Phytopathogenic Bacteria at Fort Detrick (Accession No. 60099), and the 16S rRNA sequence has been deposited into the GenBank database (Accession No. EF687663). References: (1) A. J. Caesar, Plant Dis. 78:796, 1994. (2) B. Holmes and P. Roberts, J. Appl. Bacteriol. 50:443, 1981. (3) L. W. Moore et al. Page 17 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (4) K. Ophel and A. Kerr, Int. J. Syst. Bacteriol. 40:236, 1990.

Puccinia acroptili on Russian Knapweed in Colorado, Montana, and Wyoming.

Acroptilon repens (L.) DC. (Russian knapweed) is a long-lived perennial weed from central Asia that is widely distributed in the western United States (U.S.). Recently, accessions of a rust disease were collected from Colorado (CO), Montana (MT), and Wyoming (WY) for comparison with Eurasian isolates. U.S. accessions had two-celled teliospores with slight constrictions in the middle and urediniospores with three germ pores ± equatorial in location. Urediniospores were (state, width × length, [n = 100]): CO, 16.4 to 25.7 × 19.2 to 27.0 µm; MT, 18.4 to 23.1 × 17.4 to 24.6 µm; and WY, 18.0 to 26.2 × 20.2 to 26.7 µm. These were similar to those of 16.6 to 25.7 × 21.2 to 28.0 µm from two New Mexican (NM) herbarium specimens (BPI Nos. 1107952 and 1110177) (1). Teliospores measured 19.9 to 27.7 × 29.8 to 47.4 µm, 17.4 to 26.0 × 32.4 to 44.2 µm, 16.5 to 27.5 × 29.4 to 45.7 µm, and 18.7 to 27.6 × 31.0 to 46.4 µm for CO, MT, WY, and NM accessions, respectively. These rust isolates have been identified as Puccinia acroptili Syd. on the basis of host plant record and spore morphology (2). To our knowledge, this is the first record of P. acroptili in CO, MT, and WY. Besides NM, P. acroptili has been reported in North America from California, British Columbia, and Saskatchewan. References: (1) M. E. Palm and S. G. Vesper. Plant Dis. 75:1075, 1991. (2) D. B. O. Savile. Can. J. Bot. 48:1567, 1970.

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.

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.

First Report of Leaf Spot Caused by a Cercosporella sp. on Centaurea solstitialis in Greece.

Centaurea solstitialis L. (yellow starthistle [YST]), family Asteraceae, an invasive weed in California and the western United States is targeted for biological control. During the spring of 2004, an epidemic of dying YST plants was found near Kozani, Greece (40°22'07″N, 21°52'35″E, 634 m elevation). Rosettes of YST had small, brown leaf spots on most of the lower leaves. In many cases, these spots coalesced and resulted in necrosis of many of the leaves and death of the rosette. Along the roadside where the disease was found, >100 of the YST plants 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. Diseased leaves were surface disinfested and placed on moist filter paper in petri dishes. Conidiophores and conidia were observed after 48 h. The fungal isolate, DB04-011, was isolated from these diseased leaves. Pathogenicity tests were performed by spray inoculating the foliage of 20 4-week-old YST rosettes with an aqueous suspension of 1 × 106 conidia per ml. Conidia were harvested from 2-week-old cultures grown on modified potato carrot agar (MPCA). Inoculated plants were placed in an environmental chamber at 23°C with 8 h of daily light and continuous dew for 48 h. Inoculated and control plants were moved to a 20°C greenhouse bench and watered twice per day. After 7 days, leaf spots were observed first on lower leaves. After 10-12 days, all inoculated plants showed typical symptoms of the disease. No symptoms developed on control plants. The pathogen, DB04-011, was consistently isolated from symptomatic leaves of all inoculated plants. Disease symptoms were scattered, amphigenous leaf spots in circular to subcircular spots that were 0.2 to 7 mm in diameter and brownish with distinct dark green margins. Intraepidermal stromata, 14 to 77 µm in diameter and pale yellow to brown, were formed within the spots. Conidiophores that arose from the stromata were straight, subcylindrical, simple, 70 to 95 × 2.8 to 4 µm, hyaline, smooth, and continuous or septate with conidial scars that were somewhat thickened, colorless, and refractive. Primary conidia were subcylindrical, slightly obclavate or fusiform, ovoid, 21 to 49 × 5 to 7.5 µm, 0 to 5 septate, hyaline, smooth, had a relatively rounded apex, and the hilum was slightly thickened. Conidial dimensions on MPCA were 11.2 to 39.2 × 4.2 to 7 µm (average 25.5 × 5.5 µm). Koch's postulates were repeated two more times with 20 and 16 plants. On the basis of fungal morphology, the organism was identified as a Cercosporella sp., (1,2; U. Braun and N. Ale-Agha, personal communication). To our knowledge, this is the first report of this genus of fungus parasitizing YST. Results of host range tests will establish if this isolate of Cercosporella has potential as a biological control agent of YST in the United States. A voucher specimen has been deposited with the U.S. National Fungus Collections (BPI 844247). Live cultures are being maintained at FDWSRU and European Biological Control Laboratoryt (EBCL), Greece. References: (1) U. Braun. A Monograph of Cercosporella, Ramularia and Allied Genera (Phytopathogenic Hyphomycetes) Vol. 1. IHW-Verlage, Eching-by-Munich, 1995. (2) U. Braun. A Monograph of Cercosporella, Ramularia and Allied Genera (Phytopathogenic Hyphomycetes) Vol. 2. IHW-Verlage, 1998.

First Report of Anthracnose of Crupina vulgaris Caused by a Colletotrichum sp. in Greece.

Crupina vulgaris Cass. (common crupina, family Asteraceae), an introduced invasive weed in the northwestern United States, is a target of biological control efforts. During the spring of 2002, ≈30 wilting C. vulgaris plants were found along a road from Volos to Portaria, Greece (39°22'58″N, 22°59'27″E, elevation 446 m). Wilting plants had irregular, purple, necrotic lesions extending along the main stems and petioles. In the laboratory, diseased leaves were surface disinfested and placed on moist filter paper in petri dishes. Acervuli with setae typical of a Colletotrichum sp. were observed after 2 to 5 days. A fungal isolate, DB 02-030, was isolated from these diseased leaves. Stems and leaves of 12- and 16-week-old plants (12 plants of each age) were spray inoculated with an aqueous suspension of 2 × 106 conidia per ml from 14-day-old cultures of DB 02-030 grown on acidified potato dextrose agar (APDA). Inoculated plants were placed in a dew chamber at 18 to 21°C with continuous dew and 8 h of light per day for 48 h. Plants were moved to a greenhouse bench with 8 h of light per day and watered twice daily. Symptoms developed after 7 days on 16-week-old plants (33% symptomatic) and 14 days on 12-week-old plants (17% symptomatic). No symptoms developed on control plants. By 61 days after inoculation, 67% of plants inoculated at 16 weeks of age were dead and 50% of plants inoculated at 12 weeks of age were wilted. Koch's postulates were repeated with isolates from two other plants. Isolate DB 02-030 was reisolated three times from 10 of 10 symptomatic leaves, 4 of 4 stems with necrotic lesions, and 4 of 4 stems with leaves from wilted inoculated plants. Conidia germination on water agar was 95% at 18 to 21°C with light compared with 19% in darkness or at 23 to 26°C. C. vulgaris is an annual plant that emerges during early spring and reproduces only by seeds. As a cool-temperature aggressive pathogen, isolate DB 02-030 has the potential as a biological control agent to reduce seed production and stands of C. vulgaris. This isolate fits the morphology of Colletotrichum gloeosporioides according to Sutton (2). On APDA, conidia were formed after 4 days. Conidia were hyaline, straight, cylindrical, nonseptate, and 18 to 27 × 3 to 6 µm. Setae produced in acervuli were abundant, straight, narrow, and 75 to 210 µm long × 3 µm at the base. Appressoria in vitro were subglobose to clavate and 8 to 12 µm in diameter. Nucleotide sequences were obtained for the internal transcribed spacer (GenBank Accession No. AY539806) and 28S (GenBank Accession No. AY539807) rDNA genes of this isolate. Parsimony analyses (unpublished), with sequences from GenBank and 25 isolates from established culture collections, indicate the isolate on C. vulgaris belongs to a clade of taxonomically problematic Colletotrichum spp. that are only distantly related to other isolates of C. gloeosporioides. A culture of DB 02-030 has been deposited at the Centraalbureau voor Schimmelcultures as CBS 114801. A dried culture voucher specimen has been deposited with the U.S. National Fungus Collections (BPI 843682). To our knowledge, no species of Colletotrichum has been reported previously on any Crupina spp. (1). References: (1) D. F. Farr et al. Fungal Databases. Systematic Botany and Mycology Laboratory, On-line publication. ARS, USDA, 2004. (2) B. C. Sutton. The Coelomycetes. CMI, Kew, Surrey, England, 1980.

First Report of Leaf Blight Caused by Phoma exigua on Acroptilon repens in Turkey.

Acroptilon repens (L.) DC. (Russian knapweed, synonym Centaurea repens L., family Asteraceae) is becoming a noxious weed in wheat fields in Turkey. Because it is also an invasive weed in the northwestern United States, A. repens is a target of biological control efforts. In the summer of 2002, approximately 20 dying A. repens plants were found on a roadside near Cankiri, Turkey (40°21'41″N, 33°31'8″E, elevation 699 m). No healthy plants were found in the immediate area. Dying plants had irregular, charcoal-colored, necrotic lesions at the leaf tips and margins, and frequently, whole leaves and plants were necrotic. Symptomatic leaves were air-dried and sent to the Foreign Disease-Weed Science Research Unit, USDA/ARS, Fort Detrick, MD. There, diseased leaves were surface-disinfested and placed on moist, filter paper in petri dishes. Pycnidia producing one-celled hyaline conidia were observed after 4 to 5 days. Internal transcribed spacer regions 1 and 2, including the 5.8S ribosomal DNA, were sequenced for isolate 02-059 (GenBank Accession No. AY367351). This sequence was identical to sequences in GenBank from six well-characterized strains of Phoma exigua Desmaz (1). Morphology was also consistent with P. exigua (2) with the exception that material grown on alfalfa twigs produced pycnidia with 1 to 4 ostioles with necks as much as 80 µm long. Typically, pycnidia of P. exigua produced on agar have 1 to 2 ostioles that lack necks. Conidial dimensions on alfalfa were 4.1 to 7.6 × 1.7 to 3.2 µm (average 5.5 × 2.4 µm). Images of the fungus are located at http://nt.ars-grin.gov under the section 'Fungi Online'. Stems and leaves of 20 3-week-old plants were spray inoculated with an aqueous suspension (1 × 107 conidia per ml) of conidia harvested from 25-day-old cultures grown on acidified potato dextrose agar, and placed in an environmental chamber at 25°C with constant light and continuous dew for 3 days. Plants were then moved to a greenhouse bench and watered twice daily. After 6 days, symptoms were observed on all plants. Once symptoms had progressed to the midveins of the leaves, the disease progressed rapidly on the plants, indicating the possibility of systemic infection or systemic movement of toxins. Phoma exigua was reisolated from the stems, petioles, and leaves of all inoculated plants. In a separate test, 12 plants were inoculated as described above, and 8 additional plants were sprayed with water only. After inoculation, plants were handled as described above. The first lesions developed after 3 days on all except the youngest leaves of inoculated plants. After 10 days, three inoculated plants were dead, and all other inoculated plants had large necrotic lesions. No symptoms developed on control plants. This isolate of Phoma exigua is a destructive pathogen on A. repens, and severe disease can be produced by inoculation of foliage with an aqueous suspension of conidia. These characteristics make this isolate of P. exigua a potential candidate for biological control of this weed in Turkey and the United States. To our knowledge, this is the first report of P. exigua on A. repens in Turkey. A voucher specimen has been deposited with the U.S. National Fungus Collections (BPI 843350). References: (1) E. C. A. Abeln et al. Mycol. Res. 106:419, 2002. (2) H. A. Van der Aa et al. Persoonia 17:435, 2000.