First Report of Aerial Blight of Ruth's Golden Aster (Pityopsis ruthii) Caused by Rhizoctonia solani in the United States.

Ruth's golden aster (Pityopsis ruthii (Small) Small: Asteraceae) is an endangered, herbaceous perennial that occurs only at a few sites along the Hiwassee and Ocoee rivers in Polk County, Tennessee. This species is drought, heat, and submergence tolerant and has ornamental potential as a fall flowering landscape plant. In 2012, we vegetatively propagated various genotypes and established plantings in a landscape at Poplarville, Mississippi. In June and July of 2013, during periods of hot and humid weather, several well-established plants exhibited black or brown necrotic aerial blight symptoms including desiccation of stems and leaves. Blighted leaf samples were surface sterilized (10% commercial bleach, active ingredient 8.25% sodium hypochlorite, 1 min), rinsed in sterile water, air-dried, and plated on 2% water agar amended with 3.45 mg fenpropathrin/liter (Danitol 2.4 EC, Valent Chemical, Walnut Creek, CA) and 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO). Rhizoctonia sp. was identified based on hyphal morphology and cultures were maintained on potato dextrose agar. Colonies were fast growing, consisting of light tan to brown mycelia and tufts of crystalline aerial hyphae. Within 10 days, brown exudates were present in cultures and there was no pigmented reverse to the agar. Hyphae were a mean of 5.2 µm wide (4.6 to 6.1 µm; n = 10) and each compartment contained three or more nuclei. Hyphae were constricted at septa with right angle branching and no clamp connections, which is typical for Rhizoctonia solani (1). Light- to medium-brown, oblong to irregularly shaped sclerotia measuring 1.2 mm long (0.7 to 2.1 mm) × 0.9 mm wide (0.5 to 1.2 mm; n = 20) were formed in cultures after 3 weeks of growth. Total genomic DNA was extracted from two different colonies grown in potato dextrose broth for 7 days, amplified with PCR using ITS1 and ITS4 primers for amplification of the 18S rDNA subunit (2), the products purified, and sequenced. A consensus sequence of 657 bp was deposited in GenBank (Accession Nos. KF843729 and KF843730) and was 96% identical to two R. solani Kühn ITS sequences in GenBank (HF678125 and HF678122). R. solani was grown on twice autoclaved oats for 2 weeks at 21°C and incorporated into Pro-Mix BX, low fertility soilless medium (Premier Horticulture, Rivière-du-Loup, Quebec, Canada) at 4% (w/w) to inoculate seven P. ruthii plants grown in 10 cm-diameter pots; seven additional plants were grown in the same medium amended with 4% (w/w) sterile oats. Plants were grown in a greenhouse and covered with a plastic dome to maintain high humidity. After 2 weeks, six of the seven inoculated plants exhibited the same aerial blight symptoms as did the original infected plants from the field; none of the control plants developed disease symptoms. Colony morphology and hyphal characteristics as well as the sequence for the ITS region of rDNA from the re-isolated fungus were identical to the original isolate. To our knowledge, this is the first report of R. solani infecting Ruth's golden aster. We are not aware of the disease occurring in wild populations of the plant, but may impact plants grown in the landscape or greenhouse. References: (1) B. Sneh et al. Identification of Rhizoctonia Species. The American Phytopathological Society, St Paul, MN, 1991. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

First Report of Leaf Spot caused by Bipolaris oryzae on Switchgrass in Tennessee.

Knowledge of pathogens in switchgrass, a potential biofuels crop, is limited. In December 2007, dark brown to black irregularly shaped foliar spots were observed on 'Alamo' switchgrass (Panicum virgatum L.) on the campus of the University of Tennessee. Symptomatic leaf samples were surface-sterilized (95% ethanol, 1 min; 20% commercial bleach, 3 min; 95% ethanol, 1 min), rinsed in sterile water, air-dried, and plated on 2% water agar amended with 3.45 mg fenpropathrin/liter (Danitol 2.4 EC, Valent Chemical, Walnut Creek, CA) and 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO). A sparsely sporulating, dematiaceous mitosporic fungus was observed. Fungal plugs were transferred to surface-sterilized detached 'Alamo' leaves on sterile filter paper in a moist chamber to increase spore production. Conidia were ovate, oblong, mostly straight to slightly curved, and light to olive-brown with 3 to 10 septa. Conidial dimensions were 12.5 to 17 × 27.5 to 95 (average 14.5 × 72) µm. Conidiophores were light brown, single, multiseptate, and geniculate. Conidial production was polytretic. Morphological characteristics and disease symptoms were similar to those described for Bipolaris oryzae (Breda de Haan) Shoemaker (2). Disease assays were done with 6-week-old 'Alamo' switchgrass grown from seed scarified with 60% sulfuric acid and surface-sterilized in 50% bleach. Nine 9 × 9-cm square pots with approximately 20 plants per pot were inoculated with a mycelial slurry (due to low spore production) prepared from cultures grown on potato dextrose agar for 7 days. Cultures were flooded with sterile water and rubbed gently to loosen mycelium. Two additional pots were inoculated with sterile water and subjected to the same conditions to serve as controls. Plants were exposed to high humidity by enclosure in a plastic bag for 72 h. Bags were removed, and plants were incubated at 25/20°C with 50 to 60% relative humidity. During the disease assay, plants were kept in a growth chamber with a 12-h photoperiod of fluorescent and incandescent lighting. Foliar leaf spot symptoms appeared 5 to 14 days post-inoculation for eight of nine replicates. Control plants had no symptoms. Symptomatic leaf tissue was processed and plated as described above. The original fungal isolate and the pathogen recovered in the disease assay were identified using internal transcribed spacer (ITS) region sequences. The ITS region of rDNA was amplified with PCR and primer pairs ITS4 and ITS5 (4). PCR amplicons of 553 bp were sequenced, and sequences from the original isolate and the reisolated pathogen were identical (GenBank Accession No. JQ237248). The sequence had 100% nucleotide identity to B. oryzae from switchgrass in Mississippi (GU222690, GU222691, GU222692, and GU222693) and New York (JF693908). Leaf spot caused by B. oryzae on switchgrass has also been described in North Dakota (1) and was seedborne in Mississippi (3). To our knowledge, this is the first report of B. oryzae from switchgrass in Tennessee. 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/, 28 June 2012. (2) J. M. Krupinsky et al. Can. J. Plant Pathol. 26:371, 2004. (3) M. Tomaso-Peterson and C. J. Balbalian. Plant Dis. 94:643, 2010. (4) T. J. White et al. Pages 315-322 in: PCR Protocols: a Guide to Methods and Applications. M. A. Innis et al. (eds), Acad. Press, San Diego, 1990.

First Report of Leaf Spot Caused by Alternaria alternata on Switchgrass in Tennessee.

Field-grown seedlings of 'Alamo' switchgrass (Panicum virgatum L.) from Vonore, TN exhibited light brown-to-dark brown leaf spots and general chlorosis in June 2009. Symptomatic leaf tissue was surface sterilized (95% ethanol for 1 min, 20% commercial bleach for 3 min, and 95% ethanol for 1 min), air dried on sterile filter paper, and plated on 2% water agar amended with 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO) and 5 µl/liter miticide (2.4 EC Danitol, Valent Chemical, Walnut Creek, CA). Plates were incubated at 26°C for 4 days in darkness. An asexual, dematiaceous mitosporic fungus was isolated and transferred to potato dextrose agar. Cultures were transferred to Alternaria sporulation medium (3) to induce conidial production. Club-shaped conidia were produced in chains with branching of chains present. Conidia were 27 to 50 × 10 to 15 µm, with an average of 42.5 × 12.5 µm. Morphological features and growth on dichloran rose bengal yeast extract sucrose agar were consistent with characteristics described previously for Alternaria alternata (1). Pathogenicity studies were conducted with 5-week-old 'Alamo' switchgrass plants grown from surface-sterilized seed. Nine pots with approximately 20 plants each were prepared. Plants were wounded by trimming the tops. Eight replicate pots were sprayed with a conidial spore suspension of 5.0 × 106 spores/ml sterile water and subjected to high humidity by enclosure in a plastic bag for 7 days. One pot was sprayed with sterile water and subjected to the same conditions to serve as a control. Plants were maintained in a growth chamber at 25/20°C with a 12-h photoperiod. Foliar leaf spot symptoms appeared 5 to 10 days postinoculation for all replicate pots inoculated with A. alternata. Symptoms of A. alternata infection were not observed on the control. Lesions were excised, surface sterilized, plated on water agar, and identified in the same manner as previously described. The internal transcribed spacer (ITS) region of ribosomal DNA and the mitochondrial small sub-unit region (SSU) from the original isolate and the reisolate recovered from the pathogenicity assay were amplified with PCR, with primer pairs ITS4 and ITS5 and NMS1 and NMS2, respectively. Resultant DNA fragments were sequenced and submitted to GenBank (Accession Nos. HQ130485.1 and HQ130486.1). A BLAST search (BLASTn, NCBI) was run against GenBank isolates. The ITS region sequences were 537 bp and matched 100% max identity with eight A. alternata isolates, including GenBank Accession No. AB470838. The SSU sequences were 551 bp and matched 100% max identity with seven A. alternata isolates, including GenBank Accession No. AF229648. A. alternata has been reported from switchgrass in Iowa and Oklahoma (2); however, this is the first report of A. alternata causing leaf spot on switchgrass in Tennessee. Switchgrass is being studied in several countries as a potentially important biofuel source, but understanding of the scope of its key diseases is limited. References: (1) B. Andersen et al. Mycol. Res. 105:291, 2001. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , September 22, 2011. (3) E. A. Shahin and J. F. Shepard. Phytopathology 69:618, 1979.

First Report of Leaf Spot and Necrotic Roots on Switchgrass Caused by Curvularia lunata var. aeria in the United States.

Curvularia lunata infects many grass species; however, switchgrass (Panicum virgatum L.) has not been reported as a host (2). In June 2009, small brown leaf spots and necrotic roots were observed on stunted 2-year-old 'Alamo' switchgrass on the University of Tennessee, Knoxville campus. Symptomatic leaf and root tissues were surface-sterilized in 95% ethanol for 1 min, 20% bleach for 3 min, and 95% ethanol for 1 min, and then air dried and placed on water agar amended with 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO) and 7.5 µl/liter Danitol (Valent Chemical, Walnut Creek, CA). Cultures were incubated at 25°C for 3 days. Hyphal tips were transferred to potato dextrose agar (PDA) and incubated at 25°C. Dark brown-to-black fungal colonies with black stromata formed. Conidiophores were dark brown, unbranched, septate, polytretic, sympodial, and geniculate at the apical region with rachis conidial ontogeny. Conidia were dark brown and cymbiform with three to four septations, with one or two central cells larger than the terminal cells. Spore size ranged from 17.5 to 30.0 × 8.8 to 12.5 µm (mean 21.6 × 10.8 µm). Morphological traits matched the description of C. lunata var. aeria (1). To test pathogenicity, fungal sporulation was optimized on PDA with pieces of sterile, moistened index card placed on each plate; cultures were incubated at 25°C with a 12-h photoperiod (3). After 14 days, conidia were dislodged in sterile water and the spore concentration adjusted to 8 × 104 conidia/ml. Ten pots, with about 15 plants per pot, of 6-week-old 'Alamo' switchgrass grown from surface-sterilized seed were inoculated with the spore suspension applied to the plant crown and surrounding soil with an aerosol sprayer. Prior to inoculation, roots were wounded with a sterile scalpel. Noninoculated plants (two pots), with roots also wounded, served as controls. To maintain high humidity, each pot was covered with a plastic bag and maintained in a growth chamber at 30°C with a 16-h photoperiod. Bags were removed after 3 days; plants were maintained as described for 6 weeks. Brown leaf spots and brown-to-black necrotic roots that matched symptoms on the naturally infected plants were observed in all inoculated plants; there were no symptoms of Curvularia infection on the controls. The fungus was reisolated from inoculated plants as described above. Genomic DNA was extracted from the original isolate and the reisolate from the pathogenicity test. PCR amplification of the internal transcribed spacer (ITS) regions from ribosomal DNA was performed with primers ITS4 and ITS5. PCR products of 503 bp were sequenced. There was 100% nucleotide identity for sequences of the original isolate and the re-isolate. The sequence was submitted to GenBank (Accession No. HQ130484.1). BLAST analysis of the fungal sequence resulted in 100% nucleotide sequence identity to the ITS sequences of isolates of C. affinis, C. geniculata, and C. lunata. On the basis of the smaller spore size and abundant stromata on PDA, the isolate was identified as C. lunata var. aeria. As switchgrass is developed as a biofuels crop, identification of new pathogens may warrant development of disease management strategies. References: (1) M. B. Ellis. Mycological Papers No. 106, CMI, Surrey, 1966. (2) D. F. Farr and A. Y. Rossman, Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , August 2011. (3) R. G. Pratt. Mycopathologia 162:133, 2006.

First Report of Spot Blotch and Common Root Rot Caused by Bipolaris sorokiniana on Switchgrass in Tennessee.

Light-to-dark brown, irregular-shaped leaf spots, chlorosis, necrotic roots, and severe stunting were observed on 'Alamo' switchgrass (Panicum virgatum L.) grown on the campus of the University of Tennessee in December 2007. Symptomatic leaf and root samples were surface sterilized, air dried on sterile filter paper, and plated on 2% water agar amended with 10 mg/liter of rifampicin (Sigma-Aldrich, St. Louis, MO) and 10 µl/liter of 2,4 EC Danitol miticide (Valent Chemical, Walnut Creek, CA). Plates were incubated at 25°C in darkness for 4 days. A sporulating, dematiaceous mitosporic fungus was noted and transferred to potato dextrose agar (PDA). Conidia were ovate, oblong, mostly straight, and olive to brown with three to nine septa. Conidial dimensions were 12.5 × 27.5 (17.5) to 20 × 77.5 (57) µm. Conidia were produced on single, light brown, multiseptate conidiophores that were polytretic, geniculate, and sympodial. Morphological features were as described for Bipolaris sorokiniana (Sacc.) Shoemaker (teleomorph = Cochliobolus sativus) (2,3). Disease assays were conducted with 5-week-old 'Alamo' switchgrass grown from surface-sterilized seed. Ten 9 × 9-cm2 with ~20 switchgrass seedlings were sprayed with 2.4 × 105 spores/ml of sterile water. Plants were subjected to high humidity created by enclosure in a plastic bag for 45 h. The bag was removed and plants were incubated at 25/20°C with 50 to 60% relative humidity. During the incubation, plants were maintained in growth chamber with a 12-h photoperiod of fluorescent and incandescent lighting. Foliar leaf spot symptoms appeared 6 to 10 days postinoculation for plants in all 10 replicates and necrotic lesions were observed on roots. Foliar lesions and diseased roots were surface sterilized, plated on water agar, and resultant fungal colonies were identified as B. sorokiniana. The internal transcribed spacer (ITS) and mitochondrial small subunit (SSU) regions of ribosomal DNA from the original isolate, and the isolate recovered from plants in the pathogenicity assay, were amplified with PCR, with primer pairs ITS4 and ITS5 and NMS1 and NMS2. PCR amplicons of ~551 and 571 bp were obtained with the two primer pairs, respectively. Both amplicons were obtained from both isolates and sequenced. Amplicon sequences from the original isolate and re-isolate were identical and the sequences were submitted to GenBank (Accession Nos. HQ611957 and HQ611958). The ITS sequences had 98% homology to 23 B. sorokiniana isolates, including B. sorokiniana strain DSM 62608 (GenBank Accession No. EF187908); SSU sequences had 99% homology to Cochliobolus sativus isolate AFTOL-ID 271 (GenBank Accession No. FJ190589). Spot blotch caused by B. sorokiniana has been reported on switchgrass in Iowa, Nebraska, Pennsylvania, and Virginia (1). To our knowledge, this is the first report of B. sorokiniana causing spot blotch or common root rot of switchgrass in Tennessee, which extends the current known distribution of these diseases. More recently, we isolated B. sorokiniana from switchgrass seed received from commercial sources in the United States, indicating a seedborne transmission. 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/ , 15 November 2010. (2) R. F. Nyvall and J. A. Percich. Plant Dis. 83:936, 1999. (3) A. Sivanesan and P. Holliday. CMI Descr. Pathog. Fungi bact. 71:701, 1981.

First Report of Leaf Spot Caused by Bipolaris spicifera on Switchgrass in the United States.

Light-to-dark brown leaf spots and general chlorosis were observed on 'Alamo' switchgrass (Panicum virgatum L.) grown in ornamental plantings on the campus of the University of Tennessee in Knoxville in December 2007. Disease distribution was patchy, infecting ~10% of plants. Patches had mild to severely infected plants with stunting in areas of severe infection. Symptomatic leaf tissue was surface sterilized, air dried on sterile filter paper, and plated on 2% water agar amended with 10 mg/liter of rifampicin (Sigma-Aldrich, St. Louis, MO) and 10 µl/liter of 2.4 EC Danitol miticide (Valent Chemical, Walnut Creek, CA). Plates were incubated at 26°C in darkness for 5 days. A sporulating, dematiaceous mitosporic fungus was observed and transferred to potato dextrose agar (PDA). Conidiophores were single, light brown, multiseptate, mostly straight, polytretic, geniculate, and sympodial. Conidia were 17.5 × 12 (22) to 30 × 14 (12.5) µm, oval, light brown, and distoseptate, with one to three septa and a flattened hilum on the basal cell. Conidia germinated from both poles. The causal agent was identified as Bipolaris spicifera (Bainier) Subram. Morphological features were as described for B. spicifera (2). Pathogenicity studies were conducted with 5-week-old 'Alamo' switchgrass plants grown from surface-sterilized seed in 9 × 9-cm pots containing 50% ProMix Potting and Seeding Mix (Premier Tech Horticulture, Rivière-du-Loup, Québec, Canada) and 50% Turface ProLeague (Profile Products, Buffalo Grove, IL) (vol/vol). Ten replicate pots with ~20 plants each were sprayed with a spore suspension of 4.5 × 106 spores/ml of sterile water prepared from 6-day-old cultures grown on PDA. Plants were subjected to high humidity for 45 h then incubated at 25/20°C with a 12-h photoperiod in a growth chamber. Leaf spot symptoms similar to the original disease appeared on plants in each of the 10 replicate pots 6 days postinoculation. Lesions were excised from leaves, surface sterilized, plated on water agar, and the resulting cultures were again identified as B. spicifera. The internal transcribed spacer (ITS) region of ribosomal DNA from the original isolate used for inoculation and the reisolated culture recovered from plants in the pathogenicity studies were amplified with PCR using primers ITS4 and ITS5 (3). PCR amplicons of ~560 bp were obtained from both isolates and sequenced. Amplicon sequences were identical and the sequence was submitted to GenBank (Accession No. HQ015445). The DNA sequence had 100% homology to the ITS sequence of B. spicifera strain NRRL 47508 (GenBank Accession No. GU183125.1) that had been isolated from sorghum seed. To our knowledge, leaf spot caused by B. spicifera has not been described on switchgrass (1). B. spicifera can be seedborne and has been reported on turfgrass seed exported from the United States to Korea (2). As switchgrass is transitioned from a prairie grass to a biofuels crop planted in large acreages, disease incidences and severities will likely increase, necessitating rapid disease identification and cost effective management strategies. 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/ , 4 August 2010. (2) H.-M. Koo et al. Plant Pathol. J. 19:133, 2003. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds, Academic Press, San Diego, 1990.