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 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 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 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.

First Report of Tilletia pulcherrima Bunt on Switchgrass (Panicum virgatum) in Texas.

Switchgrass seed samples of 'Blackwell' and 'Alamo' from Bailey County, TX were examined for bunt fungi. Fourteen completely bunted seeds of 'Blackwell' and four of 'Alamo' were found. No partially bunted seeds were found. Bunted seeds were darker and occasionally slightly swollen relative to noninfected seeds. Teliospores were globose to subglobose, 21 to 28 × 20 to 27 µm in diameter, dark reddish brown to nearly black, with blunt warts 1 to 1.8 µm long, enveloped in a hyaline sheath, and often with a short apiculus. Sterile cells were globose to subglobose, 17.5 to 22 µm, with smooth, laminated walls as much as 2.6 µm thick, and often with a short apiculus. This bunt was identified as Tilletia pulcherrima Ellis & Galloway on the basis of host and spore morphology (2). The internal transcribed spacer regions 1 and 2, including the 5.8S rDNA, were sequenced from bunted 'Blackwell' seeds (GenBank Accession No. EU915293, WSP 71501). The sequence was distinct from all Tilletia sequences in GenBank, including Tilletia barclayana (Bref.) Sacc. & Syd. on Panicum obtusum Kunth (GenBank Accession No. AF 310169) (1). To our knowledge, this is the first report of T. pulcherrima from switchgrass in Texas. Plant pathologists and regulatory officials should be aware of the potential for misidentification of T. pulcherrima as T. indica Mitra, the Karnal bunt pathogen of wheat that has similar spores, occurs in Texas, and has quarantine status. References: (1) R. Durán and G. W. Fischer. The Genus Tilletia, Washington State University, Pullman, WA, 1961. (2) K. Vánky, Mycotaxon 91:217, 2005.

First Report of Rust on Switchgrass (Panicum virgatum) Caused by Puccinia emaculata in Tennessee.

In the spring of 2007, switchgrass accessions and cultivars Alamo, Kanlow, SL-93-2001, and NSL 2001-1 (lowland), Blackwell (upland), and Grenville, Falcon, and Miami (unknown ploidy levels) were sown at the East Tennessee Research and Extension Center in Knoxville for evaluation and controlled hybridizations. In July and August of 2007, uredinia were observed primarily on the upper leaf surfaces, and to a lesser extent on the undersides of leaves, of switchgrass cvs. Alamo, Blackwell, Grenville, Falcon, Kanlow, and Miami. Uredinia were observed on all cultivars and accessions in 2008. Dimensions of spores are reported as mean ± standard deviation. Uredinia were epiphyllous, adaxial, caulicolous, oblong, and the color of cinnamon brown. Urediniospores were globose to broadly ellipsoid, 26.0 ± 3.0 × 23.2 ± 2.4 µm, with a wall that was cinnamon brown, 1.5 to 2.0 µm thick, finely echinulate with three to four equatorial pores, corresponding to Puccinia emaculata Schw. (3). Abundant teliospores were isolated from Grenville, Falcon, and Blackwell, with fewer teliospores isolated from Alamo. Telia were epiphyllous, adaxial, and caulicolous, densely crowded to scattered, oblong, and dark brown to black. Teliospores were dark brown, two-celled, ellipsoid to oblong, 33.6 ± 4.8 µm long with an apical cell width of 17.5 ± 1.2 µm and basal cell width of 15.9 ± 2.5 µm. Teliospore walls were 1.5 to 2.0 µm wide at the sides and 4 to 6 µm apically. Pedicels were brown or colorless and up to approximately one length of the teliospore, 28.5 ± 7.4 µm. Teliospore morphology confirmed the identification of this rust as P. emaculata (3), which has been reported to infect upland and lowland populations of switchgrass (2). A 2,109-bp fragment containing the internal transcribed spacer (ITS) 1, 5.8S, ITS 2, and D1/D2 region of the large subunit ribosomal DNA was sequenced for a specimen on 'Falcon' (GenBank Accession No. EU915294 and BPI No. 878722) from two overlapping PCR fragments amplified with primers PRITS1F (L. A. Castlebury, unpublished data) and ITS4B (1) for one fragment and Rust5.8SF (L. A. Castlebury, unpublished data) and LR7 (4) for the second fragment. No sequences of P. emaculata were available for comparison; however, BLAST searches of the ITS resulted in hits to P. asparagi DC (527 of 576, 91%) and P. andropogonis Schw. (523 of 568, 92%) placing this fungus in the genus Puccinia Pers. The alternate hosts of this rust are species of the Euphorbiaceae (2,3), which are ubiquitous in this area although the aecial stage has not been observed. To our knowledge, this is the first report of P. emaculata on switchgrass in Tennessee. Given the highly susceptible response of certain varieties of switchgrass to this rust in field plots, reduction in total biomass in large acreages is likely and long-standing fields of this perennial grass will compound the problem. References: (1) M. Gardes and T. D. Bruns. Mol. Ecol. 2:113, 1993. (2) D. M. Gustafson et al. Crop Sci. 43:755, 2003. (3) P. Ramachar and G. Cummins. Mycopathol. Mycol. Appl. 25:7, 1965. (4) R. Vilgalys and M. Hester. J. Bacteriol. 172:4238, 1990.