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.