Whorled sunflower, Helianthus verticillatus Small, is an endangered (U.S. Fish and Wildlife Service 2014) perennial sunflower species indigenous to the southern United States (Matthews et al. 2002; Ellis et al. 2008). Helianthus verticillatus has a showy yellow floral display in the Fall that attracts a diversity of insect visitors (Strange et al. 2020). Its hardiness in the landscape and late-season blooming makes it a potential ornamental (Trigiano et al. 2021). In June 2021, anthracnose-like lesions were observed on mature leaves collected from potted H. verticillatus plants grown in the nursery compound at the University of Tennessee, Knoxville, TN, USA. Irregularly shaped leaf spots with 1‒2 mm tan centers were observed on mature leaves, which later expanded to 3‒5 mm, and became dark brown- to- black surrounded by chlorotic halos (Fig.1). Lesions from three infected leaves were excised from a single potted plant, trimmed to 1.5-cm squares with green borders, and surface-sterilized (Trigiano et al. 2018). Tissues were placed onto potato dextrose agar (PDA), amended with 100 mg/ml of each streptomycin sulfate and chlorotetracycline, and incubated at 21 °C in the dark until axenic cultures were obtained. Initially, appressed white- to- pale gray mycelia were formed that turned light pinkish-orange with age (Fig. 2A). Conidia (Fig. 2B-C) were single-celled, hyaline, and cylindrical- to- fusiform with acute ends, and were similar to Colletotrichum fioriniae (Damm et al. 2012). Conidia measured 8.9 ± 1.3 μm long and 3.3 ± 0.6 μm wide (N=40). Genomic DNA was isolated with a Phire Direct Plant PCR kit (Thermo FisherScientific, Waltham, MA). The partial beta-tubulin (TUB2) gene, chitin synthase 1 (CHS-1) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and the internal transcribed spacer (ITS) region of ribosomal DNA were amplified with primers T1/BT2B, CHS-354R/CHS-79F, GDF1/GDR1, and ITS1/ITS4, respectively and sequenced (Damm et al. 2012). The resulting sequences were submitted to GenBank (TUB2, ON036471; CHS-1, ON036472; GAPDH, ON036470; and ITS, ON008206). Consensus sequences had 100% identity with C. fioriniae type culture CBS 128517 accessions JQ949943 (TUB2), JQ948953 (CHS-1), JQ948622 (GAPDH), and MH865005 (ITS rDNA). Because H. verticillatus is endangered, and the scarcity of available plant material, Koch's postulates were performed using a detached leaf assay (Boggess et al. 2022). Six healthy leaves were surface-sterilized using the previously described protocol, longitudinally bisected, and placed on 1.5% water agar in three 15 × 100 mm petri dishes. Three half leaves were inoculated with sterile, 5 mm-diameter PDA plugs (controls). The remaining three leaves were inoculated with 5 mm-diameter PDA plugs of C. fioriniae and incubated as described previously. After ten days, necrotic lesions developed on leaves inoculated with C. fioriniae and were similar to the initially observed lesions on plants. Lesions did not develop on control leaves. Colletotrichum fioriniae was re-isolated from lesions using the previously described protocol. The disease does not appear to cause mortality of H. verticillatus and does not require control measures but does reduce the aesthetic value of the plant. To the best of our knowledge, this is the first report of C. fioriniae infecting H. verticillatus in the United States.
Keywords: Colletotrichum fioriniae; Helianthus verticillatus; endangered plant; leaf anthracnose; whorled sunflower.