In January 2011, leaves of several daylily (Hemerocallis flava L.) plants in nurseries in Vitória da Conquista, northeastern Brazil, showed typical anthracnose symptoms. Reddish brown lesions with a yellow halo were first observed at the tip leaves. As the disease progressed, the lesions rapidly expanded down the leaves, resulting in severe blight. Small pieces up to 5 mm in diameter were removed from the lesion margins, surface sterilized for 1 min in 1.5% NaOCl, washed twice with sterile distilled water, and plated onto potato dextrose agar (PDA) amended with 0.5 g liter−1 streptomycin sulfate. Macroscopic colony characters and microscopic morphology characteristics of two isolates were developed after growth on PDA for 7 days at 25°C under a 12-h light/dark cycle. Colonies presented effuse mycelium, initially white and becoming pale gray, with numerous black structures like sclerotia, setae, and acervuli absent in culture media. Conidia were hyaline, aseptate, curved or slightly curved, round or somewhat acute apex, base truncate, 13.4 to 22.7 (18.2 ± 2.16) μm length, and 3.2 to 5.8 (4.24 ± 0.62) μm width, length/width ratio 4.37, and were typical of Colletotrichum spp. DNA sequencing of partial sequence of actin (ACT), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes and the internal transcribed spacer (ITS1-5.8S-ITS2 rRNA gene cluster) were conducted to accurately identify the species. Sequences of two daylily isolates were highly similar to those of C. spaethianum (Allesch.) Damm, P.F. Cannon & Crous. A phylogenetic analysis using Bayesian inference and including published ACT, CHS-1, GPDH, and ITS data for C. spaethianum and other Colletotrichum species associated with daylily anthracnose (1,3) showed that the isolated fungi belong to the C. spaethianum clade. Sequences of the isolates obtained in this study were deposited in GenBank (ACT Accession Nos. KC598114 and KC598115; CHS-1 Accession Nos. KC598116 and KC598117; GPDH Accession Nos. KC598118 and KC598119; ITS Accession Nos. KC598120 and KC598121). Cultures are deposited in the Culture Collection of Phytopathogenic Fungi of the Universidade Federal Rural de Pernambuco, Recife, Brazil (CMM1224 and CMM1225). Pathogenicity tests were conducted with the two C. spaethianum strains on daylily leaves. Mycelial plugs taken from the margin of actively growing colonies (PDA) of each isolate were applied in shallow wounds near the tip leaves. Four detached leaves were inoculated for each isolate, and PDA discs without fungal growth were used as controls. The leaves were maintained in humid chamber for 2 days at 25°C under a 12-h photoperiod. Anthracnose symptoms that closely resembled those observed in the affected nurseries were developed up to 5 days after inoculation. No symptoms developed on the control plants. C. spaethianum was successfully re-isolated from symptomatic plants to fulfill Koch's postulates. C. spaethianum was described from H. fulva and H. citrina in China, Hosta sielbodiana in Germany, and Lilium sp. in South Korea (3), and from Peucedanum praeruptorum in China (2). To our knowledge, this is the first report of C. spaethianum in Brazil and the first report on H. flava.
References: (1) U. Damm et al. Fungal Divers. 39:45, 2009. (2) M. Guo et al. Plant Dis. 97:1380, 2013. (3) Y. Yang et al. Trop. Plant Pathol. 37:165, 2012.
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