First Report of Colletotrichum sansevieriae Causing Anthracnose of Snake Plant (Dracaena trifasciata) in Ohio and its Draft Genome

Dracaena trifasciata (Prain) Mabb. is a popular houseplant in the United States. In September 2021, two diseased samples from two Ohio homeowners were received by the Ornamental Pathology Laboratory at The Ohio State University. Each sample included one or two detached leaves displaying circular gray water-soaked lesions scattered throughout the lamina and blighted areas with concentric rings bearing brown to black acervuli. Lesions covered between 25 and 50% of the leaf surface. Isolations were made by excising small portions of leaf tissue from the margin of the lesions, surface-disinfesting in 10% bleach for 45 s, rinsing in sterile water, and plating on potato dextrose agar (PDA). Plates were incubated at 23°C for one week. Two representative isolates, one per sample (FPH2021-5 and -6), were obtained by transferring hyphal tips to fresh PDA plates. Mycelia of both isolates were aerial, cottony, grayish-white, producing spores in a gelatinous orange matrix, and appeared gray to olivaceous-gray on the plate underside. Conidia produced by both isolates were cylindrical, single-celled, hyaline, measuring 12.02 to 18.11 (15.51) × 5.03 to 7.29 (6.14) μm (FPH2021-5; n=50) and 15.58 to 20.90 (18.39) × 5.63 to 8.27 (7.05) μm (FPH2021-6; n=50). Appressoria were globose to subglobose, single-celled, dark brown to sepia, measuring 6.62 to 13.98 (8.97) × 5.05 to 6.58 (6.58) μm (FPH2021-5; n=50), and 6.54 to 11.32 (8.63) × 4.54 to 8.94 (7.09) μm (FPH2021-6; n=50). Genomic DNA (gDNA) samples were extracted from both isolates and the internal transcribed spacer (ITS) region was amplified using primers ITS1F/ITS4 (Gardes and Bruns, 1993; White et al. 1990). GenBank BLAST sequence analysis resulted in 99.83% (FPH2021-5; GenBank Acc. No. OP410918.1) and 100% (FPH2021-6; OP410917.1) identity with 100% query coverage to the type strain of Colletotrichum sansevieriae Miho Nakam. & Ohzono MAFF239721 or Sa-1-2 (NR_152313.1; Nakamura et al. 2006). Whole genome sequencing was conducted for FPH2021-6 and the assembly was deposited in GenBank (JAOQIF000000000.1). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-tubulin (β-tub) regions were either extracted from the genome of FPH2021-6 (OP414603.1 and OP414601.1, respectively) or amplified from FPH2021-5 gDNA using primers GDF/GDR (OP414604.1) and Bt-2b/T1 (OP414602.1), respectively (Templeton et al. 1992; Glass and Donaldson 1995; O’Donnell and Cigelnik 1997). A multilocus partitioned analysis (Chernomor et al. 2016) based on concatenated sequences of ITS, GAPDH, and β-tub using ModelFinder (Kalyaanamoorthy et al. 2017) was performed to build a maximum likelihood tree (IQ-TREE v2.0.3; Nguyen et al. 2015), suggesting that these two isolates are phylogenetically closer to the type strain from Japan than to a previously reported isolate 1047 from Florida (Palmateer et al. 2012). To fulfill Koch’s postulates, two parallel leaf sections from one 10-inch D. trifasciata ‘Laurentii’ plant maintained in a 1.3-liter container were selected. Three wounds were made in each section using a sterile syringe needle. A 10-µl drop of either a 1×106 conidia/ml suspension of isolate FPH2021-6 or sterile water was placed on each wound. The plant was covered with a plastic bag for two days post-inoculation (DPI) and maintained in a greenhouse at 25°C with a 12- h photoperiod. The experiment was conducted twice. Grayish water-soaked lesions, acervuli, and leaf blight were observed on the inoculated sections 3, 10, and 14 DPI, respectively, while no symptoms appeared on the sections treated with sterile water. C. sansevieriae was re-isolated from the lesions and confirmed to be identical to the original isolate based on ITS sequencing and morphological examinations. To the best of our knowledge, this is the first report of C. sansevieriae on D. trifasciata in Ohio and the first genome draft of an isolate from the United States. Availability of whole-genome sequence data is paramount for resolving species identification in this highly diverse fungal genus, and a powerful tool to conduct comparative genomic analyses in the future.