First Report of Anthracnose Caused by Colletotrichum spaethianum on Iris lactea in Daqing, China.

Abstract

Iris lactea Pall. has high ornamental value and strong drought resistance, which can be used as a useful sand-stabilization and ornamental plant. In September 2021, typical anthracnose symptoms on I. lactea were found in the campus of Heilongjiang Bayi Agricultural University (125°10'45.31"E, 46°35'36.88"N), Daqing, China. Disease incidence varied from 37 to 51% at four survey sites (~20 m2 per site) of approximately 1,000 plants. Initially brown lesions with a yellow halo, and gray in the center were observed on the leaves. As disease progressed, the lesions expanded rapidly, resulting in dieback. To identify the pathogen, symptomatic tissues were excised from four infected leaves of four individual plants, surface sterilized for 1 min in 75% ethanol, washed twice with sterile water, plated on potato dextrose agar (PDA) contain 0.5 g/L streptomycin sulfate, and incubated at 25℃ for 3 to 4 days under dark conditions. Four morphologically similar fungal isolates (Irs-1 to -4) were obtained by using a single-spore isolation. After growth on PDA for 17 days at 25℃, a circular like zonation colonies with a color of greyish to gray green were observed. On the reverse side, circular like with zig-zag zonation colonies light brown to light black from the margin to the center. Conidiophores were pale brown, septate. Conidia were hyaline, aseptate, curved or slightly curved, round, or somewhat acute apex, base truncate, unicellular, whose sizes ranged at 18.5 ± 2.3 × 4.6 ± 0.7 μm (n = 170), with length/width ratio 4.04. Appressoria were in oval, star, or irregular shape, with brown color with sizes approximately at 13.3 ± 2.1 × 10.4 ± 2.1 μm (n = 30). These morphological characteristics are consistent with Colletotrichum spaethianum with curved conidia (Damm et al. 2009). For molecular identification, primers ITS1/ITS4 (Schoch et al. 2012), GDF1/GDR1 (Guerber et al. 2003), ACT-512F/ACT-783R and CHS-354R/CHS-79F (Carbone and Kohn 1999) were used to amplify the partial region of rDNA-ITS, a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a partial sequence of the actin (ACT), and chitin synthase 1 (CHS-1) from fresh mycelia of the four isolates (Irs-1 to -4), respectively. The sequences were submitted to GenBank (ITS: OM004553, OP804624, OR029392, and OR029391; GAPDH: OP883938, OP883939, OR060963, and OR060962; ACT: OP883940, OP883941, OR060965, and OR060964; CHS: OP868844, OP868845, OR046504, and OR046503). Phylogenetic analysis based on the ITS, GAPDH, ACT and CHS gene sequences indicated that isolates obtained in this study were all clustered with C. spaethianum. Pathogenicity test was conducted by inoculating mycelial plugs on the I. lactea seedlings (2 seedlings with 4 to 5 leaves per isolate). Two seedlings inoculated with sterilized PDA plugs were used as control. All inoculated plants were maintained in humid chamber at 25℃ under dark, and typical anthracnose symptoms were observed up to 6 days after inoculation, while the control leaves were asymptomatic. The same pathogen was successfully re-isolated and phenotypically identical to the original isolates to fulfill Koch's postulates. C. spaethianum has been described on Hemerocalis flava (Vieira and Michereff 2014) and Allium fistulosum (Santana et al. 2016) in Brazil, Polygonatum cyrtonema in China (Ma et al. 2020), Iris germanica in Japan (Sato et al. 2012). To our knowledge, this is the first report of C. spaethianum causing anthracnose disease on I. lactea in Daqing, China.