First Report of Colletotrichum liriopes Causing Leaf Spots on Liriope spicata in China

Abstract

Liriope spicata (Thunb.) Lour. is an evergreen herb, belonging to the genus Liriope, family Liliaceae. As a traditional Chinese medicine, L. spicata is widely cultured in China, Korea, and Japan for ornamental and medicinal purposes. In May 2018, 80 to 90% of L. spicata leaves were observed with leaf spots on the campus of Qingdao Agricultural University. The diseased leaves had oval or irregularly shaped spots with dark brown edges and yellow halos. Some lesions were observed with black acervuli with setae. One fungus was isolated consistently by placing surface-sterilized tissue pieces from the edge of the leaf spots on potato dextrose agar. The resulting colonies were initially whitish and then turned gray after 4 days of incubation at 25°C, and salmon pink spore masses without setae were also observed. Conidia were one-celled (17 to 24 × 2 to 5 μm), hyaline, and falcate. Based on the colony and conidial morphology, the isolate fungus was identified as Colletotrichum liriopes (Damm et al. 2009; Trigiano 2013). The sexual stage and sclerotia formation in the culture were not observed. Sequences of the internal transcribed spacer region of rDNA (GenBank accession no. MK131716), chitin synthase gene (MK144289), β-tubulin gene (MK144290), glyceraldehyde-3-phosphate dehydrogenase gene (MK144291), and calmodulin gene (MK144292) were amplified and analyzed from two single-spore isolates, Ls-CL-01 and Ls-CL-02 (Damm et al. 2009; Weir 2012; White et al. 1990). Because the sequences amplified from Ls-CL-02 were identical to those of Ls-CL-01, only one set of sequences was submitted to GenBank. These sequences had 99 to 100% nucleotide identity to that of C. liriopes (MG543776.1, GU228294.1, KC244160.1, KY995371.1, and HM582017.1, respectively) in GenBank. In addition, MK131716, MK144289, MK144290, and MK144291 had 99% nucleotide identity to that of C. liriopes ex-type CBS 119444 (GU227804, GU228294, GU228098, and GU228196) (Damm et al. 2009). The pathogenicity test was performed using the spray inoculation method. Spores were harvested from 14-day culture plates of Ls-CL-01 and Ls-CL-02 using sterilized distilled water with 0.025% of Tween 20 (Sigma-Aldrich). The spore suspension was adjusted to a final concentration of 5 × 10⁶ spores/ml and was sprayed over five healthy plants, and sterilized water with 0.025% of Tween 20 was used as a control treatment. The inoculated plants were incubated at 25°C and 90% relative humidity with 12-h/12-h dark/light for 3 days and then moved to normal conditions (12-h/12-h dark/light, 20% relative humidity) for 2 days. The experiment was repeated twice. After 5 days of inoculation, approximately 50% of leaves of the inoculated plants (all 15 plants) showed symptoms similar to those previously observed on the original leaves, whereas the control leaves remained asymptomatic. Three isolates were recovered from inoculated leaves. They were identical to isolate Ls-CL-01 in colonial and conidial features. To our knowledge, this is the first report that C. liriopes causes leaf spots on L. spicata. Leaf spots could greatly affect the value of L. spicata; identification of the pathogen provides scientific basis for the disease management.