Hymenocallis littoralis (Jacq.) Salisb, commonly known as spider lily, is a Amaryllidaceae species widely cultivated in southern China for ornamental and medicinal purposes (Anusha et al., 2016). In 2020, there was a devastating outbreak of leaf scorch of H. littoralis from July to September in Kunming city, Yunnan province of China (E102.8268°, N24.8371°), with 97% disease incidence. The initial spots were small and reddish-brown but gradually expanded to large irregular lesions with yellow centers. The leaves then turned yellow and withered from tip towards the petiole in the severely infected plants. For identification, leaf sections (5 × 5 mm2) cut from the margins of the lesions were surface-sterilized for 3 min with 1% sodium hypochlorite, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA), and incubated at 28°C at a 12-h photoperiod for three days. One fungus was isolated from 90% of the samples, of which three monosporic isolates were selected using a method of agar dilution lineation separation. After 4 days of incubation, colonies were white-yellowish, and changed to beige-mustard after 7 days. The pycnidia were produced after 10 days incubation on oatmeal agar (OA), and they were black-brown, subglobose, and ostiolate. Chlamydospores mainly formed in the aerial mycelia, globose, often in chains, brown or pale. The conidia were ellipsoidal or clavate, (0-)1-3-septate, 2.1 to 10.8 × 1.0 to 3.2 μm (n=60). The total genomic DNA of three isolates was extracted from mycelia. The nuclear ribosomal internal transcribed spacer region (ITS), the second largest subunit of nuclear DNA-dependent RNA polymerase II (rpb2), the 28S nuclear ribosomal large subunit rRNA gene (LSU), and beta-tubulin gene (tub2) were amplified using the ITS1/ITS4 (White et al., 1990), fRPB2-5F/fRPB2-7cR (Liu et al., 1999), LR0R/LR5 (Schoch et al., 2012), and Btub2Fd/Btub4Rd (Woudenberg et al., 2009) primer pairs, respectively. The amplicons were cloned in the pMD19-T vector (Code No. 6013, Takara, Kusatsu, Japan) and sequenced bi-directionally. These three isolates had the same nucleotide sequences, one of which was submitted to NCBI (ITS, OM279485; rpb2, OM304305; LSU, OP800249; tub2, OQ108870). BLASTn analyses showed that ITS, rpb2, LSU, and tub2 were genetically 100%, 98.49%, 99.89%, and 97.89%, respectively, identical with MN973518, MT018130, MN943724, and MT005618 genes of Didymella curtisii strain CBS 288.29. Phylogenetic tree was constructed with MEGAX based on the nucleotide sequences of ITS, rpb2, LSU, and tub2 using the maximum likelihood method. The fungus isolated from diseased leaves of H. littoralis was grouped into the same clade with D. curtisii. According to the morphology and sequence analyses, the isolate was D. curtisii (Chen et al., 2015) and was named isolate HlDc1. To confirm the causal agent of the disease, a spore suspension with 106 spores of HlDc1/mL was smeared on healthy leaves of six months old with brushes. Leaves in the control group were smeared with sterile water. All inoculated plants were incubated at 28°C under a 12-h photoperiod in a moisture chamber. The pathogenicity tests were conducted three times with six plants each time. Fifteen days post-inoculation, the leaves inoculated with HlDc1 developed red-brown lesions, whereas the control leaves remained asymptomatic. Isolate HlDc1 was re-isolated from infected leaves. To our knowledge, this is the first report of leaf scorch on H. littoralis in Yunnan province, China, caused by D. curtisii. The results laid the foundation for epidemiological forecasting and scientific control of this disease.
Keywords: Didymella curtisii; Hymenocallis littoralis; leaf scorch.