First Report of Phyllosticta capitalensis Causing Leaf Spot in Hymenocallis littoralis in China

Hymenocallis littoralis (Jacq.) Salisb. is a common ornamental plant in China. In November 2021, leaf spots were observed on H. littoralis in a public garden in Zhanjiang, Guangdong Province, China (21°17′25″N, 110°18′12″E). Disease incidence was around 60% (n = 100 investigated plants from about 1 ha). Leaf symptoms were round spots with collapse centers, surrounded by yellow halos. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces. The surfaces were disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Thereafter, the samples were rinsed thrice in sterile water, placed on PDA, and incubated at 28 °C in dark. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty pure cultures were obtained. Single-spore isolation method (Liu et al. 2021) was used to recover the cultures of three isolates (HPC-1, HPC-2, and HPC-3). Colonies of the isolates were dark green with a granular surface, and irregular white (later turning black) edge. Pycnidia were black, globose and 96 -140 μm in diameter. Conidia were single-celled, oval, 7.5 to 13.5 × 4.0 to 7.5 µm (n = 40), with a single apical appendage. Morphological characteristics of the isolates were consistent with the description of Phyllosticta capitalensis (Wikee et al. 2013). Molecular identification was performed using PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012). The internal transcribed spacer (ITS) region, translation elongation factor (TEF1), actin (ACT), and glyceradehyde-3-phosphate dehydrogenase (GAPDH) were amplified using primers ITS1/ITS4, EF1-728F/EF1-986R (Druzhinina et al. 2005), ACT-512F/ACT-783R, and Gpd1-LM/Gpd2-LM (Wikee et al. 2013), respectively. Sequences were deposited in GenBank with accession numbers OM654570 - OM654572 for ITS, OM831376 - OM831378 for tef1-α, OM831346 - OM831348 for ACT, and OM831364 - OM831366 for GAPDH. BLASTn analysis showed that these sequences were 99 to 100% similarity with those of P. capitalensis (ITS, FJ538339; TEF1, FJ538397; ACT, FJ538455; and GAPDH, JF343723). Besides, a phylogenetic tree was generated on the basis of the concatenated data from the sequences of ITS, TEF1, ACT, and GAPDH that nested within the clade containing P. capitalensis (CBS 117118, CPC20510,CPC20267, and CPC18848). From the combination of the morphological and molecular characteristics, the isolates were determined to be P. capitalensis. Pathogenicity testing was performed in a greenhouse with 80% relative humidity at 25 to 30°C. Ten healthy plants of H. littoralis (2 month old with 4 leaves) were grown in pots with one plant in each pot. Three leaves on one plant per isolate were inoculated with three mycelial plugs obtained from 7-day cultures, totaling five plants. Five plants treated with PDA plugs served as the controls. Wet cotton balls were fixed on the leaves with transparent tape for five days to keep it from drying out. The test was conducted three times. After 15 days, similar symptoms were observed in the inoculated leaves as in the garden, whereas control leaves remained asymptomatic and P. capitalensis was successfully re-isolated from the inoculated leaves. Previously, P. capitalensis has been reported to cause leaf spot disease of various host plants around the world (Wikee et al. 2013). However, to our knowledge, this is the first report of leaf spot caused by P. capitalensis on H. littoralis in China. This study provides an important reference for the control of the disease.