First Report of Leaf Spot on <i>Hymenocallis littoralis</i> Caused by <i>Muyocopron laterale</i> in Guangdong Province, China

Beach spider lily (Hymenocallis littoralis) is widely cultivated in China. We observed leaf spot symptoms on this plant in a field in Shenzhen (22°36'04"N, 113°59'07"E), China, in October 2024. An ~700 m² survey found 80 of 100 plants (80%) infected, with 20 to 50% of each plant’s leaves exhibiting spot symptoms. The earliest symptoms appeared as dry, yellow spots on the leaves, 0.3–0.9 × 0.4–0.8 cm. Spots gradually expanded into oval or irregular lesions with brown centers and reddish-brown margins with a yellow halo. The affected leaves became blighted and desiccated; however, fruiting structures were not observed. A total of 10 diseased leaves were collected for isolation of the possible fungal pathogen. Each leaf segment (5 × 5 mm²) from the lesion margins was disinfected using 1% NaClO for one min, 70% ethanol for 30 seconds, rinsed with sterile water, placed on PDA, and incubated at 25 °C. After three days, fungal colonies were isolated on PDA using the single hyphal tip method. Ten strains exhibiting similar morphology were collected, and two representative strains (MBSZU 25-042 and MBSZU 25-043) were selected for further study. After one week at 25 °C, colonies on PDA were 36 to 45 mm in diameter, circular and flat, with gray centers and white margins; the reverse side was black in the center and white at the margins. All strains produced asexual structures after two months of incubation; however, no conidiomata were observed. Conidiogenous cells were hyaline to dark brown, often ampulliform with a distinct flared collarette, smooth, and 10.6 to 14.6 × 10 to 12 µm diam. Conidia were aseptate, lunate, fusiform, curved, with a base obtuse to truncate, often guttulate, hyaline, smooth, and 11.8 to 20.9 × 4.8 to 8.8 µm (n = 50), with a filiform, unbranched appendage at each end. Morphologically, all strains were similar to Muyocopron (Hernández-Restrepo et al. 2019). The ITS, LSU, SSU, and tef1-α genes were amplified using ITS5/ITS4, LROR/LR5, NS1/NS4, and EF1-983F/EF1-2218R (Rehner and Buckley 2005; Vilgalys and Hester 1990; White et al. 1990). The sequences of ITS (PV973166 and PV973167), LSU (PV973169 and PV973170), SSU (PV973224 and PV973225), and tef1-α (PV983295 and PV983296) were deposited in GenBank. BLAST analysis revealed that the ITS, LSU, and tef1-α sequences had similarities of 99.35, 99.88, and 99.66% to M. laterale (CBS 14102), respectively. Phylogenetic analyses confirmed that all the strains belonged to M. laterale. Pathogenicity was assessed on four-months-old healthy plants using the attached leaf assay, which were grown in 10 cm pots. All leaves were disinfected using 0.1% NaClO and three rinses with sterile water. Wounds (three pores, 0.5 mm width) were made at the equator of each leaf using a sterile needle. A conidial suspension (10⁶ conidia/ml) from 8-week-old cultures was applied (15 µl) to each leaf. Control leaves were mock-inoculated with sterile water. Each treatment had 10 replicates and was repeated twice. The plants were kept in plastic bags for 24 h at 25 °C and 80–85% humidity. After seven days, all inoculated leaves developed brown lesions, while control leaves showed no symptoms. M. laterale was reisolated from the lesions and confirmed through morphological and multi-gene analyses, thereby fulfilling Koch's postulates. Jiang et al. (2022) reported that M. laterale causes leaf disease of Camellia sinensis in China. To the best of our knowledge, this is the first report of leaf spot on H. littoralis caused by M. laterale in China and worldwide.