First Report of Leaf Blight on Paris polyphylla Caused by Ceratobasidium ramicola in China.

Abstract

Paris polyphylla var. yunnanensisis a perennial herb with significant medicinal properties in anticancer, anti-inflammatory, antibacterial immunomodulatory and antispasmodic activities (Duan et al. 2018). In April 2022, leaf blight disease emerged in Xiangtan City (Hunan), affecting P. polyphylla plantings over an area of 3×104 m² (27.904°N, 112.918°E). The disease incidence reached an average of 22% of the plants in the field, with infected plants initially displaying water-soaked chlorosis, followed by dry yellow shrinkage that gradually spread from the leaf tips to the entire plant. To identify the causal agent, 20 leaf lesions (4 mm2) collected from 20 plants were surface sterilized with 75% ethanol for 10 s, 5% NaOCl for 30 s, rinsed in sterile distilled water three times, transferred to potato dextrose agar (PDA) plates with lactic acid (0.125%) , and incubated at 28 °C in the dark. Four isolates (PP21 to PP24) with similar morphologies were obtained and purified by the hyphal-tip method. Colonies on PDA initially appeared white with cottony mycelium, later turning light-yellow on the underside. Septate hyphae were branched at right angles with a small constriction at the departure of the branch point and measured 4.16 to 7.93 µm in diameter. Binucleate cells within the septate hyphae were visualized using Giemsa staining (Servicebio, China). For molecular identification, the rDNA internal transcribed spacer (ITS), the second largest subunit of nuclear DNA-directed RNA polymerase II (RPB2), and ATP synthase subunit 6 (ATP6) were amplified from genomic DNA of the isolates extracted by Fungus Genomic DNA Extraction Kit (Bioflux, China) using primers ITS1/ITS4 (White et al. 1990), bRPB2-6F/bRPB2-7.1R (Matheny 2005; Reeb et al. 2004) and ATP61/ATP62 (Kretzer and Bruns 1999), respectively. The ITS, RBP2 and ATP6 of the four isolates were sequenced and deposited in GenBank. BLASTn search of sequenced ITS (PQ187050, PQ187051, PP728052, PQ187052), RBP2 (PQ202833, PQ202834, PP735921, PQ202835) and ATP6 (PQ202837, PQ202838, PP735922, PQ202839) revealed a >99% identity with the type strain of Ceratobasidium ramicola CBS133.82 (NR138368, DQ301708, and DQ301577). For phylogenetic analysis, concatenated sequences of ITS, RBP2, and ATP6 were employed using the maximum-likelihood method in MEGA-X. Based on the morphological and molecular analyses, the isolates were classified into the C. ramicola clade (Bandoni 1979; Samuels et al. 2012; Jeong et al. 2023). To test the pathogenicity of the isolate PP23, mycelial plugs (5 mm in diameter) were placed directly on 15 healthy leaves from 15 three-year-old plants after puncturing with sterile needles. Sterile PDA plugs served as controls. All plants were kept in a greenhouse with conditions of 25°C, 80% relative humidity and a photoperiod of 12 h. After 5 days, all infected plants developed leaf blight symptoms similar to those described above, whereas the control plants remained asymptomatic. The pathogenicity test was conducted three times. C. ramicola was re-isolated from all symptomatic leaves and confirmed to be identical to the original isolate based on morphology and nucleotide sequences of ITS, RBP2, and ATP6. While C. ramicola is commonly reported to be isolated from diseased cacao, its pathogenicity to cacao remains unknown (Samuels et al. 2012). To our knowledge, this is the first report of C. ramicola causing leaf blight on P. polyphylla, a medicinal herb with significant economic importance in China.