First Report of Leaf Spot Disease Caused by <i>Alternaria burnsii</i> on <i>Polygonatum odoratum</i> in China

Polygonatum odoratum (Mill.) Druce, a traditional Chinese medicinal herb of the Polygonatum genus in the Liliaceae family. In May 2025, we observed P. odoratum leaf spot disease at the Baiyun Base in Guangzhou, Guangdong Province (23.3875°N, 113.4395°E), with an incidence rate of 90% and a disease index reaching 50 in an approximately 100 m² field plot. The symptomatic leaves initially presented as water-soaked spots or pale-yellow patches, which later developed into brown necrotic areas. Furthermore, some affected visible black, mold-like substance, which was identified as the sporulating mycelia and conidia of the pathogen. For pathogen isolation, leaf sections (5×5mm) were cut from the margin of the lesion and soaked in 75% ethanol for 45s and 3% sodium hypochlorite for 1 min, washed with sterile distilled water, dried on sterilized tissue paper, and placed on potato dextrose agar (PDA). Petri dishes were incubated at 28°C for three days. To obtain a pure spore culture, single-spore isolation was conducted, and one predominant isolate was designated as strain YZ-3 for further study. After seven days of cultivation on PDA, the colony exhibited a brown central region encircled by dense gray-white mycelia (Fig. S1E). Microscopic examination revealed hyphae that ranged from transparent to light brown, characterized by distinct dark, bamboo-like septa (Fig S1F). Mature conidia were dark brown, inverted club-shaped to spindle-shaped, possessing 3–5 transverse dark septa, a rough surface texture, and a prominent tubular beak-like extension at the apex, measuring 18.6–49.3 × 8.2–17.9 µm (n=30) (Fig S1G-H). The conidiophores were erect or curved, displaying a distinctive "Y"-shaped acute branching pattern at the apex (Fig S1I). Genomic DNA of strain YZ-3 was extracted from the mycelium employing the CTAB reference. PCR amplification was conducted utilizing the primer pairs ITS1/ITS4 (White et al., 1990), gpd1/gpd2 (Berbee et al., 1999), RPB2-5F/RPB2-7cR (Liu et al., 1999), and EF1-728F/EF1-986R (Carbone and Kohn, 1999) to target the ITS, GAPDH, RPB2, and TEF1, respectively. The sequences were deposited in the GenBank database. BLASTn analysis demonstrated high sequence homology between YZ-3 (ITS: PX048803, GAPDH: PX069088, RPB2: PX069089, TEF1: PX069090) and A. burnsii CBS 879.95. Phylogenetic analysis was performed based on the concatenated nucleotide sequences via PhyloSuite software (Zhang et al., 2020)(Fig S2), positioned strain YZ-3 firmly within the A. burnsii clade (Fig. S2). Based on morphological characteristics and sequence analysis, the isolated strain YZ-3 was identified as A. burnsii, in accordance with the taxonomic criteria established by (Woudenberg et al., 2015). To assess the pathogenicity of strain YZ-3, a spore suspension at a concentration of 10⁶ spores/mL was applied to the leaves of one-year-old healthy P. odoratum plants, with three replicates conducted. The inoculated plants were maintained at 28°C, 80% relative humidity, and subjected to a 12-hour photoperiod. Ten days post-inoculation, the treated leaves developed extensive yellow-brown lesions (Fig S1C-D). Subsequently, A. burnsii YZ-3 was successfully re-isolated from the symptomatic leaf tissue. To our knowledge, this is the first report of A. burnsii causing leaf spot on P. odoratum in Guangdong Province, China, providing important information for disease management.