First Report of Agroathelia rolfsii associated with Southern blight disease of Acorus gramineus in China

Acorus gramineus (Acoraceae) is a traditional Chinese herb frequently utilized to enhance appetite and improve memory (Liu et al. 2024). In September 2024, white mycelia first appeared on the soil surface and the infected stem base of A. gramineus. Subsequently, the underground rhizomes had turned black and rotten, accompanied by yellowing and wilting of the leaves. Yellowish-white sclerotia formed on the leaves of dead plants and on the soil surface within 15 days. The disease incidence was 10% in a 100 m² greenhouse located in the greenhouse, Maojian District, Shiyan (32º37'1"N, 110º46'46"E), Hubei, China. Five sclerotia and five mycelial fragments were collected from three symptomatic plants. The samples were disinfected with 75% ethanol for 20 s and 1.5% sodium hypochlorite for 1 min, rinsed three times with sterile water, and placed on potato dextrose agar (PDA) for individual cultivation. The colonies from the ten plates displayed consistent morphological features, characterized by villous mycelia and a significant number of yellow-white to black-brown sclerotia for about 15 days. The white mycelia exhibited a radial growth rate of 15.2-26.5 mm/day at 28°C. Under ×40 magnification, the hyphae displayed clamp connections. After 5 days, white silky mycelia covered the entire Petri dish, the formation of pale yellow sclerotia commenced, which progressively turned dark brown. By day 18, an average of 49.87 ± 17.91 (n = 60) dark brown, spherical sclerotia per Petri dish (90 mm) were cultivated, with a mean diameter of 1.40 ± 0.29 mm (n = 60). The characteristics of the mycelia and sclerotia were consistent with the descriptions of Agroathelia rolfsii (Sacc.) Redhead & Mullineux (basionym: Sclerotium rolfsii Sacc.) (Redhead and Mullineux 2023). For molecular identification, genomic DNA was extracted from two representative isolates (HJXJ4 and HJXJ5) using a DNA extraction kit (TSP101-200, Tsingke Biotech Co., Ltd.). The internal transcribed spacer (ITS) rDNA region and large subunit ribosomal (LSU) region were amplified by PCR using the primers ITS1/ITS4 (White et al. 1990) and LR0R/LR5 (Xu et al. 2010), respectively. The ITS and LSU sequences of the isolates showed 100% and over 99% homology with A. rolfsii YKY2020.02 (OM647806) and HJBJ-5 (OR591284). The sequences of A. rolfsii were submitted to GenBank with the following accession numbers: PX287180 (ITS/HJXJ4) and PX287181(ITS/HJXJ5), PX363206 (LSU/HJXJ4) and PX363207 (LSU/HJXJ5). A phylogenetic tree was constructed using the neighbor-joining method in MEGA 7.0 based on concatenated ITS and LSU gene sequences. The phylogenetic analysis consistently placed the isolates within the A. rolfsii clusters. To verify pathogenicity, ten healthy plants two-year-old were directly inoculated at the stem base on the soil of each potted plant with five sclerotia cultured for ten days (Marin et al. 2025). Control plants were not inoculated. All plants were incubated in a growth chamber at 28°C with 80% relative humidity with a 12 h/12 h light/dark photoperiod. The pathogenicity test was repeat three times. Five days after inoculation, the disease manifested at the stem base, characterized by the presence of white mycelium on the plants. This was followed by damage to the vascular system, resulting in leaf symptoms such as yellowing and wilting. The rhizomes displayed discoloration and rot, along with the formation of black-brown sclerotia on the soil surface, ultimately leading to plant death after two weeks. In contrast, the control plants remained unaffected. A. rolfsii was successfully re-isolated from the necrotic stem base and confirmed through morphological characteristics and ITS sequencing, consistently fulfilling Koch's postulates. The pathogenicity test was repeated three times. Furthermore, A. rolfsii has been responsible for severe southern blight in Alocasia (Marin et al. 2025) and Alpinia hainanensis (Zhang et al. 2025) in both the United States and China. This is the first report of A. rolfsii causing southern blight disease in A. gramineus worldwide. This finding expands the known host range of the pathogen and provides a critical warning for the protection of this valuable plant.