First Report of Agroathelia (syn. Sclerotium) rolfsii associated with Southern blight disease of Daphne tangutica (Gansu Ruixiang) in China.

Abstract

Daphne tangutica Maxim. (Thymelaeaceae) is widely cultivated in China for its medicinal properties including promoting blood circulation, relieving pain, and dispelling wind to eliminate dampness (Liu et al. 2009). In August 2022, stem rot disease was observed on 1-to 5-year-old D. tangutica (Gansu Ruixiang) plants, with a disease incidence rate of 10% in a 40 m2 greenhouse located in Shiyan City, Hubei Province, China. The average temperature ranged from 28°C to 33°C, with relative humidity was 60% to 80%. The disease occurred during the fruiting period and affected plants regardless of age. White mycelia appeared at the crown of the stem at the soil line, and extended upward along the stem. Both young and mature leaves curled, wilted, and fell prematurely. The xylem of infected plants turned dark brown and softened progressively, eventually leading to plant death. A suspected fungal pathogen was isolated from two symptomatic plants. Fragments of mycelia were transferred onto potato dextrose agar (PDA) plates with a sterile dissecting needle and incubated at 28°C for 2 to 3 days. Colonies were purified using the hyphal-tip method and transferred to fresh PDA plates for morphological observation. White mycelia grew at a rate of 17.7 to 23.7 mm/day (n=16), and hyphae were hyaline with typical clamp connection under ×40 magnification. Light yellow sclerotia began forming after five days and gradually turned dark brown. On average, 65.65 ± 4.33 (n = 50) spherical, dark brown sclerotia, each with a mean diameter of 1.64 ± 0.29 mm (n=50), developed on each Petri dish after 18 days of incubation. Based on morphological characteristics, the isolates were tentatively identified as Agroathelia (=Sclerotium) rolfsii (Sacc.) Redhead & Mullineux (Amylocorticiaceae) (Redhead and Mullineux). Two representative isolates, HJBJ-5 and HJBJ-9, were selected for molecular identification. Genomic DNA was extracted using a DNA extraction kit (TSP101-200, Tsingke Biotech Co., Ltd.), and PCR amplification was performed using the internal transcribed spacer ITS primers (ITS1 and ITS4) (White et al. 1990) and the large subunit ribosomal LSU primers (LR0R and LR5) (Xu et al. 2010). The PCR products were sequenced and analyzed using BLAST with the current available sequences in NCBI. The ITS sequences of HJBJ-5 (OR578406/667bp) and HJBJ-9 (PP231038/659bp) shared 99.1% and 98.8% similarity, respectively, with A. rolfsii A8.2 (GU080230/685bp) and A. rolfsii NB2 (MN071108/658bp). The LSU sequences of HJBJ-5 (OR591284/913bp) and HJBJ-9 (PP231039/897bp) showed 99.8% and 99.9% similarity with A. rolfsii Penampang (OP809607/942bp) and A. rolfsii MY17B-1 (OP658955/922bp). Phylogenetic analysis using the maximum likelihood method confirmed that the two isolates (HJBJ-5 and HJBJ-9) clustered with other known A. rolfsii strains. To confirm pathogenicity, healthy, asympomatic eight-month-old D. tangutica (Gansu Ruixiang) plants were transplanted into pots containing sterilized soil. Ten plants were inoculated with five dark brown sclerotia of HJBJ-5 collected from PDA, while another ten plants, inoculated with PDA plugs without fungal materials, were used as control. Either sclerotia or PDA plugs were placed base of the stem in the each pot (Nie et al. 2020), and all plants were maintained in a greenhouse at 28°C with 80% relative humidity. At 10 days post -inoculation (dpi), white mycelia and dark brown sclerotia had spread across the soil surface and up the stems. By 15 dpi, the stem base became soften and rot, followed by leaf defoliation. One week later, the mycelia had reached the upper stem, leading to plant death, symptoms consistent with those observed in the field. In contrast, no symptoms developed in the control group. The pathogenicity test was repeated three times. Pathogenic fungi were re-isolated from infected plants and confirmed through morphological characteristics and ITS sequencing (ITS1 and ITS4), fulfilling Koch's postulates. Agroathelia rolfsii has previously been reported as the causal agent of root or stem diseases in various crops, including in Amorphophallus muelleri (Gao et al. 2014), Artocarpus heterophyllus (Yi et al. 2023), Basella alba (Khoo et al. 2023) and hemp (Mersha et al. 2020) in China, Malaysia, and the United States. However, this is the first report of southern blight caused by A. rolfsii on D. tangutica Maxim. (Thymelaeaceae) in Hubei, China. These findings highlight the need for effective disease management strategies during cultivation, such as drip irrigation and chemical protection on branches before cuttage.