First Report of <i>Stemonitis</i> sp. Causing Rot Disease of Cultivated <i>Phallus indusiatus</i> in China

Phallus indusiatus Vent. is a high-value edible and medicinal mushroom widely cultivated in China. From 2023 to 2025, a severe myxomycete disease was consistently detected (50%–100% incidence) at the P. indusiatus Cultivation Base in Huangshan, Anhui Province (29°40′30″ N, 118°15′41″ E). This disease, which affects the soil layer and mulching straw used for P. indusiatus cultivation, inhibits mycelial growth and primordia formation and causes water-soaked decay of young fruiting bodies. High temperature and humidity favor disease development, with creamy-yellow plasmodia appearing on the substrate and then darkening to form clustered sporangia. Sporangia were collected and examined microscopically (Figure 1); the method for isolating and culturing samples was adapted from that described by Dai (2017). Sporangia were stalked, cylindrical, dark brown–black, 0.2–0.3 mm in diameter, and 2–3 mm in height, with an evanescent peridium and a well-developed columella. Stalk height was approximately one-third of the total height. Capillitia were delicate and attached to the columella, with the final branches united as a surface net. Spores (6–7 µm diameter) were globose and dark brown en masse, with distinct spinulose ornamentation revealed by a scanning electron microscope. On the basis of morphology, the sample was identified as Stemonitis herbatica Peck. Harvested fruiting bodies and sporangia were deposited in the Mycology Laboratory at Baicheng Normal University (BCNUZFY72, BCNUZFY72I). Genomic DNA was extracted from mature sporangia as described by Zhao et al. (2021). 18S rRNA and elongation factor 1-alpha (EF1a) genes were amplified by PCR using primer pairs S2/SSU_rev (Fiore-Donno et al. 2008; Prikhodko et al. 2023) and SMEF-F196/SMEF-R960 (Zhang et al. 2018). BLASTn analysis of 18S rRNA (PX845259) and EF1a (PZ013459) gene sequences indicated that they were most similar to Stemonitis sp. (JQ900833, 86.99%) and Physarum pezizoideum (OP631032, 90.52%) sequences, respectively. Only six S. herbatica sequences are available in the NCBI database [three 18S rDNA sequences (MK041080, MK041081, OQ658886), one ITS1 sequence (KF772261), and two unverified sequences (JQ277917, JF339223)]. We therefore constructed a phylogenetic tree using 18S rDNA sequences. All S. herbatica sequences, including the novel sequence from this study, were in the Stemonitis clade, but on distinct sub-branches (Figure 2). Thus, the causal agent of the myxomycete disease affecting P. indusiatus was identified as Stemonitis sp.. Koch’s postulates were fulfilled by inoculating three healthy P. indusiatus cultivation boxes with 10 ml spore suspension (152 spores/ml) or sterilized water (control) under controlled conditions (25 °C, 90%–95% relative humidity). After 40 days, spore-inoculated boxes had symptoms identical to those in the field, whereas control boxes were symptom-free. Sporangia from the inoculated substrate were collected and examined morphologically. The re-isolated myxomycete was confirmed as the novel sample from this study according to morphology and the 18S rDNA sequence (identical to PX845259). Several species from the family Stemonitiaceae cause slime mold disease of cultivated mushrooms (Lee et al. 2014; Zhang et al. 2018). To the best of our knowledge, this is the first report of Stemonitis sp. causing persistent rot disease of cultivated P. indusiatus in China.