First report of cobweb disease of Auricularia heimuer caused by Hypomyces mycophilus in Fujian Province, China.

Auricularia heimuer is a gelatinous fungus with great edible and medicinal values. In September 2021, a suspected cobweb disease was found in some A. heimuer farms in Fujian Province, China. The disease caused white cottony mycelium to grow on the basal surface of the A. heimuer at the beginning of infection and gradually spread along the outer edge of the fruiting body, and eventually the white pathogen mycelium covered the entire fruiting body, which eventually led to the wilting and death of about 35% of A. heimuer . Two heavily infected tissues of A. heimuer were collected and two isolates were obtained by single spore isolation and purification technique. The pathogen colonies grew 10 to 12 mm per day on potato dextrose agar (PDA), and the colonies were initially white in color and gradually changed to yellowish brown with neat margins. Well-developed mycelium with septum, Conidiophores are bottle-stemmed and whorl-shaped branches, Conidia solitary, as ovoid, colorless singletons or doublets. The chlamydospores are yellowish, smooth surface, with 2-3 septa, size 9-22 µm × 30-38 µm. These morphological features are consistent with the Hypomyces mycophilus (Gea et al. 2019; Wang et al. 2021). For molecular identification, genomic DNA of the two isolates was obtained using an extraction kit (Biocolor, Shanghai, China), internal transcribed spacers (ITS) regions and 5SrRNA were amplified using ITS1 and ITS4 primers (White et al. 1990). A 590 bp DNA fragment was obtained and the sequences were deposited in GenBank (Accession Nos. OP715875 and OP782039), A BLAST search in GenBank revealed the highest similarity (≥99%) to H. mycophilus (GenBank Accession Nos. MH857567 and KU937111) . To fulfill Koch's postulates, the isolates cultured on PDA plates for 10 days were made into a spore suspension with sterile water at a concentration of 5 × 106 conidia/ml and sprayed onto twenty healthy fruiting bodies grown to about 2 cm in diameter, and another ten healthy fruiting bodies sprayed with sterile water as control, and incubated in an artificial climate chamber at 25℃ and relative humidity of 90%-95% (Back et al. 2012). After 4 days of inoculation, the pathogen started to germinate and slowly grew white mycelium, then the white mycelium multiplied at the base of the fruiting bodies and spread from the base to the periphery, and finally the fruiting bodies were completely covered by the pathogenic mycelium and gradually wilted. The symptoms were consistent with the natural disease symptoms under cultivation conditions, while the control group had normal growth of the seeds and no disease symptoms. H. mycophilus was reisolated and purified from symptomatic cotyledons and identified by the above method, and the results of the two experiments were consistent. To our knowledge, this is the first report of H. mycophilus causing cobweb disease in A. heimuer.

First report of cobweb disease in Auricularia cornea caused by Hypomyces mycophilus in Guizhou, China.

Auricularia cornea is a widely cultivated mushroom in China, which has high medicinal values such as hemostaticity, analgesia, antioxidation and anti-tumor (Wu et al., 2019). In 2022, an investigation on edible mushroom diseases in Guizhou Province observed a suspected cobweb disease in an A. cornea growing factory, with up to 30% incidence. The pathogen first produced flocculent hyphae on the surface of the fruiting body of A. cornea, and then developed spider web-like aerial hyphae, covering the entire fruiting bodies. It hinders the normal growth of A. cornea, resulting in deformity and rot of the fruiting bodies. These symptoms seriously affect the quantity and quality of mushroom yields and cause huge economic losses. Three fungal isolates (GUCCX001, GUCCX002 and GUCCX003) were recovered from the diseased mushroom fruiting bodies and purified through single spore isolation. The colonies of three isolates spread rapidly on PDA, reaching 79-82 mm in seven days. The flocculent mycelium was whitish, and its reverse turned from yellowish to amber after 14 days. The branched conidiophores arising from aerial mycelia were septate and each cell contained several denticulate conidiogenous loci. Each denticle contained a single conidium. Conidia were observed at the tip of conidiophore branches and were 0-1-septate, oval or spherical, transparent, 5.2-11.3 × 11.7-18.7 µm (n = 35). Chlamydospores were visible as 3-4 thick-walled cells at the tip of lateral hyphal branches. Three isolates were tentatively identified as H. mycophilus based on their morphological characteristics similar to those described by Rogerson and Samuels (1993). The sequence of internal transcribed spacer (ITS) region (primers ITS5/ITS4) (Rehner and Samuels, 1994) and nuclear ribosomal large subunit (LSU) region (primers LR0R/LR5) (Vilgalys and Heste, 1990) of GUCCX001 (ITS: OP777905; LSU: OQ152071), GUCCX002 (ITS: OP862872; LSU: OQ152072) and GUCCX003 (ITS: OP862873; LSU: OP862873) were 99%-100% similar to H. mycophilus CBS 175.56 (ITS: MH857567; LSU: MH869110). Fifteen healthy fruiting bodies of A. cornea were inoculated by spraying spore suspension (106 conidia/mL) of the three isolates and five healthy fruiting bodies were sprayed with sterile water as control. All inoculated fruiting bodies were kept at 25 ℃. After three days, fruiting bodies of A. cornea treated with the spore suspension exhibited the same symptoms of cobweb as in the factory, while no symptom appeared in the control. Pathogens re-isolated from diseased fruiting bodies were confirmed to be H. mycophilus based on morphological characteristics, which fulfills the Koch's postulate. Zeng et al. (2017) reported H. mycophilus on the fruiting bodies of Auricularia sp. as a new record in Guangdong, China. H. mycophilus caused cobweb disease on A. auricula (Liu et al., 2020), A. cornea var. Li. (Cao et al., 2023) and A. heimuer (Zhang et al., 2023). To our knowledge, this is the first report of cobweb disease in A. cornea caused by H. mycophilus in Guizhou, China. Our findings will provide a basis for correct diagnosis and management of cobweb diseases on A. cornea.

First Report of Cobweb Disease in Auricularia cornea var. Li. Caused by Hypomyces mycophilus in China.

Auricularia cornea var. Li. is a natural mutation strain of A. cornea which has been preferred by consumers for its white colour, good taste and pharmacological properties (Rebecca et al. 2020). In October 2021, a disease with symptoms similar to that of cobweb disease (Carrasco et al. 2017) was observed in A. cornea var. Li. in four mushroom farms in Fangshan District (115.83°E, 39.55°N), Beijing, China, infecting 20% of the fruiting bodies (Fig. 1A-D). White cottony mycelia formed typically on the casing soil and they gradually spread to the stipes and pileus, covering the whole fruiting body, which eventually died and lost commodity value. Cultures were obtained by aseptically transferring the diseased fruiting bodies onto potato dextrose agar (PDA); they were deposited in the culture collection (ID: JZBQA3) of the Beijing Academy of Agriculture and Forestry Sciences, China. The colonies were floccose with aerial mycelium white. Purplish grey diffusing pigments occasionally formed on the reverse side of the plate at 25 °C (Fig. 2A-B). Conidiophores arising in aerial mycelium, indefinite in length, branches septate, each cell producing denticulate conidiogenous loci, each denticle bearing a single conidium. Conidia mostly oblong to ellipsoidal, smooth, (9.0-)9.9-17.0(-18.0)×(6.0-)6.9-10.2 µm (n = 60), 0~1 septate (Fig. 2C-E). Chlamydospores forming as lateral branches of hyphae were commonly observed, globose, ellipsoid or oblong, 14.8-22×14.7-19.6 µm, l/w = 1.0-1.3 (Fig. 2F-G). The morphological characteristics were consistent with that of Hypomyces mycophilus, whose anamorph was Cladobotryum polypori (Rogerson et al. 1993, Zeng et al. 2017). For further species identification, genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, USA). The internal transcribed spacer (ITS) regions and large subunit ribosomal RNA (LSU) genes were amplified using the primer pairs ITS1/ITS4 (White et al. 1990) and LROR/LR5 (Bhattacharya et al. 2000), respectively. The sequences were deposited in GenBank with accession numbers OP430530 and OP430531. BLAST nucleotide searches showed more than 99% homology with corresponding sequences of Hypomyces mycophilus HMAS 275554 and CBS 175.56. Phylogenetic trees based on ITS and LSU revealed that the strain JZBQA3 was grouped with H. mycophilus with high support value (Fig. 3). A in vivo pathogenicity test was performed using eight mushroom sticks with healthy fruiting bodies in triplicate. Each four sticks were sprayed with conidial suspension (108 spores/mL) of strain JZBQA3 or sterile distilled water, respectively, and maintained in an artificial climate chamber at 25-26°C. Cobweb-like characteristics were observed on the fruiting bodies treated with the JZBQA3 conidial suspension 2-3 days after inoculation, while those treated with sterile distilled water remained symptomless (Fig. 4 A-B). The same pathogen was re-isolated and confirmed from the infected fruiting bodies by integrated analysis of morphological characteristics and gene sequencing data, fulfilling Koch's postulates. Hypomyces mycophilus was first reported on Trametes versicolor in North Carolina (Rogerson et al. 1993), and is the causal agent of cobweb diseases on Auricularia heimuer (Zhang et al. 2023). To our knowledge, this is the first report of cobweb disease caused by H. mycophilus in A. cornea var. Li. This finding is a valuable contribution to the knowledge of cobweb disease development in edible fungi.