Inhibition mechanism of cordycepin and ergosterol from Cordyceps militaris Link. against xanthine oxidase and cyclooxygenase-2.

Cordyceps militaris Link. (C. militaris) is an entomopathogenic fungus that parasitizes the pupa or cocoon of lepidopteran insect larvae, with various bioactive compounds. Cordycepin and ergosterol are the two active components in C. militaris. This study aimed to evaluate the inhibitory activity of cordycepin and ergosterol against xanthine oxidase (XO) and cyclooxygenase-2 (COX-2), as well as investigate the inhibition mechanism. Cordycepin could better inhibit XO (IC50 = 0.014 mg/mL) and COX-2 (IC50 = 0.055 mg/mL) than ergosterol. Additionally, surface hydrophobicity and circular dichroism (CD) spectra results confirmed the conformational changes in enzymes induced by cordycepin and ergosterol. Finally, cordycepin and ergosterol significantly decreased uric acid (UA) and inflammatory factors to normal level in mice with gouty nephropathy (GN). This study could provide theoretical evidence for utilization of C. militaris in hyperuricemia-management functional foods.

First Report of Cladobotryum protrusum causing Cobweb Disease on the Edible Mushroom Coprinus comatus.

Coprinus comatus is one of the most commercially important mushrooms in China. Its fruiting body possesses rich nutritional and medicinal value. In November 2013, unusual symptoms were observed on C. comatus on a mushroom farm in Wuhan, Hubei, China. At first, fruiting bodies were covered by white and cobweb-like mycelia. Later, the cap and stipe turned brown or dark before rotting and cracking. The pathogen was isolated from infected tissue of C. comatus. Colonies of the pathogen on potato dextrose agar (PDA) medium first appeared yellowish, followed by an obvious ochraceous or pinkish color. Aerial mycelia grew along the plate wall, cottony, 1 to 4 mm high. Conidiophores were borne on the tops of hyphae, had two to four branches, and were cylindrical, long clavate, or fusiform. Conidia were borne on the tops of the branches of conidiophores, had one to two separates, and were long and clavate. The spores ranged from 15.3 to 22.1 µm long and were 5.1 to 8.3 µm wide, which was consistent with the characteristics of Cladobotryum protrusum (1). The species was identified by ribosomal internal transcribed spacer sequencing. The ribosomal ITS1-5.8S-ITS2 region was amplified from the isolated strain using primers ITS1 and ITS4. A BLAST search in GenBank revealed the highest similarity (99%) to C. protrusum (GenBank Accession Nos. FN859408.1 and FN859413.1). The pathogen was grown on PDA at 25°C for 3 days, and the inoculation suspension was prepared by flooding the agar surface with sterilized double-distilled water for spore suspension (1 × 105 conidia/ml). In one treatment, the suspension was sprayed on casing soil (106 conidia/m2) and mixed thoroughly with it, then cased with treated soil for 2 to 3 cm thickness on the surface of compost in cultivation pots (35 × 25× 12 cm), with sterile distilled water as a control (2). Eight biological replicates were included in this treatment. In the second treatment, mycelia plugs (0.3 × 0.3 cm) without spore production were added to 20 fruiting bodies. Mushrooms treated with blank agar plugs (0.3 × 0.3 cm) were used as a control. The plugs were covered with sterilized cotton balls to avoid loss of moisture. Tested cultivation pots were maintained at 18°C and 85 to 95% relative humidity. In the samples where casing soil was sprayed with conidia suspension, white mildew developed on the pileus, and a young fruiting body grew out from the casing soil. Eventually, the surface of the mushroom was overwhelmed by the mycelia of the pathogen and the pileus turned brown or black. For the other group inoculated with mycelia plugs, only the stipe and pileus inoculated with mycelia turned brown or dark; it rotted and cracked 2 to 3 days later. The symptoms were similar to those observed on the C. comatus cultivation farm. Pathogens re-isolated from pathogenic fruiting bodies were confirmed to be C. protrusum based on morphological characteristics and ITS sequence. To our knowledge, this is the first report of the occurrence of C. protrusum on the edible mushroom C. comatus (3). Based on the pathogenicity test results, C. protrusum has the ability to severely infect the fruiting body of C. comatus. References: (1) K. Põldmaa. Stud. Mycol. 68:1, 2011. (2) F. J. Gea et al. Plant Dis. 96:1067, 2012. (3) W. H. Dong et al. Plant Dis. 97:1507, 2013.

First Report of Trichoderma oblongisporum Causing Green Mold Disease on Lentinula edodes (shiitake) in China.

Lentinula edodes (shiitake) is well known for its delicious taste and valuable medical functions, and ranked as the second most important mushroom in terms of total world production. In March 2012, a serious green mold epidemic occurred on many mushroom farms in Suizhou County of China. The infected mycelia of L. edodes in cultivated bags became rotten, yellow, wilted, and finally died, with the surfaces of the cultivated bags covered with dark green fungal colonies. At a temperature above 20°C, disease incidence was nearly 100% on some mushroom farms. Three diseased cultivated bags were collected from three different mushroom farms, and two portions at the junction of the diseased and healthy portions of the bag were plated individually on potato dextrose agar (PDA) and incubated at 25°C for 4 days. Following incubation, agar discs cut from the growing front of colonies were inoculated onto fresh PDA and subcultured to obtain putative pathogens. Three purified isolates were all whitish initially, followed by the emergence of greenish conidial clusters at the outer margin of the colony. The underside of the colony appeared pale yellow. The growth rate of the isolates was about 0.95 to 1.02 cm/day in PDA at 25°C. Aerial mycelia were floccose, white, and septate. Chlamydospores were sub-globose to broadly ellipsoidal. Conidiophore branches arose at right angles, and primary branches arose singly or in pairs. Phialides were ampulliform, 3.1 to 6.7 × 2.7 to 4.0 µm, slightly constricted at the base, swollen in the middle, and narrowed abruptly at the apex. Conidia were produced on the top of the phialides with the shape varying from ellipsoidal to oblong, 3.3 to 4.7 × 2.4 to 3.2 µm. These observations were consistent with the description of Trichoderma oblongisporum by Bissett (1). The ITS and partial tef1 were amplified from the three isolates as previously reported (2) and sequenced (KM110064 to KM110069). Nucleotide alignment showed 99% sequence identity (ITS) with two T. oblongisporum isolates (FJ623268 and DQ083020), and 88% similarity (tef1) with T. oblongisporum (AY750884). Neighbor joining tree of ITS and tef1 nucleotide sequences also showed that our three isolates had the closest relationship with the aforementioned three T. oblongisporum strains. To determine pathogenicity, a sawdust substrate was sterilized for 2 h in polyethylene bags and subsequently inoculated separately with three isolates and L. edodes strain Qiu7, which was cultivated widely in Suizhou. When the mycelia of Qiu7 colonized the bags, 10 cm3 of substrate was withdrawn from each bag. The substrate was then exposed to 10 cm3 of mycelium from each pathogen in 10 bags. A parallel inoculation with 10 cm3 of sterilized sawdust substrate without pathogen mycelia was performed as a control. The inoculated cultivated bags were kept at 25°C. After 2 months, all of the mycelia in the bags became wilted and dead, and the cultivated bags became soft, rotten, and covered with dark green fungal colonies. The controls remained uninfected. The symptoms were similar to those observed on mushroom farms. Pathogens re-isolated from all the inoculated culture bags were confirmed to be T. oblongisporum through morphological characteristics, molecular identification, and phylogenetic analysis. To our knowledge, this is the first report of T. oblongisporum causing green mold disease on L. edodes in the world. References: (1) J. Bissett. Can. J. Bot. 69:2398, 1991. (2) N. Sadfi-Zouaoui et al. Can. J. Microbiol. 55:154, 2009.

Stipe Canker Caused by Trichothecium roseum on the Edible Shaggy Mane Coprinus comatus in China.

Coprinus comatus, the shaggy mane, is one of the most popular and widely cultivated edible mushrooms in China. Its young fruiting body has good nutritional and medicinal value as well as a special flavor. In July 2010, an unusual stipe rot symptom was observed in cultivation tunnels in Pingyin county of Shandong Province. The lower part of the stipe was infected and water soaked scab occurred. The scab then expanded, a few mycelia and pink spores emerged on the scab surface, and finally, the stipe decayed and the fruiting body became wilted. The pathogen was isolated from infected tissues of C. comatus and the colonies on CYM were whitish at first, then pink sorus emerged, later forming concentric rings of sporulation. Mycelia were floccose, colorless, slender, and septate. Conidiophores bore upright, nonbranched, and colorless sporogenous cells, and slightly rounded spores were borne on the top and aggregated in pink cephaloid. Conidia were obovoid or obpyriform. Almost all spores were two-celled and one septate, while one was nearly round and the other bore an apex (1). The size of spores ranged from 10 to 21 µm long and 5 to 7 µm wide, which was consistent with the characteristics of Trichothecium roseum (1). The species identification was confirmed by sequencing the ribosomal ITS sequences. The ribosomal ITS1-5.8S-ITS2 region was amplified from the isolated strain using primers ITS1 and ITS4. A BLAST search in GenBank revealed the highest similarity (99%) to T. roseum (JQ434580). Pathogenicity was tested on different parts of 20 fruiting bodies of C. comatus with or without the wound treatment. One inoculum was prepared by flooding the agar surface with sterilized double distilled water for spore suspension (6.5 × 103 conidia/ml), and the other was by 0.2 × 0.2 cm mycelial plugs without spore production on CYM at 25°C for 5 days. After 1 to 2 days, only inoculated stipes showed water soaked and slight decay on the injured surface of all 20 fruiting bodies, while control fruiting bodies remained healthy. The symptoms were similar to those observed in the cultivation tunnels. No symptom was observed on the pileus, either with or without wound treatment. Pathogens reisolated from the inoculated stipes were confirmed to be T. roseum based on morphological characteristics. Because T. roseum is generally regarded as a postharvest disease of fruits and vegetables such as apple, pear, and muskmelon (2), apples and pears were inoculated with this fungus as well using the same methods. The parts inoculated were sunken, wettish, and decayed with brown stain at 25°C and 90% relative humidity after 5 days. Thus, it was confirmed that the T. roseum from the C. comatus stipe canker could infect the fruits of apple and pear. To our knowledge, this is the first report that T. roseum can cause disease on agaric. References: (1) G. Dal Bello. Australas. Plant Dis. Notes 3:103, 2008. (2) J.-H. Kwon et al. Plant Pathol. J. 26:296, 2010.