Four new species of Cystolepiota (Agaricaceae, Agaricales) from northeastern China.

Cystolepiota is a tiny lepiotaceous fungi. During our 3 years fieldwork, we found four new species of Cystolepiota from northeastern China. A phylogenetic study of a combined dataset of ITS+nrLSU+rpb2+tef1-α revealed that Cystolepiota changbaishanensis and Cystolepiota hetieri are sister clades; Cystolepiota hongshiensis belongs to Cystolepiota seminuda complex; Cystolepiota luteosquamulosa formed a clade not closely related with any other; Cystolepiota nivalis and Cystolepiota sp. (HMJAU68235) formed a sister clade. All new species are provided with descriptions, photos of the basidiomata, and colored illustrations of the microstructures. A key for the identification of Cystolepiota species from China is also presented.

Improved Understanding of the Macrofungal Diversity of Mongolia: Species Richness, Conservation Status, and An Annotated Checklist.

In this study, we updated and revised the checklist of macrofungi, along with the distribution of phytogeographical regions and the regional conservation status in Mongolia. The checklist comprises 677 macrofungal species belonging to 284 genera and 119 families in the country. Based on previous studies, 18 species are currently invasive to Mongolia. In this checklist, only four species are endemic to Mongolia. Among the 677 species, the regional conservation status of 51 species was previously assessed as threatened in the country. Furthermore, we collected all available occurrence records from various sources. A total of 4733 occurrences of 655 species across Mongolia were analyzed for species richness based on a 0.5° × 0.5° grid cell size. We found the records to be unevenly distributed across Mongolia, where records from the northern and central parts dominate. Among these, we identified 43 grids with a high diversity of macrofungal species. Most of these grids did not reside inside by protected geographical areas.

Six New Species of Agaricus (Agaricaceae, Agaricales) from Northeast China.

Agaricus belongs to Agaricaceae and is one of the most well-known macrofungi, with many edible species. More than 300 Agaricus specimens were collected during a three-year macrofungal resource field investigation in Northeast China. Based on morphological observations and multi-gene (ITS + nrLSU + tef1-a) phylogenetic analyses, six new Agaricus species, i.e., Agaricus aurantipileatus, A. daqinggouensis, A. floccularis, A. griseopileatus, A. sinoagrocyboides, and A. velutinosus, were discovered. These new species belong to four sections within different subgenera: A. (subg. Agaricus) sect. Agaricus, A. (subg. Flavoagaricus) sect. Arvenses, A. (subg. Minores) sect. Minores, and A. (subg. Pseudochitonia) sect. Xanthodermatei. Morphological descriptions, line illustrations, and basidiomata photographs of these new species are provided, and their differences from similar species are discussed.

Modicellaguangxiensis (Mortierellomycota, Mortierellaceae), a new species from south-western karst areas of China.

Background: Modicella Kanouse (1936) is the only genus of Mortierellaceae known to produce macroscopic fruiting bodies in the form of small, whitish, round sporocarps. Specimens which belong to Modicella were collected during our field investigations in tropical karst areas of China. Based on morphological characteristics and phylogenetic analysis, a new species is introduced. New information: Modicellaguangxiensis is described as a new species from tropical karst areas of China. The main distinguishing characteristics of M.guangxiensis are the number of sporangiospores per sporangium (11-18), the size of sporangiospores (14-34 × 12-27.5 µm) and the surface of some hyphae with hemispherical tuber. The phylogenetic analyses, based on the internal transcribed spacer (ITS) and the large subunit (LSU) regions of rDNA sequences using Bayesian (BA) and Maximum Likelihood (ML) methods showed that the new taxon is closely related to M.reniformis.

Conocybe Section Pilosellae in China: Reconciliation of Taxonomy and Phylogeny Reveals Seven New Species and a New Record.

Conocybe belongs to the Bolbitiaceae. The morphological classification and molecular phylogenetics of Conocybe section Pilosellae are not in agreement. In this study, based on the specimens from China, we investigated the sect. Pilosellae and identified 17 species, including 7 new species: Conocybe pilosa, with a densely hairy pileus and stipe; C. reniformis, with reniform spores; C. ceracea, with waxy dehydration of the lamellae; C. muscicola, growing on moss; C. sinobispora, with two-spored basidia; C. hydrophila, with a hygrophanous pileus; C. rufostipes, growing on dung with a brown stipe; and C. pseudocrispa, one new record for China. A key was compiled for the sect. Pilosellae in China. Here, the sect. Pilosellae, and new species and records from China are morphologically described and illustrated. Maximum likelihood and Bayesian analyses were performed using a combined nuc rDNA internal transcribed spacer region (ITS) and nuc 28S rDNA (nrLSU), and translation elongation factor 1-alpha (tef1-α) dataset to reconstruct the relationships of this section. We found that the sect. Pilosellae was the basal clade of Conocybe, and its evolutionary features may shed light on the characteristics of Conocybe. By integrating morphological classification and phylogenetic analysis, we explored the possible phylogenetic relationships among the species of the sect. Pilosellae in China.

Rhodocybe subasyae, a new species of Rhodocybe sect. Rufobrunnea (Entolomataceae, Agaricales) from northeast China.

Rhodocybe subasyae is described from northeast China as a new species with both morphological and molecular characteristics, which is morphologically characterized by its tricholomatoid basidiomata, orange white to beige red pileus, adnexed to sinuate lamellae and long clavate branched cheilocystidia within section Rufobrunnea. The phylogenetic tree based on internal transcribed spacers of rDNA (nrITS) sequences using Bayesian methods showed that new taxon was distinguished from other species of Rhodocybe.

Phylogeny and Taxonomic Revision of the Family Discinaceae (Pezizales, Ascomycota).

Species of Discinaceae are common macrofungi with a worldwide distribution. Some of them are commercially consumed, while a few others are reported as poisonous. Two genera were accepted in the family: the epigeous Gyromitra with discoid, cerebriform to saddle-shaped ascomata and the hypogeous Hydnotrya with globose or tuberous ascomata. However, due to discrepancies in their ecological behaviors, a comprehensive investigation of their relationship was not thoroughly explored. In this study, phylogenies of Discinaceae were reconstructed using sequence analyses of combined and separate three gene partitions (internal transcribed spacer [ITS], large subunit ribosomal DNA [LSU], and translation elongation factor [TEF]) with a matrix containing 116 samples. As a result, the taxonomy of the family was renewed. Eight genera were recognized: two of them (Gyromitra and Hydnotrya) were retained, three (Discina, Paradiscina, and Pseudorhizina) were revived, and three (Paragyromitra, Pseudodiscina, and Pseudoverpa) were newly established. Nine new combinations were made in four genera. Two new species in Paragyromitra and Pseudodiscina and an un-named taxon of Discina were described and illustrated in detail based on the materials collected from China. Furthermore, a key to the genera of the family was also provided. IMPORTANCE Taxonomy of the fungal family Discinaceae (Pezizales, Ascomycota) was significantly renewed on the basis of sequence analyses of internal transcribed spacer (ITS), large subunit ribosomal DNA (LSU), and translation elongation factor (TEF). Eight genera were accepted, including three new genera; two new species were described; and nine new combinations were made. A key to the accepted genera of the family is provided. The aim of this study is to deepen the understanding of the phylogenetic relationships among genera of the group, as well as the associated generic concepts.

Morphological and phylogenetic evidence reveal three new Pseudohydnum (Auriculariales, Basidiomycota) species from North China.

Pseudohydnum is characterized by gelatinous basidiomata with hydnoid hymenophores and longitudinally septate basidia. In this study, samples of the genus from North China were examined morphologically and phylogenetically using a dataset of the internal transcribed spacer of the ribosomal RNA gene and the nuclear large subunit rDNA. This study describes three new species, namely Pseudohydnum abietinum, Pseudohydnum candidissimum, and Pseudohydnum sinobisporum. Pseudohydnum abietinum is characterized by pileate and pale clay pink basidiomata when fresh, with a rudimentary stipe base, four-celled basidia, and broadly ellipsoid to ovoid or subglobose basidiospores (6-7.5 × 5-6.3 µm). P. candidissimum is characterized by very white basidiomata when fresh, frequently four-celled basidia, and broadly ellipsoid to subglobose basidiospores (7.2-8.5 × 6-7 µm). P. sinobisporum is characterized by ivory basidiomata when fresh, two-celled basidia, ovoid to broadly ellipsoid, or subglobose basidiospores (7.5-9.5 × 5.8-7.2 µm). The main characteristics, type localities, and hosts of Pseudohydnum species are listed.

Seven New Species of the Genus Geastrum (Geastrales, Geastraceae) in China.

Geastrum belongs to Basidiomycota, Agaricomycetes, Geastrales, and Geastraceae. The genus Geastrum exoperidium normally splits at maturity into a characteristic star-like structure. It is a saprophytic fungus with great research significance. Based on morphological observation combined with phylogenetic analysis through ITS and LSU, seven new species of Geastrum belong to four sections, viz., Sect. Myceliostroma, Geastrum laneum; Sect. Exareolata, Geastrum litchi, Geastrum mongolicum; Sect. Corollina, Geastrum pseudosaccatum, Geastrum melanorhynchum, Geastrum oxysepalum; and Sect. Campestria, Geastrum microphole. Illustrated descriptions and the ecological habits of the novel species are provided.

Asproinocybaceae fam. nov. (Agaricales, Agaricomycetes) for Accommodating the Genera Asproinocybe and Tricholosporum, and Description of Asproinocybe sinensis and Tricholosporum guangxiense sp. nov.

Asproinocybe and Tricholosporum are not well known, and their placement at the family level remains undetermined. In this study, we conducted molecular phylogenetic analyses based on nuc rDNA internal transcribed spacer region (ITS) and nuc 28S rDNA (nrLSU), and a dataset with six molecular markers (ITS, LSU, RNA polymerase II largest subunit (RPB1), RNA polymerase II second largest subunit (RPB2), 18S nuclear small subunit ribosomal DNA (nrSSU), and translation elongation factor 1-alpha (TEF1-α)) using Bayesian (BA) and Maximum Likelihood (ML) methods, we found that the species of Asproinocybe and Tricholosporum formed an independent family-level clade (0.98/72). Asproinocybaceae fam. nov., a new family, is established here for accommodating this clade. Two new species, Asproinocybe sinensis and Tricholosporum guangxiense, from subtropical and tropical karst areas of China, are also described here.

DNA Barcoding Mushroom Spawn Using EF-1α Barcodes: A Case Study in Oyster Mushrooms (Pleurotus).

Oyster mushrooms (genus Pleurotus) are widespread and comprise the most commonly cultivated edible mushrooms in the world. Species identification of oyster mushroom spawn based on cultural, morphological, and cultivated characteristics is time consuming and can be extraordinarily difficult, which has impeded mushroom breeding and caused economic loss for mushroom growers. To explore a precise and concise approach for species identification, the nuclear ribosomal internal transcribed spacer (ITS), 28S rDNA, and the widely used protein-coding marker translation elongation factor 1α (EF-1α) gene were evaluated as candidate DNA barcode markers to investigate their feasibility in identifying 13 oyster mushroom species. A total of 160 sequences of the candidate loci were analyzed. Intra- and interspecific divergences and the ease of nucleotide sequence acquisition were the criteria used to evaluate the candidate genes. EF-1α showed the best intra- and interspecific variation among the candidate markers and discriminated 84.6% of the species tested, only being unable to distinguish two closely related species Pleurotus citrinopileatus and Pleurotus cornucopiae. Furthermore, EF-1α was more likely to be acquired than ITS or 28S rDNA, with an 84% success rate of PCR amplification and sequencing. For ITS and 28S rDNA, the intraspecific differences of several species were distinctly larger than the interspecific differences, and the species identification efficiency of the two candidate markers was worse (61.5 and 46.2%, respectively). In addition, these markers had some sequencing problems, with 55 and 76% success rates of sequencing, respectively. Hence, we propose EF-1α as a possible DNA barcode marker for oyster mushroom spawn.

Two new rare species of Candolleomyces with pale spores from China.

Most species of Candolleomyces have brown or dark brown spores. Although pale-spored members are rare in the genus we frequently collected two such species from many Provinces during our investigations in subtropical China from 2016-2020. As revealed by morphological characterisation and multigene phylogenetic analyses (ITS LSU ß-tub and tef-1α) these species which we have named C. subcacao and C. subminutisporus are unique and distinct from known taxa. In addition a new combination C. cladii-marisci is proposed on the basis of ITS sequence analysis of the type specimen. Detailed descriptions colour photos illustrations and a key to related species are presented.

A new genus and four new species in the /Psathyrella s.l. clade from China.

Based on traditional morphological and phylogenetic analyses (ITS, LSU, tef-1α and ß-tub) of psathyrelloid specimens collected from China, four new species are here described: Heteropsathyrella macrocystidia, Psathyrella amygdalinospora, P. piluliformoides, and P. truncatisporoides. H. macrocystidia forms a distinct lineage and groups together with Cystoagaricus, Kauffmania, and Typhrasa in the /Psathyrella s.l. clade, based on the Maximum Likelihood and Bayesian analyses. Thus, the monospecific genus Heteropsathyrella gen. nov. is introduced for the single species. Detailed descriptions, colour photos, and illustrations are presented in this paper.

Grassland fairy rings of Leucocalocybe mongolica represent the center of a rich soil microbial community.

BACKGROUND: The ecological phenomenon of fungal fairy rings is usually found in grasslands and caused by the growth of specific fairy ring fungi in soil. The fairy rings are classified into three zones (DARK, DEAD, and OUT), and they have the potential to increase crop yield. Among these fairy rings, distinct characteristics of type I fairy rings can be seen in the rings formed by Leucocalocybe mongolica (LM). Our studies addressed changes in the soil microbial structure due to LM fairy rings to enhance understand of this ecological phenomenon. METHODS: In the present study, we report the soil microbial analysis results (fungi and bacteria), including those of metabarcoding (16s rRNA, ITS), microbial quantity, and metagenomics surveys of soils collected from various fairy ring zones, of 6 LM fairy rings. All sampling sites cover the grasslands of Mongolian Plateau in China. RESULTS: First, we found through metabarcoding surveys that the difference in microbial diversity is relatively less in bacteria and that the abundance of fairy ring fungi (LM) is relatively high in DEAD zones. We also identified eight bacterial and fungal families, including Sphingobacteriaceae and Sphingomonadaceae that were enriched within the soils of fairy ring zones. Second, we found that the abundance of soil bacteria in the DEAD zones is sharply increased along with the growth of fairy ring fungi (LM). Third, we found through shotgun sequencing that fairy ring-infected zones, DARK and DEAD, exhibit greater genetic diversity than OUT zones. Finally, we showed that the fairy ring ecosystem is the center for a rich grassland microbial community. CONCLUSIONS: The reported data can improve our understanding of type I fairy rings and will be further insightful to the research on crop production.

Analyses of transcriptomes and the first complete genome of Leucocalocybe mongolica provide new insights into phylogenetic relationships and conservation.

In this study, we report a de novo assembly of the first high-quality genome for a wild mushroom species Leucocalocybe mongolica (LM). We performed high-throughput transcriptome sequencing to analyze the genetic basis for the life history of LM. Our results show that the genome size of LM is 46.0 Mb, including 26 contigs with a contig N50 size of 3.6 Mb. In total, we predicted 11,599 protein-coding genes, of which 65.7% (7630) could be aligned with high confidence to annotated homologous genes in other species. We performed phylogenetic analyses using genes form 3269 single-copy gene families and showed support for distinguishing LM from the genus Tricholoma (L.) P.Kumm., in which it is sometimes circumscribed. We believe that one reason for limited wild occurrences of LM may be the loss of key metabolic genes, especially carbohydrate-active enzymes (CAZymes), based on comparisons with other closely related species. The results of our transcriptome analyses between vegetative (mycelia) and reproductive (fruiting bodies) organs indicated that changes in gene expression among some key CAZyme genes may help to determine the switch from asexual to sexual reproduction. Taken together, our genomic and transcriptome data for LM comprise a valuable resource for both understanding the evolutionary and life history of this species.

Investigation of the possible mechanism of polysaccharides extracted from Leucocalocybe mongolica in exerting antitumor effects in H22 tumor-bearing mice.

A response surface method was used to optimize the extraction of polysaccharides from Leucocalocybe mongolica. Moreover, the preliminary structural characteristics and antitumor activity of L. mongolica polysaccharide (LMP) were investigated. The results showed that the optimized extraction technological parameters for LMP were 93°C extraction temperature, 5 hr extraction time, and 30 ml/g liquid-to-solid ratio. The LMP content extracted under the optimal conditions was 6.64%. LC-MS/MS results indicated that LMP is a neutral polysaccharide composed of d-fructose, d-mannose, dextrose anhydrate, d-xylose, trehalose, and galactose. The tumor inhibition rate was significantly improved by LMP treatment. LMP had minimal toxicity based on the significant decrease in AST and BUN levels; VEGF protein levels were also significantly decreased. In contrast, the levels of IFN-γ, IL-2, IL-6, and TNF-α were improved. The results of ELISA, H&E staining, TUNEL assay, immunohistochemistry, and western blotting indicated that the LMP exhibited antitumor activity in vivo by promoting apoptosis, mediating inflammatory responses, and inhibiting angiogenesis. PRACTICAL APPLICATIONS: As one of the main bioactive components, fungal polysaccharide has always been a hot research topic. Fungal polysaccharides are carbohydrate polymers composed of monosaccharide units bound together by glycosidic linkages, which have been found to be involved in many biological processes. In this research, the LMP structure was analyzed, and the immunohistochemical and western blot analysis confirmed that, LMP could effectively reduce the generation of tumor angiogenesis, promote apoptosis of tumor cell sand inhibit tumor growth. The results of this study can effectively provide a basis for clinical research and development of antitumor drugs, and lay a foundation for the study of the antitumor effects of wild edible and medicinal fungi.

Investigation of the possible mechanism of two kinds of sterols extracted from Leucocalocybe mongolica in inducing HepG2 cell apoptosis and exerting anti-tumor effects in H22 tumor-bearing mice.

Sterols are one of the main components of medicinal fungi with an anti-tumor effect. In this study, ergosta-4, 6, 8(14), 22-tetraen-3-one (ET) and (22E, 24R)-ergosta-7, 22-dien-3ß, 5α, 6ß-triol (ED) were obtained from Leucocalocybe mongolica and were used for the first time to study their ability to induce apoptosis in HepG2 cells and their anti-tumor effects and related mechanism in H22 tumor-bearing mice. METHOD: The chemical structures were defined by IR and NMR. In vitro, the CCK8 assay was used as a cytotoxicity assay. Flow cytometry was used for the HepG-2 cell apoptosis analysis, which was examined via annexin V-FITC/PI double staining, and the related expression levels of the apoptosis-associated proteins were determined by western blot analysis. In vivo, ICR male mice were randomly assigned to eight groups: the model group, CTX (25 mg/kg/d) group, and ET and ED groups, which were treated with three different concentrations of each compound (0.025, 0.05, and 0.1 mmol/kg/d). Relevant biochemical indicators were detected by ELISA assay, H & E staining, TUNEL assay, immunohistochemical staining and western blot. RESULTS: In vitro, ET and ED showed significant cytotoxic effects against HepG2, MCF-7, and HeLa cells, especially HepG-2 cells, and both ED and ET demonstrated a good effect in inhibiting the proliferation of HepG-2 cells. In vivo, ET and ED significantly decreased the tumor volume and VEGF levels but increased the serum cytokine levels of IFN-γ, IL-2, IL-6 and TNF-α. H & E staining, TUNEL assay, immunohistochemical analysis, and western blotting indicated that the both ET and ED exhibited anti-tumor activity in vivo by promoting apoptosis and inhibiting angiogenesis. CONCLUSION: These results indicated that both ET and ED have a strong inhibitory effect on the proliferation of HepG-2 cells in vitro and an anti-H22 tumor effect in vivo.

Preventive Effect of Small Molecular Fraction of Irpex lacteus (Agaricomycetes) Fruiting Body against Chronic Nephritis in Mice and Identification of Active Compounds.

Previous study found that the fruit body of Irpex lacteus has an effect on the prevention and treatment of chronic nephritis. In this study, we systematically investigated the preventive effect of small molecular fraction (SMF) of the fungal fruit body against chronic nephritis. In addition, we analyzed, isolated, and identified the chemical constituents of SMF, and screened the activity of three small peptides isolated in vitro. The results showed SMF significantly reduced amounts of urine protein (UP), the content of urea (BUN), creatinine (Cr), tumor necrosis factor-α (TNF-α), and maleic dialdehyde (MDA) in serum, and significantly increased superoxide dismutase (SOD) level in renal tissue homogenate (P < 0.05). Moreover, the results of hematoxylin and eosin (H&E) and Masson staining of renal tissues indicated that SMF has protective effects on renal tissues and prevents renal interstitial from fibrosis. The peptide sequences isolated from SMF were identified as WSMGPAPDSVH (SP1), QCTGNASCSPPC (SP2), and HYCCTAKYA (SP3), which were active compounds for the prevention of nephritis, and these new peptides were isolated for the first time. The cell proliferation assay showed that 10 µg/L transforming growth factor-ß1 (TGF-ß1) significantly induced the proliferation of human renal tubular epithelial cells (HK-2), compared with the control group, and the difference was statistically significant (P < 0.01). However, when combined with three small peptides, respectively, the cell proliferation was significantly inhibited (P < 0.05). These results suggest that isolated peptides can maintain the morphological stability of HK-2 cells, inhibit cell proliferation induced by TGF-ß1 to some extent, and prevent cell fibrosis.

GC-MS Analysis and Cytotoxicity Detection of Volatile Oil from the Fragrant Bracket Mushroom, Trametes suaveolens (Agaricomycetes).

Trametes suaveolens is a medicinal mushroom known as Baizhi in traditional Chinese medicine. Our previous research has found that it has some pharmacological activity in vivo. The aim of the study was to investigate the chemical compounds and cytotoxic effects of volatile oil from T. suaveolens. In this study, internal transcribed spacer (ITS) sequence analysis was used to determine wild T. suaveolens collected. To fully analyze the composition of volatile oil extracted from T. suaveolens, hydrodistillation (HD) and solid phase microextraction (SPME) were adopted simultaneously. In both cases, the analysis was carried out using gas chromatography-mass spectrometry (GC-MS) and the cytotoxic effects of T. suaveolens volatile oil on human NCI-H460 lung non-small cell carcinoma cells and MCF-7 breast adenocarcinoma cells were investigated. The results indicated that all these wild samples were identified as T. suaveolens. Thirty-one components in HD and 62 components in SPME were identified, respectively. Furthermore, the volatile compounds obtained from T. suaveolens by HD indicated that esters compounds were a major class (68.47%), followed by acids (25.06%), aldehydes (4.20%), and alcohols (1.48%). SPME found that the largest content were aldehydes (45.47%), followed by alcohols (31.42%), ketones (6.89%), and esters (6.72%). In the cytotoxic assays, the volatile oil was found to have toxic effect on NCI-H460 and MCF-7 tumor cells but not BEAS-2B and MCF-10A normal cells, and the IC50 values of NCI-H460 and MCF-7 tumor cells were 24.1 and 19.2 µg/ml, respectively. The present study shows that the composition of essential oil from T. suaveolens has potential value for the prevention and treatment of lung cancer.

New species of Retiboletus (Boletales, Boletaceae) from China based on morphological and molecular data.

Species of the genus Retiboletus in China were investigated based on morphology and phylogenetic analysis of DNA sequences from the nuclear ribosomal large subunit (nrLSU) and the translation elongation factor 1-α gene (TEF1-α). Nine species were recovered from China, including two new and seven known species. The new species, namely Retiboletus ater and R. sinogriseus, from southwestern and northeastern China respectively, are documented and illustrated in this paper. Retiboletus ater is morphologically characterized by its black to grayish black pileus, white to grayish hymenophore, black to blackish stipe and white to grayish white context. Retiboletus sinogriseus is morphologically characterized by its brown to grayish-brown pileus, yellow to grayish-yellow hymenophore, pale yellow to brownish stipe and yellow to brownish-yellow context. Descriptions and line drawings of these two novel species and their comparisons with allied taxa are presented.