Two highly conjugated ergosterols from the fungus Psathyrella rogueiana and their anti-inflammatory activity.

Two previously undescribed ergosterols containing a highly conjugated ring system, psathrosterols A and B (1 and 2), have been isolated from the fungus Psathyrella rogueiana. Their structures with absolute configurations were established by extensive spectroscopic methods, as well as single crystal X-ray diffraction. Compounds 1 and 2 showed inhibitory activity against NO production with IC50 values of 22.3 and 16.4 µM, respectively.

Identification of microRNA-like RNAs in Cordyceps guangdongensis and their expression profile under differential developmental stages.

Cordyceps guangdongensis is a well-known fungus with high nutritional and medicinal value. The metabolite profile of C. guangdongensis is similar to that of Ophiocordyceps sinensis. In plants and animals, microRNAs play important roles in regulating gene expression at the post-transcriptional level. MicroRNA-like RNAs (milRNAs) have been documented in several macro-fungi. To comprehensively investigate the milRNAs in C. guangdongensis, three small RNA libraries from the differentially developmental stages were constructed. Twenty-six conserved milRNAs were identified, and 19 novel milRNA candidates were predicted. Among them, 20 milRNAs were differentially expressed across the developmental processes, and 12 milRNAs were verified using stem-loop quantitative real-time reverse transcription polymerase chain reaction. In addition, the potential target genes of milRNA were predicted to be involved in the development of fruiting bodies and metabolite biosynthesis. This study is the first to report the milRNAs of C. guangdongensis, and provides important insights into studies of milRNA regulation pathways in ascomycete fungi.

Mitogenome of Tolypocladium guangdongense.

Tolypocladium guangdongense is a high-value edible fungus with various medicinal and food safety properties. However, its evolutionary and genetic information is still limited. Mitochondrial genomes are potential models for molecular evolution and phylogenetic studies. In this study, we sequenced the complete mitogenome of T. guangdongense, demonstrating circular sequence of 46,102 bp, containing 14 standard protein-coding genes (PCGs), 2 ribosomal RNA subunit genes, and 28 tRNA genes. Phylogenetic analysis based on mitochondrial genes indicated that T. guangdongense was clustered into the Tolypocladium genus with high support value, based on the core PCG dataset. In addition, rps3 is also a suitable marker in the phylogenetic analysis in Hypocreales. Gene rearrangement analysis indicated that the gene order of PCGs was highly consistent in Hypocreales, and tRNA rearrangement events occurred in most species of Hypocreales; however, the rearrangement rates were not taxonomically correlated. Divergence time estimation based on the old fossil record and previous reports revealed that T. guangdongense originated approximately in the middle Cenozoic (42 Mya, 95% highest posterior density interval: 43-116) with the Tolypocladium genus differentiation. Our results provided more mitogenomic information of T. guangdongense and shed new insights into evolution of the Tolypocladium genus. KEY POINTS: • The general and unique features of T. guangdongense mitogenome are firstly reported. • Phylogenetic analysis further verified the taxonomic status of T. guangdongense. • Divergence time estimation provides more evolutionary information of T. guangdongense.

Genetic diversity analysis of Hypsizygus marmoreus with target region amplification polymorphism.

Hypsizygus marmoreus is an industrialized edible mushroom. In the present paper, the genetic diversity among 20 strains collected from different places of China was evaluated by target region amplification polymorphism (TRAP) analysis; the common fragment of TRAPs was sequenced and analyzed. Six fixed primers were designed based on the analysis of H. marmoreus sequences from GenBank database. The genomic DNA extracted from H. marmoreus was amplified with 28 TRAP primer combinations, which generated 287 bands. The average of amplified bands per primer was 10.27 (mean polymorphism is 69.73%). The polymorphism information content (PIC) value for TRAPs ranged from 0.32 to 0.50 (mean PIC value per TRAP primer combination is 0.48), which indicated a medium level of polymorphism among the strains. A total of 36 sequences were obtained from TRAP amplification. Half of these sequences could encode the known or unknown proteins. According to the phylogenetic analysis based on TRAP result, the 20 strains of H. marmoreus were classified into two main groups.

A Contribution to the Phylogeny and Taxonomy of Hydnum (Cantharellales, Basidiomycota) from China.

Hydnum is a well-characterized genus in the family Hydnaceae of Cantharellales and is characterized by spinose hymenophores. In this study, an ITS phylogenetic overview and a multilocus (ITS-nrLSU-tef1) phylogenetic tree of Hydnum were carried out. On the basis of morphological characteristics and phylogenetic results, seven species from China were confirmed, described, illustrated, and compared with similar species, including three new species, i.e., H. longipes, H. microcarpum, and H. sinorepandum, and four known species, i.e., H. cremeoalbum, H. melitosarxm, H. orientalbidum, and H. pinicola were recorded for the first time in China. A key to the species of Hydnum in China was provided.

Morphology and Molecular Phylogeny Reveal Five New Species of Laccaria (Hydnangiaceae, Agaricales) from Southern China.

The genus Laccaria is a type of cosmopolitan and ecologically important fungal group. Members can form ectomycorrhizal associations with numerous trees, and some species are common edible fungi in local markets. Although some new species from China are recently published, the species diversity of Laccaria is still unclear in China. In this study, some samples of Laccaria were collected from southern China, and morphological characteristics and phylogenetic analyses based on the multilocus dataset of ITS-LSU-tef1-rpb2 confirmed five new species. Laccaria miniata, L. nanlingensis and L. neovinaceoavellanea were collected from subtropical broad-leaved forests, and L. rufobrunnea and L. umbilicata were collected from subtropical mixed forests of southwest China. Full descriptions, illustrations, comparisons with similar species and phylogenetic analysis are provided.

Diversity of Cantharellus (Cantharellales, Basidiomycota) in China with Description of Some New Species and New Records.

Cantharellus is a well-known genus of edible mushrooms, belonging to the family Hydnaceae in the class Agaricomycetes. In this study, a phylogenetic overview of Cantharellus subg. Cinnabarinus and C. subg. Parvocantharellus in China is carried out with the description of four new species. Species description are based on morphological characters of basidiomata and phylogenetic analyses of multi-locus dataset of 28S + tef1 + rpb2. Among the new species, two species, C. chrysanthus and C. sinocinnabarinus, belong to C. subg. Cinnabarinus and two new species, C. convexus and C. neopersicinus, belong to C. subg. Parvocantharellus. Species delimitation characters of the new taxa are compared with closely related species. In addition, three new records of Cantharellus are reported for China: C. albovenosus and C. citrinus of subg. Cinnabarinus and C. koreanus of subg. Parvocantharellus. A key to the species of subg. Cinnabarinus in China was provided.

Cordyceps guangdongensis lipid-lowering formula alleviates fat and lipid accumulation by modulating gut microbiota and short-chain fatty acids in high-fat diet mice.

Obesity has caused serious health and economic problems in the world. Cordyceps guangdongensis is a high-value macrofungus with broad application potential in the food and bio-medicine industry. This current study aimed to estimate the role of C. guangdongensis lipid-lowering compound formula (CGLC) in regulating fat and lipid accumulation, gut microbiota balance, short-chain fatty acid (SCFA) contents, and expression levels of genes involved in fat and lipid metabolism in high-fat diet (HFD) mice. The results showed that CGLC intervention markedly reduced body weights and fat accumulation in HFD mice, improved glucose tolerance and blood lipid levels, and decreased lipid droplet accumulation and fat vacuole levels in the liver. CGLC decreased the ratio of Firmicutes and Bacteroidetes and increased the relative abundances of Bacteroides (B. acidifaciens) and Bifidobacterium (B. pseudolongum). In addition, CGLC treatment significantly promoted the production of SCFAs and regulated the relative expression levels of genes involved in fat and lipid metabolism in liver. Association analysis showed that several species of Bacteroides and most of SCFAs were significantly associated with serum lipid indicators. These results suggested that CGLC is a novel candidate formulation for treating obesity and non-alcohol fatty liver by regulating gut microbiota, SCFAs, and genes involved in fat and lipid metabolism.

Peptide toxin components of Amanita exitialis basidiocarps.

Eight peptide toxins were isolated and purified from basidiocarps of Amanita exitialis with high performance liquid chromatography and were subjected to ultraviolet, nuclear magnetic resonance and mass spectrometry. We identified seven peptide toxins, α-amanitin, ß-amanitin, amaninamide, phallacin, phallacidin, phallisacin and desoxoviroidin. The molecular weight (729.5 Da) of the eighth compound did not match that of any reported Amanita toxins and, although the UV absorption spectrum indicated it to be a phallotoxin, further studies are required to identify this component. This is the first report of amaninamide, phallacin, phallisacin and desoxoviroidin in this lethal mushroom species.

Multigene Phylogeny and Morphology Reveal Unexpectedly High Number of New Species of Cantharellus Subgenus Parvocantharellus (Hydnaceae, Cantharellales) in China.

The genus Cantharellus, commonly known as chanterelles, has recently been divided into six subgenera; however, wider sampling approaches are needed to clarify the relationships within and between these groups. A phylogenetic overview of Cantharellus subgenus Parvocantharellus in China was inferred based on the large subunit nuclear ribosomal RNA gene (nrLSU), the DNA-directed RNA polymerase II subunit 2 (rpb2), and the transcription elongation factor 1-alpha (tef1). A total of nine species from China were assigned to the subgenus, including seven novel species, namely Cantharellusaurantinus, C. austrosinensis, C. galbanus, C. luteolus, C. luteovirens, C. minioalbus, and C. sinominior, and two known species, namely C. albus and C. zangii. The detailed descriptions and illustrations were provided based on the newly obtained data, with the comparisons to closely related species. C. zangii was restudied based on the paratype specimens and multiple new collections from the type locality. Futhermore, the Indian species C. sikkimensis was identified as a synonym of C. zangii based on the morphological and molecular analyses. A key to the Chinese species belonging to the subg. Parvocantharellus is also provided.

Two new species of Phallus (Phallaceae) with a white indusium from China.

Two new Phallus species, P.cremeo-ochraceus and P.rigidiindusiatus were discovered in southwestern and southern China, respectively. Phalluscremeo-ochraceus is morphologically characterized by its cream to ochraceous receptacle, white to very slightly pinkish indusium, white to pinkish pseudostipe and white to slightly purplish pink volva. Phallusrigidiindusiatus is characterized by a white to yellowish white receptacle, a strongly rigid indusium usually without serrated margin and smaller basidiospores than those of P.serratus. Phylogenetic positions of the two species are located in two independent lineages respectively. Detailed descriptions, color photographs, illustrations and a key to the related species are presented.

Biosynthetic Pathway and the Potential Role of Melatonin at Different Abiotic Stressors and Developmental Stages in Tolypocladium guangdongense.

Melatonin, a bioactive compound and an important signaling molecule produced in plants and animals, is involved in many biological processes. However, its function and synthetic pathways in fungi are poorly understood. Here, the samples from Tolypocladium guangdongense, a highly valued edible fungus with functional food properties, were collected under different experimental conditions to quantify the levels of melatonin and its intermediates. The results showed that the intracellular melatonin content was markedly improved by Congo red (CR), cold, and heat stresses; the levels of intracellular melatonin and its intermediates increased at the primordial (P) and fruiting body (FB) stages. However, the levels of most intermediates exhibited a notable decrease under CR stress. Several genes related to melatonin synthesis, excluding AADC (aromatic-L-amino-acid decarboxylase), were markedly upregulated at an early stage of CR stress but downregulated later. Compared to the mycelial stage, those genes were significantly upregulated at the P and FB stages. Additionally, exogenous melatonin promoted resistance to several abiotic stressors and P formation in T. guangdongense. This study is the first to report melatonin biosynthesis pathway in macro-fungi. Our results should help in studying the diversity of melatonin function and melatonin-synthesis pathways and provide a new viewpoint for melatonin applications in the edible-medicinal fungus.

Two new Inosperma (Inocybaceae) species with unexpected muscarine contents from tropical China.

An accurate identification of poisonous mushrooms and the confirmation of the toxins involved are both of great importance in the treatment of mushroom poisoning incidents. In recent years, cases of mushroom poisoning by Inosperma spp. have been repeatedly reported from tropical Asia. It is urgent to know the real species diversity of Inosperma in this region. In the present study, we proposed two new Inosperma species from tropical Asia, namely I.muscarium and I.hainanense. They were described based on morphology and multilocus phylogeny. Detailed descriptions, color photographs and the discussion with other closely related species of the two new taxa were provided. In addition, a comprehensive muscarine determination of these two new species using ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) approach has been performed. Results showed that these two species were muscarine positive, with a content of 16.03 ± 1.23 g/kg in I.muscarium and a content of 11.87 ± 3.02 g/kg in I.hainanense, much higher than the known species I.virosum. Recovery of muscarine ranged from 93.45% to 97.25%, and the average recovery is 95.56%.

Distribution, evolution and expression of GATA-TFs provide new insights into their functions in light response and fruiting body development of Tolypocladium guangdongense.

BACKGROUND: Fungal GATA-type transcription factors (GATA-TFs) are a class of transcriptional regulators involved in various biological processes. However, their functions are rarely analyzed systematically, especially in edible or medicinal fungi, such as Tolypocladium guangdongense, which has various medicinal and food safety properties with a broad range of potential applications in healthcare products and the pharmaceutical industry. METHODS: GATA-TFs in T. guangdongense (TgGATAs) were identified using InterProScan. The type, distribution, and gene structure of TgGATAs were analyzed by genome-wide analyses. A phylogenetic tree was constructed to analyze their evolutionary relationships using the neighbor-joining (NJ) method. To explore the functions of GATA-TFs, conserved domains were analyzed using MEME, and cis-elements were predicted using the PlantCARE database. In addition, the expression patterns of TgGATAs under different light conditions and developmental stages were studied using qPCR. RESULTS: Seven TgGATAs were identified. They were randomly distributed on four chromosomes and contained one to four exons. Phylogenetic analysis indicated that GATA-TFs in each subgroup are highly conserved, especially for GATA1 to GATA5. Intron distribution analyses suggested that GATA1 and GATA3 possessed the most conserved gene structures. Light treatments induced the expression levels of TgGATA1 and TgGATA5-7, but the expression levels varied depending on the duration of illumination. The predicted protein structures indicate that TgGATA1 and TgGATA2 possess typical light-responsive domains and may function as photoreceptors to regulate downstream biological processes. TgGATA3 and TgGATA5 may be involved in nitrogen metabolism and siderophore biosynthesis, respectively. TgGATA6 and TgGATA7 possess unique Zn finger loop sequences, suggesting that they may have special functions. Furthermore, gene expression analysis indicated that TgGATA1 (WC1) was notably involved in mycelial color transformation, while other genes were involved in fruiting body development to some extent. These results provide valuable information to further explore the mechanisms through which TgGATAs are regulated during fruiting body development.

Transcriptome and proteome analyses reveal the regulatory networks and metabolite biosynthesis pathways during the development of Tolypocladium guangdongense.

Tolypocladium guangdongense has a similar metabolite profile to Ophiocordyceps sinensis, a highly regarded fungus used for traditional Chinese medicine with high nutritional and medicinal value. Although the genome sequence of T. guangdongense has been reported, relatively little is known about the regulatory networks for fruiting body development and about the metabolite biosynthesis pathways. In order to address this, an analysis of transcriptome and proteome at differential developmental stages of T. guangdongense was performed. In total, 9076 genes were found to be expressed and 2040 proteins were identified. There were a large number of genes that were significantly differentially expressed between the mycelial stage and the stages. Interestingly, the correlation between the transcriptomic and proteomic data was low, suggesting the importance of the post-transcriptional processes in the growth and development of T. guangdongense. Among the genes/proteins that were both differentially expressed during the developmental process, there were numerous heat shock proteins and transcription factors. In addition, there were numerous proteins involved in terpenoid, ergosterol, adenosine and polysaccharide biosynthesis that also showed significant downregulation in their expression levels during the developmental process. Furthermore, both tryptophan and tryptamine were present at higher levels in the primordium stage. However, indole-3-acetic acid (IAA) levels continuously decreased as development proceeded, and the enzymes involved in IAA biosynthesis were also clearly differentially downregulated. These data could be meaningful in studying the molecular mechanisms of fungal development, and for the industrial and medicinal application of macro-fungi.

Selection and validation of reliable reference genes for Tolypocladium guangdongense gene expression analysis under differentially developmental stages and temperature stresses.

Tolypocladium guangdongense, formerly known as Cordyceps guangdongensis, is a widely cultivated fungus of the Cordyceps s.l. species that has been investigated over the last 12 years. It has the potential to be used in a number of applications in the health and pharmaceutical industries for it has shown its high nutritional and medicinal values according to previous animal studies. qRT-PCR (quantitative reverse transcription polymerase chain reaction) is extensively used to analyze the expression pattern and molecular mechanisms of functional genes under differentially experimental conditions. The expression stability of reference genes used for normalization determines the reliability of qRT-PCR results, indicating the importance of selection and validation of reference genes before gene expression analysis. In the present study, three statistical algorithms, geNorm, NormFinder and BestKeeper, were used for analyzing the expression stability of nineteen candidate reference genes (CRGs) in T. guangdongense. Investigation were carried out under differentially experimental conditions, which included differentially developmential stages (mycelia, primordia, young and mature fruiting bodies), different carbon sources, cold and heat stresses. The results showed that histone H4 and tubulin beta chain 2 (ß-tub2) were the most and least stable genes, respectively, across all the experimental samples. Moreover, analysis of individual data sets exhibited different stability and expression profiles of reference genes. The vacuolar protein sorting gene VPS was the most stable gene expressed under the differentially developmental stages and temperature stresses, whereas H4 was the most stably expressed gene under different carbon sources. Therefore, it can be proposed that VPS and H4 are the preferred reference genes for normalization of gene expression under different experimental conditions. The results of our present study will enable more accurate evaluation of gene expression in T. guangdongense using the optimal reference gene for qRT-PCR analysis.

Expression profiling of Cordyceps DnaJ protein family in Tolypocladium guangdongense during developmental and temperature stress processes.

DnaJ is an important molecular chaperone, with significant roles in growth, development, and stress resistance. Studies on the DnaJ gene family in macro-fungi such as Cordyceps spp. s.l. is scare. In this study, 22, 20, and 24 putative DnaJ genes were identified in Tolypocladium guangdongense, Ophiocordyceps sinensis, and C. militaris, respectively. They were classified into four groups based on the presence of the J, zinc finger, and C-terminal domains. We mainly studied the T. guangdongense DnaJ genes being located in the endoplasmic reticulum, cytoplasm, mitochondrion, and nucleus. Phylogenetic analysis revealed gene duplications during the evolutionary process. Multiple cis-elements and transcription factor binding sites were observed in the promoter, suggesting their involvement in the response to multiple stresses. qRT-PCR analysis showed that 63.63% and 45.45% of T. guangdongense DnaJ genes were differentially expressed under cold and heat stress, respectively, indicating their involvement in the response to temperature stress. Many T. guangdongense DnaJ genes in the primordium and fruiting body exhibited differential expression, in comparison to those in the mycelium, suggesting a regulatory role in its growth and development process. These findings will facilitate further functional analysis, and provide information on the classification and conservative functions of DnaJ proteins in macro-fungi.

Clitopilus, Clitocella, and Clitopilopsis in China.

Within the family Entolomataceae, many reports of Entoloma and Rhodocybe have been published, but few reports on Clitopilus and allied genera are available. In this study, Clitopilus, Clitocella, and Clitopilopsis from China were studied by morphological and phylogenetic methods. Portions of nuc 28S rDNA (28S), RNA polymerase II second largest subunit (rpb2), translation elongation factor 1-alpha (tef1), and ATPase subunit 6 (atp6), were employed to elucidate the relationships of Clitopilus and allies. Results indicate that Clitopilus should be divided into three sections, namely, sect. Clitopilus, sect. Scyphoides, and sect. Crispi, the last of which is newly described. In total, 17 species of Clitopilus, Clitocella, and Clitopilopsis are found in China; 14 species belong to Clitopilus, of which 5 are new: C. yunnanensis, C. brunniceps, C. rugosiceps, C. sinoapalus, and C. umbilicatus. Two species of Clitocella, including one new species, Clitocella orientalis, are described. One new species of Clitopilopsis, namely, Clitopilopsis albida, is proposed.

Transcriptome sequencing analysis of the MSDIN gene family encoding cyclic peptides in lethal Amanita fuligineoides.

Amanita fuligineoides, a lethal mushroom discovered in China, contains abundant cyclic peptide toxins that can cause fatal poisoning. However, the MSDIN gene family encoding for these cyclic peptides in A. fuligineoides has not been systematically studied. In this research, the transcriptome sequencing of A. fuligineoides was performed and its MSDIN family members were analyzed. A total of 4.41 Gb data containing 30833 unigenes was obtained; sequence alignments throughout several databases were done to obtain their functional annotations. Based on these annotations, MSDIN genes were found and verified by RT-PCR. A total of 29 different core peptides were obtained: 3 toxin genes, encoding ß-amanitin (ß-AMA), phalloidin (PHD), and phallacidin (PCD), and 26 genes encoding unknown cyclic peptides, 20 of which are reported for the first time and may encode for novel cyclic peptides. Analysis of the predicted precursor peptides indicated that octocyclic peptides were the main MSDIN peptides synthesized by A. fuligineoides, accounting for the 45%. A phylogenetic analysis suggested that studied precursor peptides could be clustered into 7 clades, which might represent different functionalities. Results suggested that A. fuligineoides might have a strong capacity to synthesize cyclopeptides, laying the foundation for their excavation and utilization.

Genome-Wide Analysis of the Zn(II)2Cys6 Zinc Cluster-Encoding Gene Family in Tolypocladiumguangdongense and Its Light-Induced Expression.

The Zn(II)2Cys6 zinc cluster gene family is a subclass of zinc-finger proteins, which are transcriptional regulators involved in a wide variety of biological processes in fungi. We performed genome-wide identification and characterization of Zn(II)2Cys6 zinc-cluster gene (C6 zinc gene) family in Tolypocladiumguangdongense, Cordycepsmilitaris and Ophiocordycepssinensis. Based on the structures of the C6 zinc domains, these proteins were observed to be evolutionarily conserved in ascomycete fungi. We focused on T.guangdongense, a medicinal fungus, and identified 139 C6 zinc genes which could be divided into three groups. Among them, 49.6% belonged to the fungal specific transcriptional factors, and 16% had a DUF3468 domain. Homologous and phylogenetic analysis indicated that 29 C6 zinc genes were possibly involved in the metabolic process, while five C6 zinc genes were supposed to be involved in asexual or sexual development. Gene expression analysis revealed that 54 C6 zinc genes were differentially expressed under light, including two genes that possibly influenced the development, and seven genes that possibly influenced the metabolic processes. This indicated that light may affect the development and metabolic processes, at least partially, through the regulation of C6 zinc genes in T.guangdongense. Our results provide comprehensive data for further analyzing the functions of the C6 zinc genes.