Diverse function and regulation of CmSnf1 in entomopathogenic fungus Cordyceps militaris.

SNF1/AMPK protein kinases play important roles in fungal development and activation of catabolite-repressed genes. In this study, we characterized the role of SNF1 ortholog in Cordyceps militaris (CmSnf1). The vegetative growth of a CmSnf1 deletion mutant was (ΔCmSnf1) reduced by 42.2% with arabinose as a sole carbon source. Most strikingly, the ΔCmSnf1 produced only a few conidia and exhibited delayed conidial germination. We found that CmSnf1 was necessary for mycelium to penetrate the insect cuticle to form the fruiting body on silkworm pupae, consistent with the down-regulation of chitinase- and protease-encoding genes in ΔCmSnf1. However, cordycepin content increased by more than 7 times in culture supernatants. Correspondingly, the relative expression levels of cordycepin gene cluster members were also elevated. In particular, the expression of cns4 associated with cordycepin transfer was up-regulated >10-fold. Furthermore, transcriptional analysis showed that CmSnf1 regulated the expression of genes involved in cell autophagy and oxidative stress tolerance. We speculated that under environmental stress, both the ATG and SNF1 pathways might collaborate to sustain adverse environments. Our study provides an initial framework to probe the diverse function and regulation of CmSnf1 in C. militaris, which will shed more light on the direction of molecular improvement of medicinal fungi.

Fungal Systematics and Evolution: FUSE 5.

Thirteen new species are formally described: Cortinarius brunneocarpus from Pakistan, C. lilacinoarmillatus from India, Curvularia khuzestanica on Atriplex lentiformis from Iran, Gloeocantharellus neoechinosporus from China, Laboulbenia bernaliana on species of Apenes, Apristus, and Philophuga (Coleoptera, Carabidae) from Nicaragua and Panama, L. oioveliicola on Oiovelia machadoi (Hemiptera, Veliidae) from Brazil, L. termiticola on Macrotermes subhyalinus (Blattodea, Termitidae) from the DR Congo, Pluteus cutefractus from Slovenia, Rhizoglomus variabile from Peru, Russula phloginea from China, Stagonosporopsis flacciduvarum on Vitis vinifera from Italy, Strobilomyces huangshanensis from China, Uromyces klotzschianus on Rumex dentatus subsp. klotzschianus from Pakistan. The following new records are reported: Alternaria calendulae on Calendula officinalis from India; A. tenuissima on apple and quince fruits from Iran; Candelariella oleaginescens from Turkey; Didymella americana and D. calidophila on Vitis vinifera from Italy; Lasiodiplodia theobromae causing tip blight of Dianella tasmanica 'variegata' from India; Marasmiellus subpruinosus from Madeira, Portugal, new for Macaronesia and Africa; Mycena albidolilacea, M. tenuispinosa, and M. xantholeuca from Russia; Neonectria neomacrospora on Madhuca longifolia from India; Nothophoma quercina on Vitis vinifera from Italy; Plagiosphaera immersa on Urtica dioica from Austria; Rinodina sicula from Turkey; Sphaerosporium lignatile from Wisconsin, USA; and Verrucaria murina from Turkey. Multi-locus analysis of ITS, LSU, rpb1, tef1 sequences revealed that P. immersa, commonly classified within Gnomoniaceae (Diaporthales) or as Sordariomycetes incertae sedis, belongs to Magnaporthaceae (Magnaporthales). Analysis of a six-locus Ascomycota-wide dataset including SSU and LSU sequences of S. lignatile revealed that this species, currently in Ascomycota incertae sedis, belongs to Pyronemataceae (Pezizomycetes, Pezizales).

Discovery and Characterization of the Highly Active Fungal Immunomodulatory Protein Fip-vvo82.

Volvaria volvacea (Bull. ex Fr.) Sing, an important edible and medicinal macro-fungus, has been used to remedy various diseases for hundreds of years in East Asia. To identify key proteins with the unique therapeutic activity in V. volvacea, we conducted a genomewide comparison of V. volvacea protein families and those of other edible fungi that lack therapeutic functions and identified seven fungal immunomodulatory proteins (FIPs) in V. volvacea. On the basis of the predicted physiological and biochemical characteristics of the seven FIPs, the novel Fip-vvo82 was inferred to have high immunomodulatory activity; this was confirmed by molecular and immunological experiments and further characterized by modeling the three-dimensional structure and protein-protein docking. This is the first study to show that V. volvacea has more than one FIP.

Widely targeted metabolomics analysis of Sanghuangporus vaninii mycelia and fruiting bodies at different harvest stages.

Sanghuangprous vaninii is a medicinal macrofungus cultivated extensively in China. Both the mycelia and fruiting bodies of S. vaninii have remarkable therapeutic properties, but it remains unclear whether the mycelia may serve as a substitute for the fruiting bodies. Furthermore, S. vaninii is a perennial fungus with therapeutic components that vary significantly depending on the growing year of the fruiting bodies. Hence, it is critical to select an appropriate harvest stage for S. vaninii fruiting bodies for a specific purpose. With the aid of Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), metabolomics based on ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS) was used to preliminarily determine 81 key active metabolites and 157 active pharmaceutical metabolites in S. vaninii responsible for resistance to the six major diseases. To evaluate the substitutability of the mycelia and fruiting bodies of S. vaninii and to select an appropriate harvest stage for the fruiting bodies of S. vaninii, we analyzed the metabolite differences, especially active metabolite differences, among the mycelia and fruiting bodies during three different harvest stages (1-year-old, 2-year-old, and 3-year-old). Moreover, we also determined the most prominent and crucial metabolites in each sample of S. vaninii. These results suggested that the mycelia show promise as a substitute for the fruiting bodies of S. vaninii and that extending the growth year does not necessarily lead to higher accumulation levels of active metabolites in the S. vaninii fruiting bodies. This study provided a theoretical basis for developing and using S. vaninii.

Development of conventional and nested PCR assays for the detection of Ophiocordyceps sinensis.

Ophiocordyceps sinensis, endemic to the Tibetan Plateau, is one of the most important medicinal fungi with a huge economic value. In the present study, specific primer pairs were designed based on a comprehensive ITS sequence dataset of O. sinensis and its related fungi, and tested for specificity and sensitivity through PCR experiments using 27 individuals of O. sinensis from different geographical origins and 40 other related fungal species in terms of phylogeny or ecology. A primer pair highly specific to O. sinensis was obtained, yielding a 275 bp PCR product from all the individuals of O. sinensis but no product from the other fungi tested. The detection limit of the primers was demonstrated to be 10 ng of pure O. sinensis DNA for conventional PCR and 10 pg for nested PCR in a 25 µl reaction system. Soil samples collected from the habitat of O. sinensis were also tested using this PCR assay. The results showed that the primer pair and PCR-based assays developed in this study can be applied to the rapid detection of O. sinensis in its natural habitat.

Gerhardtia tomentosa and Ossicaulis borealis (Agaricales, Lyophyllaceae)-Two new species from northeast China.

Background: Gerhardtia and Ossicaulis are two genera within the family Lyophyllaceae, which show an apparently poor species diversity worldwide. During the field investigation on wild macrofungi, six interesting collections within Gerhardtia and Ossicaulis genera are discovered in the northeastern China. Methods: To identify whether these collections of Gerhardtia and Ossicaulis are novel species, we performed phylogenetic analyzes using the following DNA regions: the internal transcribed spacer (ITS) region and the large subunit nuclear ribosomal RNA (nrLSU) region. Moreover, a traditional morphological method also be conducted based on both the macro-morphological and micro-morphological features. Results: The results indicated that these collections tested formed two independent lineages in each genus with a high support. In addition, they can easily be separated from all other taxa of the two genera in morphology. Based on the combination of morphological and molecular data, Gerhardtia tomentosa and Ossicaulis borealis, are confirmed as two new species to science. Discussions: This study provided a theoretical basis is for the two lyophylloid genera and indicated that the biodiversity resources of northeastern China might be underestimated.

Selection and validation of internal control genes for quantitative real-time RT‒qPCR normalization of Phlebopus portentosus gene expression under different conditions.

Phlebopus portentosus (Berk. and Broome) Boedijn is an attractive edible mushroom and is considered the only bolete for which artificial cultivation in vitro has been achieved. Gene expression analysis has become widely used in research on edible fungi and is important for elucidating the functions of genes involved in complex biological processes. Selecting appropriate reference genes is crucial to ensuring reliable RT‒qPCR gene expression analysis results. In our study, a total of 12 candidate control genes were selected from 25 traditional housekeeping genes based on their expression stability in 9 transcriptomes of 3 developmental stages. These genes were further evaluated using geNorm, NormFinder, and RefFinder under different conditions and developmental stages. The results revealed that MSF1 domain-containing protein (MSF1), synaptobrevin (SYB), mitogen-activated protein kinase genes (MAPK), TATA-binding protein 1 (TBP1), and SPRY domain protein (SPRY) were the most stable reference genes in all sample treatments, while elongation factor 1-alpha (EF1), actin and ubiquitin-conjugating enzyme (UBCE) were the most unstably expressed. The gene SYB was selected based on the transcriptome results and was identified as a novel reference gene in P. portentosus. This is the first detailed study on the identification of reference genes in this fungus and may provide new insights into selecting genes and quantifying gene expression.

LC/MS- and GC/MS-based metabolomic profiling to determine changes in flavor quality and bioactive components of Phlebopus portentosus under low-temperature storage.

Introduction: Low temperature is the most common method used to maintain the freshness of Phlebopus portentosus during long-distance transportation. However, there is no information regarding the nutritional changes that occur in P. portentosus preserved postharvest in low temperature. Methods: In this study, the changes in flavor quality and bioactive components in fruiting bodies stored at 4 °C for different storage periods were determined through LC/MS and GC/MS analyses. Sampling was performed at 0, 3, 5, 7, and 13 days storage. Results and Discussion: Based on the results, the metabolites present in caps and stipes were different at the same period and significantly different after 7 days of storage. A total of 583 and 500 different metabolites were detected in caps and stipes, respectively, and were mainly lipids and lipid-like molecules, organic acids and derivatives, organic oxygen compounds and others. Except for prenol lipids and nucleotides, the expression levels of most metabolites increased with longer storage time. In addition, geosmin was identified as the major contributor to earthy-musty odors, and the level of geosmin was increased when the storage time was short. Conclusion: The variations in these metabolites might cause changes in flavor quality and bioactive components in P. portentosus. Variations in these metabolites were thoroughly analyzed, and the results revealed how storage processes affect the postharvest quality of P. portentosus for the first time.

The complete mitochondrial genome of the edible mushroom Grifola frondosa.

The culinary-medicinal mushroom Grifola frondosa is widely cultivated in East Asia. In this study, the complete mitochondrial genome of G. frondosa was determined using Illumina sequencing. The circular molecule was 197,486 bp in length with a content of 25.01% GC, which was one of the largest mitochondrial genomes in the order Polyporales. A total of 39 known genes encoding 13 common mitochondrial genes, 24 tRNA genes, 1 ribosomal protein s3 gene (rps3), and 1 DNA polymerase gene (dpo) were predicted in this genome. The phylogenetic analysis showed that G. frondosa clustered together with Sparassis crispa, Laetiporus sulphureus, Wolfiporia cocos, and Taiwanofungus camphoratus. The complete mitochondrial genome reported here may provide new insight into genetic information and evolution for further studies.

Two new species of Fistulina (Agaricales, Basidiomycota) from the Northern Hemisphere.

Phylogenetic and morphological analyses on samples of Fistulina from East Asia and North America were carried out, and two new species were described, namely, Fistulina americana and Fistulina orientalis, both previously known as Fistulina hepatica. The former is characterized by lateral stipitate basidiocarps, relatively small pores (7-8 per mm), a monomitic hyphal system with both clamp connections and simple septa, and ellipsoid basidiospores of 4-4.8 × 3-3.3 µm, and the species has been found on Quercus in North-East USA. F. orientalis is characterized by lateral stipitate basidiocarps, very small pores (11-12 per mm) with pruinose dissepiments, a monomitic hyphal system with both clamp connections and simple septa, and ovoid to subglobose basidiospores of 3-4 × 2.7-3 µm, and the species has been found on Castanopsis in East Asia. Phylogenetically, samples of F. americana and F. orientalis form two new lineages nested in the Fistulina clade.

Development of microsatellite markers for Ophiocordyceps sinensis (Ophiocordycipitaceae) using an ISSR-TAIL-PCR method.

PREMISE OF THE STUDY: Microsatellite primers were developed for Ophiocordyceps sinensis, an endangered medicinal fungus endemic to the Tibetan Plateau, to investigate its genetic diversity and population structure. METHODS AND RESULTS: An inter-simple sequence repeat (ISSR)-thermal asymmetric interlaced (TAIL)-PCR method was established to develop microsatellite markers. A total of 30 perfect and imperfect microsatellites were identified in 48 individuals of O. sinensis from five provinces within China representing different populations. Seventeen loci were polymorphic with two to four alleles per locus, while 13 were monomorphic. CONCLUSIONS: The results indicate that the microsatellite markers developed here may be used in studies of population genetics and conservation biology of O. sinensis. Furthermore, the ISSR-TAIL-PCR method is a simple strategy for microsatellite marker development.

Whole-Genome and Transcriptome Sequencing of Phlebopus portentosus Reveals Its Associated Ectomycorrhizal Niche and Conserved Pathways Involved in Fruiting Body Development.

Phlebopus portentosus (Berk. and Broome) Boedijin, a widely consumed mushroom in China and Thailand, is the first species in the order Boletaceae to have been industrially cultivated on a large scale. However, to date, the lignocellulose degradation system and molecular basis of fruiting body development in P. portentosus have remained cryptic. In the present study, genome and transcriptome sequencing of P. portentosus was performed during the mycelium (S), primordium (P), and fruiting body (F) stages. A genome of 32.74 Mb with a 48.92% GC content across 62 scaffolds was obtained. A total of 9,464 putative genes were predicted from the genome, of which the number of genes related to plant cell wall-degrading enzymes was much lower than that of some saprophytic mushrooms with specific ectomycorrhizal niches. Principal component analysis of RNA-Seq data revealed that the gene expression profiles at all three stages were different. The low expression of plant cell wall-degrading genes also confirmed the limited ability to degrade lignocellulose. The expression profiles also revealed that some conserved and specific pathways were enriched in the different developmental stages of P. portentosus. Starch and sucrose metabolic pathways were enriched in the mycelium stage, while DNA replication, the proteasome and MAPK signaling pathways may be associated with maturation. These results provide a new perspective for understanding the key pathways and hub genes involved in P. portentosus development.

Deep Sequencing, Nested PCR, and Denaturing Gradient Gel Electrophoresis Reveal a Wider Distribution of Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes), in Native Soil Types.

Ophiocordyceps sinensis appears as stroma emerging from underground sclerotium enclosed by the skeleton of Thitarodes moth larvae. However, the actual distribution of the fungus in soil still remains unclarified. In this study, 40 soil samples were used for detection of O. sinensis to confirm its distribution in native habitats using denaturing gradient gel electrophoresis, nested internal transcribed spacer (ITS) PCR, and 454 pyrosequencing methods. The soil samples included six types: Os, where both stromata and host moth larvae were found; NL, representing no signs of stromata, but where moth larvae were found; NOs, where neither stroma nor moth larvae were found; BS, with bare soil without the presence of stroma of O. sinensis or moth larvae; AF, from soil surrounding the stroma; and MP, soil particles firmly wrapping the sclerotium of O. sinensis. Of 40 samples tested, 36 showed positive detection of O. sinensis by at least one of the three detection methods, with positive detection in all six sample types at all five sites. The results showed that traces of O. sinensis can be detected in locations with no macroscopically visible evidence of the fungus or its host and at least 100 m away from such locations.

RIP mutated ITS genes in populations of Ophiocordyceps sinensis and their implications for molecular systematics.

Different hypotheses have been proposed to interpret the observed unusual ITS (internal transcribed spacer) sequences in Ophiocordyceps sinensis. The coexistence of diverged ITS paralogs in a single genome was previously shown by amplifying the ITS region from mono-ascospore isolates using specific primers designed for different ITS paralog groups. Among those paralogs, are AT-biased ITS sequences which were hypothesized to result from repeat-induced point mutation (RIP). This is a process that detects and mutates repetitive DNA and frequently leads to epigenetic silencing, and these mutations have been interpreted as pseudogenes. Here we investigate the occurrence and frequency of ITS pseudogenes in populations of O. sinensis using large-scale sampling, and discusses the implications of ITS pseudogenes for fungal phylogenetic and evolutionary studies. Our results demonstrate a wide distribution of ITS pseudogenes amongst different geographic populations, and indicate how ITS pseudogenes can contribute to the reconstruction of the evolutionary history of the species.

Bacterial Profiling and Dynamic Succession Analysis of Phlebopus portentosus Casing Soil Using MiSeq Sequencing.

Phlebopus portentosus (Berk. and Broome) Boedijin is a popular edible mushroom found in China and Thailand. To date, P. portentosus is the only species in the order Boletales that can be successfully cultivated worldwide. The use of a casing layer or casing soil overlaying the substrate is a crucial step in the production of this mushroom. In this study, bacterial profiling and dynamic succession analyses of casing soil during the cultivation of P. portentosus were performed. One hundred and fifty samples were collected, and MiSeq sequencing of the V3-V4 region of the 16S rRNA gene was conducted. After performing a decontamination procedure, only 38 samples were retained, including 6 casing soil-originated samples (OS), 6 casing soil samples (FHCS) and 5 upper substrate samples (FHCU) from the period of complete colonization by mycelia; 6 casing soil samples (PCS) and 5 upper substrate samples (PCU) from the primordium period; and 6 casing soil samples (FCS) and 4 upper substrate samples (FCU) from fruit body period. The results revealed that bacterial diversity increased sharply from the hyphal to the primordium stage and then decreased during harvesting. The non-metric multidimensional scaling (NMDS) ordination and analysis of similarities (ANOSIM) analysis suggested that the community composition during different stages was significantly different in casing soil. The most abundant phyla in all of the samples were Proteobacteria, Chloroflexi, Acidobacteria, Actinobacteria, Saccharibacteria, and Bacteroidetes. Burkholderia was the most abundant genus in all the samples except the OS samples. The relative abundance of Burkholderia in the FHCS samples (55.79%) decreased to 35.14% in the PCS samples and then increased to 45.60% in the FCS samples. The abundances of Acidobacterium, Rhizobium, Acidisphaera, Bradyrhizobium, and Bacillus increased from the FHCS to PCS samples. The linear discriminant analysis (LDA) effect size (LEfSe) suggested that Acidobacterium and Acidisphaera are micromarkers for PCS, whereas Bradyrhizobium, Roseiarcus, and Pseudolabrys were associated with fruit body stages. The network analyses resulted in 23 edges, including 4 negative and 19 positive edges. Extensive mutualistic interactions may occur among casing soil bacteria. Furthermore, these bacteria play important roles in mycelial elongation, primordium formations, and the production of increased yields.

The Reliability of DNA Sequences in Public Databases Belonging to the Most Economically Important Shiitake Culinary-Medicinal Mushroom Lentinus edodes (Agaricomycetes) in Asia.

Large numbers of DNA sequences deposited in the International Nucleotide Sequence Databases (INSD) are erroneously annotated. The erroneous information may lead to misleading conclusions or cause great economic losses to farmers. Lentinus edodes (= Lentinula edodes (Berk.) Pegler) is one of the most important and popular culinary-medicinal mushrooms with a high nutritional value. In this study, experimental and in silico methods were used to correct the sequences annotated as L. edodes in the INSD. A total of 3,426 nucleotide entries were retrieved from public databases, including 140 different types of genetic sequences. Excluding 1,893 genome sequences, the most abundant signatures represented by ITS (258) and IGS1 (259) sequences accounted for 33.23% of the total entries. A total of 3,058 sequences were annotated correctly, 350 were indeterminate, and 18 were annotated erroneously based on the two methods. Correction of sequences will be beneficial for species identification and annotation. Phylogenic analysis based on ITS sequences suggested that L. edodes segregate in four clades in the tree based on ITS sequences. The isolates from China were distributed into two clades. In L. edodes, the intraspecific variation of the ITS2 sequences was much higher than that of the ITS1 sequences. In addition, the genetic diversity of the L. edodes sequences from China was much higher than that of any other regions included in this study. The northwest and southwest regions of China were L. edodes diversity centers.

Evaluation of the ribosomal DNA internal transcribed spacer (ITS), specifically ITS1 and ITS2, for the analysis of fungal diversity by deep sequencing.

The nuclear ribosomal DNA internal transcribed spacer (ITS) has been widely used to assess the fungal composition in different environments by deep sequencing. To evaluate the ITS in the analysis of fungal diversity, comparisons of the clustering and taxonomy generated by sequencing with different portions of the whole fragment were conducted in this study. For a total of 83,120 full-length ITS sequences obtained from the UNITE database, it was found that, on average, ITS1 varied more than ITS2 within the kingdom Fungi; this variation included length and GC content variations and polymorphisms, with some polymorphisms specific to particular fungal groups. The taxonomic accuracy for ITS was higher than that for ITS1 or ITS2. The commonly used operational taxonomic unit (OTU) for evaluating fungal diversity and richness assigned several species to a single OTU even with clustering at 99.00% sequence similarity. The clustering and taxonomic capacities did not differ between ITS1 and ITS2. However, the OTU commonality between ITS1 and ITS2 was very low. To test this observation further, 219,741 pyrosequencing reads, including 39,840 full-length ITS sequences, were obtained from 10 soil samples and were clustered into OTUs. The pyrosequencing results agreed with the results of the in silico analysis. ITS1 might overestimate the fungal diversity and richness. Analyses using ITS, ITS1 and ITS2 yielded several different taxa, and the taxonomic preferences for ITS and ITS2 were similar. The results demonstrated that ITS2 alone might be a more suitable marker for revealing the operational taxonomic richness and taxonomy specifics of fungal communities when the full-length ITS is not available.

The complete mitochondrial genome of the Basidiomycete edible fungus Hypsizygus marmoreus.

The complete mitochondrial genome of the edible fungus Hypsizygus marmoreus was published in this paper. It was determined using Pacbio and Illumina sequencing. The complete mitochondrial DNA (mtDNA) is 106,417 bp in length with a GC content of 31.74%, which was the fourth large mitogenome in Agaricales. The circular mitogenome encoded 67 protein-coding genes and one ribosomal RNAs (rns). Among these genes, 13 conserved protein-coding genes were determined in the genome, including 6 subunits of NAD dehydrogenase (nad1-4, 4L and 6), three cytochrome oxidases (cox1-3), one apocytochrome b (cob) and three ATP synthases (atp6, apt 8 and apt 9). The phylogenic analysis confirmed that H. marmoreus (Lyophyllaceae) clustered together with Tricholoma matsutake (Tricholomataceae).

rRNA Pseudogenes in Filamentous Ascomycetes as Revealed by Genome Data.

The nuclear ribosomal DNA (rDNA) is considered as a paradigm of concerted evolution. Components of the rDNA tandem repeats (45S) are widely used in phylogenetic studies of different organisms and the internal transcribed spacer (ITS) region was recently selected as a fungal DNA bar code. However, rRNA pseudogenes, as one kind of escape from concerted evolution, were reported in a wide range of organisms, especially in plants and animals. Moreover, large numbers of 5S rRNA pseudogenes were identified in several filamentous ascomycetes. To study whether rDNA evolves in a strict concerted manner and test whether rRNA pseudogenes exist in more species of ascomycetes, intragenomic rDNA polymorphisms were analyzed using whole genome sequences. Divergent rDNA paralogs were found to coexist within a single genome in seven filamentous ascomycetes examined. A great number of paralogs were identified as pseudogenes according to the mutation and secondary structure analyses. Phylogenetic analyses of the three rRNA coding regions of the 45S rDNA repeats, i.e., 18S, 5.8S, and 28S, revealed an interspecies clustering pattern of those different rDNA paralogs. The identified rRNA pseudogenic sequences were validated using specific primers designed. Mutation analyses revealed that the repeat-induced point (RIP) mutation was probably responsible for the formation of those rRNA pseudogenes.