Uncovering Gene Expression Profiles of Three Morphologic Mycelium Forms of the Chinese Caterpillar Mushroom Ophiocordyceps sinensis (Ascomycota) Using High-Throughput Sequencing.

The asexual form of Ophiocordyceps sinensis has been controversial, but various morphologic mycelium appeared when O. sinensis was cultured under experimental conditions. To explore the generation mechanism of morphologic mycelium, developmental transcriptomes were analyzed from three kinds of mycelium (aerial mycelium, hyphae knot, and substrate mycelium). The results showed that diameter and morphology of these three kinds of mycelium were obviously different. KEGG functional enrichment analysis showed that the differential expressed genes (DEGs) of substrate mycelium were enriched in ribosomes and peroxisomes, indicating that prophase culture was rich in nutrients and the metabolism of substrate mycelium cells was vigorous in the stage of nutrient absorption. The up-DEGs of hyphae knot were mainly enriched in the oxidative phosphorylation pathway, indicating that oxidative phosphorylation was the main energy source for mycelium formation in the stage of nutrient accumulation and reproductive transformation. The up-DEGs of aerial mycelium were mainly enriched in the synthesis and degradation pathways of valine, leucine, and isoleucine, suggesting that the occurrence of aerial mycelium was related to amino acid metabolism at the later stage of culture, and nutritional stress accelerated the reproduction of asexual spores. In addition, the important roles of mycelium formation related genes were verified by combined analysis of qRT-PCR and transcriptome sequencing. Collectively, this study will provide theoretical guidance for inhibiting the occurrence of aerogenous mycelium and promoting the development of mycelium into pinhead primordia in the culture of O. sinensis in the future.

Transcriptome Sequencing to Identify Candidate Genes for Mycelium Period Metabolism of Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes), Exposed to Light Treatment.

The Chinese caterpillar mushroom, Ophiocordyceps sinensis, is rare in traditional Chinese herbal medicine. It was reported that the development of ascospores in sexual stage of Chinese Cordyceps requires certain UV irradiation, but whether light has effect on the asexual stage of O. sinensis is unclear. It is important and necessary to identify the candidate genes involved in asexual stages (mycelium period) metabolism of O. sinensis exposed to light treatment. In this study, the isolated and purified monoascospore strains from O. sinensis were treated with 10 days light (L10) and dark as control. Transcriptome sequencing (RNA-seq) was conducted to investigate the effect of light treatment on O. sinensis at the gene level and the changes in various metabolic pathways. The results showed that the colony surface was covered with villous aerial hyphae with a yellow circular mycelium ribbon in the center of the colony, and the content of polysaccharides, urea, adenosine, and cordycepin were significantly enhanced by L10 treatment. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation analysis showed that cell structure and catalytic metabolism were the significant items classified, and crucial genes affecting the anabolic pathways of polysaccharides, urea, adenosine, and cordycepin were also identified. In addition, the important roles of photoreceptor genes CRYD, WC-1, and FRQ were verified by combined analysis of qRT-PCR and transcriptome sequencing. Collectively, this study would be helpful to better understand the influence of light on the asexual stage of Chinese Cordyceps and provide a preliminary light treatment reference for Cordyceps artificial cultivation.

Genomic Sequence and Transcriptome Analysis of the Medicinal Fungus Keithomyces neogunnii.

The filamentous fungus Keithomyces neogunnii can infect the larvae of Lepidoptera (Hepialus sp.) and form an insect-fungi complex, which is utilized as an important traditional Chinese medicine. As a valuable medicinal fungus, K. neogunnii produces diverse bioactive substances (e.g., polysaccharide, vitamins, cordycepic acid, and adenosine) under cultivation conditions. Herein, we report the first high-quality genome of the K. neogunnii single-spore isolate Cg7.2a using single-molecule real-time sequencing technology in combination with Illumina sequencing. The assembled genome was 32.6 Mb in size, containing 8,641 predicted genes and having a GC content of 52.16%. RNA sequencing analysis revealed the maximum number of differentially expressed genes in the fungus during the stroma formation stage compared with those during the mycelium stage. These data are valuable to enhance our understanding of the biology, development, evolution, and physiological metabolism of K. neogunnii.

The arbuscular mycorrhizal fungus Rhizophagus irregularis harmonizes nuclear dynamics in the presence of distinct abiotic factors.

Arbuscular mycorrhizal fungi (AMF) are widespread obligate root symbionts that assist plants in obtaining nutrients and protection against environmental stresses. In the model species Rhizophagus irregularis, heterokaryotic strains (AMF dikaryons) carry thousands of nuclei originating from two parental strains whose frequency varies depending on strains and host identity. Here, using digital droplet PCR, we demonstrate that surrounding abiotic factors (temperature, phosphorus, and pH) also change the nuclear dynamics of such strains in root organ cultures. Furthermore, when spatially separated portions of the AMF mycelium grow under different abiotic conditions, all the produced spores carry highly similar nuclear ratios. Overall, these findings demonstrate that abiotic stressors impact the nuclear organization of a widespread group of multinucleate plant symbionts, and reveal remarkable mechanisms of nuclear ratio harmonization across the mycelium in these prominent symbionts.

Transcriptomic analysis of the orchestrated molecular mechanisms underlying fruiting body initiation in Chinese cordyceps.

Chinese cordyceps, the fruiting body of the Chinese caterpillar fungus (Ophiocordyceps sinensis, syn. Cordyceps sinensis), is among the most valuable traditional Chinese medicine fungi. Transcriptomic analysis of O. sinensis has revealed several aspects of its life cycle and ecological importance. However, the molecular mechanisms involved in fruiting body initiation remain unclear. The developmental transcriptomes were analyzed from three tissues at the fruiting body initiation stage, namely, the mycelium, sclerotium and primordium. Principal component analysis showed that in the three tissues, the gene expression patterns differed from each other. The functional analysis of differentially expressed genes showed that DNA synthesis and cell division were active in the primordium. In addition, the function of the mycelium was to absorb certain substances from the environment and the sclerotium was the metabolism center of O. sinensis. Genes participating in the mitogen-activated protein kinase (MAPK) signal pathway were involved in fruiting body initiation. Two environmental sensing genes, including a pheromone receptor gene (OSIN6252) and an amino acid sensing gene (OSIN6398), were highly expressed in the primordium, suggesting their important roles in initiation. These results provided insights into the orchestrated functions and gene profiles of different O. sinensis tissues at the key stage. These findings will aid in revealing the underlying mechanisms of fruiting body initiation, which will further benefit artificial cultivation.

Necrotrophic lifestyle of Rhizoctonia solani AG3-PT during interaction with its host plant potato as revealed by transcriptome analysis.

The soil-borne pathogen Rhizoctonia solani infects a broad range of plants worldwide and is responsible for significant crop losses. Rhizoctonia solani AG3-PT attacks germinating potato sprouts underground while molecular responses during interaction are unknown. To gain insights into processes induced in the fungus especially at early stage of interaction, transcriptional activity was compared between growth of mycelium in liquid culture and the growing fungus in interaction with potato sprouts using RNA-sequencing. Genes coding for enzymes with diverse hydrolase activities were strongly differentially expressed, however with remarkably dissimilar time response. While at 3 dpi, expression of genes coding for peptidases was predominantly induced, strongest induction was found for genes encoding hydrolases acting on cell wall components at 8 dpi. Several genes with unknown function were also differentially expressed, thus assuming putative roles as effectors to support host colonization. In summary, the presented analysis characterizes the necrotrophic lifestyle of R. solani AG3-PT during early interaction with its host.

On Boolean gates in fungal colony.

A fungal colony maintains its integrity via flow of cytoplasm along mycelium network. This flow, together with possible coordination of mycelium tips propagation, is controlled by calcium waves and associated waves of electrical potential changes. We propose that these excitation waves can be employed to implement a computation in the mycelium networks. We use FitzHugh-Nagumo model to imitate propagation of excitation in a single colony of Aspergillus niger. Boolean values are encoded by spikes of extracellular potential. We represent binary inputs by electrical impulses on a pair of selected electrodes and we record responses of the colony from sixteen electrodes. We derive sets of two-inputs-on-output logical gates implementable the fungal colony and analyse distributions of the gates.

Morphological Characteristics, Molecular Identification and Antioxidant Activities of Phallus atrovolvatus (Agaricomycetes) Isolated from Thailand.

Phallus atrovolvatus is a wild edible mushroom found in Thailand. Three strains of Ph. atrovolvatus (DOAP-1, DOAP-2, and DOAP-3) were collected from forests in Central Thailand. Some requirements for mycelial growth were obtained in different media. Potato dextrose agar was determined as the best medium to support mycelial growth (83.50 mm after incubation for 7 days). Molecular phylogenetic analyses based on the sequence of internal transcribed spacers (ITS1 and ITS4) confirmed DOAP-1 species status within Phallaceae as Ph. atrovolvatus with high levels of similarity at 99.34%. Antioxidant properties of hot water extract from the fruiting body of three isolates (CMP-1, CMP-2, and CMP-3) were also evaluated. Highest free radical scavenging ability was found in CMP-1 (94.94% at 2.0 mg/mL) whereas crude mushroom extracts exhibited very strong ferrous-ion chelating effects of 99.16% at 10 mg/ mL. Results indicating that all CMP isolates from Ph. atrovolvatus possess excellent antioxidant properties from natural sources.

Phylogenetic Comparison between Italian and Worldwide Hericium Species (Agaricomycetes).

A broad literature concerns the genus Hericium, mainly regarding the medicinal properties of H. erinaceus. Congeneric species of H. erinaceus have been poorly investigated. We collected basidiomata of H. alpestre, H. coralloides and H. erinaceus in Italy and isolated the corresponding mycelia in pure culture. Analysis of the respective internal transcribed spacer regions confirmed the morphological identification of the strains. Internal transcribed spacer sequences from the Italian strains were phylogenetically compared along with 64 other sequences available from Gen-Bank, the CBS Strain Database, and the European Nucleotide Archive (ENA) for the same Hericium . Geographic origin and host plant species were cross-checked using the above data banks. Bayesian phylogenetic analysis produced a phylogram that permitted good discrimination among Hericium species. It provides an updated phylogeny within the genus Hericium and a better understanding of affinity among the species analyzed. The main Hericium clade includes the following: the H. erinaceus group and the H. alpestre/H. coralloides group, where the two species cluster separately. This study also allowed us to differentiate the H. erinaceus group on a biogeographical basis. The phylogenetic comparison further confirms the importance of a joint morphological-molecular approach to avoid misidentification and to guarantee the quality of strains for further chemical and medicinal characterization.

Profile of Ophiocordyceps sinensis transcriptome and differentially expressed genes in three different mycelia, sclerotium and fruiting body developmental stages.

Ophiocordyceps sinensis, a Chinese complementary and alternative medicine (CAM), is an entomopathogenic, fungus, parasitizing larvae of the moth genus Thitarodes. It has three stages of the life cycle, i.e., the anamorph mycelia prior to infection (Cm_Os), the mycelia sclerotium forming in the caterpillar (Te_Ca), and the fruiting bodies or stromata (Te_St). Characterization of the O. sinensis transcriptome among these stages could provide a better understanding of the underlying biology processes. Transcriptomics of the O. sinensis asexual mycelia and hyphae in deceased caterpillars and perithecial stroma was assessed by using Illumina HiSeq™ 2000 technology. A total of 14,922 unigenes were identified and categorized into 46 sub-categories under three gene ontology categories ("biological process", "cellular component", and "molecular function"). Of these genes, 5520 were differentially expressed among the libraries of these three groups of samples (P < 0.05), and 391 genes occurred in all three groups. Compared to the anamorph stage, there were 3049 differentially expressed genes (DEGs) in the teleomorph stage, but only 1023 DEGs occurred within the teleomorph groups (Te_St vs. Te_Ca). Collectively, this study provides a novel resource to further investigate O. sinensis and their three different development stages.

Productivity and Antioxidant Activity of Wild, Reconstituted, and Hybrid Strains of the Pink Oyster Mushroom, Pleurotus djamor (Agaricomycetes), from Mexico.

The genus Pleurotus is the third most commonly produced edible fungi in the world. In addition, species of genus Pleurotus have functional properties such as anticancer, antiviral, antimicrobial, anti-inflammatory, and antioxidant activities, which are mainly attributed to phenolic compounds. For these reasons, this study evaluated the productivity and antioxidant activity (AA) of 2 wild strains (white and pink), 2 reconstituted strains (called "BB" and "RR"), and 4 hybrid strains (H1, H2, H3, and H4) of P. djamor from monokaryotic components (neohaplonts). The results showed that the white wild-type strain and the reconstituted strains exhibited the best production potential, expressed as biological efficiency and mycelial growth rate. The carpophores of hybrid strains H1 and H3 had the greatest AA, as evaluated with DPPH radical scavenging and reducing power assays, respectively. The H3 strain had the highest total phenol (TP) content. Pearson correlations led us to conclude that the mycelial growth rate has a regular inverse correlation with TP and a regular direct correlation with AA of methanolic extracts from carpophores and myce-lia. This is, to our knowledge, the first report in the literature about the effect of Pleurotus strain hybridization through a chemical de-dikaryotization process on TP content.

Profiling of Intra- and Extracellular Enzymes Involved in Fructification of the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

Fruiting bodies of Ganoderma lucidum have been widely used as a source of potent nutraceutical products. However, the key proteins involved in fructifying G. lucidum, to our knowledge, have not yet been reported. We evaluated the protein profile of fruiting and nonfruiting G. lucidum strains at various developmental stages: mycelia, spawn running, pinning, and fruiting body. Four strains of G. lucidum (GL-I to GL-IV) were grown in both liquid medium (mushroom minimal medium broth) and bags of wheat straw, after which the biomass and fruiting bodies were harvested. Enzyme studies revealed enhanced intracellular and extracellular enzymatic activities during the spawn run stage compared with that during mycelial growth in broth. The esterase and peroxidase activities increased significantly during the pinning of the fruiting cultures, thus indicating their positive role in fructification. Fourier transform infrared spectroscopy of proteins at 3 stages of cultivation-spawn run, pin head formation, and fruiting-exhibited the presence of hydrophobic amino acids and an ordered protein structure in fruiting strains (GL-I and GL-II), indicating the presence of hydrophobin proteins and their role in mushroom fructification. However, basic and aromatic amino acids predominated in the nonfruiting strain GL-IV, and an unordered protein structure was present, which indicate the positive role of hydrophobic amino acids and hydrophobin proteins in mushroom fructification.

Brown Mycelial Mat as an Essential Morphological Structure of the Shiitake Medicinal Mushroom Lentinus edodes (Agaricomycetes).

We show here, to our knowledge for the first time, that the brown mycelial mat of the xylotrophic shiitake medicinal mushroom, Lentinus edodes, not only performs a protective function owing to significant changes in the ultrastructure (thickening of the cell wall, increased density, and pigmentation of the fungal hyphae) but also is a metabolically active stage in the development of the mushroom. The cells of this morphological structure exhibit repeated activation of expression of the genes lcc4, tir, exp1, chi, and exg1, coding for laccase, tyrosinase, a specific transcription factor, chitinase, and glucanase, which are required for fungal growth and morphogenesis. This study revealed the maximum activity of functionally important proteins with phenol oxidase and lectin activities, and the emergence of additional laccases, tyrosinases, and lectins, which are typical of only this stage of morphogenesis and have a regulatory function in the development and formation of fruiting bodies.

The Impact of Steroidal Glycoalkaloids on the Physiology of Phytophthora infestans, the Causative Agent of Potato Late Blight.

Steroidal glycoalkaloids (SGAs) are plant secondary metabolites known to be toxic to animals and humans and that have putative roles in defense against pests. The proposed mechanisms of SGA toxicity are sterol-mediated disruption of membranes and inhibition of cholinesterase activity in neurons. It has been suggested that phytopathogenic microorganisms can overcome SGA toxicity by enzymatic deglycosylation of SGAs. Here, we have explored SGA-mediated toxicity toward the invasive oomycete Phytophthora infestans, the causative agent of the late blight disease in potato and tomato, as well as the potential for SGA deglycosylation by this species. Our growth studies indicate that solanidine, the nonglycosylated precursor of the potato SGAs α-chaconine and α-solanine, has a greater physiological impact than its glycosylated forms. All of these compounds were incorporated into the mycelium, but only solanidine could strongly inhibit the growth of P. infestans in liquid culture. Genes encoding several glycoside hydrolases with potential activity on SGAs were identified in the genome of P. infestans and were shown to be expressed. However, we found no indication that deglycosylation of SGAs takes place. We present additional evidence for apparent host-specific adaptation to potato SGAs and assess all results in terms of future pathogen management strategies.

Antifungal Activity of Eucalyptus Oil against Rice Blast Fungi and the Possible Mechanism of Gene Expression Pattern.

Eucalyptus oil possesses a wide spectrum of biological activity, including anti-microbial, fungicidal, herbicidal, acaricidal and nematicidal properties. We studied anti-fungal activities of the leaf oil extracted from Eucalyptus. grandis × E. urophylla. Eleven plant pathogenic fungi were tested based on the mycelium growth rates with negative control. The results showed that Eucalyptus oil has broad-spectrum inhibitory effects toward these fungi. Remarkable morphological and structural alterations of hypha have been observed for Magnaporthe grisea after the treatment. The mRNA genome array of M. grisea was used to detect genes that were differentially expressed in the test strains treated by the Eucalyptus oil than the normal strains. The results showed 1919 genes were significantly affected, among which 1109 were down-regulated and 810 were up-regulated (p < 0.05, absolute fold change >2). According to gene ontology annotation analysis, these differentially expressed genes may cause abnormal structures and physiological function disorders, which may reduce the fungus growth. These results show the oil has potential for use in the biological control of plant disease as a green biopesticide.

Characterization of Three New Glutaredoxin Genes in the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis: Putative Role of RiGRX4 and RiGRX5 in Iron Homeostasis.

Glutaredoxins (GRXs) are small ubiquitous oxidoreductases involved in the regulation of the redox state in living cells. In an attempt to identify the full complement of GRXs in the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis, three additional GRX homologs, besides the formerly characterized GintGRX1 (renamed here as RiGRX1), were identified. The three new GRXs (RiGRX4, RiGRX5 and RiGRX6) contain the CXXS domain of monothiol GRXs, but whereas RiGRX4 and RiGRX5 belong to class II GRXs, RiGRX6 belongs to class I together with RiGRX1. By using a yeast expression system, we observed that the newly identified homologs partially reverted sensitivity of the GRX deletion yeast strains to external oxidants. Furthermore, our results indicated that RiGRX4 and RiGRX5 play a role in iron homeostasis in yeast. Gene expression analyses revealed that RiGRX1 and RiGRX6 were more highly expressed in the intraradical (IRM) than in the extraradical mycelium (ERM). Exposure of the ERM to hydrogen peroxide induced up-regulation of RiGRX1, RiGRX4 and RiGRX5 gene expression. RiGRX4 expression was also up-regulated in the ERM when the fungus was grown in media supplemented with a high iron concentration. These data indicate the two monothiol class II GRXs, RiGRX4 and RiGRX5, might be involved in oxidative stress protection and in the regulation of fungal iron homeostasis. Increased expression of RiGRX1 and RiGRX6 in the IRM suggests that these GRXs should play a key role in oxidative stress protection of R. irregularis during its in planta phase.

[Influence of diethyl sulfate (DES) mutagenesis on growth properties and pigment secondary metabolites of Phellinus igniarius].

The diethyl sulfate (DES) mutagenesis was chosen for the mutagenic treatment to Phellinus igniarius, and the relationship of mutagenesis time and death rate was investigated with 0.5% DES. The differences of mycelial growth speed, liquid fermentation mycelia biomass, morphology and pigment classes of secondary metabolites production speed and antioxidant activities of metabolite products were discussed. The study displayed that DES mutagenesis could change mycelial morphology without obvious effect on mycelium growth, and the DES mutagenesis improved antioxidant activities of the active ingredients of P. igniarius and had more antioxidant activity of hypoxia/sugar PC12 nerve cells than that of P. igniarius.

Isolation of the Entomopathogenic Fungal Strain Cod-MK1201 from a Cicada Nymph and Assessment of Its Antibacterial Activities.

The entomopathogenic fungus Cod-MK1201 was isolated from a dead cicada nymph. Three regions of ribosomal nuclear DNA, the internal transcribed spacers of nuclear ribosomal DNA repeats (ITS), the partial small subunit of rDNA (nrSSU) , and the partial large subunit of rDNA (nrLSU), and two protein-coding regions, the elongation factor 1α (EF-1α), and the largest subunit of the RNA polymerase II (rpb1) gene, were sequenced and used for fungal identification. The phylogenetic analysis of the ITS and the combined data set of the five genes indicated that the fungal isolate Cod-MK1201 is a new strain of Cordyceps sp. that is closely related to Cordyceps nipponica and C. kanzashiana. Crude extracts of mycelium-cultured Cod-MK1201 were obtained using distilled water and 50% (v/v) ethanol, and the antibacterial activity of each was determined. Both extracts had activity against Gram-positive and Gram-negative bacteria, but the ethanol extract was the more potent of the two. The antibacterial activity of the protein fractions of these extracts was also determined. The protein fraction from the ethanol extract was more antibacterial than the protein fraction from the aqueous extract. Three antibacterial constituents including adenosine, the total phenolic content (TPC), and the total flavonoid content (TFC) was also determined. The results showed that the adenosine content, the TPC, and the TFC of the ethanol extract were more active than those of the aqueous extract. Moreover, synergism was detected between these antibacterial constituents. In conclusion, the entomopathogenic fungal isolate Cod-MK1201 represents a natural source of antibacterial agents.

Involvement of threonine deaminase FgIlv1 in isoleucine biosynthesis and full virulence in Fusarium graminearum.

In this study we characterized FgIlv1, a homologue of the Saccharomyces cerevisiae threonine dehydratase (TD) from the important Fusarium head blight fungus Fusarium graminearum. TD catalyzes the first step in the biosynthesis pathway of isoleucine (Ile) for conversion of threonine (Thr) to 2-ketobutyrate (2-KB). The FgILV1 deletion mutant ΔFgIlv1-3 was unable to grow on minimal medium or fructose gelatin agar which lacked Ile. Exogenous supplementation of Ile or 2-KB but not Thr rescued the mycelial growth defect of ΔFgIlv1-3, indicating the involvement of FgIlv1 in the conversion of Thr to 2-KB in Ile biosynthesis. Additionally, exogenous supplementation of Methionine (Met) could also rescue the mycelial growth defect of ΔFgIlv1-3, indicating a crosstalk between Ile biosynthesis and Met catabolism in F. graminearum. Deletion of FgILV1 also caused defects in conidial formation and germination. In addition, ΔFgIlv1-3 displayed decreased virulence on wheat heads and a low level of deoxynivalenol (DON) production in wheat kernels. Taken together, results of this study indicate that FgIlv1 is an essential component in Ile biosynthesis and is required for various cellular processes including mycelial and conidial morphogenesis, DON biosynthesis, and full virulence in F. graminearum. Our data indicate the potential of targeting Ile biosynthesis for anti-FHB management.

Effect of revulsive cultivation on the yield and quality of newly formed sclerotia in medicinal Wolfiporia cocos.

Wolfiporia cocos is a well-known medicinal mushroom widely used in China, Japan and other Asiatic countries for its various therapeutic effects. 'Revulsive cultivation' is a newly developed method for promoting sclerotia growth in W. cocos field cultivation in China. In this report, we have systematically examined the effects of 'revulsive cultivation' on the yield and quality of newly formed sclerotia. The results showed that the genetic differences between the cultivated strain and the revulsive strain of T1 used in this study did not affect the formation process of new, large sclerotia in which the mycelia of the cultivated strain grew on pine logs directionally assembled on the revulsive strain. Additionally, 'revulsive cultivation', in which the cultivated strain and the revulsive strain used had the same or different genotypes, could remarkably increase the yield, lower the water content, and increase the water-soluble polysaccharide content of the newly formed sclerotia. Moreover, we observed that the changes in the values of the tested economic traits obtained from different genotype combinations through 'revulsive cultivation' were dissimilar. The correlations of these changes with the original sclerotium-forming ability of the cultivated strains and the genetic differences between the cultivated strain and the revulsive strain were not significant. These results will broaden our knowledge regarding the field cultivation of this medical fungus, stimulate new thinking on the study of sclerotium formation in some sclerotium-forming fungi, and promote further studies on the mechanism of sclerotium formation in W. cocos.