A new Schizophyllum commune strain as a potential biocontrol agent against blueberry root rot.

In recent years, blueberry root rot has been caused mainly by Fusarium commune, and there is an urgent need for a green and efficient method to control this disease. To date, research on Schizophyllum commune has focused on antioxidant mechanisms, reactive dye degradation, etc., but the mechanism underlying the inhibition of pathogenic microorganisms is still unclear. Here, the control effects of S. commune on F. commune and blueberry root rot were studied using adversarial culture, tissue culture, and greenhouse pot experiments. The results showed that S. commune can dissolve insoluble phosphorus and secrete various extracellular hydrolases. The results of hyphal confrontation and fermentation broth antagonism experiments showed that S. commune had a significant inhibitory effect on F. commune, with inhibition rates of 70.30% and 22.86%, respectively. Microscopy results showed distortion of F. commune hyphae, indicating that S. commune is strongly parasitic. S. commune had a significant growth-promoting effect on blueberry tissue-cultured seedlings. After inoculation with S. commune, inoculation with the pathogenic fungus, or inoculation at a later time, the strain significantly reduced the root rot disease index in the potted blueberry seedlings, with relative control effects of 79.14% and 62.57%, respectively. In addition, S. commune G18 significantly increased the antioxidant enzyme contents in the aboveground and underground parts of potted blueberry seedlings. We can conclude that S. commune is a potential biocontrol agent that can be used to effectively control blueberry root rot caused by F. commune in the field.

Rapid Accumulation of Mutations in Growing Mycelia of a Hypervariable Fungus Schizophyllum commune.

The basidiomycete Schizophyllum commune has the highest level of genetic polymorphism known among living organisms. In a previous study, it was also found to have a moderately high per-generation mutation rate of 2×10-8, likely contributing to its high polymorphism. However, this rate has been measured only in an experiment on Petri dishes, and it is unclear how it translates to natural populations. Here, we used an experimental design that measures the rate of accumulation of de novo mutations in a linearly growing mycelium. We show that S. commune accumulates mutations at a rate of 1.24×10-7 substitutions per nucleotide per meter of growth, or ∼2.04×10-11 per nucleotide per cell division. In contrast to what has been observed in a number of species with extensive vegetative growth, this rate does not decline in the course of propagation of a mycelium. As a result, even a moderate per-cell-division mutation rate in S. commune can translate into a very high per-generation mutation rate when the number of cell divisions between consecutive meiosis is large.

Enhancement of schizophyllan production in Schizophyllum commune using microparticles in medium.

Schizophyllum commune is a wood-rotting filamentous fungus that secrets a homopolysaccharide called as schizophyllan. Schizophyllan has several applications such as enhanced oil recovery, pharmaceutical materials and an anti-cancer drug carrier. Biomass growth and schizophyllan production increase the viscosity of the cultivation medium, thus resulting in mass transfer limitation for the substrate. In this study, adding talc and aluminium oxide microparticles into the cultivation medium was studied to improve the fungal growth and morphology. The response surface methodology and one factor at a time were applied to find the effects of microparticles with different sizes and concentrations on the schizophyllan production. The optimum concentration and size of aluminium oxide microparticles were obtained as 20 g L-1 and < 30 µm, respectively. Aluminium oxide microparticles in shake flask culture caused to increase the schizophyllan production from 10 to 15 g L-1 and decrease the cultivation time from 10 to 7 days. The production yield also increased from 0.11 to 0.30 g of schizophyllan/g glucose. Bioreactor cultivation showed a twofold increase in schizophyllan production from 1.5 to 3 g L-1. The results of this study suggested a significant increase in the production of schizophyllan using a low-cost "microparticle-enhanced cultivation" without any further optimization of the culture medium.

The regulator of G-protein signalling Thn1 links pheromone response to volatile production in Schizophyllum commune.

The regulator of G-protein signalling, Thn1, is involved in sexual development through pheromone signalling in the mushroom forming basidiomycete Schizophyllum commune affecting hyphal morphology and mating interactions. Thn1 plays a key role in coordinating sesquiterpene production, pheromone response and sexual development. The gene thn1 is transcriptionally regulated in response to mating with a role in clamp cell development and hydrophobin gene transcription. Further, it negatively regulates cAMP signalling and secondary metabolism. Disruption of thn1 affects dikaryotization by reducing clamp fusion and development with predominant non-fused pseudoclamps. Enhanced protein kinase A (PKA) activities in Δthn1 strains indicate that Thn1 regulates pheromone signalling by de-activating G-protein α subunits, which control cAMP-dependent PKA. The repressed formation of aerial hyphae could be linked to a reduced metabolic activity and to a transcriptional down-regulation of hyd6 and sc3 hydrophobin genes. Thn1 was also shown to be necessary for the biosynthesis of sesquiterpenes and an altered spectrum of sesquiterpenes in Δthn1 is linked to transcriptional up-regulation of biosynthesis genes. Proteome analysis indicated changes in cytoskeletal structure affecting actin localization, linking the major regulator Thn1 to growth and development of S. commune. The results support a role for Thn1 in G-protein signalling connecting development and secondary metabolism.

Smelling the difference: Transcriptome, proteome and volatilome changes after mating.

Mushrooms, such as Schizophyllum commune, have a specific odor. Whether this is linked to mating, prerequisite for mushroom formation, or also found in monokaryotic, unmated strains, was investigated with a comprehensive study on the transcriptome and proteome of this model organism. Mating interactions were investigated using a complete, cytosolic proteome map for unmated, monokaryotic, as well as for mated, dikaryotic mycelia. The regulations of the proteome were compared to transcriptional changes upon mating and to changes in smell by volatilome studies. We could show a good overlap between proteome and transcriptome data, but extensive posttranslational regulation was identified for more than 80% of transcripts. This suggests down-stream regulation upon interaction of mating partners and formation of the dikaryon that is competent to form fruiting bodies. The volatilome was shown to respond to mating by a broader spectrum of volatiles and increased emission of the mushroom smell molecules 3-octanone and 1-octen-3-ol, as well as ethanol and ß-bisabolol in the dikaryon. Putatively involved biosynthetic proteins like alcohol dehydrogenases, Ppo-like oxygenases, or sesquiterpene synthases showed correlating transcriptional regulation depending on either mono- or dikaryotic stages.

First report on cutaneous infectious granuloma caused by Schizophyllum commune.

BACKGROUND: Schizophyllum commune, a basidiomycetous fungus, is a common invader of rotten wood. This fungus rarely causes mycotic disease in humans, especially cutaneous infection. In this paper, we describe the first case of cutaneous granuloma caused by S. commune in a Chinese woman. CASE PRESENTATION: A 25-year-old female with a two-year history of erythema, papules, nodules, and scales on her sole of left foot was presented to our outpatient center. Samples were obtained by the scraping of lesion and for light microscopy. Hyphae were observed by microscopic examination. We carried out a skin tissue biopsy, which showed multiple granulomatous nodules. Biopsy specimens were also inoculated onto media. After being cultured on SDA at 27 °C for 7 days, spreading-woolly-white colonies grew on the inoculation sites of media containing chloramphenicol only and there,s no other colonies grew. S. commune was identified by morphology methods, biochemical tests, and PCR sequencing. Pathological findings also aided in diagnosing cutaneous fungal granuloma. Oral itraconazole was applied. After 1 month of therapy, rashes on her left foot and pain were improved. CONCLUSION: We describe the first case of cutaneous granuloma caused by Schizophyllum commune, which illustrates the importance of recognizing uncommon pathogenic fungal infections.

First isolation of Schizophyllum commune in a harbor seal (Phoca vitulina).

To date, Schizophyllum commune infection has been identified in only humans and dogs. A 7-year-old female harbor seal (Phoca vitulina) died after exhibiting corneal opacity, anorexia, and labored respiration. At necropsy, phthisis of the left eyeball was detected, and multiple nodular lesions were observed in the thoracic and abdominal regions, especially in the lung, heart, and lymph nodes. Histopathologically, numerous hyphae were seen in granulomatous lesions in the eyes, lung, heart, and lymph nodules. An isolate on potato dextrose agar from the eyes, lung, and sputum yielded a rapidly growing white woolly mycelia with basidiocarps (fruiting bodies) at 37°C. A suitable temperature for mycelial growth was obtained at 25°C, although sustained growth also occurred at 37°C. The fungal isolate, KH-JPN15-011, had distinctive features including hyphae bearing spicules and clamp connections, which were consistent with the characteristics of basidiomycete fungus. The sequence of the internal transcribed spacer region of nuclear ribosomal DNA showed 99.67% (617 bp) similarity with those of S. commune Phylogenetic analysis showed that the present isolate is most closely related to the samples from the Old World. This is the first report of a fatal disease caused by S. commune in exotic animals. Previously reported human and canine infections have not included granulomatous endophthalmitis and myocarditis. After considering these and previous findings, there is a possibility that S. commune from the Old World may include numerous highly pathogenic strains.

Analysis of the sexual development-promoting region of Schizophyllum commune TRP1 gene.

This study aims to elucidate the mechanism of sexual development of basidiomycetous mushrooms from mating to fruit body formation. Sequencing analysis showed the TRP1 gene of basidiomycete Schizophyllum commune encoded an enzyme with three catalytic regions of GAT (glutamine amidotransferase), IGPS (indole-3-glycerol phosphate synthase), and PRAI (5-phosphoribosyl anthranilate isomerase); among these three regions, the trp1 mutant (Trp(-)) had a missense mutation (L→F) of a 338th amino acid residue of the TRP1 protein within the IGPS region. To investigate the function of IGPS region related to sexual development, dikaryons with high, usual, and no expression of the IGPS region of TRP1 gene were made. The dikaryotic mycelia with high expression of the IGPS formed mature fruit bodies earlier than those with usual and no expression of the IGPS. These results showed that the IGPS region in TRP1 gene promoted sexual development of S. commune.

The efficacy of six elite isolates of the fungus Chondrostereum purpureum against the sprouting of European aspen.

The sprouting of broad-leaved trees after cutting is problematic in forest regeneration areas, along roads and railways, under electric power and above gas pipe lines. In Finland, one of the most difficult species to control in these areas is the European aspen (Populus tremula), which produces both stump sprouts and root suckers after saplings have been cut. In this study, we investigated whether a decay fungus of broad-leaved trees, Chondrostereum purpureum, could be used as a biological control agent against aspen sprouting. The efficacy of six elite strains of C. purpureum (improved earlier in a breeding process) was investigated on aspen for three years. The most efficient C. purpureum strain, R53, tested earlier on birch (Betula pendula and B. pubescens), was efficient in causing mortality of aspen stumps and preventing the development of root suckers. With this strain, stump mortality was 78%, while significantly lower in control stumps which were cut only (47%). Aspen trees in the vicinity of the treatments (within a 10 m radius around each sapling) decreased the efficacy of C. purpureum. This study shows that the decay fungus C. purpureum can successfully be used in the sprout control of aspen saplings.

A central role for Ras1 in morphogenesis of the basidiomycete Schizophyllum commune.

Fungi have been used as model systems to define general processes in eukaryotes, for example, the one gene-one enzyme hypothesis, as well as to study polar growth or pathogenesis. Here, we show a central role for the regulator protein Ras in a mushroom-forming, filamentous basidiomycete linking growth, pheromone signaling, sexual development, and meiosis to different signal transduction pathways. ras1 and Ras-specific gap1 mutants were generated and used to modify the intracellular activation state of the Ras module. Transformants containing constitutive ras1 alleles (ras1(G12V) and ras1(Q61L)), as well as their compatible mating interactions, did show strong phenotypes for growth (associated with Cdc42 signaling) and mating (associated with mitogen-activated protein kinase signaling). Normal fruiting bodies with abnormal spores exhibiting a reduced germination rate were produced by outcrossing of these mutant strains. Homozygous Δgap1 primordia, expected to experience increased Ras signaling, showed overlapping phenotypes with a block in basidium development and meiosis. Investigation of cyclic AMP (cAMP)-dependent protein kinase A indicated that constitutively active ras1, as well as Δgap1 mutant strains, exhibit a strong increase in Tpk activity. Ras1-dependent, cAMP-mediated signal transduction is, in addition to the known signaling pathways, involved in fruiting body formation in Schizophyllum commune. To integrate these analyses of Ras signaling, microarray studies were performed. Mutant strains containing constitutively active Ras1, deletion of RasGap1, or constitutively active Cdc42 were characterized and compared. At the transcriptome level, specific regulation highlighting the phenotypic differences of the mutants is clearly visible.

The regulatory network of the White Collar complex during early mushroom development in Schizophyllum commune.

Blue light is an important signal for fungal development. In the mushroom-forming basidiomycete Schizophyllum commune, blue light is detected by the White Collar complex, which consists of WC-1 and WC-2. Most of our knowledge on this complex is derived from the ascomycete Neurospora crassa, where both WC-1 and WC-2 contain GATA zinc-finger transcription factor domains. In basidiomycetes, WC-1 is truncated and does not contain a transcription factor domain, but both WC-1 and WC-2 are still important for development. We show that dimerization of WC-1 and WC-2 happens independent of light in S. commune, but that induction by light is required for promoter binding by the White Collar complex. Furthermore, the White Collar complex is a promoter of transcription, but binding of the complex alone is not always sufficient to initiate transcription. For its function, the White Collar complex associates directly with the promoters of structural genes involved in mushroom development, like hydrophobins, but also promotes the expression of other transcription factors that play a role in mushroom development.

The blue light receptor complex WC-1/2 of Schizophyllum commune is involved in mushroom formation and protection against phototoxicity.

Blue light is necessary for initiation of mushroom formation in Schizophyllum commune. The genome of this basidiomycete contains homologues of the blue light receptor genes wc-1 and wc-2 of Neurospora crassa. Here, it is shown that inactivation of either or both of these genes in S. commune results in a blind phenotype. Mushroom formation was abolished in dikaryons and they formed symmetrical instead of asymmetrical colonies. Development was restored in a temperature dependent way in a Δwc-2Δwc-2 strain by introducing a construct encompassing the wc-2 gene under control of the promoter of the heat shock gene hsp3. A genome-wide expression analysis showed that the transcription factor genes c2h2 and hom1 as well as many hydrophobin genes are downregulated in light-grown colonies of the Δwc-2Δwc-2 mutant when compared with the wild-type dikaryon. Inactivation of wc-1 and/or wc-2 also resulted in sensitivity of the mycelium to intense light. Monokaryotic mutant strains only survived exposure to 6500 lux of light by growing into the agar. Expression analysis indicates that the photosensitivity of the Δwc-1 and Δwc-2 strains is due to lower levels of photolyase and ferrochelatase.

Transcriptome and functional analysis of mating in the basidiomycete Schizophyllum commune.

In this study, we undertook a functional characterization and transcriptome analysis that enabled a comprehensive study of the mating type loci of the mushroom Schizophyllum commune. Induced expression of both the bar2 receptor and the bap2(2) pheromone gene within 6 to 12 h after mates' contact was demonstrated by quantitative real-time PCR. Similar temporal expression patterns were confirmed for the allelic bbr1 receptor and bbp1 pheromone-encoding genes by Northern hybridization. Interestingly, the fusion of clamp connections to the subterminal cell was delayed in mating interactions in which one of the compatible partners expressed the bar2 receptor with a truncated C terminus. This developmental delay allowed the visualization of a green fluorescent protein (Gfp)-labeled truncated receptor at the cell periphery, consistent with a localization in the plasma membrane of unfused pseudoclamps. This finding does not support hypotheses envisioning a receptor localization to the nuclear membrane facilitating recognition between the two different nuclei present in each dikaryotic cell. Rather, Gfp fluorescence observed in such pseudoclamps indicated a role of receptor-pheromone interaction in clamp fusion. Transcriptome changes associated with mating interactions were analyzed in order to identify a role for pheromone-receptor interactions. We detected a total of 89 genes that were transcriptionally regulated in a mating type locus A-dependent manner, employing a cutoff of 5-fold changes in transcript abundance. Upregulation in cell cycle-related genes and downregulation of genes involved in metabolism were seen with this set of experiments. In contrast, mating type locus B-dependent transcriptome changes were observed in 208 genes, with a specific impact on genes related to cell wall and membrane metabolism, stress response, and the redox status of the cell.

Biochemical constituents of a wild strain of Schizophyllum commune isolated from Achanakmar-Amarkantak Biosphere Reserve (ABR), India.

A wild strain of Schizophyllum commune (MTCC 9670) isolated from Achanakmar-Amarkantak Biosphere Reserve of Central India was evaluated for the production of bioactive compounds. The chemical constituents of wild and in vitro grown cultures were compared. Under optimized conditions, different organic and aqueous extracts from mycelia and fruiting bodies were used to extract chemical components from the cultures grown in vitro. The gas chromatography combined wih mass spectrometry analysis of extracts identified two phenolic compounds, namely Phenyl benzoate (C13H10O2) and 4-(phenyl methoxy) phenol (C13H12O2) in the ethanolic extract of in vitro grown fruiting bodies and one antibacterial compound Pyrrolo (1, 2-a) piperazine-3, 6-dione (C7H10O2N2) in the methanolic extract of mycelia. High-performance liquid chromatography analysis revealed that the gallic acid and L-ascorbic acid were identifiable antioxidant components in the extracts possessing high free radical scavenging activity. The findings suggest that the wild strain of S. commune may serve as the source of novel bioactive compounds with effective antimicrobial and antioxidant activities.

Transcription factor genes of Schizophyllum commune involved in regulation of mushroom formation.

Mushrooms represent the most conspicuous structures of fungi. Their development is being studied in the model basidiomycete Schizophyllum commune. The genome of S. commune contains 472 genes encoding predicted transcription factors. Of these, fst3 and fst4 were shown to inhibit and induce mushroom development respectively. Here, we inactivated five additional transcription factor genes. This resulted in absence of mushroom development (in the case of deletion of bri1 and hom2), in arrested development at the stage of aggregate formation (in the case of c2h2) and in the formation of more but smaller mushrooms (in the case of hom1 and gat1). Moreover, strains in which hom2 and bri1 were inactivated formed symmetrical colonies instead of irregular colonies like the wild type. A genome-wide expression analysis identified several gene classes that were differentially expressed in the strains in which either hom2 or fst4 was inactivated. Among the genes that were downregulated in these strains were c2h2 and hom1. Based on these results, a regulatory model of mushroom development in S. commune is proposed. This model most likely also applies to other mushroom-forming fungi and will serve as a basis to understand mushroom formation in nature and to enable and improve commercial mushroom production.

Protective effects of fermented Citrus unshiu peel extract against ultraviolet-A-induced photoageing in human dermal fibrobolasts.

The aqueous extracts of Citrus unshiu peel containing flavonoid glycosides was used as co-substrate with Schizophyllum commune mycelia producing ß-glucosidase and its biological activities were studied. ß-glucosidase-produced S. commune mycelia converted the glycosides (narirutin and hesperidin) into aglycones (naringenin and hesperetin). The photoprotective potential of fermented C. unshiu peel extract with S. commune (S-CPE) was tested in human dermal fibroblasts (HDFs) exposed to UVA. It was revealed that S-CPE had an inhibitory effect on human interstitial collagenase (matrix metalloproteinase, MMP-1) expression in UVA-irradiated HDFs. The treatment of UVA-irradiated HDFs with S-CPE resulted in a dose-dependent decrease in the expression level of MMP-1 mRNA. The UVA irradiation raised the proportion of senescence-associated ß-galactosidase (SA-ß-gal) positive cells in comparison with the normal control group. The treatment of UVA-irradiated HDFs with S-CPE was shown to decrease the level of SA-ß-gal (by approximately 45% at an S-CPE concentration 0.1%, w/v) compared with the UVA-irradiated HDFs. It was found that S-CPE containing hesperetin has notable collagen biosynthetic activity for fibroblasts, indicating that S-CPE can be promising cosmetic ingredients.

Ras and Rho small G proteins: insights from the Schizophyllum commune genome sequence and comparisons to other fungi.

Unlike in animal cells and yeasts, the Ras and Rho small G proteins and their regulators have not received extensive research attention in the case of the filamentous fungi. In an effort to begin to rectify this deficiency, the genome sequence of the basidiomycete mushroom Schizophyllum commune was searched for all known components of the Ras and Rho signalling pathways. The results of this study should provide an impetus for further detailed investigations into their role in polarized hyphal growth, sexual reproduction and fruiting body development. These processes have long been the targets for genetic and cell biological research in this fungus.

Holocellulase activity from Schizophyllum commune grown on bamboo: a comparison with different substrates.

The natural biodiversity that is found in tropical areas offers countless biotechnological opportunities; especially if we take in account that many biomolecules from several microorganisms have supported for many years, different industrial applications in areas such as pharmacology, agro-industry, bioprocess, environmental technology, and bioconversion. In order to find new lignocellulolytic enzymes and evaluate bamboo fibers as substrate, Schizophyllum commune a fungus with broad distribution was isolated and grown during 15 days in liquid culture medium containing 1% lignocellulosic fibers from bamboo, banana stem, and sugarcane bagasse. The enzymatic activity of xylanase, mannanase, polygalacturonase, CMCase, FPase, and avicelase were evaluated. Sugarcane bagasse and banana stem showed to induce higher hollocellulase activity when compared with bamboo as the main carbon source. The physical mechanism that the fungus uses to degrade bamboo was observed not only in fibers naturally infected but also in healthy fibers that were treated and untreated with enzyme solution. SEM analysis showed the structural disruption and invasion of the vascular bundles, parenchyma cells, and parenchymatous tissues as a consequence of the presence of this fungus and the catalytic action of its enzymes into the plant tissue.

Chitin-glucan complex production by Schizophyllum commune submerged cultivation.

Chitin-glucan complex is a fungal origin copolymer that finds application in medicine and cosmetics. Traditionally, the mycelium of Micromycetes is considered as an industrial chitin-glucan complex source. Basidiomycete Schizophyllum commune submerged cultivation for chitin-glucan complex production was studied. In different S. commune strains chitin-glucan complex composed 15.2 +/- 0.4 to 30.2 +/- 0.2% of mycelium dry weight. Optimized conditions for chitin-glucan complex production (nutrient medium composition in g/l: sucrose - 35, yeast extract - 4, Na2HPO4*12H2O - 2.5, MgSO4*7 H2O - 0.5; medium initial pH 6.5; aeration intensity 21 of air per 11 of medium; 144 hours of cultivation) resulted in 3.5 +/- 0.3 g/l complex yield. Redirection of fungal metabolism from exopolysaccharide synthesis to chitin-glucan complex accumulation was achieved most efficiently by aeration intensity increase. Chitin-glucan complex from S. commune had the structure of microfibers with diameter 1-2 microm, had water-swelling capacity of 18 g/g, and was composed of 16.63% chitin and 83.37% glucan with a degree of chitin deacetylation of 26.9%. S. commune submerged cultivation is a potent alternative to Micromycetes for industrial-scale chitin-glucan complex production.

H3K4me2 ChIP-Seq reveals the epigenetic landscape during mushroom formation and novel developmental regulators of Schizophyllum commune.

Mushroom formation represents the most complex multicellular development in fungi. In the model mushroom Schizophyllum commune, comparative genomics and transcriptomics have previously resulted in a regulatory model of mushroom development. However, little is known about the role of epigenetic regulation. We used chromatin immunoprecipitation sequencing (ChIP-Seq) to determine the distribution of dimethylation of lysine 4 on histone H3 (H3K4me2), a mark for transcriptionally active genes, during monokaryotic and dikaryotic development. We identified a total of 6032 and 5889 sites during monokaryotic and dikaryotic development, respectively. The sites were strongly enriched near translation initiation sites of genes. Although the overall epigenetic landscape was similar between both conditions, we identified 837 sites of differential enrichment during monokaryotic or dikaryotic development, associated with 965 genes. Six transcription factor genes were enriched in H3K4me2 during dikaryotic development, indicating that these are epigenetically regulated during development. Deletion of two of these genes (fst1 and zfc7) resulted in arrested development of fruiting bodies, resulting in immature mushrooms. Together these results indicate that H3K4me2 ChIP-Seq is a powerful new tool to map the restructuring of the epigenetic landscape during mushroom development. Moreover, it can be used to identify novel developmental regulators.