Wood Extractives of Silver Fir and Their Antioxidant and Antifungal Properties.

The chemical composition of extractives in the sapwood (SW), heartwood (HW), knotwood (KW), and branchwood (BW of silver fir (Abies alba Mill.) was analyzed, and their antifungal and antioxidant properties were studied. In addition, the variability of extractives content in a centripetal direction, i.e., from the periphery of the stem towards the pith, was investigated. The extracts were analyzed chemically with gravimetry, spectrophotometry, and chromatography. The antifungal and antioxidative properties of the extracts were evaluated by the agar well diffusion method and the diphenyl picrylhydrazyl radical scavenging method. Average amounts of hydrophilic extractives were higher in KW (up to 210.4 mg/g) and BW (148.6 mg/g) than in HW (34.1 mg/g) and SW (14.8 mg/g). Extractives identified included lignans (isolariciresinol, lariciresinol, secoisolariciresinol, pinoresinol, matairesinol) phenolic acids (homovanillic acid, coumaric acid, ferulic acid), and flavonoids epicatechin, taxifolin, quercetin). Secoisolariciresinol was confirmed to be the predominant compound in the KW (29.8 mg/g) and BW (37.6 mg/g) extracts. The largest amount of phenolic compounds was extracted from parts of knots (281.7 mg/g) embedded in the sapwood and from parts of branches (258.9 mg/g) adjacent to the stem. HW contained more lignans in its older sections. Hydrophilic extracts from knots and branches inhibited the growth of wood-decaying fungi and molds. KW and BW extracts were better free radical scavengers than HW extracts. The results of the biological activity tests suggest that the protective function of phenolic extracts in silver fir wood can also be explained by their antioxidative properties. The results of this study describe BW as a potential source of phenolic extractives in silver fir.

Effect of surfactants on the production of polysaccharides from Schizophyllum commune through submerged fermentation.

Schizophyllum commune (S. commune) polysaccharides are biomacromolecules with multiple biological activities and wide applications. In this study, polysaccharide production through submerged fermentation of S. commune using different surfactants was investigated. The addition of 1 g/L of polyoxyethylene sorbitan monooleate (Tween 80) at the beginning of the fermentation showed the best promotional effects on collective exopolysaccharide (EPS) production (which increased by 37.17%) while shortening the production cycle by 2 days. The monosaccharide composition of the EPS produced when the added Tween 80 was similar to that of the control; however, the molecular weight (Mw) was lower. Notably, the addition of Tween 80 significantly increased the ATP levels and the transcription levels of phosphoglucomutase and ß-glucan synthase genes in the polysaccharide synthesis pathway. The addition of Tween 80 reduced the pellet size of the mycelium compared to that of the control, but did not significantly change the microstructure of the mycelial cells. This study proposes an efficient strategy for the production of polysaccharides through submerged fermentation of S. commune, and elucidates the detailed mechanism of using Tween 80 as a fermentation stimulatory reagent.

Cloning, expression, and characterization of a GH 19-type chitinase with antifungal activity from Lysobacter sp. MK9-1.

The chitin-assimilating gram-negative bacterium, Lysobacter sp. MK9-1, was isolated from soil and was the source of a glycoside hydrolase family 19-type chitinase (Chi19MK) gene that is 933-bp long and encodes a 311-residue protein. The deduced amino acid sequence of Chi19MK includes a signal peptide, an uncharacterized sequence, a carbohydrate-binding module family 12-type chitin binding domain, and a catalytic domain. The catalytic domain of Chi19MK is approximately 60% similar to those of ChiB from Burkholderia gladioli CHB101, chitinase N (ChiN) from Chitiniphilus shinanonensis SAY3T, ChiF from Streptomyces coelicolor A3(2), Chi30 from Streptomyces olivaceoviridisis, ChiA from Streptomyces cyaneus SP-27, and ChiC from Streptomyces griseus HUT6037. Chi19MK lacking the signal and uncharacterized sequences (Chi19MKΔNTerm) was expressed in Escherichia coli Rosetta-gami B(DE3), resulting in significant chitinase activity in the soluble fraction. Purified Chi19MKΔNTerm hydrolyzed colloidal chitin and released disaccharide. Furthermore, Chi19MKΔNTerm inhibited hyphal extension in Trichoderma reesei and Schizophyllum commune. Based on quantitative antifungal activity assays, Chi19MKΔNTerm inhibits the growth of Trichoderma viride with an IC50 value of 0.81 µM.

Fungal mycelium classified in different material families based on glycerol treatment.

Fungal mycelium is an emerging bio-based material. Here, mycelium films are produced from liquid shaken cultures that have a Young's modulus of 0.47 GPa, an ultimate tensile strength of 5.0 MPa and a strain at failure of 1.5%. Treating the mycelial films with 0-32% glycerol impacts the material properties. The largest effect is observed after treatment with 32% glycerol decreasing the Young's modulus and the ultimate tensile strength to 0.003 GPa and 1.8 MPa, respectively, whereas strain at failure increases to 29.6%. Moreover, glycerol treatment makes the surface of mycelium films hydrophilic and the hyphal matrix absorbing less water. Results show that mycelium films treated with 8% and 16-32% glycerol classify as polymer- and elastomer-like materials, respectively, while non-treated films and films treated with 1-4% glycerol classify as natural material. Thus, mycelium materials can cover a diversity of material families.

[Fungal sinus infection by Schizophyllum commune: Report of two clinical cases]. / Infección fúngica sinusal por Schizophyllum commune: a propósito de dos casos clínicos.

BACKGROUND: Schizophyllum commune is a basidiomycete fungus which is widely distributed in nature. Its role as responsible for disease in humans is not well known, partly due to its difficult identification. The incorporation of mass spectrometry techniques (MALDI-TOF) and molecular biology to the laboratories has allowed the description of a greater number of cases. CASE REPORT: In this paper, we present two cases in which S. commune was identified as the causative agent of disease: in the first case an immunocompetent patient suffered from chronic rhinosinusitis, and in the second one a sphenoid sinus infection was diagnosed in an immunocompromised patient. In both cases, S. commune was isolated. Its identification was possible by means of MALDI-TOF and this was confirmed in both patients by amplification and sequencing of the ITS region. CONCLUSIONS: In conclusion, S. commune should be considered a potential causative agent of fungal disease. Currently, MALDI-TOF and sequencing techniques are necessary for its identification.

The central role of septa in the basidiomycete Schizophyllum commune hyphal morphogenesis.

The purpose of the present research was to observe in the filamentous basidiomycete Schizophyllum commune, the connection between the nuclear division and polymerization of the contractile actin ring with subsequent formation of septa in living hyphae. The filamentous actin was visualized using Lifeact-mCherry and the nuclei with EGFP tagged histone 2B (H2B). Time-lapse fluorescence microscopy confirmed that in monokaryotic and dikaryotic hyphae, the first signs of the contractile actin ring occur at the site of the nuclear division, in one to two minutes after division. At this stage, the telophase nuclei have moved tens of micrometers from the division site. The actin ring is replaced by the septum in six minutes. The apical cells treated with filamentous actin disrupting drug latrunculin A, had swollen tips but the cells were longer than in control samples due to the absence of the actin rings. The nuclear pairing and association with clamp cell development as well as the clamp cell fusion with the subapical cell was disrupted in latrunculin-treated dikaryotic hyphae, indicating that actin filaments are involved in these processes, also regulated by the A and B mating-type genes. This suggests that the actin cytoskeleton may indirectly be a target for mating-type genes.

Metal release and sequestration from black slate mediated by a laccase of Schizophyllum commune.

Schizophyllum commune is a filamentous basidiomycete which can degrade complex organic macromolecules like lignin by the secretion of a large repertoire of enzymes. One of these white rot enzymes, laccase, exhibits a broad substrate specificity and is able to oxidize a variety of substances including carbonaceous rocks. To investigate the role of laccase in bioweathering, laccase gene lcc2 was overexpressed, and the influence on weathering of black slate, originating from a former alum mine in Schmiedefeld, Germany, was examined. The metal release from the rock material was enhanced, associated with a partial metal accumulation into the mycelium. A sequestration of metals could be shown with fluorescent staining methods, and an accumulation of Zn, Cd, and Pb was visualized in different cell organelles. Additionally, we could show an increased metal resistance of the laccase overexpressing strain.

Epidural abscess caused by Schizophyllum commune: A rare case of rhinogenic cranial complication by a filamentous basidiomycete.

Central nervous system (CNS) infections due to filamentous basidiomycetes are extremely rare. We encountered a case of epidural abscess due to Schizophyllum commune that extended from sinusitis. A 53-year-old Japanese man presented at our hospital with a headache. Computed tomography (CT) of the cranium and sinuses showed ethmoid and sphenoid sinusitis with no intracranial abnormalities. The patient was diagnosed with acute sinusitis and underwent antibiotic treatment. However, the symptoms deteriorated, and the patient came to our hospital again with consciousness disturbance. CT scan of the cranium and sinuses showed no improvement of sinusitis after antibiotic therapy and an epidural abscess emerged in the middle cranial fossa. Therefore, emergency craniotomy and endoscopic sinus fenestration were performed. Filamentous fungal elements were observed in both rhinorrhoea and epidural abscess. The symptoms improved after the operation and administration of liposomal amphotericin B. The clinical isolate was identified as S. commune by a molecular-based method. To our knowledge, this is the first report of epidural abscess due to this fungus. Although rare, clinicians should be aware that S. commune could be a causative agent of CNS infections.

Fungal keratitis due to Schizophyllum commune: an emerging pathogenic fungus.

Fungal keratitis due to Schizophyllum commune is very rare. In this study, we report the clinical and microbiological profile of five patients with fungal keratitis due to S. commune. Direct microscopic examination of corneal scrapings from all five patients showed septate branching hyaline fungal filaments. Similarly, in all five patients Sabouraud dextrose agar (SDA) plates inoculated with corneal scrapings showed white, cottony colonies on the second day of incubation. Lactophenol cotton blue stained wet preparation of 7-day-old colonies on SDA revealed clamp connections and no spores. The fungus was identified by its characteristic clamp connections, fan-shaped bracket fruiting body with pinkish-grey longitudinally split-radiating gills. The phenotypic identification of one of the five isolates further conformed by ITS sequencing. Treatment outcome was available for two of the five patients; in these two patients, the keratitis resolved with topical natamycin.

Schizophyllum radiatum, an Emerging Fungus from Human Respiratory Tract.

Schizophyllum is an important genus of basidiomycetes that, apart from being of genetic and biotechnological interest, is also reported to be a plant and animal pathogen. Schizophyllum commune is the best-known species and the only one reported from clinical specimens thus far, being recovered mainly from the respiratory tract. The aim of this study was to determine the species diversity of 23 clinical isolates of Schizophyllum from the United States using multilocus phylogenetic analysis and their in vitro susceptibilities to six drugs. The markers used for sequencing were the internal transcribed spacer (ITS), a portion of the nuclear large subunit (LSU) of ribosomal DNA, the RNA polymerase II second-largest subunit (RPB2), and the translation elongation factor 1α (EF-1α) gene. The analyses revealed that 22 of the clinical isolates were in the Schizophyllum radiatum clade with high support values and 1 isolate was in the S. commune clade. This is the first report of this species in clinical samples. The two species mentioned above showed very similar morphological features in culture (i.e., white, cottony, unsporulated colonies composed of hyphae with clamp connections), making morphological discrimination between the two impossible. An epitype is designed for S. radiatum, and its sequences have been deposited in GenBank. The antifungal that showed the greatest in vitro activity against the strains tested was shown to be amphotericin B. In general, the strains of S. radiatum showed higher MICs than S. commune.

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.

Biosynthesis and Secretion of Indole-3-Acetic Acid and Its Morphological Effects on Tricholoma vaccinum-Spruce Ectomycorrhiza.

Fungus-derived indole-3-acetic acid (IAA), which is involved in development of ectomycorrhiza, affects both partners, i.e., the tree and the fungus. The biosynthesis pathway, excretion from fungal hyphae, the induction of branching in fungal cultures, and enhanced Hartig net formation in mycorrhiza were shown. Gene expression studies, incorporation of labeled compounds into IAA, heterologous expression of a transporter, and bioinformatics were applied to study the effect of IAA on fungal morphogenesis and on ectomycorrhiza. Tricholoma vaccinum produces IAA from tryptophan via indole-3-pyruvate, with the last step of this biosynthetic pathway being catalyzed by an aldehyde dehydrogenase. The gene ald1 was found to be highly expressed in ectomycorrhiza and induced by indole-3-acetaldehyde. The export of IAA from fungal cells is supported by the multidrug and toxic extrusion (MATE) transporter Mte1 found in T. vaccinum. The addition of IAA and its precursors induced elongated cells and hyphal ramification of mycorrhizal fungi; in contrast, in saprobic fungi such as Schizophyllum commune, IAA did not induce morphogenetic changes. Mycorrhiza responded by increasing its Hartig net formation. The IAA of fungal origin acts as a diffusible signal, influencing root colonization and increasing Hartig net formation in ectomycorrhiza.

Characterization of α-1,3-glucanase isozyme from Paenibacillus glycanilyticus FH11 in a new subgroup of family 87 α-1,3-glucanase.

Two α-1,3-glucanase isozymes, designated as α-1,3-glucanase 1 (Agl-FH1) and α-1,3-glucanase 2 (Agl-FH2), were purified from the culture medium of Paenibacillus glycanilyticus FH11. Agl-FH1 and Agl-FH2 exhibited similar characteristics such as optimal pH, pH stability, optimal temperature, thermostability, and molecular masses on SDS-PAGE. However, their hydrolysis products of α-1,3-glucan varied somewhat. Agl-FH1 hydrolyzed α-1,3-glucan into a mixture of maltotriose and maltotetraose, and maltotetraose was the major hydrolysis product of Agl-FH2. N-terminal amino acid sequence analysis and LC-MS/MS analysis of trypsin digested fragments revealed several differences between the amino acid sequences of Agl-FH1 and Agl-FH2. Genes of Agl-FH1 and Agl-FH2 were subcloned into an expression plasmid, and both enzymes were successfully expressed in Escherichia coli. The recombinant Agl-FH1 and Agl-FH2 exhibited the same enzymatic properties as those of each wild-type enzyme, and both of the recombinants showed the activity on the protoplast formation of Schizophyllum commune mycelia. A great diversity was detected in the C-terminal region of family 87 α-1,3-glucanases. Compared with Agl-FH2 which is highly sequence-related to the known α-1,3-glucanases, the C-terminal region of Agl-FH1 has only slight similarity to them (approximately 20% identity). Our analysis revealed that Agl-FH1 was the first member of a new subgroup of family 87 α-1,3-glucanases.

Molecular characterization and in vitro antifungal susceptibility profile of Schizophyllum commune, an emerging basidiomycete in bronchopulmonary mycoses.

Schizophyllum commune (n = 30) showed lowest geometric mean MICs of isavuconazole (0.19 µg/ml), itraconazole (0.2 µg/ml), voriconazole (0.24 µg/ml), and amphotericin B (0.29 µg/ml) and high geometric mean MICs of fluconazole (19.39 µg/ml) and flucytosine (17.28 µg/ml). Five cases (of 8) of allergic bronchopulmonary mycosis that were treated with itraconazole had no recrudescence after 6 to 24 months of follow-up. One case each of invasive pulmonary mycosis and fungal ball were treated successfully with voriconazole and itraconazole.

Schizophyllum commune as an emerging fungal pathogen: a review and report of two cases.

We report Schizophyllum commune as the aetiological agent of one case each of allergic broncho-pulmonary mycosis (ABPM) and pulmonary fungal ball, and present a literature review. The fungus was characterised by clamp connections, hyphal spicules, and formation of basidiocarps with basidiospores. The phenotypic identification was confirmed by sequencing of the ITS region. To-date, ABPM and pulmonary fungal ball to S. commune have been reported exclusively from Japan and North America respectively. Of the 71 globally reported cases due to S. commune, 45 (63%) were bronchopulmonary, 22 (31%) sinusitis and 4 extrapulmonary. Taken together, cases of bronchopulmonary disease and sinusitis numbered 67 (94%), indicating the respiratory tract as the primary target of disease. Concerning the country-wise distribution, Japan topped the list with 33 cases (46%), followed by Iran - 7 cases (10%), U.S.A. - 6 cases (9%), and a lower prevalence of 1.4-6% for the remaining 12 countries. The preponderance of the disease in Japan may be attributed to its greater awareness vis-à-vis that in other countries rather than to any geographical/climatic factors. We believe that the burden of S. commune-incited disease is currently underestimated, warranting comprehensive prospective studies to determine its prevalence.

Molecular identification of Schizophyllum commune as a cause of allergic fungal sinusitis.

Schizophyllum commune, a basidiomycetous fungus, rarely causes disease in humans. We report a rare case of allergic fungal sinusitis caused by S. commune in a 14-yr-old girl. The patient presented with nasal obstruction and a purulent nasal discharge. Materials obtained during endoscopic surgery of the frontal recess revealed allergic mucin and a few fungal hyphae. A potato dextrose agar (PDA) culture from the allergic mucin yielded a rapidly growing white woolly mold. Although no distinctive features including hyphae bearing spicules or a clamp connection were present, the case isolate disclosed compatible mycological features including growth at 37℃, susceptibility to cycloheximide, and production of a tart and disagreeable smell. S. commune was confirmed by sequence analysis of the internal transcribed spacer region and D1/D2 regions of the 26S ribosomal DNA. We believe this is the first report of allergic fungal sinusitis caused by S. commune in Korea. Moreover, this report highlights the value of gene sequencing as an identification tool for non-sporulating isolates of S. commune.

Phleomycin increases transformation efficiency and promotes single integrations in Schizophyllum commune.

Phleomycin is mutagenic by introducing double-strand breaks in DNA. The ble gene of Streptoalloteychus hindustanus, which confers resistance to this substance, is widely used as a selection marker for transformation. Schizophyllum commune grows on 25 microg of phleomycin ml(-1) after introduction of a resistance cassette based on the ble gene. However, we here report that growth of resistant colonies on this concentration of phleomycin resulted in aberrant colony morphologies. Apparently, phleomycin was mutagenic despite acquired resistance. Therefore, a new selection system was developed based on resistance to the antibiotic nourseothricin. However, the transformation efficiency was tenfold lower than that obtained with phleomycin as a selection agent. This low transformation efficiency could be rescued by addition of a nonselective concentration of phleomycin during protoplast regeneration. This was accompanied by a higher incidence of single-copy integrations and with an increase of expression of key genes involved in double-strand break repair. Taken together, we conclude that the effect of a nonselective concentration of phleomycin strongly resembles the effect of restriction enzyme-mediated integration (REMI) but, unlike REMI, it does not depend on the presence of a target restriction site.