Changes in volatile production during interspecific interactions between four wood rotting fungi growing in artificial media

Effects of volatile organic compounds (VOCs) produced during interspecific mycelial interactions were examined by measuring extension rate of ‘target’ fungi growing in agar plates taped above two interacting mycelia – Bjerkandera adusta, Hypholoma fasciculare, Stereum gausapatum and Trametes versicolor in all combinations. Extension rates of T. versicolor, S. gausapatum and H. fasciculare above self-pairings were not significantly different (P > 0.05) to growth above agar controls, but the extension rate of B. adusta was significantly (P ≤ 0.05) greater. Extension rates of T. versicolor and B. adusta were often significantly greater above inter-specific interactions and above other species compared with growth above self or agar controls. VOC production was quantified and qualified, by gas chromatography–mass spectrometry (GC/MS), over the course of interactions with T. versicolor replacing S. gausapatum, deadlocking with B. adusta and replaced by H. fasciculare. VOC production was species- and interaction-specific. It varied over the time course of interactions, and changes in production were correlated with production of pigments in interactions involving S. gausapatum. VOCs included 3 monoterpenes, benzoic acid, alkenols of different chain lengths, two long-chain hydrocarbons and a quinolinium-like compound. Their possible roles are discussed.     

Changes in oxidative enzyme activity during interspecific mycelial interactions involving the white-rot fungus Trametes versicolor

Interspecific fungal antagonism leads to biochemical changes in competing mycelia, including up-regulation of oxidative enzymes. Laccase, manganese peroxidase (MnP), manganese-repressed peroxidase (MRP) and lignin peroxidase (LiP) gene expression and enzyme activity were compared during agar interactions between Trametes versicolor and five other wood decay fungi resulting in a range of interaction outcomes from deadlock to replacement of one fungus by another. Increased laccase and Mn-oxidising activities were detected at all interaction zones, but there were few changes in activity in regions away from the interaction zone in T. versicolor mycelia compared to self-pairings. Whilst no LiP activity was detected in any pairing, low level LiP gene expression was detected. MnP activity was detected but not expression of MnP genes; instead, MRP could explain the observed activity. No relationship was found between extent of enzyme activity increase and interaction outcome. Similarities between patterns of gene expression and enzyme activity are discussed.     

Are stag beetles fungivorous?

Stag beetle larvae generally feed on decaying wood; however, it was unknown whether they can use wood-rotting fungi alone as food. Here, to clarify this, newly hatched larvae of Dorcus rectus (Motschulsky) (Coleoptera: Lucanidae) were reared for 14 days on artificial diets containing a fixed amount of freeze-dried mycelia of the following fungi: Bjerkandera adusta, Trametes versicolor, Pleurotus ostreatus, and Fomitopsis pinicola. The mean incremental gain in larval body mass was greatest on diets containing B. adusta, followed by T. versicolor, P. ostreatus, and F. pinicola. The growth rate of body mass correlated positively with mycelial nitrogen content of the different fungi. It also correlated positively with the mycelial content of B. adusta in the diet. Addition of antibiotics to diets with mycelia nearly halved larval growth, indicating that larvae were able to use fungal mycelia as food without the assistance of associated microbes although the microbes positively affected larval growth. Four newly hatched larvae reared on artificial diets containing B. adusta mycelia developed to the second instar in 21-34 days; and one developed to the third (=final) instar. This study provides evidence that fungi may constitute the bulk of the diet of D. rectus larvae.     

Biodegradation of azo and phthalocyanine dyes by Trametes versicolor and Bjerkandera adusta

Eighteen fungal strains, known for their ability to degrade lignocellulosic material or lignin derivatives, were screened for their potential to decolorize commercially used reactive textile dyes. Three azo dyes, Reactive Orange 96, Reactive Violet 5 and Reactive Black 5, and two phthalocyanine dyes, Reactive Blue 15 and Reactive Blue 38, were chosen as representatives of commercially used reactive dyes. From the 18 tested fungal strains only Bjerkandera adusta, Trametes versicolor and Phanerochaete chrysosporium were able to decolorize all the dyes tested. During degradation of the nickel-phthalocyanine complex, Reactive Blue 38, by B. adusta and T. versicolor respectively, the toxicity of this dye to Vibrio fischeri was significantly reduced. In the case of Reactive Violet 5, a far-reaching detoxification was achieved by treatment with B. adusta. Reactive Blue 38 and Reactive Violet 5 were decolorized by crude exoenzyme preparations from T. versicolor and B. adusta in a H₂O₂-dependent reaction. Specific activities of the exoenzyme preparations with the dyes were determined and compared to oxidation rates by commercial horseradish peroxidase. Received: 3 February 1997 / Received revision: 9 April 1997 / Accepted: 13 April 1997     

Isolation and evaluation of terrestrial fungi with algicidal ability from Zijin Mountain,Nanjing, China

Approximately 60 fungal isolates from Zijin Mountain (Nanjing, China) were screened to determine their algicidal ability. The results show that 8 fungi belonging to Ascomycota and 5 belonging to Basidiomycota have algicidal ability. Of these fungi, Irpex lacteus T2b, Trametes hirsuta T24, Trametes versicolor F21a, and Bjerkandera adusta T1 showed strong algicidal ability. The order of fungal chlorophyll-a removal efficiency was as follows: T. versicolor F21a > I. lacteus T2b > B. adusta T1 > T. hirsuta T24. In particular, T. versicolor F21a completely removed algal cells within 30 h, showing the strongest algicidal ability. The results also show that all 4 fungal species degraded algal cells through direct attack. In addition, most of the tested fungi from the order Polyporales of Basidiomycota exhibited strong algicidal activity, suggesting that most fungi that belong to this order have algicidal ability. The findings of this work could direct the search for terrestrial fungi for bloom control.     

Detection,quantification and identification of fungal extracellular laccases using polyclonal antibody and mass spectrometry

This study presents a combined method to analyze extracellular fungal laccases using a new anti-laccase antibody together with the identification of tryptic laccase peptides by mass spectrometry (nanoLC–ESI–MS/MS). The polyclonal anti-laccase antibody LccCbr2 was raised against peptides designed from the copper binding region II of fungal laccases using in silico data obtained from GenBank database. As a consequence, detection requires denaturation of the enzymes due to the stable conformation of the copper binding region II. The specificity of the antibody was shown with denatured laccase Lcc1 of Coprinopsis cinerea and laccase of Hypholoma fasciculare. LccCbr2 detected amounts as low as 5 ng of highly purified laccase, indicating a possible use of the antibody for quantification of laccase proteins. Denatured extracellular laccases from culture supernatants of the basidiomycetes C. cinerea, H. fasciculare, Lentinula edodes, Mycena sp., Piriformospora indica, Pleurotus cornucopiae, Pleurotus ostreatus, Pycnoporus cinnabarinus, Trametes versicolor and furthermore the ascomycete Verpa conica were detected with apparent molecular masses between 60 and 70 kDa by LccCbr2. The identity of extracellular laccases from C. cinerea, H. fasciculare, P. ostreatus, P. cinnabarinus and T. versicolor were verified by tryptic peptides using nanoLC–ESI–MS/MS.     

In-vitro compatibility evaluation of fungal mixed culture for bioconversion of domestic wastewater sludge

Six different fungal strains/isolates were selected after conducting a series of experiments of isolation and screening to evaluate their successful adaptation and growth to domestic wastewater sludge and its efficient bioconversion into compost. Two different fungi were grown in the same petri dish 4 cm apart in two culture media, potato dextrose agar (PDA) and malt extract agar (MEA). Fifteen different in-vitro interactions were studied and summarized according to five possible outcomes, i.e., mutual intermingling, partial mutual intermingling, inhibition at contact point, inhibition at a distance and replacement. The interaction of Trichoderma hazianum ^s Rifai with Phanerochaete chrysosporium 2094 was identified as mutual intermingling. The partial mutual intermingling of T. hazianum ^s with Mucor hiemalis Wehmer suggested compatibility of the two strains without showing any abnormal effects. Perhaps these two combinations may interact mutually in any mixed culture programme. The fungal strain Aspergillus versicolor Vuill performed as a strong repellent and all interactions exhibited deadlock/inhibition at a certain distance. The isolate RW-Pl 512 from the gill of a basidiomycete from a rotten wood stub actively replaced the strain M. hiemalis in in-vitro culture.     

Modification of wheat straw lignin by solid state fermentation with white-rot fungi

The potential of crude enzyme extracts, obtained from solid state cultivation of four white-rot fungi (Trametes versicolor, Bjerkandera adusta, Ganoderma applanatum and Phlebia rufa), was exploited to modify wheat straw cell wall. At different fermentation times, manganese-dependent peroxidase (MnP), lignin peroxidase (LiP), laccase, carboxymethylcellulase (CMCase), avicelase, xylanase and feruloyl esterase activities were screened and the content of lignin as well as hydroxycinnamic acids in fermented straw were determined. All fungi secreted feruloyl esterase while LiP was only detected in crude extracts from B. adusta. Since no significant differences (P > 0.05) were observed in remaining lignin content of fermented straw, LiP activity was not a limiting factor of enzymatic lignin removal process. The levels of esterified hydroxycinnamic acids degradation were considerably higher than previous reports with lignocellulosic biomass. The data show that P. rufa, may be considered for more specific studies as higher ferulic and p-coumaric acids degradation was observed for earlier incubation times.     

Fungal control of early-stage bacterial community development in decomposing wood

The earliest stages of bacterial colonisation of wood have received little attention, particularly with respect to how the colonisation process may be affected by the presence of wood-decay fungi. This study used 16s rRNA gene sequencing to examine the bacterial community in wood that had been incubated in the field for 14 or 84 d, either in wood uncolonised by fungi or pre-colonised by Vuilleminia comedens, Trametes versicolor or Hypholoma fasciculare. All three fungal species significantly delayed bacterial colonisation of the wood. V. comedens and H. fasciculare also reduced bacterial OTU richness and altered bacterial community composition, increasing the relative abundance of Burkholderiales and reducing the proportion of Enterobacteriaceae and Bacteroidetes. Wood that had not been pre-colonised showed seasonal differences between autumn and spring: bacterial richness increased between 14 d and 84 d in the spring, but not in the autumn. Community composition at 84 d in spring was also different to the other time points, with reduced dominance of Gamma-proteobacteria. Archaea were also detected in nearly a third of samples, but with no apparent pattern, and always at low abundances.     

Comparison of ligninolytic activities of selected white-rot fungi

Summary Six fast growing ligninolytic white-rot fungi were compared with Phanerochaete chrysosporium. The results showed that the fungi have similar ligninolytic systems, although minor differences exist. Like in P. chrysosporium the ligninolytic system could be induced by veratryl alcohol in Coriolus versicolor and Chrysosporium pruinosum. These three lignin peroxidase producing fungi were the fastest lignin degraders in stationary cultures, whereas in agitated cultures Bjerkandera adusta showed highest lignin degradation rates. Metabolites accumulating during the degradation of veratryl alcohol were analyzed and compared. Peroxidase production seems to be a common feature of all the tested fungi. Polyclonal antibodies against the lignin peroxidase with pl of 4.65 from P. chrysosporium reacted with the extracellular peroxidases of C. pruinosum, C. versicolor and B. adusta, but not with those of Pleurotus ostreatus.Dedicated to Professor Dr. Hans-Jürgen Rehm on the occasion of his 60th birthday     

Positive relationship between wood size and basidiocarp production of polypore fungi in Alnus acuminata forest

Polypores play a major role in wood decomposition. Based on presence/absence of basidiocarps, it has been shown that richness of polypores in forests is strongly affected by the size of logs. However, no study has addressed the relationship between the log size and basidiocarp production. Here, we examined the relationship between log diameter and number of basidiocarps and volume of the fructification (as surrogate of biomass) of the polypore community in Andean Alder forests from Northwest Argentina. We found a positive relationship between log diameter and basidiocarp production in the whole community analysis (fructifications of all species). This pattern was also followed by dominant species (Bjerkandera adusta, Trametes cubensis and T. versicolor) analyzed individually. The relationship was generally higher for volume of fructification than for number of basidiocarps. Through these effects on basidiocarp production, higher log diameter could promote higher sexual spore production and dispersal hence a higher genetic variability and viable populations of wood-decay species.     

Ribosomal proteins. 33. Location of amino-acid replacements in protein S12 isolated from Escherichia coli mutants resistant to streptomycin

Ribosomal protein S12, the protein coded by the strA ciatron, was isolated from nine streptomycin-resistant mutants originating from various Escherichia coli strains. Analysis of the tryptic peptides revealed that each mutant had a single amino-acid replacement in one of two peptides: in mutants belonging to the allele types strA1, strA2 and strA60 the lysine residue in position 42 (peptide T6) of protein S12 is replaced by one of three ammo acids (asparagine, threonine or arginine) whereas the mutants belonging to allele type strA40 have a replacement of lysine by arginine in peptide T15. There is a good agreement between our protein-chemical data and earlier genetic data on streptomycin-resistant mutants.     

Specific Detection of Bjerkandera adusta by Polymerase Chain Reaction and Its Incidence in Fungus-Associated Chronic Cough

Chronic cough is a common symptom at outpatient care. An uncontrollable cough with difficulty in treatment is called chronic idiopathic cough. Recent reports have demonstrated that the presence of basidiomycetous fungi in sputum is an important clinical finding that assists in clarifying the cause of chronic cough in some cases. Research has suggested that Bjerkandera adusta is related to fungus-associated chronic cough (FACC). FACC is defined as a chronic cough associated with basidiomycetous fungi found in induced sputum and can be treated with antifungal medication. B. adusta is one of the basidiomycetous fungi that exist in cosmopolitan environments. The aim of this study was to develop a B. adusta detection method using polymerase chain reaction (PCR) with a specific primer set and to research the incidence of B. adusta in FACC. The new method successfully detected B. adusta from FACC patients. The incidence of B. adusta in FACC was 42.86 %. Antifungal drugs were effective in most cases. Significant differences in treatment duration between B. adusta patients and non-B. adusta patients were observed. It is therefore suggested that the presence of B. adusta may be one of the allergic intractable factors of chronic cough. This finding may provide identifiable differences in clinical manifestations between B. adusta and non-B. adusta in FACC and lead to possible differing remedies to treat the two forms. PCR can specifically detect B. adusta from patients suffering from chronic cough and provides a new diagnosis for FACC associated with B. adusta.     

The influence of fungal food quality on the growth and fecundity of Folsomia candida (Collembola: Isotomidae)

Summary The Basidiomycete fungi Coriolus versicolor and Hypholoma fasciculare were grown in liquid media containing 2, 20, 200 and 2,000 ppm nitrogen (as asparagine) and fed to cultures of Folsomia candida. Collembola feeding on both species of fungi exhibited trends of increased moulting and egg laying rates up to 200 ppm N and an inhibition of growth and fecundity at 2,000 ppm N. The differences in moulting rates between individual treatments were small for both species of fungi and not all the pair wise comparisons of treatments were significantly different. Egg laying rates of collembola fed C. versicolor showed a highly significant response to all levels of N in the growth medium and egg production at 200 ppm N was over three times higher than at 2 ppm N. Collembola fed H. fasciculare showed a less marked fecundity response to the different nitrogen levels and egg production at 200 ppm was approximately 1.5 times higher than at 2 ppm N. Both moulting and egg laying rates were significantly affected by the species of fungus presented as food to the collembola. The patterns of growth and reproduction of starved control groups of F. candida as well as those fed the test fungi demonstrate the adaptability of this species to changes in the quality and quantity of available food.     

Discovery and characterization of new O-methyltransferase from the genome of the lignin-degrading fungus Phanerochaete chrysosporium for enhanced lignin degradation

Using bioinformatic homology search tools, this study utilized sequence phylogeny, gene organization and conserved motifs to identify members of the family of O-methyltransferases from lignin-degrading fungus Phanerochaete chrysosporium. The heterologous expression and characterization of O-methyltransferases from P. chrysosporium were studied. The expressed protein utilized S-(5′-adenosyl)-l-methionine p-toluenesulfonate salt (SAM) and methylated various free-hydroxyl phenolic compounds at both meta and para site. In the same motif, O-methyltransferases were also identified in other white-rot fungi including Bjerkandera adusta, Ceriporiopsis (Gelatoporia) subvermispora B, and Trametes versicolor. As free-hydroxyl phenolic compounds have been known as inhibitors for lignin peroxidase, the presence of O-methyltransferases in white-rot fungi suggested their biological functions in accelerating lignin degradation in white-rot basidiomycetes by converting those inhibitory groups into non-toxic methylated phenolic ones.     

Overproduction of Trametes versicolor laccase by making glucose starvation using yeast

In the present paper, overproduction of laccase by microbe interaction was studied. When Trametes versicolor was co-cultured with Candida sp. HSD07A in submerged fermentation, laccase activity could be improved significantly and reached 10500 ± 160 U/l, 11.8 times more than that of the contrast group. Fermentation tests of the yeast indicated that it could produce amylase and cellulase, but couldn’t excrete laccase and the overproductive laccase was produced by T. versicolor; the interaction mechanism between T. versicolor and Candida sp. HSD07A was investigated and the results showed that amylase and cellulose could hydrolyze cell walls of T. versicolor; however, the degree of hydrolysis was at a very low level, could not lead to overproduction of laccase; glucose starvation state made by the yeast was the real reason why T. versicolor could overproduce laccase; moreover, this study also proved that making glucose starvation using the yeast was a novel and effective method.     

Effect of Residual Lignin Type and Amount on Bleaching of Kraft Pulp by Trametes versicolor

The white rot fungus Trametes (Coriolus) versicolor can delignify and brighten unbleached hardwood kraft pulp within a few days, but softwood kraft pulps require longer treatment. To determine the contributions of higher residual lignin contents (kappa numbers) and structural differences in lignins to the recalcitrance of softwood kraft pulps to biobleaching, we tested softwood and hardwood pulps cooked to the same kappa numbers, 26 and 12. A low-lignin-content (overcooked) softwood pulp resisted delignification by T. versicolor, but a high-lignin-content (lightly cooked) hardwood pulp was delignified at the same rate as a normal softwood pulp. Thus, the longer time taken by T. versicolor to brighten softwood kraft pulp than hardwood pulp results from the higher residual lignin content of the softwood pulp; possible differences in the structures of the residual lignins are important only when the lignin becomes highly condensed. Under the conditions used in this study, when an improved fungal inoculum was used, six different softwood pulps were all substantially brightened by T. versicolor. Softwood pulps whose lignin contents were decreased by extended modified continuous cooking or oxygen delignification to kappa numbers as low as 15 were delignified by T. versicolor at the same rate as normal softwood pulp. More intensive O₂ delignification, like overcooking, decreased the susceptibility of the residual lignin in the pulps to degradation by T. versicolor.     

Solid-phase treatment with the fungus Trametes versicolor substantially reduces pharmaceutical concentrations and toxicity from sewage sludge

For safe biosolid-land-applying, sludge should be contaminant-free. However, it may contain important amounts of micropollutants, not removed in the wastewater-treatment-processes. An alternative treatment with the fungus Trametes versicolor was applied in sterile solid-phase systems consisting of sludge and a lignocellulosic substrate. Fungal colonization and activity were demonstrated during the process, according to monitoring of ergosterol, laccase activity and the naproxen-degradation test (ND24). Fourteen out of 43 analyzed pharmaceuticals were found in the raw sludge. After treatment, phenazone, bezafibrate, fenofibrate, cimetidine, clarithromycin, sulfamethazine and atenolol were completely removed, while removals between 42% and 80% were obtained for the remaining pharmaceuticals. Toxicological analyses (Daphnia magna, Vibrio fischeri and seed germination) showed an important reduction in sludge toxicity after treatment. Results suggest that a solid-phase treatment with T. versicolor may reduce the ecotoxicological impact of micropollutants present in sewage sludge. This is the first report of a fungal-approach for elimination of emerging pollutants from biosolids.     

Cooperation of dendritic cells with naïve lymphocyte populations to induce the generation of antigen-specific antibodies in vitro

The production of monoclonal antibodies by hybridoma technology is dependent on lymphocytes taken from vertebrates which have to be immunized against the corresponding antigen. We present here our first experiments which should allow the replacement of this in vivo immunization step by an in vitro immunization procedure. This work provides new possibilities for the specific activation of immune cells in order to use them for the generation of antibodies which are not of murine origin. Bone marrow-derived dendritic cells were loaded with antigen and co-cultured with naïve T and B lymphocytes of non-immunized mice. The interaction and activation of the different cell types were investigated by measuring the expression of specific cell surface markers, the release of activation-dependent interleukins and the secretion of antigen-specific antibodies. We could demonstrate that dendritic cells process and present antigen fragments and activate T cells, that T cells proliferate and release activation-induced interleukins, and that B cells maturate under the influence of activated T cells and secrete antigen-specific antibodies.