Optimized fungal defibrillation of wood using selected strains of <Emphasis Type="Italic">Gloeophyllum trabeum</Emphasis> and <Emphasis Type="Italic">Resinicium bicolor</Emphasis>

We studied the capacity of a selected strain of Gloeophyllum trabeum, alone or in combination with Resinicium bicolor, to defibrillate non treated deciduous wood at a semi industrial composting scale. Inoculum amount, aeration of the composted wood, type (freshly cut wood and wood stored since several years) and the quantity of wood used were analysed. The remaining cellular cohesion, lignin and holocellulose, as well as fungal biomass content in the wood after various treatments were determined. Results showed that G. trabeum rapidly colonised the non-sterile substrate and caused greater biodefibrillation compared with the control (non inoculated wood). Effects of the various treatments on biodefibrillation were compared and are discussed.     

Diffuse competition for heterogeneous substrate in soil among six species of wood-decomposing basidiomycetes

Competition among six wood decay fungi was studied using 15×15 mm wood blocks placed in 250×250 mm plastic trays filled with unsterilized sand or clay. The wood blocks were preinoculated with Heterobasidion annosum (Fr.) Bref., Resinicium bicolor (Alb. & Schw. ex Fr.) Parm., Phanerochaete sanguinea (Fr.) Hjortstam, Coniophora sp. DC. ex Me"rat, Armillaria borealis Marxmuller and Korhonen and Hypholoma capnoides (Fr.) Kummer before they were combined in all possible combinations in the trays. Two methods were used, one with all wood blocks inoculated, and one with sterilized non-inoculated wood blocks distributed between the inoculated ones. Wood blocks preinoculated with the six species were also used in a pairwise competition test. Following incubation for 9 months in darkness at 21°C, mycelia were reisolated and identified. R. bicolor was most successful at invading through the soil and replacing other species in the wood blocks. P. sanguinea, Coniophora sp. and H. capnoides also had some success.     

Assessment of saccharification efficacy in the cellulase system of the brown rot fungus Gloeophyllum trabeum

Brown rot fungi uniquely degrade wood by creating modifications thought to aid in the selective removal of polysaccharides by an incomplete cellulase suite. This naturally successful mechanism offers potential for current bioprocessing applications. To test the efficacy of brown rot cellulases, southern yellow pine wood blocks were first degraded by the brown rot fungus Gloeophyllum trabeum for 0, 2, 4, and 6 weeks. Characterization of the pine constituents revealed brown rot decay patterns, with selective polysaccharide removal as lignin compositions increased. G. trabeum liquid and solid state cellulase extracts, as well as a commercial Trichoderma reesei extract (Celluclast 1.5 L), were used to saccharify this pretreated material, using β-glucosidase amendment to remove limitation of cellobiose-to-glucose conversion. Conditions varied according to source and concentration of cellulase extract and to pH (3.0 vs. 4.8). Hydrolysis yields were maximized using solid state G. trabeum extracts at a pH of 4.8. However, the extent of glucose release was low and was not significantly altered when cellulase loading levels were increased threefold. Furthermore, Celluclast 1.5 L continually outperformed G. trabeum cellulase extracts, although extent of glucose release never exceeded 22.0%. Results suggest methodological advances for utilizing crude G. trabeum cellulases and imply that the suboptimal hydrolysis levels obtained with G. trabeum and Celluclast 1.5 L cellulases, even at high loading levels, may be due to brown rot modifications insufficiently distributed throughout the pretreated material.     

The isolation and immunolocalization of iron-binding compounds produced by Gloeophyllum trabeum

Summary Low molecular weight iron-binding compounds are produced by the brown-rot fungus Gloeophyllum trabeum. These chelators may function in scavenging transition metals for fungal metabolism and extracellular enzyme production. Because of the low molecular mass of the chelate-metal complex (below 1000 Da), and the oxidizing potential of the bound transition metals, certain chelating compounds could also play a role in the early stages of cellulose depolymerization by brown-rot fungi. High-affinity iron-binding compounds were isolated and partially purified from both liquid cultures of the brown-rot Gloeophyllum trabeum and from infected wood. Chelating compounds purified by thin-layer chromatography were used to prepare specific antibodies. These antibodies were shown to detect the chelator in infected wood and liquid fungal cultures by enzyme-linked immunosorbent assay and could be used in immunotransmission electron microscopy to visualize the high-affinity iron-binding compounds in situ. Elucidating the physiological roles of fungal chelate-metal complexes and determining their function in lignocellulose depolymerization will help us to better understand the mechanism of wood biodegradation.Publication no. 1549 Maine Agricultural Experiment Station Offprint requests to: J. Jellison     

Progressive changes in cell wall components of Pinus radiata during decay

Progressive changes in solubility characteristics and lignin content of Pinus radiata sapwood were assessed when small blocks were subjected to decay by brown (Gloeophyllum trabeum) and white (Perenniporia tephropora) rot fungi. The brown rot species removed lignin in approximate proportion to weight loss up to 10%; thereafter the amount of lignin altered little. In contrast, the decline in lignin content was near linear for P. tephropora. Increases in solubility (particularly with hot water and dilute alkali), in sugar content and in the acidity of aqueous extracts were recorded in wood blocks decayed to 15–20% weight loss. While these effects were more pronounced in samples decayed by G. trabeum, it appears that with both organisms structural components were degraded faster than the products could be utilised. In this case, cell wall chemistry may not have been a major determinant of weight losses.     

Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, <Emphasis Type="Italic">Gloeophyllum</Emphasis><Emphasis Type="Italic">trabeum</Emphasis>

A pyranose 2-oxidase gene from the brown-rot basidiomycete Gloeophyllum trabeum was isolated using homology-based degenerate PCR. The gene structure was determined and compared to that of several pyranose 2-oxidases cloned from white-rot fungi. The G. trabeum pyranose 2-oxidase gene consists of 16 coding exons with canonical promoter CAAT and TATA elements in the 5′UTR. The corresponding G. trabeum cDNA was cloned and contains an ORF of 1,962 base pairs encoding a 653 amino acid polypeptide with a predicted molecular weight of 72 kDa. A Hisx6 tagged recombinant G. trabeum pyranose 2-oxidase was generated and expressed heterologously in Escherichia coli yielding 15 U enzyme activity per ml of induced culture. Structural alignment and phylogenetic analysis were performed and are discussed.     

Impact of Norway spruce pre-degradation stages induced by Gloeophyllum trabeum on fungal and bacterial communities

In forest ecosystems, fungi are the key actors in wood decay. They have the capability to degrade lignified substrates and the woody biomass of coniferous forests, with brown rot fungi being common colonizers. Brown rots are typically involved in the earliest phase of lignocellulose breakdown, which therefore influences colonization by other microorganisms. However, few studies have focused on the impact of introducing decayed wood into forest environments to gauge successional colonization by natural bacterial and fungal communities following partial decay. This study aimed to address this issue by investigating the bacterial and fungal colonization of Norway spruce (Picea abies) wood, after intermediate and advanced laboratory-based, pre-decay, by the brown rot fungus Gloeophyllum trabeum. Using Illumina metabarcoding, the in situ colonization of the wood blocks was monitored 70 days after the blocks were placed on the forest floor and covered with litter. We observed significant changes in the bacterial and fungal communities associated with the pre-decayed stage. Further, the wood substrate condition acted as a gatekeeper by reducing richness for both microbial communities and diversity of fungal communities. Our data also suggest that the growth of some fungal and bacterial species was driven by similar environmental conditions.     

Pronounced ecological separation between two closely related lineages of the polyporous fungus Gloeoporus taxicola

Gloeoporus taxicola is a saprotrophic polypore that produces annual fruit bodies, in Scandinavia exclusively on coniferous wood. In this study, we demonstrate the existence of two ecotypes of G. taxicola in Norway; a coastal lowland form fruiting on dead logs and dying branches of standing Pinus sylvestris, and a highland form largely fruiting on trunks of Picea abies in moist, old-growth forests. Thus, a distinct ecological separation has happened between the two forms both concerning substrate and environmental requirements, leading to a rather distinct difference in distribution. Genetically, there seems to be a more stepwise transition between the two forms, as revealed by sequence analyses of two DNA regions. Genetic variation was significantly higher in the highland form compared with the lowland form, which could be due to disparate immigration histories in Scandinavia. Different evolutionary scenarios may explain the observed pattern, including a former allopatric distribution in isolated glacial refugia, succeeded by immigration into the same region. The partition of the genetic variation among the two forms suggests a recent or ongoing speciation event and possibly some ongoing introgression. Our findings have implications on the conservations status of the taxon, as their susceptibility to modern forestry seems to be different.     

Characterization of main sulfur source of wood-degrading basidiomycetes by S K-edge X-ray absorption near edge spectroscopy (XANES)

The main wood degraders in aerobic terrestrial ecosystems belong to the white- and brown-rot fungi, where their biomass can be created on wood decay only. However, total sulfur (S) concentration in wood is very low and only little is known about the different sulfur compounds in wood today. Sulfur-starved brown-rot fungi Gloeophyllum trabeum and Oligoporus placenta were incubated on sterilized pine wood blocks whereas Lentinus cyathiformis and the white-rot fungi Trametes versicolor were incubated on sterilized beech wood blocks. After 19 weeks of incubation, the S oxidation status was analyzed in wood, in degraded wood, and in biomass of wood-degrading fungi by synchrotron based S K-edge XANES, and total S and sulfate were quantified. Total sulfur and sulfate content in pine wood blocks were approximately 50 and 1 ??g g⁻¹, respectively, while in beech wood approximately 100 and 20 ??g g⁻¹ were found, respectively. Sulfur in beech was dominated by sulfate-esters. In contrast, pine wood also contained larger amounts of reduced S. Three out of four selected fungi caused a reduction of the S oxidation state in wood from oxidized S (sulfate-ester, sulfate) to intermediate S (sulfonate, sulfoxide) or reduced S (thiols, e.g., proteins, peptides, enzyme cofactors). Only O. placenta shifted thiol to sulfonate. Growth experiments of these fungi on selective minimal media showed that in particular cysteine (thiol), sulfonates, and sulfate enhanced total mycelium growth. Consequently, wood-degrading fungi were able to utilize a large variety of different wood S sources for growth but preferentially transformed in vivo sulfate-esters and thiol into biomass structures.     

Grazing by Folsomia candida (Collembola) differentially affects mycelial morphology of the cord-forming basidiomycetes Hypholoma fasciculare, Phanerochaete velutina and Resinicium bicolor

Cord-forming basidiomycetes are important decomposers of dead wood in forest ecosystems but the impact of mycophagous soil invertebrates on their mycelia are little known. Here we investigate the effects of different grazing intensities of Collembola (Folsomia candida) on mycelial foraging patterns of the saprotrophic cord-forming basidiomycetes Hypholoma fasciculare, Phanerochaete velutina and Resinicium bicolor growing from beech (Fagus sylvatica) wood block inocula in dishes of non-sterile soil. Mycelial extension rate and hyphal coverage decreased with increased grazing intensity. R. bicolor was most affected, high grazing density resulting in only a few major cords remaining. Grazing of H. fasciculare often resulted in points of more rapid outgrowth as cords with a fanned margin. In grazed mycelia of P. velutina the main cords had fanned tips and lateral cords became branched. These results suggest that mycophagy by Collembola may hinder the growth of cord-forming fungi in woodlands, which might impact on the ability of these fungi to forage for and decompose dead organic material.     

An NADH:Quinone Oxidoreductase Active during Biodegradation by the Brown-Rot Basidiomycete Gloeophyllum trabeum

The brown-rot basidiomycete Gloeophyllum trabeum uses a quinone redox cycle to generate extracellular Fenton reagent, a key component of the biodegradative system expressed by this highly destructive wood decay fungus. The hitherto uncharacterized quinone reductase that drives this cycle is a potential target for inhibitors of wood decay. We have identified the major quinone reductase expressed by G. trabeum under conditions that elicit high levels of quinone redox cycling. The enzyme comprises two identical 22-kDa subunits, each with one molecule of flavin mononucleotide. It is specific for NADH as the reductant and uses the quinones produced by G. trabeum (2,5-dimethoxy-1,4-benzoquinone and 4,5-dimethoxy-1,2-benzoquinone) as electron acceptors. The affinity of the reductase for these quinones is so high that precise kinetic parameters were not obtainable, but it is clear that k_cat/K_m for the quinones is greater than 10⁸ M⁻¹ s⁻¹. The reductase is encoded by a gene with substantial similarity to NAD(P)H:quinone reductase genes from other fungi. The G. trabeum quinone reductase may function in quinone detoxification, a role often proposed for these enzymes, but we hypothesize that the fungus has recruited it to drive extracellular oxyradical production.     

Biodeterioration of brazilwood Caesalpinia echinata Lam. (Leguminosae—Caesalpinioideae) by rot fungi and termites

The heartwood of Caesalpinia echinata Lam. (Leguminosae) (commonly called brazilwood) is used for violin bow manufacture due to the unique vibrational and physical properties found in the wood. In the present work, the effects of Pycnoporus sanguineus (white-rot fungus), Gloeophyllum trabeum (brown-rot fungus), Chaetomium globosum (soft-rot fungus), and Cryptotermes brevis (dry-wood termite) on weight losses and chemical composition of extractives and cell-wall polysaccharides of C. echinata wood were investigated under laboratory conditions and compared to those obtained for Anadenanthera macrocarpa, Eucalyptus grandis, and Pinus elliottii. The heartwood of C. echinata was found to be as resistant as A. macrocarpa to the decay fungi tested and to the attack of the dry-wood termite. Pinitol and galactopinitol A were the main sugar alcohols found in the extractives of wood of C. echinata, their presence, however, did not appear related to the resistance to fungal decay. Although only incipient stages of decay were found, the modifications in cell-wall polysaccharide composition of heartwood of C. echinata by rot fungi were related to decrease in polymers other than xylans. The high resistance of C. echinata to xylophages is probably due to the presence of toxic extractives in the wood.     

Field Evaluations of Subterranean Termite Preference for Sap-Stain Inoculated Wood

Few studies have focused on interactions between subterranean termites and the ophiostomatoid fungal associates of pine bark beetles or root feeding weevils. Field stake tests were employed at four locations throughout Mississippi to determine the feeding preference of subterranean termites for blue-stained, unstained, and partially decayed southern pine sapwood stakes. This study also utilized wood decayed by Gloeophyllum trabeum, a fungus previously shown to elicit a positive subterranean termite feeding response, as a positive control. Stakes inoculated with G. trabeum received significantly more attacks than all other treatments after 16 weeks. Of the stakes attacked by subterranean termites, stakes inoculated with Ophiostoma minus were degraded faster than any other treatment. Subterranean termite preference for stakes treated with either of two Leptographium spp. and the untreated negative controls did not differ; however, each was fed upon less than all other treatments. The feeding rate on stakes inoculated with O. ips and G. trabeum being fed upon by subterranean termites was not significantly different. These results represent the first evidence of wood containing non-structurally degrading fungi (O. ips and O. minus) eliciting a feeding preference from subterranean termites greater than that of decayed wood. The implications of these results are particularly relevant to pine forest ecology, nutrient cycling, subterranean termite control, and the utilization of blue-stained southern pine building products in the southeastern U.S.     

Fungicidal properties of individual components of copper–ethanolamine-based wood preservatives

The importance of copper–ethanolamine-based wood preservatives is increasing. These preservatives usually consist of copper as a fungicide, ethanolamine as a fixative, and secondary fungicides (boron, triazoles) and other additives (water repellents, fixatives, wax emulsions, etc.). Questions arise as to how each of these ingredients interacts with wood-decay fungi, and whether there are any synergistic effects between the components. In order to elucidate these questions, Norway spruce wood specimens were impregnated with five different aqueous solutions consisting of one single component only and of complete formulation of five different concentrations. These specimens were exposed to two brown-rot fungi, Antrodia vaillantii and Gloeophyllum trabeum, as well as to the white-rot fungus Trametes versicolor for 8 weeks according to mini block procedure. In parallel, petri dishes with nutrient medium containing different quantities of ingredients and of complete wood preservative were inoculated with the same fungal species, and their growth was compared with growth on media without chemicals. The results showed that both experimental methods give similar results. In general, there was no synergistic effect determined. Ethanolamine did not decrease fungicidal properties of the system, while on the other hand octanoic acid has a positive effect on the growth of brown-rot fungi. The minimal effective concentration of tested copper–ethanolamine preservative was determined by the minimum effective concentration of the most fungi-toxic ingredient.     

Synergy between pretreatment lignocellulose modifications and saccharification efficiency in two brown rot fungal systems

Brown rot wood-degrading fungi distinctly modify lignocellulose and completely hydrolyze polysaccharides (saccharification), typically without secreting an exo-acting glucanase and without removing lignin. Although each step of this two-step approach evolved within the same organism, it is unknown if the early lignocellulose modifications are made to specifically facilitate their own abbreviated enzyme system or if enhancements are more general. Because commercial pretreatments are typically approached as an isolated step, answering this question has immense implication on bioprocessing. We pretreated spruce and pine blocks with one of two brown rot fungi, Gloeophyllum trabeum or Fomitopsis pinicola. Wood harvested at weeks 1, 2, 4, and 8 showed a progression of weight loss from time zero due to selective carbohydrate removal. Hemicellulose losses progressed faster than cellulose loss. This “pretreated” material was then saccharified with commercially relevant Trichoderma reesei cellulases or with cellulases from the brown rot fungi responsible for degrading the wood to test for synergy. With increased decay, a significant increase in saccharification efficiency was apparent but not limited to same-species enzyme sources. We also calculated total sugar yields, and calculations that compensate for sugars consumed by fungi suggest a shorter residence time for fungal colonization than calculations based solely on saccharification yields.     

Comparison of methods to evaluate the potential of fungal growth on decay of spruce wood after short-time treatment

Several analytical methods were compared to evaluate characteristic wood decaying fungi for their potential to depolymerise lignin on spruce wood particles. Wood samples were treated with the white rot fungi Phlebia brevispora, Ceriporiopsis subvermispora, Merulius tremellosus, Pycnoporus sanguineus, Trametes pubescens and with the brown rot fungus Gloeophyllum trabeum. The UV absorbancies of crude ethanol extracts, total extractives content from sequential extraction, ligninolytic enzyme activities, lignin solubilisation and decrease of lignin content were compared. It was shown, that, in early decay stages, UV absorbancies of crude ethanol extracts and total extractives content correlate well with lignin degradation, increase of acid soluble lignin and increased production of ligninolytic enzymes (total peroxidase). Lignin content was determined using FT-NIR spectroscopy as well as by wet-chemical analysis, indicating a very good correlation between the two methods. According to the different analytical methods, the tested fungi can be classified into three categories based on their characteristic behaviour: brown rot, “slow” and “fast” white rot.     

Wood durability of home-garden teak against brown-rot and white-rot fungi

Natural decay resistance of teak wood grown in home-garden forestry and the factors influencing decay resistance were determined in comparison with that of a typical forest plantation. Accelerated laboratory tests were conducted on 1800 wood samples drawn from 15 trees of three planted sites. Analysis of variance based on a univariate mixed model showed that planted site, fungal species, and their interaction terms were important sources of variation in decay resistance. With increasing decay resistance from centre to periphery of the heartwood, radial position was a critical factor and the interaction effect of fungal species × radial position was significant in influencing the durability. No significant differences were found in decay resistance either between the opposite radii or due to the various possible interaction terms of radii with the site, fungal species and radial position. There were significant differences in decay resistance against brown-rot fungi between wet and dry sites of home-garden teak although differences against white-rot fungi were non-significant among the three planted sites. Polyporus palustris was the more aggressive brown-rot fungus than Gloeophyllum trabeum. The higher susceptibility of wet site home-garden teak to brown-rot decay was associated with a paler colour of the wood and lower extractive content.     

Induction of polygalacturonase and the formation of oxalic acid by pectin in brown-rot fungi

Extracellular polygalacturonase (PG) production was estimated in vitro, using liquid cultures of three species of brown-rot decay fungi (Postia placenta, Gloeophyllum trabeum and Serpula incrassata), by cup-plate assay, assay of reducing sugars, and decrease in viscosity. Although all three experimental assays demonstrated that PG was induced by pectin in all three fungi, decrease in viscosity gave the best correlation with decay capacity in soil block tests. PG activity, determined as an increase in reducing sugar activity, was greatest in G. trabeum and weakest in S. incrassata. The optimum pH for PG activity was between pH 2.5 and 4.5. Oxalic acid production was also enhanced by pectin and functioned synergistically with PG activity. We conclude that these fungi produce PG that is best induced by pectin and that PG activity exceeds production of xylanase and endoglucanase activity in vitro. Polygalacturonase is likely to act synergistically with oxalic acid to solubilize and hydrolyse the pectin in pit membranes and middle lamellae. Thus, production of PG and oxalic acid should facilitate early spread of hyphae and enhance the lateral flow of wood-decay enzymes and agents into adjacent tracheids and the wood cell wall, thus initiating the diffuse decay caused by brown-rot fungi.The Forest Products Laboratory is maintained in co-operation with the University of Wisconsin. This article was written and prepared by US Government employees on official time, and it is therefore in the public domain and not subject to copyright.     

Contrasting Effects of Elevated Temperature and Invertebrate Grazing Regulate Multispecies Interactions between Decomposer Fungi

Predicting the influence of biotic and abiotic factors on species interactions and ecosystem processes is among the primary aims of community ecologists. The composition of saprotrophic fungal communities is a consequence of competitive mycelial interactions, and a major determinant of woodland decomposition and nutrient cycling rates. Elevation of atmospheric temperature is predicted to drive changes in fungal community development. Top-down regulation of mycelial growth is an important determinant of, and moderator of temperature-driven changes to, two-species interaction outcomes. This study explores the interactive effects of a 4 °C temperature increase and soil invertebrate (collembola or woodlice) grazing on multispecies interactions between cord-forming basidiomycete fungi emerging from colonised beech (Fagus sylvatica) wood blocks. The fungal dominance hierarchy at ambient temperature (16 °C; Phanerochaete velutina > Resinicium bicolor > Hypholoma fasciculare) was altered by elevated temperature (20 °C; R. bicolor > P. velutina > H. fasciculare) in ungrazed systems. Warming promoted the competitive ability of the fungal species (R. bicolor) that was preferentially grazed by all invertebrate species. As a consequence, grazing prevented the effect of temperature on fungal community development and maintained a multispecies assemblage. Decomposition of fungal-colonised wood was stimulated by warming, with implications for increased CO₂ efflux from woodland soil. Analogous to aboveground plant communities, increasing complexity of biotic and abiotic interactions appears to be important in buffering climate change effects on soil decomposers.     

Differences in crystalline cellulose modification due to degradation by brown and white rot fungi

Wood-decaying basidiomycetes are some of the most effective bioconverters of lignocellulose in nature, however the way they alter wood crystalline cellulose on a molecular level is still not well understood. To address this, we examined and compared changes in wood undergoing decay by two species of brown rot fungi, Gloeophyllum trabeum and Meruliporia incrassata, and two species of white rot fungi, Irpex lacteus and Pycnoporus sanguineus, using X-ray diffraction (XRD) and ¹³C solid-state nuclear magnetic resonance (NMR) spectroscopy. The overall percent crystallinity in wood undergoing decay by M. incrassata, G. trabeum, and I. lacteus appeared to decrease according to the stage of decay, while in wood decayed by P. sanguineus the crystallinity was found to increase during some stages of degradation. This result is suggested to be potentially due to the different decay strategies employed by these fungi. The average spacing between the 200 cellulose crystal planes was significantly decreased in wood degraded by brown rot, whereas changes observed in wood degraded by the two white rot fungi examined varied according to the selectivity for lignin. The conclusions were supported by a quantitative analysis of the structural components in the wood before and during decay confirming the distinct differences observed for brown and white rot fungi. The results from this study were consistent with differences in degradation methods previously reported among fungal species, specifically more non-enzymatic degradation in brown rot versus more enzymatic degradation in white rot.