Saprotrophic Basidiomycetes Research Articles

Transcriptomic and protein analysis of Trametes versicolor interacting with a Hypholoma fasciculare mycelium foraging in soil

The decomposition of large woody material is an important process in forest carbon cycling and nutrient release. Cord-forming saprotrophic basidiomycete fungi create non-resource limited mycelial networks between decomposing branches, logs and tree stumps on the forest floor where colonisation of new resources is often associated with the replacement of incumbent decay communities. To date, antagonism experiments have mostly placed competing fungi in direct contact, while in nature cord-forming saprobes encounter colonised wood as mycelia in a network. Transcriptomic and peptide analyses were conducted on soil-based microcosms were foraging cord-forming Hypholoma fasciculare encountered a wood block colonised by Trametes versicolor. Protein turnover featured strongly for both species and genes putatively involved in secondary metabolite production were identified. H. fasciculare demonstrated an exploitative profile with increased transcription of genes associated with carbohydrate metabolism and RNA and ribosome processing. T. versicolor showed a shift in signalling, energy generation and amino acid metabolism. By identifying genes and proteins putatively involved in this fungal interaction, this work may help guide the discovery of bioactive molecules and mechanisms underpinning community succession.

Hyphal connection increases net resource utilization of saprotrophic fungal mycelia by improving local performance: A case study of the cord-forming basidiomycete, Phanerochaete velutina

To identify the functional advantages of hyphal connections in fungal mycelial networks, we evaluated the wood decaying abilities of the mycelia of a saprotrophic basidiomycete, Phanerochaete velutina, in soil dishes with wood baits. The dishes were surrounded by water, allowing the mycelia to access water. Dishes with two or three baits were placed adjacent to or distant from each other, representing the ease of creating hyphal bridges between the two dishes. After 8 months of incubation, the weight loss of the wood bait was significantly larger in the adjacent pairs of dishes compared to the distant pairs. Mycelia in the adjacent dishes had higher chances to access the water and thus increase water content of the wood, which was the primary factor related to wood weight loss. These results suggest that hyphal connections increase decay ability of P. velutina possibly due to water transfer across the mycelial network.

Visualising long distance sugar transport in fungi using infrared fluorescence scanning imaging

Mycelia of saprotrophic basidiomycetes can cover large areas in nature that are typified by their heterogeneous nutrient availability. This heterogeneity is overcome by long distance transport of nutrients within the hyphal network to sites where they are needed. It is therefore key to be able to study nutrient transport and its underlying mechanisms. An IRDye-conjugate was used for the first time for imaging transport in fungi. A method was set up for time-lapse, high spatial resolution infrared imaging of IRDye-labelled deoxyglucose (IRDye-DG) in Schizophyllum commune and Agaricus bisporus. Scanning imaging visualised the tracer in individual hyphae as well as deeper tissues in mushrooms (mm-cm depth). The advantage of using fluorescence scanning imaging of IRDye in contrast to radiolabelled tracers studies, is that a higher spatial resolution and higher sensitivity (244 fg/ml) can be obtained. Moreover, it has a large field of view (25 × 25 cm) compared to microscopy (µm-mm range), allowing relatively fast and detailed imaging of large dimension samples.

Activity-based protein profiling reveals dynamic substrate-specific cellulase secretion by saprotrophic basidiomycetes

BackgroundFungal saccharification of lignocellulosic biomass occurs concurrently with the secretion of a diverse collection of proteins, together functioning as a catalytic system to liberate soluble sugars from insoluble composite biomaterials. How different fungi respond to different substrates is of fundamental interest to the developing biomass saccharification industry. Among the cornerstones of fungal enzyme systems are the highly expressed cellulases (endo-β-glucanases and cellobiohydrolases). Recently, a cyclophellitol-derived activity-based probe (ABP-Cel) was shown to be a highly sensitive tool for the detection and identification of cellulases.ResultsHere we show that ABP-Cel enables endo-β-glucanase profiling in diverse fungal secretomes. In combination with established ABPs for β-xylanases and β-d-glucosidases, we collected multiplexed in-gel fluorescence activity-based protein profiles of 240 secretomes collected over ten days from biological replicates of ten different basidiomycete fungi grown on maltose, wheat straw, or aspen pulp. Our results reveal the remarkable dynamics and unique enzyme fingerprints associated with each species substrate combination. Chemical proteomic analysis identifies significant arsenals of cellulases secreted by each fungal species during growth on lignocellulosic biomass. Recombinant production and characterization of a collection of probe-reactive enzymes from GH5, GH10, and GH12 confirm that ABP-Cel shows broad selectivity towards enzymes with endo-β-glucanase activity.ConclusionUsing small-volume samples with minimal sample preparation, the results presented here demonstrate the ready accessibility of sensitive direct evidence for fungal enzyme secretion during early stages of growth on complex lignocellulosic substrates.

Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae.

Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. Saccharomyces cerevisiae is a preferred host for engineering fungal laccases. To assist the difficult secretion of active enzymes by yeast, the native signal peptide is usually replaced by the preproleader of S. cerevisiae alfa mating factor (MFα1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in S. cerevisiae of laccases fused to the α-factor preproleader, we demonstrated that mutations accumulated in the signal peptide notably raised enzyme secretion. Here we describe different protein engineering approaches carried out to enhance the laccase activity detected in the liquid extracts of S. cerevisiae cultures. We demonstrate the improved secretion of native and engineered laccases by using the fittest mutated α-factor preproleader obtained through successive laccase evolution campaigns in our lab. Special attention is also paid to the role of protein N-glycosylation in laccase production and properties, and to the introduction of conserved amino acids through consensus design enabling the expression of certain laccases otherwise not produced by the yeast. Finally, we revise the contribution of mutations accumulated in laccase coding sequence (CDS) during previous directed evolution campaigns that facilitate enzyme production.

Ligninolytic Enzyme Production and Decolorization Capacity of Synthetic Dyes by Saprotrophic White Rot, Brown Rot, and Litter Decomposing Basidiomycetes.

An extensive screening of saprotrophic Basidiomycetes causing white rot (WR), brown rot (BR), or litter decomposition (LD) for the production of laccase and Mn-peroxidase (MnP) and decolorization of the synthetic dyes Orange G and Remazol Brilliant Blue R (RBBR) was performed. The study considered in total 150 strains belonging to 77 species. The aim of this work was to compare the decolorization and ligninolytic capacity among different ecophysiological and taxonomic groups of Basidiomycetes. WR strains decolorized both dyes most efficiently; high decolorization capacity was also found in some LD fungi. The enzyme production was recorded in all three ecophysiology groups, but to a different extent. All WR and LD fungi produced laccase, and the majority of them also produced MnP. The strains belonging to BR lacked decolorization capabilities. None of them produced MnP and the production of laccase was either very low or absent. The most efficient decolorization of both dyes and the highest laccase production was found among the members of the orders Polyporales and Agaricales. The strains with high MnP activity occurred across almost all fungal orders (Polyporales, Agaricales, Hymenochaetales, and Russulales). Synthetic dye decolorization by fungal strains was clearly related to their production of ligninolytic enzymes and both properties were determined by the interaction of their ecophysiology and taxonomy, with a more relevant role of ecophysiology. Our screening revealed 12 strains with high decolorization capacity (9 WR and 3 LD), which could be promising for further biotechnological utilization.

Fluorescent protein expression in the ectomycorrhizal fungus Laccaria bicolor: a plasmid toolkit for easy use of fluorescent markers in basidiomycetes.

For long time, studies on ectomycorrhiza (ECM) have been limited by inefficient expression of fluorescent proteins (FPs) in the fungal partner. To convert this situation, we have evaluated the basic requirements of FP expression in the model ECM homobasidiomycete Laccaria bicolor and established eGFP and mCherry as functional FP markers. Comparison of intron-containing and intronless FP-expression cassettes confirmed that intron-processing is indispensable for efficient FP expression in Laccaria. Nuclear FP localization was obtained via in-frame fusion of FPs between the intron-containing genomic gene sequences of Laccaria histone H2B, while cytosolic FP expression was produced by incorporating the intron-containing 5' fragment of the glyceraldehyde-3-phosphate dehydrogenase encoding gene. In addition, we have characterized the consensus Kozak sequence of strongly expressed genes in Laccaria and demonstrated its boosting effect on transgene mRNA accumulation. Based on these results, an Agrobacterium-mediated transformation compatible plasmid set was designed for easy use of FPs in Laccaria. The four cloning plasmids presented here allow fast and highly flexible construction of C-terminal in-frame fusions between the sequences of interest and the two FPs, expressed either from the endogenous gene promoter, allowing thus evaluation of the native regulation modes of the gene under study, or alternatively, from the constitutive Agaricus bisporus gpdII promoter for enhanced cellular protein localization assays. The molecular tools described here for cell-biological studies in Laccaria can also be exploited in studies of other biotrophic or saprotrophic basidiomycete species susceptible to genetic transformation.

Spatial distribution of genets in populations of saprotrophic basidiomycetes, Mycetinis alliaceus, Marasmius rotula and Gymnopus androsaceus, from Serbian and Montenegrin forests

Saprotrophic basidiomycetes play a crucial role in leaf-litter decomposition, especially in nitrogen-limited boreal and temperate forests. Populations of this group of fungi have been inadequately investigated. We examined the populations of three different saprotrophic species (Mycetinis alliaceus, Marasmius rotula and Gymnopus androsaceus) in forests in Serbia and Montenegro. To determine the distribution of genets at each of the three investigated sites, molecular analysis was conducted using the inter-simple sequence repeats (ISSR) method. Seven to fifteen genets (genotypes, individuals) were identified on each site and the majority of them were represented by a single sporocarp. The sizes of the genets with two or more sporocarps were estimated to range from 0.3 to 4.0 m. Results obtained in this study suggest that populations of these three species can consist of numerous and relatively small genets. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. III43002]

Exotic litter of the invasive plantLigustrum lucidumalters enzymatic production and lignin degradation by selected saprotrophic fungi

Chemical changes in leaf input to forest soils have been reported to affect decay processes. In this work, litter mass loss and decomposition constants (k) during 200 days in solid-state fermentation of the native tree Celtis tala Gill. ex Planch. and the exotic one Ligustrum lucidum Ait. with three common litter saprotrophic basidiomycetes were compared. Alterations in litter quality were characterized by solid-state13C NMR spectroscopy, pH, soluble sugars, ammonium, proteins, and phenol content determination and were associated with extracellular lignocellulolytic enzyme production. Differences in substrate decomposition related to litter type were observed for Leratiomyces ceres, achieving a higher k in the exotic L. lucidum litter, which might be attributed to the induction of manganese peroxidase activity. Substrate preference for alkyl C and more degradation of lignified compounds were found in such substrates. Although no statistical differences in mass loss were observed for the rest of the fungi assayed, we detected changes in several of the parameters evaluated. This suggests that exotic invasions may alter ecosystem functioning by accelerating decomposition processes through an increased fungal ligninolytic activity.

Growing evidence for facultative biotrophy in saprotrophic fungi: data from microcosm tests with 201 species of wood-decay basidiomycetes.

Ectomycorrhizal (ECM) symbioses have evolved a minimum of 78 times independently from saprotrophic lineages, indicating the potential for functional overlap between ECM and saprotrophic fungi. ECM fungi have the capacity to decompose organic matter, and although there is increasing evidence that some saprotrophic fungi exhibit the capacity to enter into facultative biotrophic relationships with plant roots without causing disease symptoms, this subject is still not well studied. In order to determine the extent of biotrophic capacity in saprotrophic wood-decay fungi and which systems may be useful models, we investigated the colonization of conifer seedling roots invitro using an array of 201 basidiomycete wood-decay fungi. Microtome sectioning, differential staining and fluorescence microscopy were used to visualize patterns of root colonization in microcosm systems containing Picea abies or Pinus sylvestris seedlings and each saprotrophic fungus. Thirty-four (16.9%) of the tested fungal species colonized the roots of at least one tree species. Two fungal species showed formation of a mantle and one showed Hartig net-like structures. These features suggest the possibility of an active functional symbiosis between fungus and plant. The data indicate that the capacity for facultative biotrophic relationships in free-living saprotrophic basidiomycetes may be greater than previously supposed.

You are what you get from your fungi: nitrogen stable isotope patterns in Epipactis species.

Partially mycoheterotrophic plants are enriched in 13 C and 15 N compared to autotrophic plants. Here, it is hypothesized that the type of mycorrhizal fungi found in orchid roots is responsible for variation in 15 N enrichment of leaf tissue in partially mycoheterotrophic orchids. The genus Epipactis was used as a case study and carbon and nitrogen isotope abundances of eight Epipactis species, fungal sporocarps of four Tuber species and autotrophic references were measured. Mycorrhizal fungi were identified using molecular methods. Stable isotope data of six additional Epipactis taxa and ectomycorrhizal and saprotrophic basidiomycetes were compiled from the literature. The 15 N enrichment of Epipactis species varied between 3·2 ± 0·8 ‰ ( E. gigantea ; rhizoctonia-associated) and 24·6 ± 1·6 ‰ ( E. neglecta ; associated with ectomycorrhizal ascomycetes). Sporocarps of ectomycorrhizal ascomycetes (10·7 ± 2·2 ‰) were significantly more enriched in 15 N than ectomycorrhizal (5·2 ± 4·0 ‰) and saprotrophic basidiomycetes (3·3 ± 2·1 ‰). As hypothesized, it is suggested that the observed gradient in 15 N enrichment of Epipactis species is strongly driven by 15 N abundance of their mycorrhizal fungi; i.e. ɛ 15 N in Epipactis spp. associated with rhizoctonias < ɛ 15 N in Epipactis spp. with ectomycorrhizal basidiomycetes < ɛ 15 N in Epipactis spp. with ectomycorrhizal ascomycetes and basidiomycetes < ɛ 15 N in Epipactis spp. with ectomycorrhizal ascomycetes.

Mycoremediation of hydrocarbons with basidiomycetes—a review

ABSTRACTThe literature on hydrocarbon remediation with basidiomycetes was reviewed. Two ecological groups are considered for bioremediation, the saprotrophic basidiomycetes (white-rot and brown-rot fungi) and the ectomycorrhizal basidiomycetes. A remarkable capacity of basidiomycetes for in vitro degradation of simple and recalcitrant hydrocarbons, such as PAH, persistent organic pollutants (POPs), halogenated HC, aromatic HC and phenols, explosives and dyes was reported for many species. However, there is a need for more studies on the practical feasibility of field applications with basidiomycetes.

Bacteria induce pigment formation in the basidiomycete Serpula lacrymans.

Basidiomycete fungi are characterized ecologically for their vital functional role in ecosystem carbon recycling and chemically for their capacity to produce a diverse array of small molecules. Chromophoric natural products derived from the quinone precursor atromentin, such as variegatic acid and involutin, have been shown to function in redox cycling. Yet, in the context of an inter-kingdom natural system these pigments are still elusive. Here, we co-cultured the model saprotrophic basidiomycete Serpula lacrymans with an ubiquitous terrestrial bacterium, either Bacillus subtilis, Pseudomonas putida, or Streptomyces iranensis. For each, there was induction of the gene cluster encoding a non-ribosomal peptide synthetase-like enzyme (atromentin synthetase) and an aminotransferase which together produce atromentin. Correspondingly, during co-culturing there was an increase in secreted atromentin-derived pigments, i.e., variegatic, xerocomic, isoxerocomic, and atromentic acid. Bioinformatic analyses from 14 quinone synthetase genes, twelve of which are encoded in a cluster, identified a common promoter motif indicating a general regulatory mechanism for numerous basidiomycetes.

Impact of Phanerochaete chrysosporium on the Functional Diversity of Bacterial Communities Associated with Decaying Wood.

Bacteria and fungi naturally coexist in various environments including forest ecosystems. While the role of saprotrophic basidiomycetes in wood decomposition is well established, the influence of these fungi on the functional diversity of the wood-associated bacterial communities has received much less attention. Based on a microcosm experiment, we tested the hypothesis that both the presence of the white-rot fungus Phanerochaete chrysosporium and the wood, as a growth substrate, impacted the functional diversity of these bacterial communities. Microcosms containing sterile sawdust were inoculated with a microbial inoculum extracted from a forest soil, in presence or in absence of P. chrysosporium and subsequently, three enrichment steps were performed. First, bacterial strains were isolated from different microcosms previously analyzed by 16S rRNA gene-based pyrosequencing. Strains isolated from P. chrysosporium mycosphere showed less antagonism against this fungus compared to the strains isolated from the initial forest soil inoculum, suggesting a selection by the fungus of less inhibitory bacterial communities. Moreover, the presence of the fungus in wood resulted in a selection of cellulolytic and xylanolytic bacterial strains, highlighting the role of mycospheric bacteria in wood decomposition. Additionally, the proportion of siderophore-producing bacteria increased along the enrichment steps, suggesting an important role of bacteria in iron mobilization in decaying-wood. Finally, taxonomic identification of 311 bacterial isolates revealed, at the family level, strong similarities with the high-throughput sequencing data as well as with other studies in terms of taxonomic composition of the wood-associated bacterial community, highlighting that the isolated strains are representative of the wood-associated bacterial communities.

Mushrooms as Rainmakers: How Spores Act as Nuclei for Raindrops.

Millions of tons of fungal spores are dispersed in the atmosphere every year. These living cells, along with plant spores and pollen grains, may act as nuclei for condensation of water in clouds. Basidiospores released by mushrooms form a significant proportion of these aerosols, particularly above tropical forests. Mushroom spores are discharged from gills by the rapid displacement of a droplet of fluid on the cell surface. This droplet is formed by the condensation of water on the spore surface stimulated by the secretion of mannitol and other hygroscopic sugars. This fluid is carried with the spore during discharge, but evaporates once the spore is airborne. Using environmental electron microscopy, we have demonstrated that droplets reform on spores in humid air. The kinetics of this process suggest that basidiospores are especially effective as nuclei for the formation of large water drops in clouds. Through this mechanism, mushroom spores may promote rainfall in ecosystems that support large populations of ectomycorrhizal and saprotrophic basidiomycetes. Our research heightens interest in the global significance of the fungi and raises additional concerns about the sustainability of forests that depend on heavy precipitation.

Thermal acclimation in widespread heterotrophic soil microbes

Respiration by plants and microorganisms is primarily responsible for mediating carbon exchanges between the biosphere and atmosphere. Climate warming has the potential to influence the activity of these organisms, regulating exchanges between carbon pools. Physiological 'down-regulation' of warm-adapted species (acclimation) could ameliorate the predicted respiratory losses of soil carbon under climate change scenarios, but unlike plants and symbiotic microbes, the existence of this phenomenon in heterotrophic soil microbes remains controversial. Previous studies using complex soil microbial communities are unable to distinguish physiological acclimation from other community-scale adjustments. We explored the temperature-sensitivity of individual saprotrophic basidiomycete fungi growing in agar, showing definitively that these widespread heterotrophic fungi can acclimate to temperature. In almost all cases, the warm-acclimated individuals had lower growth and respiration rates at intermediate temperatures than cold-acclimated isolates. Inclusion of such microbial physiological responses to warming is essential to enhance the robustness of global climate-ecosystem carbon models.

Extracellular Enzymes of the White-Rot Fungus Fomes fomentarius and Purification of 1,4-β-Glucosidase

Production of the lignocellulose-degrading enzymes endo-1,4-β-glucanase, 1,4-β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, 1,4-β-xylosidase, Mn peroxidase, and laccase was characterized in a common wood-rotting fungus Fomes fomentarius, a species able to efficiently decompose dead wood, and compared to the production in eight other fungal species. The main aim of this study was to characterize the 1,4-β-glucosidase produced by F. fomentarius that was produced in high quantities in liquid stationary culture (25.9 U ml(-1)), at least threefold compared to other saprotrophic basidiomycetes, such as Rhodocollybia butyracea, Hypholoma fasciculare, Irpex lacteus, Fomitopsis pinicola, Pleurotus ostreatus, Piptoporus betulinus, and Gymnopus sp. (between 0.7 and 7.9 U ml(-1)). The 1,4-β-glucosidase enzyme was purified to electrophoretic homogeneity by both anion-exchange and size-exclusion chromatography. A single 1,4-β-glucosidase was found to have an apparent molecular mass of 58 kDa and a pI of 6.7. The enzyme exhibited high thermotolerance with an optimum temperature of 60 °C. Maximal activity was found in the pH range of 4.5-5.0, and K (M) and V (max) values were 62 μM and 15.8 μmol min(-1) l(-1), respectively, when p-nitrophenylglucoside was used as a substrate. The enzyme was competitively inhibited by glucose with a K (i) of 3.37 mM. The enzyme also acted on p-nitrophenylxyloside, p-nitrophenylcellobioside, p-nitrophenylgalactoside, and p-nitrophenylmannoside with optimal pH values of 6.0, 3.5, 5.0, and 4.0-6.0, respectively. The combination of relatively low molecular mass and low K (M) value make the 1,4-β-glucosidase a promising enzyme for biotechnological applications.

Interactions between saprotrophic basidiomycete mycelia and mycophagous soil fauna

Saprotrophic cord-forming basidiomycetes are functionally important components of temperate woodland ecosystems, being major agents of organic matter decomposition and affecting the distribution of nutrients within soils. They also represent the primary nutrient source for soil-dwelling animals. We review the interactions between cord-forming basidiomycetes and soil invertebrates, and their implications for the soil-decomposer community and woodland ecosystem functioning. Top-down grazing pressures can influence mycelial growth and physiology with implications for fungal nutrient distribution and mineralisation. Bottom-up effects also determine the survival, population growth and behaviour of invertebrates. These top-down and bottom-up processes are intrinsically linked and interact to determine decomposer communities and their functioning.

Charcoal filter paper improves the viability of cryopreserved filamentous ectomycorrhizal and saprotrophic Basidiomycota and Ascomycota

We assessed viability of 18 strains of filamentous ectomycorrhizal and saprotrophic basid-iomycetes and ascomycetes after cryopreservation with a novel technique based on charcoal filter paper strips (CFS). The results indicate that axenic fungal cultures grown on CFS recovered from freezing within a few days, even though none survived cryopreservation by the conventional straw method. Fungal growth on CFS was more vigorous, with morphological differentiations such as rhizomorphs and an increased amount of aerial mycelia compared to the unamended culture media. Accordingly CFS allows the cryopreservation of a wide range of rare and important ectomycorrhizal and saprotrophic fungi, which hitherto were difficult to revive from liquid nitrogen storage with the conventional and widely applied straw technique.

Impacts of grazing soil fauna on decomposer fungi are species-specific and density-dependent

The grazing impacts of different densities of woodlice, collembola and millipedes on the foraging and distribution of two saprotrophic cord-forming basidiomycetes were investigated in soil microcosms. Effects of all three invertebrate species were density-dependent, with larger populations limiting mycelial development to a greater extent. Impacts were, however, species-specific; grazing pressures exerted by low-density woodlouse populations outweighed those of high-density millipede or collembola populations. The varying abilities of soil invertebrates to influence mycelial foraging and distribution indicate that invertebrate species composition and diversity may be key factors regulating saprotrophic basidiomycete functioning in woodland soil.