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Although the commonly used internal transcribed spacer region of rDNA (ITS) is well suited for taxonomic identification of fungi, the information on the relative abundance of taxa and diversity is negatively affected by the multicopy nature of rDNA and the existence of ITS paralogues. Moreover, due to high variability, ITS sequences cannot be used for phylogenetic analyses of unrelated taxa. The part of single-copy gene encoding the second largest subunit of RNA polymerase II (rpb2) was thus compared with first spacer of ITS as an alternative marker for the analysis of fungal communities in spruce forest topsoil, and their applicability was tested on a comprehensive mock community. In soil, rpb2 exhibited broad taxonomic coverage of the entire fungal tree of life including basal fungal lineages. The gene exhibited sufficient variation for the use in phylogenetic analyses and taxonomic assignments, although it amplifies also paralogues. The fungal taxon spectra obtained with rbp2 region and ITS1 corresponded, but sequence abundance differed widely, especially in the basal lineages. The proportions of OTU counts and read counts of major fungal groups were close to the reality when rpb2 was used as a molecular marker while they were strongly biased towards the Basidiomycota when using the ITS primers ITS1/ITS4. Although the taxonomic placement of rbp2 sequences is currently more difficult than that of the ITS sequences, its discriminative power, quantitative representation of community composition and suitability for phylogenetic analyses represent significant advantages.

Bacterial communities living in forest soils contribute to the decomposition of organic matter and the recycling of nutrients in these ecosystems and form one of the most diverse habitats on Earth. Unfortunately, due to difficulty in culturing soil bacteria, the understanding of their ecology is still limited. In the case of temperate deciduous forests, soil microbial communities face large seasonal variations in environmental conditions, such as temperature or moisture. Moreover, the supply of nutrients also differs due to seasonal processes, such as the allocation of photosynthates into soil by the roots of primary producers or the seasonal input of fresh litter. The aim of this study was to reveal how the bacterial community responds to these seasonal processes in the litter and soil of a Quercus petraea forest. Bacterial communities from litter and from the organic and mineral horizons of soil were analyzed during the four seasons of the year by 16S rRNA gene pyrosequencing. The results revealed that the composition of the bacterial community is horizon specific. The litter horizon had a higher relative abundance of Proteobacteria and Bacteroidetes than soil, while the organic and mineral horizons had a higher abundance of Acidobacteria, Firmicutes and Actinobacteria than litter. Moreover, the bacterial community was significantly affected by seasonality in all horizons. Bacterial communities in the litter showed significant differences between the vegetation season (May and July) and the autumn and winter seasons (October, February). In mineral soil, bacterial community composition was specific in the summer, when it was significantly different from all other seasons, with a larger number of taxa described as rhizosphere and mycorrhizosphere inhabitants. The results indicate that litter decomposition is the main driver of bacterial community composition in litter horizon. In contrast to reports on fungal communities, bacterial community composition in mineral soil responds to the seasonal peaks of rhizodeposition in the summer.

Forty-nine white-rot strains belonging to 38 species of Basidiomycota were evaluated for olive-mill wastewater (OMW) degradation. Almost all fungi caused high total phenolics (>60%) and color (⩽ 70%) reduction, while COD and phytotoxicity decreased to a lesser extent. Culture extracts from selected Agrocybe cylindracea, Inonotus andersonii, Pleurotus ostreatus and Trametes versicolor strains showed non-altered physicochemical and enzymatic activity profiles when applied to raw OMW in the presence or absence of commercial catalase, indicating no interaction of the latter with fungal enzymes and no competition for H2O2. Hydrogen peroxide's addition resulted in drastic OMW's decolorization, with no effect on phenolic content, suggesting that oxidation affects colored components, but not necessarily phenolics. When fungal extracts were heat-treated, no phenolics decrease was observed demonstrating thus their enzymatic rather than physicochemical oxidation. Laccases added to OMW were reversibly inhibited by the effluent's high phenolic load, while peroxidases were stable and active during the entire process.

Several previous studies indicated that Actinobacteria may be enriched in soils with elevated content of heavy metals. In this study, we have developed a method for the in-depth analysis of actinobacterial communities in soil through phylum-targeted high-throughput sequencing and used it to address this question and examine the community composition in grassland soils along a gradient of heavy metal contamination (Cu, Zn, Cd, Pb). The use of the 16Sact111r primer specific for Actinobacteria resulted in a dataset obtained by pyrosequencing where over 98 % of the sequences belonged to Actinobacteria. The diversity within the Actinobacterial community was not affected by the heavy metals, but the contamination was the most important factor affecting community composition. The most significant changes in community composition were due to the content of Cu and Pb, while the effects of Zn and Cd were relatively minor. For the most abundant actinobacterial taxa, the abundance of taxa identified as members of the genera Acidothermus, Streptomyces, Pseudonocardia, Janibacter and Microlunatus increased with increasing metal content, while those belonging to Jatrophihabitans and Actinoallomurus decreased. The genus Ilumatobacter contained operational taxonomic units (OTUs) that responded to heavy metals both positively and negatively. This study also confirmed that Actinobacteria appear to be less affected by heavy metals than other bacteria. Because several Actinobacteria were also identified in playing a significant role in cellulose and lignocellulose decomposition in soil, they potentially represent important decomposers of organic matter in such environments.

In forest ecosystems, trees represent the major primary producers and affect the chemical composition and microbial processes in the ecosystem via specific litter chemistry and rhizodeposition. Effects of trees on the abundance of soil microorganisms have been previously observed but the extent to which trees affect the composition of microbial communities remains unknown. Here we analyse the factors affecting the composition of bacterial and fungal communities in forest litter and soil under seven tree species studied at twenty-eight spatially independent sites of similar age developed on the same initial substrate. Microbial communities differed between litter and soil. Bacterial communities were more diverse than fungal communities, especially in litter, and exhibited higher evenness. Eighty percent of the bacterial sequences belonged to the 200–250 most dominant operational taxonomic units (OTUs), and 80% of the fungal sequences were composed of only 23–28 OTUs. The effect of tree species on the microbial-community composition was significant in both litter and soil for fungi as well as bacteria. In bacteria, the tree effect was likely partly mediated by litter and soil pH. Fungal taxa showed a greater tendency to be tree-specific: 35–37% of the dominant fungal OTUs but only 0–3% of the bacterial OTUs were restricted to 1 or 2 trees, and 15–45% of the fungi and 80% of the bacteria were common under 6 or 7 trees. Microbial taxa were demonstrated to associate with less trees than would be expected based on the patterns of their abundance in samples and the tree identity thus affects their occurrence. The numbers of observed dominant fungal OTUs in the study area increased faster with an increasing numbers of trees, indicating high β-diversity. Although the proportion of the arbuscular mycorrhizal and ectomycorrhizal fungi differed among trees, the tree-specific fungal taxa were both root-symbiotic and saprotrophic. The effect of trees on the composition of microbial community was demonstrated to be stronger than other soil properties and to explain a large proportion of variation in community composition, especially in fungi.

One hundred and eleven strains of Basidiomycota, 39 strains of Ascomycota and 2 strains of Mucoromycotina belonging to wood decomposers that cause white-rot (WR) or brown-rot (BR), other wood associated saprotrophs (WA), litter decomposing cord-forming Basidiomycota (LDF), and saprotrophic microfungi (SA), were screened for the production of hydrolytic enzymes and laccase. The presence of enzyme-encoding genes was also analysed in the published genomes of saprotrophic fungi. Several genes, including those for acidic phosphatase, β-glucosidase and N-acetylglucosaminidase, were common in the genomes with enzyme activity widely displayed by fungi, while other enzymes, such as certain hemicellulases or laccase, were produced less frequently. Enzyme production by saprotrophic fungi was shaped by the combination of their ecophysiology and taxonomy. Basidiomycota exhibited higher activities of all enzymes, except alkaline phosphatase, α-glucosidase, N-acetylglucosaminidase, α-mannosidase and α-fucosidase, than Ascomycota. The SA and BR fungi showed distinct enzyme production patterns, while the enzyme production by WR, LDF and WA was similar. Differences among species were typically reflected in the level of enzyme activity rather than in the absence of enzymes. Enzyme screening results showed that in several cases, fungi exhibited enzyme activity without the presence of the corresponding gene and vice versa . This indicates that the use of genome-derived information for the prediction of potential enzyme production has substantial limitations and cannot replace functional screening of fungal cultures.

Precise knowledge of the fungal succession in the litter of coniferous forests will facilitate understanding litter decomposition, in which fungi play a major role. We investigated the development of a fungal community during 3 yr of Picea abies litter decomposition in three control forest sites and three sites where bark-beetle attacks had killed adult trees and stopped the yearly input of fresh litter, using both cultivation from needles and terminal restriction-fragment length polymorphism analysis. The two methods revealed similar dominant species during the fungal succession. Members of the Dothideales, Eurotiales and Helotiales predominated during the initial stage of decay, whereas members of Agaricales appeared only occasionally during this stage. The onset of the latter began from the seventh month, with a peak occurring after 1 yr. Bark-beetle attacks hastened litter decomposition and decreased fungal diversity only during the initial stages of decomposition.

Motivation Modern molecular methods have increased our ability to describe microbial communities. Along with the advances brought by new sequencing technologies, we now require intensive computational resources to make sense of the large numbers of sequences continuously produced. The software developed by the scientific community to address this demand, although very useful, require experience of the command-line environment, extensive training and have steep learning curves, limiting their use. We created SEED 2, a graphical user interface for handling high-throughput amplicon-sequencing data under Windows operating systems. Results SEED 2 is the only sequence visualizer that empowers users with tools to handle amplicon-sequencing data of microbial community markers. It is suitable for any marker genes sequences obtained through Illumina, IonTorrent or Sanger sequencing. SEED 2 allows the user to process raw sequencing data, identify specific taxa, produce of OTU-tables, create sequence alignments and construct phylogenetic trees. Standard dual core laptops with 8 GB of RAM can handle ca. 8 million of Illumina PE 300 bp sequences, ca. 4 GB of data. Availability and implementation SEED 2 was implemented in Object Pascal and uses internal functions and external software for amplicon data processing. SEED 2 is a freeware software, available at http://www.biomed.cas.cz/mbu/lbwrf/seed/ as a self-contained file, including all the dependencies, and does not require installation. Supplementary data contain a comprehensive list of supported functions. Supplementary information Supplementary data are available at Bioinformatics online.

Forest management practices often severely affect forest ecosystem functioning. Tree removal by clearcutting is one such practice, producing severe impacts due to the total reduction of primary productivity. Here, we assessed changes to fungal community structure and decomposition activity in the soil, roots and rhizosphere of a Picea abies stand for a 2-year period following clearcutting compared to data from before tree harvest. We found that the termination of photosynthate flow through tree roots into soil is associated with profound changes in soil, both in decomposition processes and fungal community composition. The rhizosphere, representing an active compartment of high enzyme activity and high fungal biomass in the living stand, ceases to exist and starts to resemble bulk soil. Decomposing roots appear to separate from bulk soil and develop into hotspots of decomposition and important fungal biomass pools. We found no support for the involvement of ectomycorrhizal fungi in the decomposition of roots, but we found some evidence that root endophytic fungi may have an important role in the early stages of this process. In soil, activity of extracellular enzymes also decreased in the long term following the end of rhizodeposition by tree roots.

Deadwood represents a source of nutrients, carbon and water for metabolism within forest ecosystem. Nutrients are mobilized due to the decomposition of wood, which is a long-term process that can be best studied by analysing environmental data on a temporary scale. Our study provides physico-temporal data on the downed logs of three major tree species in European temperate forests: Abies alba Mill., Fagus sylvatica L. and Picea abies (L.) Karst. Time since death was obtained using tree censuses (repeated for 40 years) and dendrochronology for each single downed log, the oldest being 75 years old. Standard laboratory methods were used for the determination of wood density and moisture changes. F. sylvatica was decomposed rapidly in the initial phase – mass loss was 50% during the 5 years after death, while A. alba and P. abies lost 13% and 16%, respectively. Downed logs of F. sylvatica contained 391 kg of water per m3, while these of P. abies 279 kg. A log-transformed linear model was created that shows the dependence of time since death on mass loss. According to the model, F. sylvatica had the shortest total decomposition time (39 years), followed by A. alba (58 years) and P. abies (86 years).

SUMMARY The ecology of forest soils is an important field of research due to the role of forests as carbon sinks. Consequently, a significant amount of information has been accumulated concerning their ecology, especially for temperate and boreal forests. Although most studies have focused on fungi, forest soil bacteria also play important roles in this environment. In forest soils, bacteria inhabit multiple habitats with specific properties, including bulk soil, rhizosphere, litter, and deadwood habitats, where their communities are shaped by nutrient availability and biotic interactions. Bacteria contribute to a range of essential soil processes involved in the cycling of carbon, nitrogen, and phosphorus. They take part in the decomposition of dead plant biomass and are highly important for the decomposition of dead fungal mycelia. In rhizospheres of forest trees, bacteria interact with plant roots and mycorrhizal fungi as commensalists or mycorrhiza helpers. Bacteria also mediate multiple critical steps in the nitrogen cycle, including N fixation. Bacterial communities in forest soils respond to the effects of global change, such as climate warming, increased levels of carbon dioxide, or anthropogenic nitrogen deposition. This response, however, often reflects the specificities of each studied forest ecosystem, and it is still impossible to fully incorporate bacteria into predictive models. The understanding of bacterial ecology in forest soils has advanced dramatically in recent years, but it is still incomplete. The exact extent of the contribution of bacteria to forest ecosystem processes will be recognized only in the future, when the activities of all soil community members are studied simultaneously.

Background Evergreen coniferous forests contain high stocks of organic matter. Significant carbon transformations occur in litter and soil of these ecosystems, making them important for the global carbon cycle. Due to seasonal allocation of photosynthates to roots, carbon availability changes seasonally in the topsoil. The aim of this paper was to describe the seasonal differences in C source utilization and the involvement of various members of soil microbiome in this process. Results Here, we show that microorganisms in topsoil encode a diverse set of carbohydrate-active enzymes, including glycoside hydrolases and auxiliary enzymes. While the transcription of genes encoding enzymes degrading reserve compounds, such as starch or trehalose, was high in soil in winter, summer was characterized by high transcription of ligninolytic and cellulolytic enzymes produced mainly by fungi. Fungi strongly dominated the transcription in litter and an equal contribution of bacteria and fungi was found in soil. The turnover of fungal biomass appeared to be faster in summer than in winter, due to high activity of enzymes targeting its degradation, indicating fast growth in both litter and soil. In each enzyme family, hundreds to thousands of genes were typically transcribed simultaneously. Conclusions Seasonal differences in the transcription of glycoside hydrolases and auxiliary enzyme genes are more pronounced in soil than in litter. Our results suggest that mainly fungi are involved in decomposition of recalcitrant biopolymers in summer, while bacteria replace them in this role in winter. Transcripts of genes encoding enzymes targeting plant biomass biopolymers, reserve compounds and fungal cell walls were especially abundant in the coniferous forest topsoil. Electronic supplementary material The online version of this article (10.1186/s40168-017-0340-0) contains supplementary material, which is available to authorized users.

spatial scale from which they are inferred. We show that the common assumption that climate is a predominant control on decomposition is supported only when local-scale variation is aggregated into mean values. Disaggregated data instead reveal that local-scale factors explain 73% of the variation in wood decomposition, and climate only 28%. Further, the temperature sensitivity of decomposition estimated from local versus mean analyses is 1.3-times greater. Fundamental issues with mean correlations were highlighted decades ago 9,10 , yet mean climate‐decomposition relationships are used to generate simulations that inform management and adaptation under environmental change. Our results suggest that to predict accurately how decomposition will respond to climate change, models must account for local-scale factors that control regional dynamics. Climate is traditionally thought to be the predominant control on decomposition rates at global and regional scales, with biotic factors controlling only local rates 2,4 . Biotic factors are divided into decomposer organisms, such as soil microbes, and the quality (for example, chemical composition) of the plant litter they decompose. Recent work suggests that litter quality may be more important than climate in controlling decomposition rates across biomes worldwide 3,11 , but the influence of decomposer organisms is still assumed limited across broad climate gradients 12 . A core reason for this assumption is that climate is considered a primary control on the activity of decomposers. As such, across climate gradients, mean temperature and moisture availability are assumed to explain much of the variation in the activity of decomposer organisms and hence decomposition rates of organic matter. These climatedecomposition relationships are used to parameterize and evaluate Earth-system models 13 . It is therefore important to test the assumption that climate drives decomposer activities because proper understanding of these activities is needed to inform model projections such as carbon cycleclimate

The availability of dead wood and ability to colonise these ephem- eral structures are crucial for sustaining vital populations of saproxylic insects. These insects locate suitable resources mostly visually and via olfactory cues emitted by dead wood and/or wood-decaying fungi. 2. For the conservation of saproxylic species of Heteroptera, a poorly studied group with a high proportion of threatened species, it is crucial to know which dead-wood structures are needed and how they are detected and colonised. 3. In a field experiment, we exposed different amounts of dead-wood logs and branches of the main tree species of montane beech forests (Abies alba, Fagus sylvatica) on sunny and shady forest plots. We sampled saproxylic heter- opterans and sporocarps of wood-decaying fungi in two consecutive years to test the hypothesis that early-successional saproxylic heteropterans are more attracted to wood-decaying fungi than to wood itself. 4. The activity densities of saproxylic heteropterans measured with flight- interception traps increased with increasing surface of coarse woody debris and was higher under sunny conditions. Tree species, fine woody debris and abun- dance of sporocarps had no significant effect. 5. Our results suggest that during the early-successional forest stage, dead wood provides more important cues than fungi in the search of saproxylic heteropterans for suitable hosts despite assumed close associations of the insects and certain fungal species. To improve habitats for saproxylic heteropterans, we recommend increasing the supply of dead wood of large diameter (>30 cm) in montane beech forests, particularly in sunny gaps. This can easily be realised during logging operations by gap felling.

The aim of this study was to describe the decomposition of litter along a successive series of sites developed at a post-mining overburden deposit over 12, 21 and 45 years, representing the early, mid and late stages of succession. Litter decomposition was largely dependent on the initial composition of the litters. The tree litter of the mid and late stages decomposed faster than the grass litter of the early stage, with 64, 60 and 35 % of mass lost over 2 years, respectively. The contents of hot-water-soluble C and N, which were the best predictors of litter decay rates, were relatively stable over time in all litters. Neither the nutrient content nor the plant biopolymer composition exhibited convergence during decay, indicating that the litter-derived soil organic matter most likely carries a legacy of the original vegetation. In contrast to the litter chemistry, the development of the microbial community was largely specific to the decay stage and consistent among the litters, showing decreasing fungal dominance in time. Extracellular enzymes were found to be of limited value in the prediction of litter decay rates, chemical transformation or microbial community composition.

Molecular methods for the analysis of biomolecules have undergone rapid technological development in the last decade. The advent of next-generation sequencing methods and improvements in instrumental resolution enabled the analysis of complex transcriptome, proteome and metabolome data, as well as a detailed annotation of microbial genomes. The mechanisms of decomposition by model fungi have been described in unprecedented detail by the combination of genome sequencing, transcriptomics and proteomics. The increasing number of available genomes for fungi and bacteria shows that the genetic potential for decomposition of organic matter is widespread among taxonomically diverse microbial taxa, while expression studies document the importance of the regulation of expression in decomposition efficiency. Importantly, high-throughput methods of nucleic acid analysis used for the analysis of metagenomes and metatranscriptomes indicate the high diversity of decomposer communities in natural habitats and their taxonomic composition. Today, the metaproteomics of natural habitats is of interest. In combination with advanced analytical techniques to explore the products of decomposition and the accumulation of information on the genomes of environmentally relevant microorganisms, advanced methods in microbial ecophysiology should increase our understanding of the complex processes of organic matter transformation.

Microbial activity in forest soils is driven by the dynamics of ecosystem processes, largely dependent on trees as the major primary producers. Diurnal variation of root activity, seasonality of photosynthate production or recalcitrance of decomposing plant biomass all affect microbial abundance, composition of their communities and activity. Due to low N content, fungi appear to be the major decomposers of complex plant biomass: litter and deadwood and to largely shape associated bacterial communities and their activity. On the other hand, bacteria are important in decomposition of fungal mycelia and N-cycle processes including N-fixation. Microbial activity is also affected in the short term by climatic events and in the long-term by ecosystem development after disturbances.

The effects of tree species differing in foliage and litter chemistry on the chemical, micro-morphological, and biological properties of soil were studied on post-mining sites afforested with one of six tree species (spruce, pine, larch, oak, lime, and alder) and also on sites left to natural succession (dominated by willow). The sites were located on a large colliery spoil heap that had been produced by the mining of coal in alkaline tertiary clays near the city of Sokolov, Czech Republic. Because no topsoil had been applied to the sites, soil development resulted from in situ interactions among the deposited overburden (spoil), trees, and soil biota. Soil formation differed markedly among sites afforested with different tree species. On sites with trees producing litter with a low C/N ratio (the deciduous species), the organic Oe layer was narrow or absent and a thick organomineral A layer was evident. On sites with trees producing litter with a high C/N ratio (the evergreen species), in contrast, a thick Oe layer and a thin A layer were evident. Besides C/N ratio, earthworm abundance and earthworm bioturbation activity (measured as the amount of earthworm casts in the topsoil) were the strongest predictors of A layer thickness and C accumulation in the mineral topsoil. Sites with higher C accumulation in mineral soil had higher microbial biomass and lower microbial respiration, which may have contributed to the higher C storage. The gradient of bioturbation was correlated with changes in the composition of the bacterial community and other soil biota, but partial correlation showed that the effects of litter quality and bioturbation were largely independent. Overall, the results indicate that the effect of tree species on soil development is substantially mediated by soil fauna activity and especially by earthworm bioturbation.

The relative contribution of top-down and bottom-up processes regulating primary decomposers can influence the strength of the link between the soil animal community and ecosystem functioning. Although soil bacterial communities are regulated by bottom-up and top-down processes, the latter are considered to be less important in structuring the diversity and functioning of fungal-dominated ecosystems. Despite the huge diversity of mycophagous (fungal-feeding) soil fauna, and their potential to reverse the outcomes of competitive fungal interactions, top-down grazing effects have never been found to translate to community-level changes. We constructed soil mesocosms to investigate the potential of isopods grazing on cord-forming basidiomycete fungi to influence the community composition and functioning of a complex woodland soil microbial community. Using metagenomic sequencing we provide conclusive evidence of direct top-down control at the community scale in fungal-dominated woodland soil. By suppressing the dominant cord-forming basidiomycete fungi, isopods prevented the competitive exclusion of surrounding litter fungi, increasing diversity in a community containing several hundred fungal species. This isopod-induced modification of community composition drove a shift in the soil enzyme profile, and led to a restructuring of the wider mycophagous invertebrate community. We highlight characteristics of different soil ecosystems that will give rise to such top-down control. Given the ubiquity of isopods and basidiomycete fungi in temperate and boreal woodland ecosystems, such top-down community control could be of widespread significance for global carbon and nutrient cycling.

The aim of this study was to compare how litter and excrements of Bibio marci larvae affect respiration. We measured changes in microbial respiration over a two-week period to ascertain the effects of added leaf litter and excrements produced from the same litter by larvae of St Mark's fly (B. marci). At the same time, we measured the effect of litter and excrement introduction into soil on soil respiration. We used litter pieces and ground litter to evaluate the effect of fragmentation. Two tree species (alder and oak) and two soil types (with high and low organic carbon content) were used to investigate the interaction between litter quality and soil C content. Bacterial counts were obtained and ergosterol content was measured at the end of the cultivation period. The soil with high organic matter content exhibited higher respiration than the soil with low organic matter content. In the absence of soil, both alder litter and excrements induced higher respiration than oak litter and excrements. For both tree species, litter pieces elicited higher respiration than excrements. Concerning the effect of substrate addition, ground litter of alder induced higher respiration than litter pieces. The opposite applied to oak litter, most likely because of a high content of phenolics in oak litter that were made more available in the ground litter. When the substrates were introduced into soil, litter (both in pieces and ground) increased microbial respiration more than excrements. The increase due to respiration was much higher in the soil with low organic matter content. Bacterial counts increased more in the soil with low organic matter content, while the opposite was the case for ergosterol content. Organic matter mineralization of macrofauna excrements was lower compared to intact litter. This was particularly true when litter or excrements were introduced into soil. A high-pH substrate and biomass with few microbes probably induces a large priming effect in low-carbon soils.

Fungi are important in soils as both decomposers and plant symbionts, and an understanding of the composition of their complex communities is thus indispensable to answer a variety of ecological questions. 454 Pyrosequencing is currently the method of choice for the in-depth analysis of fungal communities. However, the interpretation of its results is complicated by differences in data analysis approaches that make inter-study comparisons difficult. The pyrosequencing studies published so far have also used variable molecular targets in fungal rDNA. Although the ITS region and, in particular, ITS1 appear to be the most frequent sequencing targets, the use of various primers with different coverages of fungal groups remains a serious problem. Sequence length limits also vary widely across studies, and in many studies, length differences may negatively affect sequence similarity clustering or identification. Unfortunately, many studies neglect the need to correct for method-dependent errors, such as pyrosequencing noise or chimeric sequences. Even when performed, error rates in sequences may be high, and consensus sequences created by sequence clustering therefore better represent operational taxonomic units. We recommend a data analysis workflow that includes sequence denoising, chimera removal, sequence trimming before clustering and random resampling before calculating diversity parameters. The newly developed free pipeline (SEED) introduced here can be used to perform all the required analytical steps. The improvement and unification of data analysis procedures should make future studies both more reliable and comparable and allow meta-studies to be performed to provide more general views on fungal diversity, biogeography or ecology.

The contents of fungal biomass markers were analysed in the fruit bodies of dominant basidiomycetes from an ectomycorrhiza-dominated coniferous forest, and used to estimate the fungal biomass content in the litter and soil. The content of ergosterol (3.8 ± 2.0 mg g−1 dry fungal biomass) and the phospholipid fatty acid 18:2ω6,9 (11.6 ± 4.3 mg g−1) showed less variation than the internal transcribed spacer (ITS) copy numbers (375 ± 294 × 109 copies g−1). A high level of variation in the ITS copy numbers (per ng DNA) was also found among fungal taxa. The content of fungal biomass in the litter and soil, calculated using the mean contents, varied between 0.66 and 6.24 mg g−1 fungal biomass in the litter, and 0.22 and 0.68 mg g−1 in the soil. The ratio of fungal biomass in the litter to that in the soil varied greatly among the markers. The estimates of fungal biomass obtained with different biomarkers are not exactly comparable, and caution should be used when analysing taxon abundance using PCR amplification of fungal rDNA.

The activities of extracellular enzymes that participate in the decomposition of litter and organic matter in forest soils depend on, among other factors, temperature and soil moisture content and also reflect the quality of litter, which changes dramatically after a short litterfall period. Here, we explored the effects of soil temperature and seasonality on the sizes of extracellular enzyme pools and activities in a temperate hardwood forest soil with dominant Quercus petraea (cambisol, mean annual temperature 9.3 °C). We hypothesized that the most significant variation of enzyme activity would occur in the litter, which faces greater variations in temperature, moisture content and chemical quality during the season, which decrease with soil depth. The site exhibited relatively large seasonal temperature differences and moderate changes in soil moisture content. Enzyme activity, microbial biomass, soil moisture content, temperature and pH were monitored for three years in the litter (L), organic horizon (O) and upper mineral horizon (Ah). Enzyme activity in vitro strongly increased with temperature until 20–25 °C, the highest temperatures recorded in situ. While no significant differences in the pools of most extracellular enzymes and in the content of microbial biomass were found among the seasons, enzyme activity typically increased during the warm period of the year, especially in the O and Ah horizons. Approximately 63%, 64%, and 69% of total annual activity was recorded during the warm period of the year in the L, O, and Ah horizons, respectively. Significant positive correlations were observed between soil moisture content and fungal biomass, but not bacterial biomass, indicating a decrease of the fungal/bacterial biomass ratio under dry conditions. The effect of moisture on enzyme activities was not significant except for endoxylanase in the litter. If soil temperature rises as predicted due to global climate change, enzyme activity is predicted to rise substantially in this ecosystem, especially in winter, when decomposition is not limited by drought and fresh litter that can decompose rapidly is present.

The aim of this work was to quantify the effects of vegetation on the activity of extracellular enzymes in the litter and soil. To achieve this, we investigated a set of post-mining sites in a brown-coal mine deposit area near Sokolov, Czech Republic. The sites were 22–33 years old and had been established on the same initial substrate by planting with six tree genera or leaving for spontaneous revegetation, with four replicate sites per vegetation type. The activity of extracellular hydrolytic and oxidative enzymes and the microbial community composition of the litter and topsoil were compared in the spring, summer and autumn using the dominant tree, pH, soil nutrient content and soil moisture as the explanatory variables. Sites under individual trees exhibited significant differences in the chemical properties of both the litter and soil, and the tree effect was identified as the most important factor affecting the activity of extracellular enzymes either directly or in the interaction with seasonal effects, although not all pairs of tree species were significantly different from each other. Seasonal effects on enzyme activity were only important in the litter. The effects of dominant trees and of seasons contributed equally to the variation in the microbial community composition at individual sites. Only a minor part of the tree effect could be explained by differences in the litter or soil chemistry. Among the chemical variables, the N content most affected the microbial biomass content, increasing fungal (but not bacterial) biomass in the litter and bacterial (but not fungal) biomass in the soil. The results indicate that other factors, such as nutrient quality or the specific association of microorganisms with rhizospheres of different trees or the understory, are likely important mediators of the vegetation effects. When comparing the revegetated sites with sites under spontaneous succession, the enzyme activities and microbial biomass were similar except for the sites revegetated with Alnus which may indicate similar rates of soil development at revegetated and succession sites. Spontaneous succession in temperate Europe may thus be a suitable option for land restoration.

Globally, forests represent highly productive ecosystems that act as carbon sinks where soil organic matter is formed from residuals after biomass decomposition as well as from rhizodeposited carbon. Forests exhibit a high level of spatial heterogeneity and the importance of trees, the dominant primary producers, for their structure and functioning. Fungi, bacteria and archaea inhabit various forest habitats: foliage, the wood of living trees, the bark surface, ground vegetation, roots and the rhizosphere, litter, soil, deadwood, rock surfaces, invertebrates, wetlands or the atmosphere, each of which has its own specific features, such as nutrient availability or temporal dynamicy and specific drivers that affect microbial abundance, the level of dominance of bacteria or fungi as well as the composition of their communities. However, several microorganisms, and in particular fungi, inhabit or even connect multiple habitats, and most ecosystem processes affect multiple habitats. Forests are dynamic on a broad temporal scale with processes ranging from short-term events over seasonal ecosystem dynamics to long-term stand development after disturbances such as fires or insect outbreaks. The understanding of these processes can be only achieved by the exploration of the complex 'ecosystem microbiome' and its functioning using focused, integrative microbiological and ecological research performed across multiple habitats.

Forests are recognised as spatially heterogeneous ecosystems. However, knowledge of the small-scale spatial variation in microbial abundance, community composition and activity is limited. Here, we aimed to describe the heterogeneity of environmental properties, namely vegetation, soil chemical composition, fungal and bacterial abundance and community composition, and enzymatic activity, in the topsoil in a small area (36 m2) of a highly heterogeneous regenerating temperate natural forest, and to explore the relationships among these variables. The results demonstrated a high level of spatial heterogeneity in all properties and revealed differences between litter and soil. Fungal communities had substantially higher beta-diversity than bacterial communities, which were more uniform and less spatially autocorrelated. In litter, fungal communities were affected by vegetation and appeared to be more involved in decomposition. In the soil, chemical composition affected both microbial abundance and the rates of decomposition, whereas the effect of vegetation was small. Importantly, decomposition appeared to be concentrated in hotspots with increased activity of multiple enzymes. Overall, forest topsoil should be considered a spatially heterogeneous environment in which the mean estimates of ecosystem-level processes and microbial community composition may confound the existence of highly specific microenvironments.

The decomposition of dead plant biomass contributes to the carbon cycle and is one of the key processes in temperate forests. While fungi in litter decomposition drive the chemical changes occurring in litter, the bacterial community appears to be important as well, especially later in the decomposition process when its abundance increases. In this paper, we describe the bacterial community composition in live Quercus petraea leaves and during the subsequent two years of litter decomposition. Members of the classes Alpha-, Beta- and Gammaproteobacteria and the phyla Actinobacteria, Bacteroidetes and Acidobacteria were dominant throughout the experiment. Bacteria present in the oak phyllosphere were rapidly replaced by other taxa after leaf senescence. There were dynamic successive changes in community composition, in which the early-stage (months 2-4), mid-stage (months 6-8) and late-stage (months 10-24) decomposer communities could be distinguished, and the diversity increased with time. Bacteria associated with dead fungal mycelium were important during initial decomposition, with sequence relative abundances of up to 40% of the total bacterial community in months 2 and 4 when the highest fungal biomass was observed. Cellulose-decomposing bacteria were less frequent, with abundance ranging from 4% to 15%. The bacterial community dynamics reflects changes in the availability of possible resources either of the plant or microbial origin.

Community-level physiological profiling (CLPP) analyses from very diverse environments are frequently used with the aim of characterizing the metabolic versatility of whole environmental bacterial communities. While the limitations of the methodology for the characterization of whole communities are well known, we propose that CLPP combined with high-throughput sequencing and qPCR can be utilized to identify the copiotrophic, fast-growing fraction of the bacterial community of soil environments, where oligotrophic taxa are usually dominant. In the present work we have used this approach to analyze samples of litter and soil from a coniferous forest in the Czech Republic using BIOLOG GN2 plates. Monosaccharides and amino acids were utilized significantly faster than other C substrates, such as organic acids, in both litter and soil samples. Bacterial biodiversity in CLPP wells was significantly lower than in the original community, independently of the carbon source. Bacterial communities became highly enriched in taxa that typically showed low abundance in the original soil, belonging mostly to the Gammaproteobacteria and the genus Pseudomonas, indicating that the copiotrophic strains, favoured by the high nutrient content, are rare in forest litter and soil. In contrast, taxa abundant in the original samples were rarely found to grow at sufficient rates under the CLPP conditions. Our results show that CLPP is useful to detect copiotrophic bacteria from the soil environments and that bacterial growth is substrate specific.

Identification of bacteria that produce carbohydrolytic enzymes is extremely important given the increased demand for these enzymes in many industries. Twenty lignocellulose-degrading bacterial isolates from Algerian compost and different soils were screened for their potential to produce different enzymes involved in biomass deconstruction. Based on 16S rRNA gene sequencing, the isolates belonged to Proteobacteria and Actinobacteria. Differences among species were reflected both as the presence/absence of enzymes or at the level of enzyme activity. Among the most active species, Bosea sp. FBZP-16 demonstrated cellulolytic activity on both amorphous cellulose (CMC) and complex lignocellulose (wheat straw) and was selected for whole-genomic sequencing. The genome sequencing revealed the presence of a complex enzymatic machinery required for organic matter decomposition. Analysis of the enzyme-encoding genes indicated that multiple genes for endoglucanase, xylanase, β-glucosidase and β-mannosidase are present in the genome with enzyme activities displayed by the bacterium, while other enzymes, such as certain cellobiohydrolases, were not detected at the genomic level. This indicates that a combination of functional screening of bacterial cultures with the use of genome-derived information is important for the prediction of potential enzyme production. These results provide insight into their possible exploitation for the production of fuels and chemicals derived from plant biomass.

Background The search for new enzymes and microbial strains to degrade plant biomass is one of the most important strategies for improving the conversion processes in the production of environment-friendly chemicals and biofuels. In this study, we report a new Paenibacillus isolate, O199, which showed the highest efficiency for cellulose deconstruction in a screen of environmental isolates. Here, we provide a detailed description of the complex multi-component O199 enzymatic system involved in the degradation of lignocellulose. Results We examined the genome and the proteome of O199 grown on complex lignocellulose (wheat straw) and on microcrystalline cellulose. The genome contained 476 genes with domains assigned to carbohydrate-active enzyme (CAZyme) families, including 100 genes coding for glycosyl hydrolases (GHs) putatively involved in cellulose and hemicellulose degradation. Moreover, 31 % of these CAZymes were expressed on cellulose and 29 % on wheat straw. Proteomic analyses also revealed a complex and complete set of enzymes for deconstruction of cellulose (at least 22 proteins, including 4 endocellulases, 2 exocellulases, 2 cellobiohydrolases and 2 β-glucosidases) and hemicellulose (at least 28 proteins, including 5 endoxylanases, 1 β-xylosidase, 2 xyloglucanases, 2 endomannanases, 2 licheninases and 1 endo-β-1,3(4)-glucanase). Most of these proteins were secreted extracellularly and had numerous carbohydrate-binding domains (CBMs). In addition, O199 also secreted a high number of substrate-binding proteins (SBPs), including at least 42 proteins binding carbohydrates. Interestingly, both plant lignocellulose and crystalline cellulose triggered the production of a wide array of hydrolytic proteins, including cellulases, hemicellulases, and other GHs. Conclusions Our data provide an in-depth analysis of the complex and complete set of enzymes and accessory non-catalytic proteins—GHs, CBMs, transporters, and SBPs—implicated in the high cellulolytic capacity shown by this bacterial strain. The large diversity of hydrolytic enzymes and the extracellular secretion of most of them supports the use of Paenibacillus O199 as a candidate for second-generation technologies using paper or lignocellulosic agricultural wastes. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0518-x) contains supplementary material, which is available to authorized users.

Evidence shows that bacteria contribute actively to the decomposition of cellulose and hemicellulose in forest soil; however, their role in this process is still unclear. Here we performed the screening and identification of bacteria showing potential cellulolytic activity from litter and organic soil of a temperate oak forest. The genomes of three cellulolytic isolates previously described as abundant in this ecosystem were sequenced and their proteomes were characterized during the growth on plant biomass and on microcrystalline cellulose. Pedobacter and Mucilaginibacter showed complex enzymatic systems containing highly diverse carbohydrate-active enzymes for the degradation of cellulose and hemicellulose, which were functionally redundant for endoglucanases, β-glucosidases, endoxylanases, β-xylosidases, mannosidases and carbohydrate-binding modules. Luteibacter did not express any glycosyl hydrolases traditionally recognized as cellulases. Instead, cellulose decomposition was likely performed by an expressed GH23 family protein containing a cellulose-binding domain. Interestingly, the presence of plant lignocellulose as well as crystalline cellulose both trigger the production of a wide set of hydrolytic proteins including cellulases, hemicellulases and other glycosyl hydrolases. Our findings highlight the extensive and unexplored structural diversity of enzymatic systems in cellulolytic soil bacteria and indicate the roles of multiple abundant bacterial taxa in the decomposition of cellulose and other plant polysaccharides.

Biogeochemical processes and ecosystemic functions are mostly driven by soil microbial communities. However, most methods focus on evaluating the total microbial community and fail to discriminate its active fraction which is linked to soil functionality. Precisely, the activity of the microbial community is strongly limited by the availability of organic carbon (C) in soils under arid and semi-arid climate. Here, we provide a complementary genomic and metaproteomic approach to investigate the relationships between the diversity of the total community, the active diversity and ecosystem functionality across a dissolved organic carbon (DOC) gradient in southeast Spain. DOC correlated with the ecosystem multifunctionality index composed by soil respiration, enzyme activities (urease, alkaline phosphatase and β-glucosidase) and microbial biomass (phospholipid fatty acids, PLFA). This study highlights that the active diversity (determined by metaprotoemics) but not the diversity of the whole microbial community (evaluated by amplicon gene sequencing) is related to the availability of organic C and it is also connected to the ecosystem multifunctionality index. We reveal that DOC shapes the activities of bacterial and fungal populations in Mediterranean semi-arid soils and determines the compartmentalization of functional niches. For instance, Rhizobales thrived at high-DOC sites probably fuelled by metabolism of one-C compounds. Moreover, the analysis of proteins involved in the transport and metabolism of carbohydrates revealed that Ascomycota and Basidiomycota occupied different nutritional niches. The functional mechanisms for niche specialization were not constant across the DOC gradient.

Direct extraction and characterization of microbial community DNA through PCR amplicon surveys and metagenomics has revolutionized the study of environmental microbiology and microbial ecology. In particular, metagenomic analysis of nucleic acids provides direct access to the genomes of the “uncultivated majority.” Accelerated by advances in sequencing technology, microbiologists have discovered more novel phyla, classes, genera, and genes from microorganisms in the first decade and a half of the twenty-first century than since these “many very little living animalcules” were first discovered by van Leeuwenhoek. The unsurpassed diversity of soils promises continued exploration of a range of industrial, agricultural, and environmental functions. The ability to explore soil microbial communities with increasing capacity offers the highest promise for answering many outstanding who, what, where, when, why, and with whom questions such as: Which microorganisms are linked to which soil habitats? How do microbial abundances change with changing edaphic conditions? How do microbial assemblages interact and influence one another synergistically or antagonistically? What is the full extent of soil microbial diversity, both functionally and phylogenetically? What are the dynamics of microbial communities in space and time? How sensitive are microbial communities to a changing climate? What is the role of horizontal gene transfer in the stability of microbial communities? Do highly diverse microbial communities confer resistance and resilience in soils?, La lista completa de autores que integran el documento puede consultarse en el archivo., Facultad de Ciencias Exactas

Marine-to-terrestrial transition represents one of the most fundamental shifts in microbial life. Understanding the distribution and drivers of soil microbial communities across coastal ecosystems is critical given the roles of microbes in soil biogeochemistry and their multifaceted influence on landscape succession. Here, we studied the fungal community dynamics in a well-established salt marsh chronosequence that spans over a century of ecosystem development. We focussed on providing high-resolution assessments of community composition, diversity and ecophysiological shifts that yielded patterns of ecological succession through soil formation. Notably, despite containing 10- to 100-fold lower fungal internal transcribed spacer abundances, early-successional sites revealed fungal richnesses comparable to those of more mature soils. These newly formed sites also exhibited significant temporal variations in beta-diversity that may be attributed to the highly dynamic nature of the system imposed by the tidal regime. The fungal community compositions and ecophysiological assignments changed substantially along the successional gradient, revealing a clear signature of ecological replacement and gradually transforming the environment from a marine into a terrestrial system. Moreover, distance-based linear modelling revealed soil physical structure and organic matter to be the best predictors of the shifts in fungal beta-diversity along the chronosequence. Taken together, our study lays the basis for a better understanding of the spatiotemporally determined fungal community dynamics in salt marshes and highlights their ecophysiological traits and adaptation in an evolving ecosystem.

Stable isotope probing (SIP) provides the opportunity to label decomposer microorganisms that build their biomass on a specific substrate. In combination with high-throughput sequencing, SIP allows for the identification of fungal community members involved in a particular decomposition process. Further information can be gained through gene-targeted metagenomics and metatranscriptomics, opening the possibility to describe the pool of genes catalyzing specific decomposition reactions in situ and to identify the diversity of genes that are expressed. When combined with gene descriptions of fungal isolates from the same environment, specific biochemical reactions involved in decomposition can be linked to individual fungal taxa. Here we describe the use of these methods to explore the cellulolytic fungal community in forest litter and soil.

The precise characterization of the behavior of individual microorganisms in the presence of increased mercury contents in soil is necessary for better elucidation of the fate of mercury in the soil environment. In our investigation, resistant bacterial strains isolated from two mercury contaminated soils, represented by Paenibacillus alginolyticus, Burkholderia glathei, Burkholderia sp., and Pseudomonas sp., were used. Two differently contaminated soils (0.5 and 7 mg kg(-1) total mercury) were chosen. Preliminary soil analysis showed the presence of methylmercury and phenylmercury with the higher soil mercury level. Modified rhizobox experiments were performed to assess the ability of mercury accumulating strains to deplete the mobile and mobilizable mercury portions in the soil by modification; microbial agar cultures were used rather than the plant root zone. A sequential extraction procedure was performed to release the following mercury fractions: water soluble, extracted in acidic conditions, bound to humic substances, elemental, and bound to complexes, HgS and residual. Inductively coupled plasma mass spectrometry (ICP-MS) and a single-purpose atomic absorption spectrometer (AMA-254) were applied for mercury determination in the samples and extracts. Gas chromatography coupled to atomic fluorescence spectrometry (GC-AFS) was used for the determination of organomercury compounds. The analysis of the microbial community at the end of the experiment showed a 42% abundance of Paenibacillus sp. followed by Acetivibrio sp., Brevibacillus sp., Cohnella sp., Lysinibacillus sp., and Clostridium sp. not exceeding 2% abundance. The results suggest importance of Paenibacillus sp. in Hg transformation processes. This genus should be tested for potential bioremediation use in further research.

The feasibility of decontaminating creosote-treated wood (CTW) by co-composting with agricultural wastes was investigated using two bulking agents, grass cuttings (GC) and broiler litter (BL), each employed at a 1:1 ratio with the matrix. The initial concentration of total polycyclic aromatic hydrocarbons (PAHs) in CTW (26,500 mg kg(-1)) was reduced to 3 and 19% after 240 d in GC and BL compost, respectively. PAH degradation exceeded the predicted bioaccesible threshold, estimated through sequential supercritical CO2 extraction, together with significant detoxification, assessed by contact tests using Vibrio fisheri and Hordeum vulgare. GC composting was characterized by high microbial biomass growth in the early phases, as suggested by phospholipid fatty acid analyses. Based on the 454-pyrosequencing results, fungi (mostly Saccharomycetales) constituted an important portion of the microbial community, and bacteria were characterized by rapid shifts (from Firmicutes (Bacilli) and Actinobacteria to Proteobacteria). However, during BL composting, larger amounts of prokaryotic and eukaryotic PLFA markers were observed during the cooling and maturation phases, which were dominated by Proteobacteria and fungi belonging to the Ascomycota and those putatively related to the Glomeromycota. This work reports the first in-depth analysis of the chemical and microbiological processes that occur during the co-composting of a PAH-contaminated matrix.

Stable isotope probing (SIP) provides the opportunity to label decomposer microorganisms that build their biomass on a specific substrate. In combination with high-throughput sequencing, SIP allows for the identification of fungal community members involved in a particular decomposition process. Further information can be gained through gene-targeted metagenomics and metatranscriptomics, opening the possibility to describe the pool of genes catalyzing specific decomposition reactions in situ and to identify the diversity of genes that are expressed. When combined with gene descriptions of fungal isolates from the same environment, specific biochemical reactions involved in decomposition can be linked to individual fungal taxa. Here we describe the use of these methods to explore the cellulolytic fungal community in forest litter and soil.

Fungi are considered the primary decomposers of dead plant biomass in terrestrial ecosystems. However, current knowledge regarding the successive changes in fungal communities during litter decomposition is limited. Here we explored the development of the fungal community over 24 months of litter decomposition in a temperate forest with dominant Quercus petraea using 454-pyrosequencing of the fungal internal transcribed spacer (ITS) region and cellobiohydrolase I (cbhI) genes, which encode exocellulases, to specifically address cellulose decomposers. To quantify the involvement of phyllosphere fungi in litter decomposition, the fungal communities in live leaves and leaves immediately before abscission were also analysed. The results showed rapid succession of fungi with dramatic changes in the composition of the fungal community. Furthermore, most of the abundant taxa only temporarily dominated in the substrate. Fungal diversity was lowest at leaf senescence, increased until month 4 and did not significantly change during subsequent decomposition. Highly diverse community of phyllosphere fungi inhabits live oak leaves 2 months before abscission, and these phyllosphere taxa comprise a significant share of the fungal community during early decomposition up to the fourth month. Sequences assigned to the Ascomycota showed highest relative abundances in live leaves and during the early stages of decomposition. In contrast, the relative abundance of sequences assigned to the Basidiomycota phylum, particularly basidiomycetous yeasts, increased with time. Although cellulose was available in the litter during all stages of decomposition, the community of cellulolytic fungi changed substantially over time. The results indicate that litter decomposition is a highly complex process mediated by various fungal taxa.

Traditionally, the models of fungal cellulose hydrolysis have included the actions of endo- and exo-cleaving hydrolytic enzymes, with some involvement of nonspecific cellulose hydrolysis by radical-based oxidative reactions. Only recently, the GH61 glycosyl hydrolases have been demonstrated to be copper-containing enzymes – polysaccharide monooxygenases (PMOs) – internally cleaving cellulose molecules in an oxidative way in synergy with cellobiose dehydrogenase and other cellulolytic enzymes. PMOs can be found in most genomes of ascomycetous and basidiomycetous fungi, including plant pathogens, litter decomposers, white-rot and brown-rot fungi and mycorrhizal fungi. Although their real importance in lignocellulose decomposition is still unknown, their high expression level in the presence of lignocelluloses and substantial synergistic enhancement of cellulose hydrolysis in cooperation with other cellulolytic enzymes seems to indicate their active involvement in cellulose hydrolysis. In a recent report, polysaccharide monooxygenase genes have been reported from litter metatranscriptomes, and their involvement in litter decomposition thus seems likely.

Thirty-nine white-rot fungi belonging to nine species of Agaricomycotina (Basidiomycota) were initially screened for their ability to decrease olive-mill wastewater (OMW) phenolics. Four strains of Ganoderma australe, Ganoderma carnosum, Pleurotus eryngii and Pleurotus ostreatus, were selected and further examined for key-aspects of the OMW biodegradation process. Fungal growth in OMW-containing batch cultures resulted in significant decolorization (by 40-46% and 60-65% for Ganoderma and Pleurotus spp. respectively) and reduction of phenolics (by 64-67% and 74-81% for Ganoderma and Pleurotus spp. respectively). COD decrease was less pronounced (12-29%). Cress-seeds germination increased by 30-40% when OMW was treated by Pleurotus strains. Toxicity expressed as inhibition of Aliivibrio fischeri luminescence was reduced in fungal-treated OMW samples by approximately 5-15 times compared to the control. As regards the pertinent enzyme activities, laccase and Mn-independent peroxidase were detected for Ganoderma spp. during the entire incubation period. In contrast, Pleurotus spp. did not exhibit any enzyme activities at early growth stages; instead, high laccase (five times greater than those of Ganoderma spp.) and Mn peroxidases activities were determined at the end of treatment. OMW decolorization by Ganoderma strains was strongly correlated to the reduction of phenolics, whereas P. eryngii laccase activity was correlated with the effluent's decolorization.

Turnover of fungal biomass in forest litter and soil represents an important process in the environment. To date, knowledge of mycelial decomposition has been derived primarily from short-term studies, and the guild of mycelium decomposers has been poorly defined. Here, we followed the fate of the fruiting bodies of an ectomycorrhizal fungus in litter and soil of a temperate forest over 21 wk. The community of associated microbes and enzymatic processes in this specific substrate were described. The decomposition of fungal fruiting bodies exhibited biphasic kinetics. The rapid initial phase, which included the disappearance of DNA, was followed by a slower turnover of the recalcitrant fraction. Compared with the surrounding litter and soil, the mycelium represented a hotspot of activity of several biopolymer-degrading enzymes and high bacterial biomass. Specific communities of bacteria and fungi were associated with decomposing mycelium. These communities differed between the initial and late phases of decomposition. The bacterial community associated with decomposing mycelia typically contained the genera Pedobacter, Pseudomonas, Variovorax, Chitinophaga, Ewingella and Stenotrophomonas, whereas the fungi were mostly nonbasidiomycetous r-strategists of the genera Aspergillus, Penicillium, Mortierella, Cladosporium and several others. Decomposing ectomycorrhizal fungal mycelium exhibits high rates of decomposition and represents a specific habitat supporting a specific microbial community.

Decomposition of Picea abies needles and production of extracellular enzymes involved in decomposition of lignin, cellulose, hemicelluloses and other organic compounds were studied in fungal strains of interior needle colonizers isolated from needles in different stages of decomposition (attached to trees, and early and late decomposition stages in the litter horizon). In total, 12 strains of ascomycetes (members of Helotiales, Hypocreales, Dothideales, Diaporthales and Eurotiales) and four basidiomycetes (Polyporales, Agaricales and Russulales) were tested. Significant decomposition of needles was recorded for all fungal isolates. All isolates produced cellobiohydrolase, β-glucosidase, β-xylosidase, N-acetylglucosaminidase, α-glucosidase, phosphatase and arylsulfatase and most fungi also produced endocellulase, endoxylanase and laccase in needle litter. In addition, other hemicellulases were produced by all strains. Mn-peroxidase was only produced by two basidiomycetes. Although enzyme activities varied, fungi associated with needles on fallen trees exhibited enzyme production comparable with later litter colonizers, and there was no significant difference in enzyme production between ascomycete and basidiomycete strains.

Chemical composition of litter has previously been reported to affect in situ decomposition. To identify its effects on a single species level, the saprotrophic basidiomycete Hypholoma fasciculare was grown on 11 types of litter with variable chemical composition (N content of 3.4–28.9 mg g−1), and the mass loss of litter and lignin, production of extracellular enzymes and fungal biomass were followed. After 12 weeks, mass loss ranged from 16 % to 34 %. During early decomposition stages, litter mass loss, fungal biomass production (estimated by ergosterol content) as well as fungal substrate use efficiency all increased with increasing initial N content of the litter. The initial litter decomposition rate was significantly positively correlated with the activities of arylsulfatase, cellobiohydrolase, endoxylanase and phosphatase. Contrary to expectations, the lignin content did not affect litter mass loss, when covariation with N content was accounted for. The ratio of lignin loss to total mass loss depended on the litter type and did not reflect the activities of ligninolytic enzymes.

Extracellular enzymes produced by heterotrophic microorganisms in the soil are responsible for the decomposition of organic compounds. Basidiomycete fungi are the primary decomposer agents in temperate wooded ecosystems and contribute extensively to extracellular enzyme activity and nutrient mineralisation within soils. Growth and development of basidiomycete mycelia is influenced by soil-dwelling invertebrate grazers with potential implications for fungal activity and ecosystem functioning. The impacts of four invertebrate taxa belonging to Isopoda, Myriapoda, Collembola and Nematoda on the production of eight hydrolytic enzymes by four saprotrophic basidiomycetes (Phanerochaete velutina, Resinicium bicolor and two strains of Hypholoma fasciculare) were compared in a factorial microcosm study. Grazing generally increased enzyme production but invertebrates had species-specific impacts on enzyme activity. The magnitude of grazing influenced enzyme activity; macrofauna (woodlice and millipedes) induced the greatest responses. Enzymatic responses varied markedly between fungi. Grazing enhanced enzyme activity in the exploitative mycelial networks of P. velutina and H. fasciculare, while the opposite effects were observed in the explorative R. bicolor networks. The impacts of soil fauna on nutrient mineralisation depend on fungal community composition. β-glucosidase, cellobiohydrolase, N-acetylglucosaminidase, acid phosphatase and phosphodiesterase activities were affected most frequently by grazing and invertebrate activity, and thus had direct consequences for carbon, nitrogen and phosphorous cycling. The results indicate that invertebrate diversity and community composition may influence the spatial distribution and activity of extracellular enzymes with direct implications for nutrient mineralisation and trunover in woodland soils.

The unmanaged Norway spruce montane forests in the Bohemian Forest National Park (Czech Republic) suffered from repeated large-scale bark beetle outbreaks in last decade. In this study, the diversity of culturable foliar endophyte microfungi in needles originating from eleven recently wind-fallen trees in this area was surveyed. Our aims were to describe their diversity and to determine the relative host and organ specificities of isolated endophyte species to estimate the species pool and abundance of foliar endophytes before the forest degradation. Microfungi were isolated from surface-sterilized needles, and the outgrowing strains were identified based on morphological and molecular characteristics (analyses of ITS1, ITS2 and partial 28S rDNA). Fungal communities in the needles were diverse, with ascomycetes (mostly anamorphs of Helotiales) dominating basidiomycetes. The most frequent species (Phacidiopycnis sp., Cistella acuum, Sirococcus sp. and two species of Chalara) did not correspond with those recorded in previous studies of Picea spp. For example, the widely distributed Lophodermium piceae was rarely recorded in this study. This pattern may be caused by different methods of sterilization and cultivation or by physiological characteristics of the needles, or it may reflect the species distribution in the studied area. Members of the Helotiales, along with sequences from GenBank, showed substantial overlap in host affinities, most prominently between Pinus and Picea, and also among species from distant plant lineages.

Laccases are oxidases that contain several copper atoms, and catalyse single-electron oxidations of phenolic compounds with concomitant reduction of oxygen to water. The enzymes are particularly widespread in ligninolytic basidiomycetes, but also occur in certain prokaryotes, insects and plants. Depending on the species, laccases are involved in various biosynthetic processes contributing to carbon recycling in land ecosystems and the morphogenesis of biomatrices, wherein low-molecular-weight naturally occurring phenols serve as key enzyme substrates. Studies of these in vivo synthetic pathways have afforded new insights into fungal laccase applicability in green synthetic chemistry. Thus, we here review fungal laccase-catalysed oxidations of naturally occurring phenols that are particularly relevant to the synthesis of fine organic chemicals, and we discuss how the discovered synthetic strategies mimic laccase-involved in vivo pathways, thus enhancing the green nature of such reactions. Laccase-catalysed in vivo processes yield several types of biopolymers, including those of cuticles, lignin, polyflavonoids, humus and the melanin pigments, using natural mono- or poly-phenols as building blocks. The in vivo synthetic pathways involve either phenoxyl radical-mediated coupling or cross-linking reactions, and can be adapted to the design of in vitro oxidative processes involving fungal laccases in organic synthesis; the laccase substrates and the synthetic mechanisms reflect in vivo processes. Notably, such in vitro synthetic pathways can also reproduce physicochemical properties (e.g. those of chromophores, and radical-scavenging, hydration and antimicrobial activities) found in natural biomaterials. Careful study of laccase-associated in vivo metabolic pathways has been rewarded by the discovery of novel green applications for fungal laccases. This review comprehensively summarizes the available data on laccase-catalysed biosynthetic pathways and associated applications in fine chemical syntheses.

Changes in the abundance of bacteria and fungi and in the composition of bacterial communities during primary succession were investigated in a brown coal mine deposit area near Sokolov, the Czech Republic, using phospholipid fatty acids analysis, microarray and 16S rRNA gene sequencing. The study considered a chronosequence of sites undergoing spontaneous succession: 6-, 12-, 21- and 45-year-old and a 21-year-old site revegetated with Alnus glutinosa . During succession, organic carbon and the total nitrogen content increased while the pH and the C/N ratio decreased. Microbial biomass and bacterial diversity increased until 21 years and decreased later; bacteria dominated over fungi in the initial and late phases of succession. Bacterial community composition of the 6-year-old site with no vegetation cover largely differed from the older sites, especially by a higher content of Gammaproteobacteria,Cyanobacteria and some Alphaproteobacteria . Bacteria belonging to the genera Acidithiobacillus,Thiobacillus and related taxa, the CO2 and N2 fixers, dominated the community at this site. In the later phases, bacterial community development seemed to reflect more the changes in soil nutrient content and pH than vegetation with a decrease of Actinobacteria and an increase of Acidobacteria . The site revegetated with A. glutinosa resembled the 45-year-old primary succession site and exhibited an even lower pH and C/N ratio, indicating that recultivation is able to accelerate soil development.