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Abstract While the succession of terrestrial plant communities is well studied, less is known about succession on dead wood, especially how it is affected by environmental factors. While temperate forests face increasing canopy mortality, which causes considerable changes in microclimates, it remains unclear how canopy openness affects fungal succession. Here, we used a large real-world experiment to study the effect of closed and opened canopy on treatment-based alpha and beta fungal fruiting diversity. We found increasing diversity in early and decreasing diversity at later stages of succession under both canopies, with a stronger decrease under open canopies. However, the slopes of the diversity versus time relationships did not differ significantly between canopy treatments. The community dissimilarity remained mainly stable between canopies at ca. 25% of species exclusively associated with either canopy treatment. Species exclusive in either canopy treatment showed very low number of occupied objects compared to species occurring in both treatments. Our study showed that canopy loss subtly affected fungal fruiting succession on dead wood, suggesting that most species in the local species pool are specialized or can tolerate variable conditions. Our study indicates that the fruiting of the fungal community on dead wood is resilient against the predicted increase in canopy loss in temperate forests.

Abstract Background Grasslands provide fundamental ecosystem services that are supported by their plant diversity. However, the importance of plant taxonomic diversity for the diversity of other taxa in grasslands remains poorly understood. Here, we studied the associations between plant communities, soil chemistry and soil microbiome in a wooded meadow of Čertoryje (White Carpathians, Czech Republic), a European hotspot of plant species diversity. Results High plant diversity was associated with treeless grassland areas with high primary productivity and high contents of soil nitrogen and organic carbon. In contrast, low plant diversity occurred in grasslands near solitary trees and forest edges. Fungal communities differed between low-diversity and high-diversity grasslands more strongly than bacterial communities, while the difference in arbuscular mycorrhizal fungi (AMF) depended on their location in soil versus plant roots. Compared to grasslands with low plant diversity, high-diversity plant communities had a higher diversity of fungi including soil AMF, a different fungal and soil AMF community composition and higher bacterial and soil AMF biomass. Root AMF composition differed only slightly between grasslands with low and high plant diversity. Trees dominated the belowground plant community in low-diversity grasslands, which influenced microbial diversity and composition. Conclusions The determinants of microbiome abundance and composition in grasslands are complex. Soil chemistry mainly influenced bacterial communities, while plant community type mainly affected fungal (including AMF) communities. Further studies on the functional roles of microbial communities are needed to understand plant-soil-microbe interactions and their involvement in grassland ecosystem services.

IntroductionRevegetation of barren substrates is often determined by the composition and distance of the nearest plant community, serving as a source of colonizing propagules. Whether such dispersal effect can be observed during the development of soil microbial communities, is not clear. In this study, we aimed to elucidate which factors structure plant and soil bacterial and fungal communities during primary succession on a limestone quarry spoil heap, focusing on the effect of distance to the adjoining xerophilous grassland.MethodsWe established a grid of 35 plots covering three successional stages – initial barren substrate, early successional community and late successional grassland ecosystem, the latter serving as the primary source of soil colonization. On these plots, we performed vegetation surveys of plant community composition and collected soil cores to analyze soil chemical properties and bacterial and fungal community composition.ResultsThe composition of early successional plant community was significantly affected by the proximity of the source late successional community, however, the effect weakened when the distance exceeded 20 m. Early successional microbial communities were structured mainly by the local plant community composition and soil chemical properties, with minimal contribution of the source community proximity.DiscussionThese results show that on small spatial scales, species migration is an important determinant of plant community composition during primary succession while the establishment of soil microbial communities is not limited by dispersal and is primarily driven by local biotic and abiotic conditions.

Fungi are an integral part of the nitrogen and phosphorus cycling in trophic networks, as they participate in biomass decomposition and facilitate plant nutrition through root symbioses. Nutrient content varies considerably between the main fungal habitats, such as soil, plant litter or decomposing dead wood, but there are also large differences within habitats. While some soils are heavily loaded with N, others are limited by N or P. One way in which nutrient availability can be reflected in fungi is their content in biomass. In this study, we determined the C, N, and P content (in dry mass) of fruiting bodies of 214 fungal species to inspect how phylogeny and membership in ecological guilds (soil saprotrophs, wood saprotrophs, and ectomycorrhizal fungi) affect the nutrient content of fungal biomass. The C content of fruiting bodies (415 ± 25 mg g–1) showed little variation (324–494 mg g–1), while the range of N (46 ± 20 mg g–1) and P (5.5 ± 3.0 mg g–1) contents was within one order of magnitude (8–103 mg g–1 and 1.0–18.9 mg g–1, respectively). Importantly, the N and P contents were significantly higher in the biomass of soil saprotrophic fungi compared to wood saprotrophic and ectomycorrhizal fungi. While the average C/N ratio in fungal biomass was 11.2, values exceeding 40 were recorded for some fungi living on dead wood, typically characterized by low N content. The N and P content of fungal mycelium also showed a significant phylogenetic signal, with differences in nutrient content being relatively low within species and genera of fungi. A strong correlation was found between N and P content in fungal biomass, while the correlation of N content and the N-containing fungal cell wall biopolymer—chitin showed only weak significance. The content of macronutrients in fungal biomass is influenced by the fungal life style and nutrient availability and is also limited by phylogeny.

Abstract Background Below-ground microbes mediate key ecosystem processes and play a vital role in plant nutrition and health. Understanding the composition of the belowground microbiome is therefore important for maintaining ecosystem stability. The structure of the belowground microbiome is largely determined by individual plants, but it is not clear how far their influence extends and, conversely, what the influence of other plants growing nearby is. Results To determine the extent to which a focal host plant influences its soil and root microbiome when growing in a diverse community, we sampled the belowground bacterial and fungal communities of three plant species across a primary successional grassland sequence. The magnitude of the host effect on its belowground microbiome varied among microbial groups, soil and root habitats, and successional stages characterized by different levels of diversity of plant neighbours. Soil microbial communities were most strongly structured by sampling site and showed significant spatial patterns that were partially driven by soil chemistry. The influence of focal plant on soil microbiome was low but tended to increase with succession and increasing plant diversity. In contrast, root communities, particularly bacterial, were strongly structured by the focal plant species. Importantly, we also detected a significant effect of neighbouring plant community composition on bacteria and fungi associating with roots of the focal plants. The host influence on root microbiome varied across the successional grassland sequence and was highest in the most diverse site. Conclusions Our results show that in a species rich natural grassland, focal plant influence on the belowground microbiome depends on environmental context and is modulated by surrounding plant community. The influence of plant neighbours is particularly pronounced in root communities which may have multiple consequences for plant community productivity and stability, stressing the importance of plant diversity for ecosystem functioning.

Abstract Wood decomposition is a central process contributing to global carbon and nutrient cycling. Quantifying the role of the major biotic agents of wood decomposition, i.e. insects and fungi, is thus important for a better understanding of this process. Methods to quantify wood decomposition, such as dry mass loss, suffer from several shortcomings, such as destructive sampling or subsampling. We developed and tested a new approach based on computed tomography (CT) scanning and semi-automatic image analysis of logs from a field experiment with manipulated beetle communities. We quantified the volume of beetle tunnels in wood and bark and the relative wood volume showing signs of fungal decay and compared both measures to classic approaches. The volume of beetle tunnels was correlated with dry mass loss and clearly reflected the differences between beetle functional groups. Fungal decay was identified with high accuracy and strongly correlated with ergosterol content. Our data show that this is a powerful approach to quantify wood decomposition by insects and fungi. In contrast to other methods, it is non-destructive, covers entire deadwood objects and provides spatially explicit information opening a wide range of research options. For the development of general models, we urge researchers to publish training data.

ABSTRACT Microorganisms dominate all ecosystems on Earth and play a key role in the turnover of organic matter. By producing enzymes, they degrade complex carbohydrates, facilitating the recycling of nutrients and controlling the carbon cycle. Despite their importance, our knowledge regarding microbial carbohydrate utilization has been limited to genome-sequenced taxa and thus heavily biased to specific groups and environments. Here, we used the Genomes from Earth’s Microbiomes (GEM) catalog to describe the carbohydrate utilization potential in >7000 bacterial and archaeal taxa originating from a range of terrestrial, marine and host-associated habitats. We show that the production of carbohydrate-active enzymes (CAZymes) is phylogenetically conserved and varies significantly among microbial phyla. High numbers of carbohydrate-active enzymes were recorded in phyla known for their versatile use of carbohydrates, such as Firmicutes, Fibrobacterota, and Armatimonadota, but also phyla without cultured representatives whose carbohydrate utilization potential was so far unknown, such as KSB1, Hydrogenedentota, Sumerlaeota, and UBP3. Carbohydrate utilization potential reflected the specificity of various habitats: the richest complements of CAZymes were observed in MAGs of plant microbiomes, indicating the structural complexity of plant biopolymers. IMPORTANCE This study expanded our knowledge of the phylogenetic distribution of carbohydrate-active enzymes across prokaryotic tree of life, including new phyla where the carbohydrate-active enzymes composition have not been described until now and demonstrated the potential for carbohydrate utilization of numerous yet uncultured phyla. Profiles of carbohydrate-active enzymes are largely habitat-specific and reflect local carbohydrate availability by selecting taxa with appropriate complements of these enzymes. This information should aid in the prediction of functions in microbiomes of known taxonomic composition and helps to identify key components of habitat-specific carbohydrate pools. In addition, these findings have a high relevance for the understanding of carbohydrate utilization and carbon cycling in the environment, the process that is closely link to the carbon storage potential of Earth habitats and the production of greenhouse gasses.

Measurement(s) metagenomic data • metatranscriptomic data • microbiome • RNA-seq of total RNA Technology Type(s) DNA sequencing • RNA-seq of total RNA • amplicon sequencing • RNA sequencing Factor Type(s) time of decomposition Sample Characteristic - Organism Fungi • Bacteria Sample Characteristic - Environment wood Sample Characteristic - Location Narodni prirodni rezervace Zofinsky prales Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.14821752

Threats to global biodiversity are increasingly recognised by scientists and the public as a critical challenge. Molecular sequencing technologies offer means to catalogue, explore, and monitor the richness and biogeography of life on Earth. However, exploiting their full potential requires tools that connect biodiversity infrastructures and resources. As a research infrastructure developing services and technical solutions that help integrate and coordinate life science resources across Europe, ELIXIR is a key player. To identify opportunities, highlight priorities, and aid strategic thinking, here we survey approaches by which molecular technologies help inform understanding of biodiversity. We detail example use cases to highlight how DNA sequencing is: resolving taxonomic issues; Increasing knowledge of marine biodiversity; helping understand how agriculture and biodiversity are critically linked; and playing an essential role in ecological studies. Together with examples of national biodiversity programmes, the use cases show where progress is being made but also highlight common challenges and opportunities for future enhancement of underlying technologies and services that connect molecular and wider biodiversity domains. Based on emerging themes, we propose key recommendations to guide future funding for biodiversity research: biodiversity and bioinformatic infrastructures need to collaborate closely and strategically; taxonomic efforts need to be aligned and harmonised across domains; metadata needs to be standardised and common data management approaches widely adopted; current approaches need to be scaled up dramatically to address the anticipated explosion of molecular data; bioinformatics support for biodiversity research needs to be enabled and sustained; training for end users of biodiversity research infrastructures needs to be prioritised; and community initiatives need to be proactive and focused on enabling solutions. For sequencing data to deliver their full potential they must be connected to knowledge: together, molecular sequence data collection initiatives and biodiversity research infrastructures can advance global efforts to prevent further decline of Earth’s biodiversity.

Fine woody debris (FWD) represents the majority of the deadwood stock in managed forests and serves as an important biodiversity hotspot and refuge for many organisms, including deadwood fungi. Wood decomposition in forests, representing an important input of nutrients into forest soils, is mainly driven by fungal communities that undergo continuous changes during deadwood decomposition. However, while the assembly processes of fungal communities in long-lasting coarse woody debris have been repeatedly explored, similar information for the more ephemeral habitat of fine deadwood is missing. Here, we followed the fate of FWD of Fagus sylvatica and Abies alba in a Central European forest to describe the assembly and diversity patterns of fungal communities over 6 years. Importantly, the effect of microclimate on deadwood properties and fungal communities was addressed by comparing FWD decomposition in closed forests and under open canopies because the large surface-to-volume ratio of FWD makes it highly sensitive to temperature and moisture fluctuations. Indeed, fungal biomass increases and pH decreases were significantly higher in FWD under closed canopy in the initial stages of decomposition indicating higher fungal activity and hence decay processes. The assembly patterns of the fungal community were strongly affected by both tree species and microclimatic conditions. The communities in the open/closed canopies and in each tree species were different throughout the whole succession with only limited convergence in time in terms of both species and ecological guild composition. Decomposition under the open canopy was characterized by high sample-to-sample variability, showing the diversification of fungal resources. Tree species-specific fungi were detected among the abundant species mostly during the initial decomposition, whereas fungi associated with certain canopy cover treatments were present evenly during decomposition. The species diversity of forest stands and the variability in microclimatic conditions both promote the diversity of fine woody debris fungi in a forest.

Mycorrhizal symbioses have evolved repeatedly in diverse fungal lineages. A large phylogenomic analysis sheds light on genomic changes associated with transitions from saprotrophy to symbiosis, including divergent genetic innovations underlying the convergent origins of the ectomycorrhizal guild.

The authors assemble and analyse previously generated mycobiome data linked to geographical locations across the world. They describe the distribution of fungal taxa and show that climate is an important driver of fungal biogeography and that fungal diversity appears to be concentrated at high latitudes.

Summary Cistus ladanifer scrublands, traditionally considered as unproductive, have nonetheless been observed to produce large quantities of king bolete (Boletus edulis) fruitbodies. These pyrophytic scrublands are prone to wildfires, which severely affect fungi, hence the need for fire prevention in producing C. ladanifer scrublands. In addition, B. edulis productions have severely decreased in the last years. A deeper understanding of the B. edulis life cycle and of biotic and abiotic factors influencing sporocarp formation is needed to implement management practices that facilitate B. edulis production. For example, some bacteria likely are involved in sporocarp production, representing a key part in the triple symbiosis (plant–fungus–bacteria). In this study, we used soil DNA metabarcoding in C. ladanifer scrublands to (i) assess the effect of site history and fire prevention treatment on bacterial richness and community composition; (ii) test if there was any correlation between various taxonomic groups of bacteria and mycelial biomass and sporocarp production of B. edulis; and to (iii) identify indicator bacteria associated with the most productive B. edulis sites. Our results show that site history drives bacterial richness and community composition, while fire prevention treatments have a weaker, but still detectable effect, particularly in the senescent plots. Sporocarp production correlated positively with genera in Verrucomicrobia. Several genera, e.g. Azospirillum and Gemmatimonas, were identified as indicators of the most productive sites, suggesting a potential biological role in B. edulis fructification. This study provides a better understanding of the triple symbiosis (plant–fungus–bacteria) involved in C. ladanifer–B. edulis systems.

The diverse chemical, biological, and microbial properties of litter and organic matter (OM) in forest soil along an altitudinal gradient are potentially important for nutrient cycling. In the present study, we sought to evaluate soil chemical, biological, microbial, and enzymatic characteristics at four altitude levels (0, 500, 1,000, and 1,500 m) in northern Iran to characterize nutrient cycling in forest soils. The results showed that carbon (C) and nitrogen (N) turnover changed with altitude along with microbial properties and enzyme activity. At the lowest altitude with mixed forest and no beech trees, the higher content of N in litter and soil, higher pH and microbial biomass nitrogen (MBN), and the greater activities of aminopeptidases affected soil N cycling. At elevations above 1,000 m, where beech is the dominant tree species, the higher activities of cellobiohydrolase, arylsulfatase, β-xylosidase, β-galactosidase, endoglucanase, endoxylanase, and manganese peroxidase (MnP) coincided with higher basal respiration (BR), substrate-induced respiration (SIR), and microbial biomass carbon (MBC) and thus favored conditions for microbial entropy and C turnover. The low N content and high C/N ratio at 500-m altitude were associated with the lowest microbial and enzyme activities. Our results support the view that the plain forest with mixed trees (without beech) had higher litter quality and soil fertility, while forest dominated by beech trees had the potential to store higher C and can potentially better mitigate global warming.

Microbial diversity plays an important role in the decomposition of soil organic matter. However, the pattern and drivers of the relationship between microbial diversity and decomposition remain unclear. In this study, we followed the decomposition of organic matter in soils where microbial diversity was experimentally manipulated. To produce a gradient of microbial diversity, we used soil samples at two sites of the same chronosequence after brown coal mining in Sokolov, Czech Republic. Soils were X-ray sterilized and inoculated by two densities of inoculum from both soils and planted with seeds of six local plant species. This created two soils each with four levels of microbial diversity characterized by next-generation sequencing. These eight soils were supplied, or not, by litter of the bushgrass Calamagrostis epigejos, and microbial respiration was measured to assess the rate of decomposition. A strong positive correlation was found between microbial diversity and decomposition of organic matter per gram of carbon in soil, which suggests that microbial diversity supports decomposition if the microbial community is limited by available carbon. In contrast, microbial respiration per gram of soil negatively correlated with bacterial diversity and positively with fungal biomass, suggesting that in the absence of a carbon limitation, decomposition rate is controlled by the amount of fungal biomass. Soils with the addition of grass litter showed a priming effect in the initial stage of decomposition compared to the samples without the addition of litter. Thus, the relationship between microbial diversity and the rate of decomposition may be complex and context dependent.

Elucidating dynamics of soil microbial communities after disturbance is crucial for understanding ecosystem restoration and sustainability. However, despite the widespread practice of swidden agriculture in tropical forests, knowledge about microbial community succession in this system is limited. Here, amplicon sequencing was used to investigate effects of soil ages (spanning at least 60 years) after disturbance, geographic distance (from 0.1 to 10 km) and edaphic property gradients (soil pH, conductivity, C, N, P, Ca, Mg, and K), on soil bacterial and fungal communities along a chronosequence of sites representing the spontaneous succession following swidden agriculture in lowland forests in Papua New Guinea. During succession, bacterial communities (OTU level) as well as its abundant (OTU with relative abundance > 0.5%) and rare (

The bacterial genus Sodalis is represented by insect endosymbionts as well as free-living species. While the former have been studied frequently, the distribution of the latter is not yet clear. Here, we present a description of a free-living strain, Sodalis ligni sp. nov., originating from decomposing deadwood. The favored occurrence of S. ligni in deadwood is confirmed by both 16S rRNA gene distribution and metagenome data. Pangenome analysis of available Sodalis genomes shows at least three groups within the Sodalis genus: deadwood-associated strains, tsetse fly endosymbionts and endosymbionts of other insects. This differentiation is consistent in terms of the gene frequency level, genome similarity and carbohydrate-active enzyme composition of the genomes. Deadwood-associated strains contain genes for active decomposition of biopolymers of plant and fungal origin and can utilize more diverse carbon sources than their symbiotic relatives. Deadwood-associated strains, but not other Sodalis strains, have the genetic potential to fix N2, and the corresponding genes are expressed in deadwood. Nitrogenase genes are located within the genomes of Sodalis, including S. ligni, at multiple loci represented by more gene variants. We show decomposing wood to be a previously undescribed habitat of the genus Sodalis that appears to show striking ecological divergence.

Deadwood decomposition is responsible for a significant amount of carbon (C) turnover in natural forests. While fresh deadwood contains mainly plant compounds and is extremely low in nitrogen (N), fungal biomass and N content increase during decomposition. Here, we examined 18 genome-sequenced bacterial strains representing the dominant deadwood taxa to assess their adaptations to C and N utilization in deadwood. Diverse gene sets for the efficient decomposition of plant and fungal cell wall biopolymers were found in Acidobacteria, Bacteroidetes, and Actinobacteria. In contrast to these groups, Alphaproteobacteria and Gammaproteobacteria contained fewer carbohydrate-active enzymes and depended either on low-molecular-mass C sources or on mycophagy. This group, however, showed rich gene complements for N2 fixation and nitrate/nitrite reduction—key assimilatory and dissimilatory steps in the deadwood N cycle. We show that N2 fixers can obtain C independently from either plant biopolymers or fungal biomass. The succession of bacteria on decomposing deadwood reflects their ability to cope with the changing quality of C-containing compounds and increasing N content.

Dead wood represents an important pool of carbon and nitrogen in forest ecosystems. This source of soil organic matter has diverse ecosystem functions that include, among others, carbon and nitrogen cycling. However, information is limited on how deadwood properties such as chemical composition, decomposer abundance, community composition, and age correlate and affect decomposition rate. Here, we targeted coarse dead wood of beech, spruce, and fir, namely snags and tree trunks (logs) in an old-growth temperate forest in central Europe; measured their decomposition rate as CO2 production in situ; and analyzed their relationships with other measured variables. Respiration rate of dead wood showed strong positive correlation with acid phosphatase activity and negative correlation with lignin content. Fungal biomass (ergosterol content) and moisture content were additional predictors. Our results indicate that dead wood traits, including tree species, age, and position (downed/standing), affected dead wood chemical properties, microbial biomass, moisture condition, and enzyme activity through changes in fungal communities and ultimately influenced the decomposition rate of dead wood.

Abstract Background The impacts of man-made chemicals, in particular of persistent organic pollutants, are multifactorial as they may affect the integrity of ecosystems, alter biodiversity and have undesirable effects on many organisms. We have previously demonstrated that the belowground mycobiota of forest soils acts as a buffer against the biocide pollutant pentachlorophenol. However, the trade-offs made by mycobiota to mitigate this pollutant remain cryptic. Results Herein, we demonstrate using a culture-dependent approach that exposure to pentachlorophenol led to alterations in the composition and functioning of the metacommunity, many of which were not fully alleviated when most of the biocide was degraded. Proteomic and physiological analyses showed that the carbon and nitrogen metabolisms were particularly affected. This dysregulation is possibly linked to the higher pathogenic potential of the metacommunity following exposure to the biocide, supported by the secretion of proteins related to pathogenicity and reduced susceptibility to a fungicide. Our findings provide additional evidence for the silent risks of environmental pollution, particularly as it may favour the development of pathogenic trade-offs in fungi, which may impose serious threats to animals and plant hosts.

ABSTRACT Fungal-bacterial interactions play a key role in the functioning of many ecosystems. Thus, understanding their interactive dynamics is of central importance for gaining predictive knowledge on ecosystem functioning. However, it is challenging to disentangle the mechanisms behind species associations from observed co-occurrence patterns, and little is known about the directionality of such interactions. Here, we applied joint species distribution modeling to high-throughput sequencing data on co-occurring fungal and bacterial communities in deadwood to ask whether fungal and bacterial co-occurrences result from shared habitat use (i.e., deadwood’s properties) or whether there are fungal-bacterial interactive associations after habitat characteristics are taken into account. Moreover, we tested the hypothesis that the interactions are mainly modulated through fungal communities influencing bacterial communities. For that, we quantified how much the predictive power of the joint species distribution models for bacterial and fungal community improved when accounting for the other community. Our results show that fungi and bacteria form tight association networks (i.e., some species pairs co-occur more frequently and other species pairs co-occur less frequently than expected by chance) in deadwood that include common (or opposite) responses to the environment as well as (potentially) biotic interactions. Additionally, we show that information about the fungal occurrences and abundances increased the power to predict the bacterial abundances substantially, whereas information about the bacterial occurrences and abundances increased the power to predict the fungal abundances much less. Our results suggest that fungal communities may mainly affect bacteria in deadwood. IMPORTANCE Understanding the interactive dynamics between fungal and bacterial communities is important to gain predictive knowledge on ecosystem functioning. However, little is known about the mechanisms behind fungal-bacterial associations and the directionality of species interactions. Applying joint species distribution modeling to high-throughput sequencing data on co-occurring fungal-bacterial communities in deadwood, we found evidence that nonrandom fungal-bacterial associations derive from shared habitat use as well as (potentially) biotic interactions. Importantly, the combination of cross-validations and conditional cross-validations helped us to answer the question about the directionality of the biotic interactions, providing evidence that suggests that fungal communities may mainly affect bacteria in deadwood. Our modeling approach may help gain insight into the directionality of interactions between different components of the microbiome in other environments.

ABSTRACT Forests accumulate and store large amounts of carbon (C), and a substantial fraction of this stock is contained in deadwood. This transient pool is subject to decomposition by deadwood-associated organisms, and in this process it contributes to CO2 emissions. Although fungi and bacteria are known to colonize deadwood, little is known about the microbial processes that mediate carbon and nitrogen (N) cycling in deadwood. In this study, using a combination of metagenomics, metatranscriptomics, and nutrient flux measurements, we demonstrate that the decomposition of deadwood reflects the complementary roles played by fungi and bacteria. Fungi were found to dominate the decomposition of deadwood and particularly its recalcitrant fractions, while several bacterial taxa participate in N accumulation in deadwood through N fixation, being dependent on fungal activity with respect to deadwood colonization and C supply. Conversely, bacterial N fixation helps to decrease the constraints of deadwood decomposition for fungi. Both the CO2 efflux and N accumulation that are a result of a joint action of deadwood bacteria and fungi may be significant for nutrient cycling at ecosystem levels. Especially in boreal forests with low N stocks, deadwood retention may help to improve the nutritional status and fertility of soils. IMPORTANCE Wood represents a globally important stock of C, and its mineralization importantly contributes to the global C cycle. Microorganisms play a key role in deadwood decomposition, since they possess enzymatic tools for the degradation of recalcitrant plant polymers. The present paradigm is that fungi accomplish degradation while commensalist bacteria exploit the products of fungal extracellular enzymatic cleavage, but this assumption was never backed by the analysis of microbial roles in deadwood. This study clearly identifies the roles of fungi and bacteria in the microbiome and demonstrates the importance of bacteria and their N fixation for the nutrient balance in deadwood as well as fluxes at the ecosystem level. Deadwood decomposition is shown as a process where fungi and bacteria play defined, complementary roles.

Belowground litter derived from tree roots has been shown as a principal source of soil organic matter in coniferous forests. Fate of tree root necromass depends on fungal communities developing on the decaying roots. Local environmental conditions which affect composition of tree root mycobiome may also influence fungal communities developing on decaying tree roots. Here, we assessed fungal communities associated with decaying roots of Picea abies decomposing in three microhabitats: soil with no vegetation, soil with ericoid shrubs cover, and P. abies deadwood, for a 2-year period. Forest microhabitat showed stronger effect on structuring fungal communities associated with decaying roots compared to living roots. Some ericoid mycorrhizal fungi showed higher relative abundance on decaying roots in soils under ericoid shrub cover, while saprotrophic fungi had higher relative abundance in roots decomposing inside deadwood. Regardless of the studied microhabitat, we observed decline of ectomycorrhizal fungi and increase of endophytic fungi during root decomposition. Interestingly, we found substantially more fungal taxa with unknown ecology in late stages of root decomposition, indicating that highly decomposed roots may represent so far overlooked niche for soil fungi. Our study shows the importance of microhabitats on the fate of the decomposing spruce roots.

Unveiling the relationship between taxonomy and function of the microbiome is crucial to determine its contribution to ecosystem functioning. However, while there is a considerable amount of information on microbial taxonomic diversity, our understanding of its relationship to functional diversity is still scarce. Here, we used a meta-analysis of completely annotated extant genomes of 377 taxonomically distinct fungal species to predict the total fungal microbiome functionality on Earth with accumulation curves (ACs) of all known functions from the level 3 of KEGG Orthology using both parametric and non-parametric estimates in an explorative data-mining approach. The unsaturated model extrapolating functional diversity as a function of species richness described the ACs significantly better than the saturated model that assumed a limited total number of functions, which suggested the presence of widespread and rare functions. Based on previous estimates of 3.8 million fungal species on Earth, we propagated the unsaturated model to predict a total of 42.4 ± 0.5 million KEGG level 3 functions of which only 0.06% are known today. Our approach not only highlights the presence of widespread and rare functions but points toward the necessity of novel and more sophisticated methods to unveil the entirety of functions to fully understand the involvement of the fungal microbiome in ecosystem functioning.

Along with recent developments in high-throughput sequencing (HTS) technologies and thus fast accumulation of HTS data, there has been a growing need and interest for developing tools for HTS data processing and communication. In particular, a number of bioinformatics tools have been designed for analysing metabarcoding data, each with specific features, assumptions and outputs. To evaluate the potential effect of the application of different bioinformatics workflow on the results, we compared the performance of different analysis platforms on two contrasting high-throughput sequencing data sets. Our analysis revealed that the computation time, quality of error filtering and hence output of specific bioinformatics process largely depends on the platform used. Our results show that none of the bioinformatics workflows appears to perfectly filter out the accumulated errors and generate Operational Taxonomic Units, although PipeCraft, LotuS and PIPITS perform better than QIIME2 and Galaxy for the tested fungal amplicon dataset. We conclude that the output of each platform requires manual validation of the OTUs by examining the taxonomy assignment values.

Deadwood represents an important carbon stock and contributes to climate change mitigation. Wood decomposition is mainly driven by fungal communities. Their composition is known to change during decomposition, but it is unclear how environmental factors such as wood chemistry affect these successional patterns through their effects on dominant fungal taxa. We analysed the deadwood of Fagus sylvatica and Abies alba across a deadwood succession series of >40 years in a natural fir-beech forest in the Czech Republic to describe the successional changes in fungal communities, fungal abundance and enzymatic activities and to link these changes to environmental variables. The fungal communities showed high levels of spatial variability and beta diversity. In young deadwood, fungal communities showed higher similarity among tree species, and fungi were generally less abundant, less diverse and less active than in older deadwood. pH and the carbon to nitrogen ratio (C/N) were the best predictors of the fungal community composition, and they affected the abundance of half of the dominant fungal taxa. The relative abundance of most of the dominant taxa tended to increase with increasing pH or C/N, possibly indicating that acidification and atmospheric N deposition may shift the community composition towards species that are currently less dominant.

Abstract 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.

Due to the ability of soil bacteria to solubilize minerals, fix N2 and mobilize nutrients entrapped in the organic matter, their role in nutrient turnover and plant fitness is of high relevance in forest ecosystems. Although several authors have already studied the organic matter decomposing enzymes produced by soil and plant root-interacting bacteria, most of the works did not account for the activity of cell wall-attached enzymes. Therefore, the enzyme deployment strategy of three bacterial collections (genera Luteibacter, Pseudomonas and Arthrobacter) associated with Quercus spp. roots was investigated by exploring both cell-bound and freely-released hydrolytic enzymes. We also studied the potential of these bacterial collections to produce enzymes involved in the transformation of plant and fungal biomass. Remarkably, the cell-associated enzymes accounted for the vast majority of the total activity detected among Luteibacter strains, suggesting that they could have developed a strategy to maintain the decomposition products in their vicinity, and therefore to reduce the diffusional losses of the products. The spectrum of the enzymes synthesized and the titres of activity were diverse among the three bacterial genera. While cellulolytic and hemicellulolytic enzymes were rather common among Luteibacter and Pseudomonas strains and less detected in Arthrobacter collection, the activity of lipase was widespread among all the tested strains. Our results indicate that a large fraction of the extracellular enzymatic activity is due to cell wall-attached enzymes for some bacteria, and that Quercus spp. root bacteria could contribute at different levels to carbon (C), phosphorus (P) and nitrogen (N) cycles.

Two long-term contaminated soils differing in contents of Pb, Zn, As, Cd were compared in a microcosm experiment for changes in microbial community structure and respiration after various treatments. We observed that the extent of long-term contamination (over 200 years) by toxic elements did not change the total numbers and diversity of bacteria but influenced their community composition. Namely, numbers of Actinobacteria determined by phylum specific qPCR increased and also the proportion of Actinobacteria and Chloroflexi increased in Illumina sequence libraries in the more contaminated soil. In the experiment, secondary disturbance by supplemented cadmium (doses from double to 100-fold the concentration in the original soil) and organic substrates (cellobiose or straw) increased bacterial diversity in the less contaminated soil and decreased it in the more contaminated soil. Respiration in the experiment was higher in the more contaminated soil in all treatments and correlated with bacterial numbers. Considering the most significant changes in bacterial community, it seemed that particularly Actinobacteria withstand contamination by toxic elements. The results proved higher resistance to secondary disturbance in terms of both, respiration and bacterial community structure in the less contaminated soil.

Revealing the relationship between taxonomy and function in microbiomes is critical to discover their contribution to ecosystem functioning. However, while the relationship between taxonomic and functional diversity in bacteria and fungi is known, this is not the case for archaea. Here, we used a meta-analysis of 417 completely annotated extant and taxonomically unique archaeal genomes to predict the extent of microbiome functionality on Earth contained within archaeal genomes using accumulation curves of all known level 3 functions of KEGG Orthology. We found that intergenome redundancy as functions present in multiple genomes was inversely related to intragenome redundancy as multiple copies of a gene in one genome, implying the tradeoff between additional copies of functionally important genes or a higher number of different genes. A logarithmic model described the relationship between functional diversity and species richness better than both the unsaturated and the saturated model, which suggests a limited total number of archaeal functions in contrast to the sheer unlimited potential of bacteria and fungi. Using the global archaeal species richness estimate of 13,159, the logarithmic model predicted 4164.1 ± 2.9 KEGG level 3 functions. The non-parametric bootstrap estimate yielded a lower bound of 2994 ± 57 KEGG level 3 functions. Our approach not only highlighted similarities in functional redundancy but also the difference in functional potential of archaea compared to other domains of life.

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.

In temperate forests, climate seasonality restricts the photosynthetic activity of primary producers to the warm season from spring to autumn, while the cold season with temperatures below the freezing point represents a period of strongly reduced plant activity. Although soil microorganisms are active all-year-round, their expressions show seasonal patterns. This is especially visible on the ectomycorrhizal fungi, the most abundant guild of fungi in coniferous forests. We quantified the production of fungal mycelia using ingrowth sandbags in the organic layer of soil in temperate coniferous forest and analysed the composition of fungal communities in four consecutive seasons. We show that fungal biomass production is as low as 0.029 µg g−1 of sand in December–March, while it reaches 0.122 µg g−1 in June–September. The majority of fungi show distinct patterns of seasonal mycelial production, with most ectomycorrhizal fungi colonising ingrowth bags in the spring or summer, while the autumn and winter colonisation was mostly due to moulds. Our results indicate that fungal taxa differ in their seasonal patterns of mycelial production. Although fungal biomass turnover appears all-year-round, its rates are much faster in the period of plant activity than in the cold season.

Soil formation in post-mining sites is crucial for restoring ecosystem function, and soil formation depend on the accumulation of soil organic matter and the development of an active microbial community. In this study, we used substrate-induced respiration (SIR) and Biolog plates to characterize microbial catabolic profiles in a chronosequence of soil samples from 15 unreclaimed post-mining sites in Sokolov, Czech Republic. The sites had been undergoing spontaneous succession for 3 to 45 years. Biolog ECO plates included 31 substrates. Of substrates used for SIR (glucose, chitin, cellulose, Tween 80, phenylethylamine, N-acetyl-D-glucosamine, L-asparagine, D-mannitol, D-galacturonic acid, α-cyclodextrin, and 4-hydroxy benzoic acid), eight were also used for the Biolog plates. Soil respiration, total bacteria number, and culturable bacteria number were also measured. The total and culturable number of bacteria increased with site age (p < 0.01 and p < 0.05, respectively). The percentage of culturable bacteria decreased with site age (p < 0.01). Biolog analysis indicated that average well-color development (AWCD), evenness, and richness increased with site age. SIR data indicated that only average activities tended to increase with site age (p = 0.06). According to redundancy analysis (RDA), the eight substrates, which were commonly used in both methods (SIR and BIOLOG) explained 74.4% of the variation of data from all Biolog substrates. Among the eight substrates common to both methods, only data for N-acetyl-D-glucosamine were positively correlated (p < 0.01) between Biolog and SIR. Both methods revealed microbial catabolic profile changed along the chronosequence. PCA indicated that site age, soil carbon, and pH were the most important drivers of microbial catabolic profiles.

Recent advances in Next-Generation Sequencing (NGS) make comparative analyses of the composition and diversity of whole microbial communities possible at a far greater depth than ever before. This brings new challenges, such as an increased dependence on computation to process these huge datasets. The demand on system resources usually requires migrating from Windows to Linux-based operating systems and prior familiarity with command-line interfaces. To overcome this barrier, we developed a fully automated and easy-to-install package as well as a complete, easy-to-follow pipeline for microbial metataxonomic analysis operating in the Windows Subsystem for Linux (WSL)—Bioinformatics Through Windows (BTW). BTW combines several open-access tools for processing marker gene data, including 16S rRNA, bringing the user from raw sequencing reads to diversity-related conclusions. It includes data quality filtering, clustering, taxonomic assignment and further statistical analyses, directly in WSL, avoiding the prior need of migrating from Windows to Linux. BTW is expected to boost the use of NGS amplicon data by facilitating rapid access to a set of bioinformatics tools for Windows users. Moreover, several Linux command line tools became more reachable, which will enhance bioinformatics accessibility to a wider range of researchers and practitioners in the life sciences and medicine. BTW is available in GitHub (https://github.com/vpylro/BTW). The package is freely available for noncommercial users.

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.

While it is known that several Actinobacteria produce enzymes that decompose polysaccharides or phenolic compounds in dead plant biomass, the occurrence of these traits in the environment remains largely unclear. The aim of this work was to screen isolated actinobacterial strains to explore their ability to produce extracellular enzymes that participate in the degradation of polysaccharides and their ability to cometabolically transform phenolic compounds of various complexities. Actinobacterial strains were isolated from meadow and forest soils and screened for their ability to grow on lignocellulose. The potential to transform (14)C-labelled phenolic substrates (dehydrogenation polymer (DHP), lignin and catechol) and to produce a range of extracellular, hydrolytic enzymes was investigated in three strains of Streptomyces spp. that possessed high lignocellulose degrading activity. Isolated strains showed high variation in their ability to produce cellulose- and hemicellulose-degrading enzymes and were able to mineralise up to 1.1% and to solubilise up to 4% of poplar lignin and to mineralise up to 11.4% and to solubilise up to 64% of catechol, while only minimal mineralisation of DHP was observed. The results confirm the potential importance of Actinobacteria in lignocellulose degradation, although it is likely that the decomposition of biopolymers is limited to strains that represent only a minor portion of the entire community, while the range of simple, carbon-containing compounds that serve as sources for actinobacterial growth is relatively wide.

16S ribosomal RNA currently represents the most important target of study in bacterial ecology. Its use for the description of bacterial diversity is, however, limited by the presence of variable copy numbers in bacterial genomes and sequence variation within closely related taxa or within a genome. Here we use the information from sequenced bacterial genomes to explore the variability of 16S rRNA sequences and copy numbers at various taxonomic levels and apply it to estimate bacterial genome and DNA abundances. In total, 7,081 16S rRNA sequences were in silico extracted from 1,690 available bacterial genomes (1-15 per genome). While there are several phyla containing low 16S rRNA copy numbers, in certain taxa, e.g., the Firmicutes and Gammaproteobacteria, the variation is large. Genome sizes are more conserved at all tested taxonomic levels than 16S rRNA copy numbers. Only a minority of bacterial genomes harbors identical 16S rRNA gene copies, and sequence diversity increases with increasing copy numbers. While certain taxa harbor dissimilar 16S rRNA genes, others contain sequences common to multiple species. Sequence identity clusters (often termed operational taxonomic units) thus provide an imperfect representation of bacterial taxa of a certain phylogenetic rank. We have demonstrated that the information on 16S rRNA copy numbers and genome sizes of genome-sequenced bacteria may be used as an estimate for the closest related taxon in an environmental dataset to calculate alternative estimates of the relative abundance of individual bacterial taxa in environmental samples. Using an example from forest soil, this procedure would increase the abundance estimates of Acidobacteria and decrease these of Firmicutes. Using the currently available information, alternative estimates of bacterial community composition may be obtained in this way if the variation of 16S rRNA copy numbers among bacteria is considered.

Threats to global biodiversity are increasingly recognised by scientists and the public as a critical challenge. Molecular sequencing technologies offer means to catalogue, explore, and monitor the richness and biogeography of life on Earth. However, exploiting their full potential requires tools that connect biodiversity infrastructures and resources. As a research infrastructure developing services and technical solutions that help integrate and coordinate life science resources across Europe, ELIXIR is a key player. To identify opportunities, highlight priorities, and aid strategic thinking, here we survey approaches by which molecular technologies help inform understanding of biodiversity. We detail example use cases to highlight how DNA sequencing is: resolving taxonomic issues; Increasing knowledge of marine biodiversity; helping understand how agriculture and biodiversity are critically linked; and playing an essential role in ecological studies. Together with examples of national biodiversity programmes, the use cases show where progress is being made but also highlight common challenges and opportunities for future enhancement of underlying technologies and services that connect molecular and wider biodiversity domains. Based on emerging themes, we propose key recommendations to guide future funding for biodiversity research: biodiversity and bioinformatic infrastructures need to collaborate closely and strategically; taxonomic efforts need to be aligned and harmonised across domains; metadata needs to be standardised and common data management approaches widely adopted; current approaches need to be scaled up dramatically to address the anticipated explosion of molecular data; bioinformatics support for biodiversity research needs to be enabled and sustained; training for end users of biodiversity research infrastructures needs to be prioritised; and community initiatives need to be proactive and focused on enabling solutions. For sequencing data to deliver their full potential they must be connected to knowledge: together, molecular sequence data collection initiatives and biodiversity research infrastructures can advance global efforts to prevent further decline of Earth’s biodiversity.

Threats to global biodiversity are increasingly recognised by scientists and the public as a critical challenge. Molecular sequencing technologies offer means to catalogue, explore, and monitor the richness and biogeography of life on Earth. However, exploiting their full potential requires tools that connect biodiversity infrastructures and resources. As a research infrastructure developing services and technical solutions that help integrate and coordinate life science resources across Europe, ELIXIR is a key player. To identify opportunities, highlight priorities, and aid strategic thinking, here we survey approaches by which molecular technologies help inform understanding of biodiversity. We detail example use cases to highlight how DNA sequencing is: resolving taxonomic issues; Increasing knowledge of marine biodiversity; helping understand how agriculture and biodiversity are critically linked; and playing an essential role in ecological studies. Together with examples of national biodiversity programmes, the use cases show where progress is being made but also highlight common challenges and opportunities for future enhancement of underlying technologies and services that connect molecular and wider biodiversity domains. Based on emerging themes, we propose key recommendations to guide future funding for biodiversity research: biodiversity and bioinformatic infrastructures need to collaborate closely and strategically; taxonomic efforts need to be aligned and harmonised across domains; metadata needs to be standardised and common data management approaches widely adopted; current approaches need to be scaled up dramatically to address the anticipated explosion of molecular data; bioinformatics support for biodiversity research needs to be enabled and sustained; training for end users of biodiversity research infrastructures needs to be prioritised; and community initiatives need to be proactive and focused on enabling solutions. For sequencing data to deliver their full potential they must be connected to knowledge: together, molecular sequence data collection initiatives and biodiversity research infrastructures can advance global efforts to prevent further decline of Earth’s biodiversity.