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Abstract The methodical developments in the fields of molecular biology and analytical chemistry significantly increased the level of detail that we achieve when exploring soils and their microbial inhabitants. High-resolution description of microbial communities, detection of taxa with minor abundances, screening of gene expression or the detailed characterization of metabolomes are nowadays technically feasible. Despite all of this, our understanding of soil is limited in many ways. The imperfect tools to describe microbial communities and limited possibilities to assign traits to community members make it difficult to link microbes to functions. Also the analysis of processes exemplified by enzyme activity measurements is still imperfect. In the future, it is important to look at soil at a finer detail to obtain a better picture on the properties of individual microbes, their in situ interactions, metabolic rates and activity at a scale relevant to individual microbes. Scaling up is needed as well to get answers at ecosystem or biome levels and to enable global modelling. The recent development of novel tools including metabolomics, identification of genomes in metagenomics sequencing datasets or collection of trait data have the potential to bring soil ecology further. It will, however, always remain a highly demanding scientific discipline. Despite the significant achievements in soil microbial ecology over the past years, many common methodical approaches have important limitations that need to be addressed as well as our way of thinking about soil as the ecosystem component.

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.

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.

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.

Forests are essential biomes for global biogeochemical cycles, and belowground microorganisms have a key role in providing relevant ecosystem services. To predict the effects of environmental changes on these ecosystem services requires a comprehensive understanding of how biotic and abiotic factors drive the composition of microbial communities in soil. However, microorganisms are not homogeneously distributed in complex environments such as soil, with different features affecting microbes at different extent depending on the niche they occupy. Indeed, this spatial heterogeneity hampers the extrapolation of microbial diversity study results from particular habitats to the ecosystem level, even if the resolution of the more recent studies has increased significantly after the standardization of high-throughput sequencing techniques. The present work intends to give a comprehensive view of the knowledge accumulated until date defining the more important drivers determining the structure of forest soil microbial communities from fine to continental scales.

Fungi are major ecological players in both terrestrial and aquatic environments by cycling organic matter and channelling nutrients across trophic levels. High-throughput sequencing (HTS) studies of fungal communities are redrawing the map of the fungal kingdom by hinting at its enormous — and largely uncharted — taxonomic and functional diversity. However, HTS approaches come with a range of pitfalls and potential biases, cautioning against unwary application and interpretation of HTS technologies and results. In this Review, we provide an overview and practical recommendations for aspects of HTS studies ranging from sampling and laboratory practices to data processing and analysis. We also discuss upcoming trends and techniques in the field and summarize recent and noteworthy results from HTS studies targeting fungal communities and guilds. Our Review highlights the need for reproducibility and public data availability in the study of fungal communities. If the associated challenges and conceptual barriers are overcome, HTS offers immense possibilities in mycology and elsewhere.

Abstract 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 specific ecosystems comprising a multitude of microbial habitats with specific properties, such as 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 that are dynamic on a broad temporal scale with ecosystem processes ranging from short-term events over seasonal ones to long-term stand development where fungi, bacteria and other organisms composing the ‘forest microbiome’ play important roles.

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.

Fungi are the agents primarily responsible for the transformation of plant-derived carbon in terrestrial ecosystems. However, little is known of their responses to the seasonal changes in resource availability in deciduous forests, including photosynthate allocation below ground and seasonal inputs of fresh litter. Vertical stratification of and seasonal changes in fungal abundance, activity and community composition were investigated in the litter, organic and upper mineral soils of a temperate Quercus petraea forest using ergosterol and extracellular enzyme assays and amplicon 454-pyrosequencing of the rDNA-ITS region. Fungal activity, biomass and diversity decreased substantially with soil depth. The highest enzyme activities were detected in winter, especially in litter, where these activities were followed by a peak in fungal biomass during spring. The litter community exhibited more profound seasonal changes than did the community in the deeper horizons. In the litter, saprotrophic genera reached their seasonal maxima in autumn, but summer typically saw the highest abundance of ectomycorrhizal taxa. Although the composition of the litter community changes over the course of the year, the mineral soil shows changes in biomass. The fungal community is affected by season. Litter decomposition and phytosynthate allocation represent important factors contributing to the observed variations.

This article is a Commentary on Smith et al., 215: 747–755.