Feeding on fungi: genomic and proteomic analysis of the enzymatic machinery of bacteria decomposing fungal biomass.

Dead fungal biomass is an abundant source of nutrition in both litter and soil of temperate forests largely decomposed by bacteria. Here, we have examined the utilization of dead fungal biomass by the five dominant bacteria isolated from the in situ decomposition of fungal mycelia using a multiOMIC approach. The genomes of the isolates encoded a broad suite of carbohydrate-active enzymes, peptidases and transporters. In the extracellular proteome, only Ewingella americana expressed chitinases while the two Pseudomonas isolates attacked chitin by lytic chitin monooxygenase, deacetylation and deamination. Variovorax sp. expressed enzymes acting on the side-chains of various glucans and the chitin backbone. Surprisingly, despite its genomic potential, Pedobacter sp. did not produce extracellular proteins to decompose fungal mycelia but presumably feeds on simple substrates. The ecological roles of the five individual strains exhibited complementary features for a fast and efficient decomposition of dead fungal biomass by the entire bacterial community.

Drivers of microbial community structure in forest soils.

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.

Differential sensitivity of total and active soil microbial communities to drought and forest management.

Climate change will affect semiarid ecosystems through severe droughts that increase the competition for resources in plant and microbial communities. In these habitats, adaptations to climate change may consist of thinning-that reduces competition for resources through a decrease in tree density and the promotion of plant survival. We deciphered the functional and phylogenetic responses of the microbial community to 6 years of drought induced by rainfall exclusion and how forest management affects its resistance to drought, in a semiarid forest ecosystem dominated by Pinus halepensis Mill. A multiOMIC approach was applied to reveal novel, community-based strategies in the face of climate change. The diversity and the composition of the total and active soil microbiome were evaluated by 16S rRNA gene (bacteria) and ITS (fungal) sequencing, and by metaproteomics. The microbial biomass was analyzed by phospholipid fatty acids (PLFAs), and the microbially mediated ecosystem multifunctionality was studied by the integration of soil enzyme activities related to the cycles of C, N, and P. The microbial biomass and ecosystem multifunctionality decreased in drought-plots, as a consequence of the lower soil moisture and poorer plant development, but this decrease was more notable in unthinned plots. The structure and diversity of the total bacterial community was unaffected by drought at phylum and order level, but did so at genus level, and was influenced by seasonality. However, the total fungal community and the active microbial community were more sensitive to drought and were related to ecosystem multifunctionality. Thinning in plots without drought increased the active diversity while the total diversity was not affected. Thinning promoted the resistance of ecosystem multifunctionality to drought through changes in the active microbial community. The integration of total and active microbiome analyses avoids misinterpretations of the links between the soil microbial community and climate change.

Microbial genomics, transcriptomics and proteomics: new discoveries in decomposition research using complementary methods.

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.

Variation of carbon, nitrogen and phosphorus content in fungi reflects their ecology and phylogeny.

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.

Changes induced by Trichoderma harzianum in suppressive compost controlling Fusarium wilt.

The addition of species of Trichoderma to compost is a widespread technique used to control different plant diseases. The biological control activity of these species is mainly attributable to a combination of several mechanisms of action, which may affect the microbiota involved in the suppressiveness of compost. This study was therefore performed to determine the effect of inoculation of Trichoderma harzianum (T. harzianum) on compost, focusing on bacterial community structure (16S rRNA) and chitinase gene diversity. In addition, the ability of vineyard pruning waste compost, amended (GCTh) or not (GC) with T. harzianum, to suppress Fusarium wilt was evaluated. The addition of T. harzianum resulted in a high relative abundance of certain chitinolytic bacteria as well as in remarkable protection against Fusarium oxysporum comparable to that induced by compost GC. Moreover, variations in the abiotic characteristics of the media, such as pH, C, N and iron levels, were observed. Despite the lower diversity of chitinolytic bacteria found in GCTh, the high relative abundance of Streptomyces spp. may be involved in the suppressiveness of this growing media. The higher degree of compost suppressiveness achieved after the addition of T. harzianum may be due not only to its biocontrol ability, but also to changes promoted in both abiotic and biotic characteristics of the growing media.

Investigating the Bacterial and Fungal Communities Involved in Dead Biomass Degradation in Forest Soils.

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 microbial community members involved in a particular decomposition process. Further information can be gained (in SIP experiments) 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 and/or bacterial isolates from the same environment, specific biochemical reactions involved in decomposition can be linked to individual microbial taxa. Here, we describe the use of these methods to explore the decomposer community of fungi and bacteria in forest litter and soil.

Forest microbiome and global change.

Forests influence climate and mitigate global change through the storage of carbon in soils. In turn, these complex ecosystems face important challenges, including increases in carbon dioxide, warming, drought and fire, pest outbreaks and nitrogen deposition. The response of forests to these changes is largely mediated by microorganisms, especially fungi and bacteria. The effects of global change differ among boreal, temperate and tropical forests. The future of forests depends mostly on the performance and balance of fungal symbiotic guilds, saprotrophic fungi and bacteria, and fungal plant pathogens. Drought severely weakens forest resilience, as it triggers adverse processes such as pathogen outbreaks and fires that impact the microbial and forest performance for carbon storage and nutrient turnover. Nitrogen deposition also substantially affects forest microbial processes, with a pronounced effect in the temperate zone. Considering plant-microorganism interactions would help predict the future of forests and identify management strategies to increase ecosystem stability and alleviate climate change effects. In this Review, we describe the impact of global change on the forest ecosystem and its microbiome across different climatic zones. We propose potential approaches to control the adverse effects of global change on forest stability, and present future research directions to understand the changes ahead.

Global Distribution of Carbohydrate Utilization Potential in the Prokaryotic Tree of Life.

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.

Bacterial community in soil and tree roots of Picea abies shows little response to clearcutting.

Clearcutting represents a standard management practice in temperate forests with dramatic consequences for the forest ecosystem. The removal of trees responsible for the bulk of primary production can result in a complex response of the soil microbiome. While studies have shown that tree root-symbiotic ectomycorrhizal fungi disappear from soil and decomposing fine roots of trees become a hotspot for fungal decomposition, the fate of the bacterial component of the soil microbiome following clearcutting is unclear. Here, we investigated the response of bacterial community composition for 2 years following clearcutting of a Picea abies stand in soil, rhizosphere and tree roots, by 16S rRNA amplicon sequencing. While in the first few months after clearcutting there was no significant response of bacterial community composition in the rhizosphere and soil, bacterial communities associated with tree roots underwent more profound changes over time. Acidobacteria were abundant in rhizosphere and soil, while Firmicutes were strongly represented in the roots. In addition, bacterial communities on decomposing roots were significantly different from those on pre-clearcut live roots. Compared with fungi, the response of bacterial communities to clearcutting was much less pronounced, indicating independent development of the two microbial domains.

Analysis of subgroup C of fungal chitinases containing chitin-binding and LysM modules in the mycoparasite Trichoderma atroviride.

Fungi have a plethora of chitinases, which can be phylogenetically divided into three subgroups (A, B and C). Subgroup C (sgC) chitinases are especially interesting due to their multiple carbohydrate-binding modules, but they have not been investigated in detail yet. In this study, we analyzed sgC chitinases in the mycoparasites Trichoderma atroviride and Trichoderma virens. The expression of sgC chitinase genes in T. atroviride was induced during mycoparasitism of the fungal prey Botrytis cinerea, but not Rhizoctonia solani and correspondingly only by fungal cell walls of the former. Interestingly, only few sgC chitinase genes were inducible by chitin, suggesting that non-chitinous cell wall components can act as inducers. In contrast, the transcriptional profile of the most abundantly expressed sgC chitinase gene tac6 indicated a role of the protein in hyphal network formation. This shows that sgC chitinases have diverse functions and are not only involved in the mycoparasitic attack. However, sequence analysis and 3D modelling revealed that TAC6 and also its ortholog in T. virens have potentially detrimental deletions in the substrate-binding site and are thus probably not catalytically active enzymes. Genomic analysis showed that the genes neighboring sgC chitinases often encode proteins that are solely composed of multiple LysM modules, which were induced by similar stimuli as their neighboring sgC chitinase genes. This study provides first insights into fungal sgC chitinases and their associated LysM proteins.

Combined ozonation and solarization for the removal of pesticides from soil: Effects on soil microbial communities.

Pesticides have been used extensively in agriculture to control pests and soil-borne diseases. Most of these pesticides can persist in soil in harmful concentrations due to their intrinsic characteristics and their interactions with soil. Soil solarization has been demonstrated to enhance pesticide degradation under field conditions. Recently, ozonation has been suggested as a feasible method for reducing the pesticide load in agricultural fields. However, the effects of ozonation in the soil microbial community have not been studied so far. Here, we evaluate the combined effects of solarization and ozonation on the microbial community of a Mediterranean soil. For this purpose, soil physico-chemical characteristics and enzyme activities and the biomass (through analysis of microbial fatty acids) and diversity (through 16S rRNA and ITS amplicon sequencing) of soil microbial communities were analyzed in a 50-day greenhouse experiment. The degradation of the pesticides was increased by 20%, 28%, and 33% in solarized soil (S), solarized soil with surface ozonation (SOS), and solarized soil with deep ozonation (SOD), respectively, in comparison to control (untreated) soil. Solarization and its combination with ozonation (SOS and SOD) increased the ammonium content as well as the electrical conductivity, while enzyme activities and soil microbial biomass were negatively affected. Despite the biocidal character of ozone, several microbial populations with demonstrated pesticide-degradation capacity showed increases in their relative abundance. Overall, the combination of solarization plus ozone did not exacerbate the effects of solarization on the soil chemistry and microbial communities, but did improve pesticide degradation.

Metagenomes, metatranscriptomes and microbiomes of naturally decomposing deadwood.

Deadwood represents significant carbon (C) stock in a temperate forests. Its decomposition and C mobilization is accomplished by decomposer microorganisms - fungi and bacteria - who also supply the foodweb of commensalist microbes. Due to the ecosystem-level importance of deadwood habitat as a C and nutrient stock with significant nitrogen fixation, the deadwood microbiome composition and function are critical to understanding the microbial processes related to its decomposition. We present a comprehensive suite of data packages obtained through environmental DNA and RNA sequencing from natural deadwood. Data provide a complex picture of the composition and function of microbiome on decomposing trunks of European beech (Fagus sylvatica L.) in a natural forest. Packages include deadwood metagenomes, metatranscriptomes, sequences of total RNA, bacterial genomes resolved from metagenomic data and the 16S rRNA gene and ITS2 metabarcoding markers to characterize the bacterial and fungal communities. This project will be of use to microbiologists, environmental biologists and biogeochemists interested in the microbial processes associated with the transformation of recalcitrant plant biomass.

Complementary Roles of Wood-Inhabiting Fungi and Bacteria Facilitate Deadwood Decomposition.

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.

Organic amendments exacerbate the effects of silver nanoparticles on microbial biomass and community composition of a semiarid soil.

Increased utilization of silver nanoparticles (AgNPs) can result in an accumulation of these particles in the environment. The potential detrimental effects of AgNPs in soil may be associated with the low fertility of soils in semiarid regions that are usually subjected to restoration through the application of organic amendments. Microbial communities are responsible for fundamental processes related to soil fertility, yet the potential impacts of low and realistic AgNPs concentrations on soil microorganisms are still unknown. We studied the effects of realistic citrate-stabilized AgNPs concentrations (0.015 and 1.5 µg kg-1) at two exposure times (7 and 30 days) on a sandy clay loam Mediterranean soil unamended (SU) and amended with compost (SA). We assessed soil microbial biomass (microbial fatty acids), soil enzyme activities (urease, ß-glucosidase, and alkaline phosphatase), and composition of the microbial community (bacterial 16S rRNA gene and fungal ITS2 sequencing) in a microcosm experiment. In the SA, the two concentrations of AgNPs significantly decreased the bacterial biomass after 7 days of incubation. At 30 days of incubation, only a significant decrease in the Gram+ was observed at the highest AgNPs concentration. In contrast, in the SU, there was a significant increase in bacterial biomass after 30 days of incubation at the lowest AgNPs concentration. Overall, we found that fungal biomass was more resistant to AgNPs than bacterial biomass, in both SA and SU. Further, the AgNPs changed the composition of the soil bacterial community in SA, the relative abundance of some bacterial taxa in SA and SU, and fungal richness in SU at 30 days of incubation. However, AgNPs did not affect the activity of extracellular enzymes. This study demonstrates that the exposure time and organic amendments modulate the effects of realistic concentrations of AgNPs in the biomass and composition of the microbial community of a Mediterranean soil.

Environmentally relevant concentrations of silver nanoparticles diminish soil microbial biomass but do not alter enzyme activities or microbial diversity.

The increasing use of silver nanoparticles (AgNPs) due to their well-known antimicrobial activity, has led to their accumulation in soil ecosystems. However, the impact of environmental realistic concentrations of AgNPs on the soil microbial community has been scarcely studied. In this work, we have assessed the impact of AgNPs, that mimic real concentrations in nature, on tropical soils cultivated with Coffea arabica under conventional and organic management systems. We evaluated the biomass, extracellular enzyme activities, and diversity of the soil microbial community, in a microcosm experiment as a function of time. After seven days of incubation, we found an increase in microbial biomass in an AgNPs-concentration-independent manner. In contrast, after 60-day-incubation, there was a decrease in Gram+ and actinobacterial biomass, in both soils and all AgNPs concentrations. Soil physico-chemical properties and enzyme activities were not affected overall by AgNPs. Regarding the microbial community composition, only some differences in the relative abundance at phylum and genus level in the fungal community were observed. Our results suggest that environmental concentrations of AgNPs affected microbial biomass but had little impact on microbial diversity and may have little effects on the soil biogeochemical cycles mediated by extracellular enzyme activities.

Author Correction: GlobalFungi, a global database of fungal occurrences from high-throughput-sequencing metabarcoding studies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

GlobalFungi, a global database of fungal occurrences from high-throughput-sequencing metabarcoding studies.

Fungi are key players in vital ecosystem services, spanning carbon cycling, decomposition, symbiotic associations with cultivated and wild plants and pathogenicity. The high importance of fungi in ecosystem processes contrasts with the incompleteness of our understanding of the patterns of fungal biogeography and the environmental factors that drive those patterns. To reduce this gap of knowledge, we collected and validated data published on the composition of soil fungal communities in terrestrial environments including soil and plant-associated habitats and made them publicly accessible through a user interface at https://globalfungi.com . The GlobalFungi database contains over 600 million observations of fungal sequences across > 17 000 samples with geographical locations and additional metadata contained in 178 original studies with millions of unique nucleotide sequences (sequence variants) of the fungal internal transcribed spacers (ITS) 1 and 2 representing fungal species and genera. The study represents the most comprehensive atlas of global fungal distribution, and it is framed in such a way that third-party data addition is possible.

Lignocellulolytic systems of soil bacteria: A vast and diverse toolbox for biotechnological conversion processes.

Lignocellulose from plant biomass represents an abundant and renewable source for the production of environmentally friendly chemicals and biofuels. However, its recalcitrant nature entails the use of complex biochemical reactions that are still challenging. Since the degradation of lignocellulose is the current bottleneck of the conversion processes, the search for novel enzymes and microbial strains for degrading plant biomass is of high importance. Currently, bacteria are in the spotlight as promising candidates for novel conversion strategies due to their wide functional diversity and versatility. Here, we review the lines of evidence that show the high potential of bacterial strains from soil for biomass conversion ranging from strain characterization to metagenome and metatranscriptome analysis. Substantial and diverse fractions of soil bacteria are able to decompose the major lignocellulose components. To do that, bacteria evolved structurally variable and often highly complex lignocellulolytic systems composed of enzymes as well as proteins involved in efficient substrate binding. Both as individual components or in combination, bacterial enzymes, and accessory proteins appear to be promising tools in the biotechnological valorization of lignocellulose.

Land use shapes the resistance of the soil microbial community and the C cycling response to drought in a semi-arid area.

The aim of this study was to understand the responses of the microbial community of soil under different land uses to drought in a semi-arid Mediterranean area. In a laboratory incubation, soil samples from different land uses (natural forest, drip-irrigated orchard, rain-fed almond tree cultivation and abandoned area) were maintained at 20% and 60% of the WHC. The microbial biomass and potential enzyme activities were determined after four and fifty days of soil incubation. The diversity and composition of the microbial community were studied after 50 days of incubation. The total mineralisation of soil organic C (SOC), as well as, the mineralisation of fresh organic matter (FOM) and the "priming effect" were analysed after addition of 13C-enriched plant tissue. Both land use and drought had significant effects in the soil microbial community, but the effect of land use was stronger than that of drought. The PLFA content (microbial biomass) of the forests soil was greater under drought. After 50 days of soil incubation, the microbial biomass and most of potential enzyme activities of the almond tree and abandoned soil samples were not significantly affected by drought contrary to those in orchard soil. The total and FOM mineralisation were on average lower in soil under drought than under optimal moisture for all land uses. However, the responses of the priming effect to drought were dependent on the land use. Overall, we conclude that the resistance to drought of the soil microbial community from an agroecosystem having a semi-arid climate is strongly influenced by the previous land use.