Functional genomics gives new insights into the ectomycorrhizal degradation of chitin.

Ectomycorrhizal (EcM) fungi play a crucial role in the mineral nitrogen (N) nutrition of their host trees. While it has been proposed that several EcM species also mobilize organic N, studies reporting the EcM ability to degrade N-containing polymers, such as chitin, remain scarce. Here, we assessed the capacity of a representative collection of 16 EcM species to acquire 15 N from 15 N-chitin. In addition, we combined genomics and transcriptomics to identify pathways involved in exogenous chitin degradation between these fungal strains. Boletus edulis, Imleria badia, Suillus luteus, and Hebeloma cylindrosporum efficiently mobilized N from exogenous chitin. EcM genomes primarily contained genes encoding for the direct hydrolysis of chitin. Further, we found a significant relationship between the capacity of EcM fungi to assimilate organic N from chitin and their genomic and transcriptomic potentials for chitin degradation. These findings demonstrate that certain EcM fungal species depolymerize chitin using hydrolytic mechanisms and that endochitinases, but not exochitinases, represent the enzymatic bottleneck of chitin degradation. Finally, this study shows that the degradation of exogenous chitin by EcM fungi might be a key functional trait of nutrient cycling in forests dominated by EcM fungi.

Warming drives a 'hummockification' of microbial communities associated with decomposing mycorrhizal fungal necromass in peatlands.

Dead fungal mycelium (necromass) represents a critical component of soil carbon (C) and nutrient cycles. Assessing how the microbial communities associated with decomposing fungal necromass change as global temperatures rise will help in determining how these belowground organic matter inputs contribute to ecosystem responses. In this study, we characterized the structure of bacterial and fungal communities associated with multiple types of decaying mycorrhizal fungal necromass incubated within mesh bags across a 9°C whole ecosystem temperature enhancement in a boreal peatland. We found major taxonomic and functional shifts in the microbial communities present on decaying mycorrhizal fungal necromass in response to warming. These changes were most pronounced in hollow microsites, which showed convergence towards the necromass-associated microbial communities present in unwarmed hummocks. We also observed a high colonization of ericoid mycorrhizal fungal necromass by fungi from the same genera as the necromass. These results indicate that microbial communities associated with mycorrhizal fungal necromass decomposition are likely to change significantly with future climate warming, which may have strong impacts on soil biogeochemical cycles in peatlands. Additionally, the high enrichment of congeneric fungal decomposers on ericoid mycorrhizal necromass may help to explain the increase in ericoid shrub dominance in warming peatlands.

Sapwood and heartwood affect differentially bacterial and fungal community structure and successional dynamics during Quercus petraea decomposition.

In forests, bacteria and fungi are key players in wood degradation. Still, studies focusing on bacterial and fungal successions during the decomposition process depending on the wood types (i.e. sapwood and heartwood) remain scarce. This study aimed to understand the effect of wood type on the dynamics of microbial ecological guilds in wood decomposition. Using Illumina metabarcoding, bacterial and fungal communities were monitored every 3 months for 3 years from Quercus petraea wood discs placed on forest soil. Wood density and microbial enzymes involved in biopolymer degradation were measured. We observed rapid changes in the bacterial and fungal communities and microbial ecological guilds associated with wood decomposition throughout the experiment. Bacterial and fungal succession dynamics were very contrasted between sapwood and heartwood. The initial microbial communities were quickly replaced by new bacterial and fungal assemblages in the sapwood. Conversely, some initial functional guilds (i.e. endophytes and yeasts) persisted all along the experiment in heartwood and finally became dominant, possibly limiting the development of saprotrophic fungi. Our data also suggested a significant role of bacteria in nitrogen cycle during wood decomposition.

Root presence modifies the long-term decomposition dynamics of fungal necromass and the associated microbial communities in a boreal forest.

Recent studies have highlighted that dead fungal mycelium represents an important fraction of soil carbon (C) and nitrogen (N) inputs and stocks. Consequently, identifying the microbial communities and the ecological factors that govern the decomposition of fungal necromass will provide critical insight into how fungal organic matter (OM) affects forest soil C and nutrient cycles. Here, we examined the microbial communities colonising fungal necromass during a multiyear decomposition experiment in a boreal forest, which included incubation bags with different mesh sizes to manipulate both plant root and microbial decomposer group access. Necromass-associated bacterial and fungal communities were taxonomically and functionally rich throughout the 30 months of incubation, with increasing abundances of oligotrophic bacteria and root-associated fungi (i.e., ectomycorrhizal, ericoid mycorrhizal and endophytic fungi) in the late stages of decomposition in the mesh bags to which they had access. Necromass-associated ß-glucosidase activity was highest at 6 months, while leucine aminopeptidase peptidase was highest at 18 months. Based on an asymptotic decomposition model, root presence was associated with an initial faster rate of fungal necromass decomposition, but resulted in higher amounts of fungal necromass retained at later sampling times. Collectively, these results indicate that microbial community composition and enzyme activities on decomposing fungal necromass remain dynamic years after initial input, and that roots and their associated fungal symbionts result in the slowing of microbial necromass turnover with time.

Interactions between Hg and soil microbes: microbial diversity and mechanisms, with an emphasis on fungal processes.

Mercury (Hg) is a highly toxic metal with no known biological function, and it can be highly bioavailable in terrestrial ecosystems. Although fungi are important contributors to a number of soil processes including plant nutrient uptake and decomposition, little is known about the effect of Hg on fungi. Fungi accumulate the largest amount of Hg and are the organisms capable of the highest bioaccumulation of Hg. While referring to detailed mechanisms in bacteria, this mini-review emphasizes the progress made recently on this topic and represents the first step towards a better understanding of the mechanisms underlying Hg tolerance and accumulation in fungal species and hence on the role of fungi within the Hg cycle at Hg-contaminated sites. KEY POINTS: • The fungal communities are more resilient than bacterial communities to Hg exposure. • The exposure to Hg is a threat to microbial soil functions involved in both C and nutrient cycles. • Fungal (hyper)accumulation of Hg may be important for the Hg cycle in terrestrial environments. • Understanding Hg tolerance and accumulation by fungi may lead to new remediation biotechnologies.

First evidences that the ectomycorrhizal fungus Paxillus involutus mobilizes nitrogen and carbon from saprotrophic fungus necromass.

Fungal succession in rotting wood shows a surprising abundance of ectomycorrhizal (EM) fungi during the late decomposition stages. To better understand the links between EM fungi and saprotrophic fungi, we investigated the potential capacities of the EM fungus Paxillus involutus to mobilize nutrients from necromass of Postia placenta, a wood rot fungus, and to transfer these elements to its host tree. In this aim, we used pure cultures of P. involutus in the presence of labelled Postia necromass (15 N/13 C) as nutrient source, and a monoxenic mycorrhized pine experiment composed of labelled Postia necromass and P. involutus culture in interaction with pine seedlings. The isotopic labelling was measured in both experiments. In pure culture, P. involutus was able to mobilize N, but C as well, from the Postia necromass. In the symbiotic interaction experiment, we measured high 15 N enrichments in all plant and fungal compartments. Interestingly, 13 C remains mainly in the mycelium and mycorrhizas, demonstrating that the EM fungus transferred essentially N from the necromass to the tree. These observations reveal that fungal organic matter could represent a significant N source for EM fungi and trees, but also a C source for mycorrhizal fungi, including in symbiotic lifestyle.

Microbial Enzymatic Activities and Community-Level Physiological Profiles (CLPP) in Subsoil Layers Are Altered by Harvest Residue Management Practices in a Tropical Eucalyptus grandis Plantation.

Harvest residue management is a key issue for the sustainability of Eucalyptus plantations established on poor soils. Soil microbial communities contribute to soil fertility by the decomposition of the organic matter (OM), but little is known about the effect of whole-tree harvesting (WTH) in comparison to stem only harvesting (SOH) on soil microbial functional diversity in Eucalyptus plantations. We studied the effects of harvest residue management (branches, leaves, bark) of Eucalyptus grandis trees on soil enzymatic activities and community-level physiological profiles in a Brazilian plantation. We measured soil microbial enzymatic activities involved in OM decomposition and we compared the community level physiological profiles (CLPP) of the soil microbes in WTH and SOH plots. WTH decreased enzyme activities and catabolic potential of the soil microbial community. Furthermore, these negative effects on soil functional diversity were mainly observed below the 0-5 cm layer (5-10 and 10-20 cm), suggesting that WTH can be harmful to the soil health in these plantations.

N-Acetylglucosaminidase activity, a functional trait of chitin degradation, is regulated differentially within two orders of ectomycorrhizal fungi: Boletales and Agaricales.

Chitin is one of the most abundant nitrogen-containing polymers in forest soil. Ability of ectomycorrhizal (EM) fungi to utilize chitin may play a key role in the EM symbiosis nutrition and soil carbon cycle. In forest, EM fungi exhibit high diversity, which could be based on function partitioning and trait complementarity. Although it has long been recognized that closely related species share functional characteristics, the phylogenetic conservatism of functional traits within microorganisms remains unclear. Because extracellular N-acetylglucosaminidase activity has been proposed as functional trait of chitin degradation, we screened this activity on 35 EM fungi species with or without chitin in the growth medium to (i) describe the functional diversity of EM fungi and (ii) identify potential links between this functional trait and EM fungal phylogeny. We observed large variations of the extracellular N-acetylglucosaminidase activities among the fungal strains. Furthermore, our results revealed two regulation patterns of extracellular N-acetylglucosaminidase activities. Indeed, these chitinolytic activities were stimulated or repressed in the presence of chitin, in comparison to the control treatment. These profiles of extracellular N-acetylglucosaminidase stimulation/repression might be conserved at a high phylogenetic level in the Basidiomycota phylum, as illustrated by the opposite patterns of regulation between Boletales and Agaricales. Finally, the downregulation of this activity by chitin, for some EM fungal groups, might suggest another chitin degradation pathway.

Environmental Metabarcoding Reveals Contrasting Belowground and Aboveground Fungal Communities from Poplar at a Hg Phytomanagement Site.

Characterization of microbial communities in stressful conditions at a field level is rather scarce, especially when considering fungal communities from aboveground habitats. We aimed at characterizing fungal communities from different poplar habitats at a Hg-contaminated phytomanagement site by using Illumina-based sequencing, network analysis approach, and direct isolation of Hg-resistant fungal strains. The highest diversity estimated by the Shannon index was found for soil communities, which was negatively affected by soil Hg concentration. Among the significant correlations between soil operational taxonomic units (OTUs) in the co-occurrence network, 80% were negatively correlated revealing dominance of a pattern of mutual exclusion. The fungal communities associated with Populus roots mostly consisted of OTUs from the symbiotic guild, such as members of the Thelephoraceae, thus explaining the lowest diversity found for root communities. Additionally, root communities showed the highest network connectivity index, while rarely detected OTUs from the Glomeromycetes may have a central role in the root network. Unexpectedly high richness and diversity were found for aboveground habitats, compared to the root habitat. The aboveground habitats were dominated by yeasts from the Lalaria, Davidiella, and Bensingtonia genera, not detected in belowground habitats. Leaf and stem habitats were characterized by few dominant OTUs such as those from the Dothideomycete class producing mutual exclusion with other OTUs. Aureobasidium pullulans, one of the dominating OTUs, was further isolated from the leaf habitat, in addition to Nakazawaea populi species, which were found to be Hg resistant. Altogether, these findings will provide an improved point of reference for microbial research on inoculation-based programs of tailings dumps.

Dendrochemical assessment of mercury releases from a pond and dredged-sediment landfill impacted by a chlor-alkali plant.

Although current Hg emissions from industrial activities may be accurately monitored, evidence of past releases to the atmosphere must rely on one or more environmental proxies. We used Hg concentrations in tree cores collected from poplars and willows to investigate the historical changes of Hg emissions from a dredged sediment landfill and compared them to a nearby control location. Our results demonstrated the potential value of using dendrochemistry to record historical Hg emissions from past industrial activities.

Primer pairs, PCR conditions, and peptide nucleic acid clamps affect fungal diversity assessment from plant root tissues.

High-throughput sequencing has become a prominent tool to assess plant-associated microbial diversity. Still, some technical challenges remain in characterising these communities, notably due to plant and fungal DNA co-amplification. Fungal-specific primers, Peptide Nucleic Acid (PNA) clamps, or adjusting PCR conditions are approaches to limit plant DNA contamination. However, a systematic comparison of these factors and their interactions, which could limit plant DNA contamination in the study of plant mycobiota, is still lacking. Here, three primers targeting the ITS2 region were evaluated alone or in combination with PNA clamps both on nettle (Urtica dioica) root DNA and a mock community. PNA clamps did not improve the richness or diversity of the fungal communities but increased the number of fungal reads. Among the tested factors, the most significant was the primer pair. Specifically, the 5.8S-Fun/ITS4-Fun pair exhibited a higher OTU richness but fewer fungal reads. Our study demonstrates that the choice of primers is critical for limiting plant and fungal DNA co-amplification. PNA clamps increase the number of fungal reads when ITS2 is targeted but do not result in higher fungal diversity recovery at high sequencing depth. At lower read depths, PNA clamps might enhance microbial diversity quantification for primer pairs lacking fungal specificity.

Differential effects of tree species identity on rhizospheric bacterial and fungal community richness and composition across multiple trace element-contaminated sites.

Soil microbial communities play a key role in plant nutrition and stress tolerance. This is particularly true in sites contaminated by trace metals, which often have low fertility and stressful conditions for woody plants in particular. However, we have limited knowledge of the abiotic and biotic factors affecting the richness and composition of microbial communities inhabiting the rhizosphere of plants in contaminated sites. Using high-throughput amplicon sequencing, we studied the rhizospheric bacterial and fungal community structures of 14 woody plant families planted in three contrasting sites contaminated by metals (Pb, Cd, Zn, Mn, Fe, S). The rhizospheric bacterial communities in the given sites showed no significant difference between the various woody species but did differ significantly between sites. The Proteobacteria phylum was dominant, accounting for over 25 % of the overall relative abundance, followed by Actinobacteria, Bacteroidetes and Gemmatimonadetes. Site was also the main driver of fungal community composition, yet unlike bacteria, tree species identity significantly affected fungal communities. The Betulaceae, Salicaceae and Fagaceae families had a high proportion of Basidiomycota, particularly ectomycorrhizal fungi, and the lowest diversity and richness. The other tree families and the unplanted soil harboured a greater abundance of Ascomycota and Mucoromycota. Consequently, for both bacteria and fungi, the site effect significantly impacted their community richness and composition, while the influence of plants on the richness and composition of rhizospheric microbial communities stayed consistent across sites and was dependent on the microbial kingdom. Finally, we highlighted the importance of considering this contrasting response of plant rhizospheric microbial communities in relation to their host identity, particularly to improve assisted revegetation efforts at contaminated sites.

Fungi rather than bacteria drive early mass loss from fungal necromass regardless of particle size.

Microbial necromass is increasingly recognized as an important fast-cycling component of the long-term carbon present in soils. To better understand how fungi and bacteria individually contribute to the decomposition of fungal necromass, three particle sizes (>500, 250-500, and <250 µm) of Hyaloscypha bicolor necromass were incubated in laboratory microcosms inoculated with individual strains of two fungi and two bacteria. Decomposition was assessed after 15 and 28 days via necromass loss, microbial respiration, and changes in necromass pH, water content, and chemistry. To examine how fungal-bacterial interactions impact microbial growth on necromass, single and paired cultures of bacteria and fungi were grown in microplates containing necromass-infused media. Microbial growth was measured after 5 days through quantitative PCR. Regardless of particle size, necromass colonized by fungi had higher mass loss and respiration than both bacteria and uninoculated controls. Fungal colonization increased necromass pH, water content, and altered chemistry, while necromass colonized by bacteria remained mostly unaltered. Bacteria grew significantly more when co-cultured with a fungus, while fungal growth was not significantly affected by bacteria. Collectively, our results suggest that fungi act as key early decomposers of fungal necromass and that bacteria may require the presence of fungi to actively participate in necromass decomposition.

Knowns and unknowns of the soil fungal necrobiome.

Dead microbial cells, commonly referred to as necromass, are increasingly recognized as an important source of both persistent carbon as well as nutrient availability in soils. Studies of the microbial communities associated with decomposing fungal necromass have accumulated rapidly in recent years across a range of different terrestrial ecosystems. Here we identify the primary ecological patterns regarding the structure and dynamics of the fungal necrobiome as well as highlight new research frontiers that will likely be key to gaining a full understanding of fungal necrobiome composition and its associated role in soil biogeochemical cycling. Because many members of the fungal necrobiome are culturable, combining laboratory functional assays with field-based surveys and experiments will allow ongoing studies of the fungal necrobiome to move from largely descriptive to increasingly predictive.

Defining a core microbial necrobiome associated with decomposing fungal necromass.

Despite growing interest in fungal necromass decomposition due to its importance in soil carbon retention, whether a consistent group of microorganisms is associated with decomposing necromass remains unresolved. Here, we synthesize knowledge on the composition of the bacterial and fungal communities present on decomposing fungal necromass from a variety of fungal species, geographic locations, habitats, and incubation times. We found that there is a core group of both bacterial and fungal genera (i.e. a core fungal necrobiome), although the specific size of the core depended on definition. Based on a metric that included both microbial frequency and abundance, we demonstrate that the core is taxonomically and functionally diverse, including bacterial copiotrophs and oligotrophs as well as fungal saprotrophs, ectomycorrhizal fungi, and both fungal and animal parasites. We also show that the composition of the core necrobiome is notably dynamic over time, with many core bacterial and fungal genera having specific associations with the early, middle, or late stages of necromass decomposition. While this study establishes the existence of a core fungal necrobiome, we advocate that profiling the composition of fungal necromass decomposer communities in tropical environments and other terrestrial biomes beyond forests is needed to fill key knowledge gaps regarding the global nature of the fungal necrobiome.

Fungal necromass presents a high potential for Mercury immobilization in soil.

Past industrial activities have generated many contaminated lands from which Mercury (Hg) escapes, primarily by volatilization. Current phytomanagement techniques aim to limit Hg dispersion by increasing its stabilization in soil. Although soil fungi represent a source of Hg emission associated with biovolatilization mechanisms, there is limited knowledge about how dead fungal residues (i.e., fungal necromass) interact with soil Hg. This study determined the Hg biosorption potential of fungal necromass and the chemical drivers of passive Hg binding with dead mycelia. Fungal necromass was incubated under field conditions with contrasting chemical properties at a well-characterized Hg phytomanagement experimental site in France. After four months of incubation in soil, fungal residues passively accumulated substantial quantities of Hg in their recalcitrant fractions ranging from 400 to 4500 µg Hg/kg. In addition, infrared spectroscopy revealed that lipid compounds explained the amount of Hg biosorption to fungal necromass. Based on these findings, we propose that fungal necromass is likely an important factor in Hg immobilization in soil.

Melanization slows the rapid movement of fungal necromass carbon and nitrogen into both bacterial and fungal decomposer communities and soils.

Microbial necromass contributes significantly to both soil carbon (C) persistence and ecosystem nitrogen (N) availability, but quantitative estimates of C and N movement from necromass into soils and decomposer communities are lacking. Additionally, while melanin is known to slow fungal necromass decomposition, how it influences microbial C and N acquisition as well as elemental release into soils remains unclear. Here, we tracked decomposition of isotopically labeled low and high melanin fungal necromass and measured 13C and 15N accumulation in surrounding soils and microbial communities over 77 d in a temperate forest in Minnesota, USA. Mass loss was significantly higher from low melanin necromass, corresponding with greater 13C and 15N soil inputs. A taxonomically and functionally diverse array of bacteria and fungi was enriched in 13C and/or 15N at all sampling points, with enrichment being consistently higher on low melanin necromass and earlier in decomposition. Similar patterns of preferential C and N enrichment of many bacterial and fungal genera early in decomposition suggest that both microbial groups co-contribute to the rapid assimilation of resource-rich soil organic matter inputs. While overall richness of taxa enriched in C was higher than in N for both bacteria and fungi, there was a significant positive relationship between C and N in co-enriched taxa. Collectively, our results demonstrate that melanization acts as a key ecological trait mediating not only fungal necromass decomposition rate but also necromass C and N release and that both elements are rapidly co-utilized by diverse bacterial and fungal decomposers in natural settings. IMPORTANCE Recent studies indicate that microbial dead cells, particularly those of fungi, play an important role in long-term carbon persistence in soils. Despite this growing recognition, how the resources within dead fungal cells (also known as fungal necromass) move into decomposer communities and soils are poorly quantified, particularly in studies based in natural environments. In this study, we found that the contribution of fungal necromass to soil carbon and nitrogen availability was slowed by the amount of melanin present in fungal cell walls. Further, despite the overall rapid acquisition of carbon and nitrogen from necromass by a diverse range of both bacteria and fungi, melanization also slowed microbial uptake of both elements. Collectively, our results indicate that melanization acts as a key ecological trait mediating not only fungal necromass decomposition rate, but also necromass carbon and nitrogen release into soil as well as microbial resource acquisition.

Partial overlap of fungal communities associated with nettle and poplar roots when co-occurring at a trace metal contaminated site.

Stinging nettle (Urtica dioica L.) raises growing interest in phytomanagement because it commonly grows under poplar Short Rotation Coppices (SRC) set up at trace-metal (TM) contaminated sites and provides high-quality herbaceous fibres. The mycobiome of this non-mycorhizal plant and its capacity to adapt to TM-contaminated environments remains unknown. This study aimed at characterizing the mycobiome associated with nettle and poplar roots co-occurring at a TM-contaminated site. Plant root barcoding using the fungi-specific ITS1F-ITS2 primers and Illumina MiSeq technology revealed that nettle and poplar had distinct root fungal communities. The nettle mycobiome was dominated by Pezizomycetes from known endophytic taxa and from the supposedly saprotrophic genus Kotlabaea (which was the most abundant). Several ectomycorrhizal fungi such as Inocybe (Agaricomycetes) and Tuber (Pezizomycetes) species were associated with the poplar roots. Most of the Pezizomycetes taxa were present in the highly TM-contaminated area whereas Agaricomycetes tended to be reduced. Despite being a known non-mycorrhizal plant, nettle was associated with a significant proportion of ectomycorrhizal OTU (9.7%), suggesting some connexions between the poplar and the nettle root mycobiomes. Finally, our study raised the interest in reconsidering the fungal networking beyond known mycorrhizal interactions.

Functional convergence in the decomposition of fungal necromass in soil and wood.

Understanding the post-senescent fate of fungal mycelium is critical to accurately quantifying forest carbon and nutrient cycling, but how this organic matter source decomposes in wood remains poorly studied. In this study, we compared the decomposition of dead fungal biomass (a.k.a. necromass) of two species, Mortierella elongata and Meliniomyces bicolor, in paired wood and soil plots in a boreal forest in northern Minnesota, USA. Mass loss was quantified at four time points over an 8-week incubation and the richness and composition of the fungal communities colonizing fungal necromass were characterized using high-throughput sequencing. We found that the structure of fungal decomposer communities in wood and soil differed, but, in both habitats, there was relatively rapid decay (∼30% remaining after 56 days). Mass loss was significantly faster in soil and for high-quality (i.e. high nitrogen and low melanin) fungal necromass. In both habitats, there was a clear trajectory of early colonization by opportunistic fungal taxa followed by colonization of fungi with greater enzymatic capacities to degrade more recalcitrant compounds, including white-rot and ectomycorrhizal fungi. Collectively, our results indicate that patterns emerging regarding substrate quality effects on fungal necromass decomposition in soil and leaf litter can be largely extended to fungal necromass decomposition in wood.