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The impacts of plant species on the microbial communities and physico-chemical characteristics of soil are well documented for many herbs, grasses and legumes but much less so for tree species. Here, we investigate by rRNA and ITS amplicon sequencing the diversity of microorganisms from the three domains of life (Archaea, Bacteria and Eukaryota:Fungi) in soil samples taken from the forest experimental site of Breuil-Chenue (France). We discovered significant differences in the abundance, composition and structure of the microbial communities associated with two phylogenetically distant tree species of the same age, deciduous European beech (Fagus sylvatica) and coniferous Norway spruce (Picea abies Karst), planted in the same soil. Our results suggest a significant effect of tree species on soil microbiota though in different ways for each of the three microbial groups. Fungal and archaeal community structures and compositions are mainly determined according to tree species, whereas bacterial communities differ to a great degree between rhizosphere and bulk soils, regardless of the tree species. These results were confirmed by quantitative PCR, which revealed significant enrichment of specific bacterial genera, such as Burkholderia and Collimonas, known for their ability to weather minerals within the tree root vicinity.

Abstract Background In forest ecosystems, communities of ectomycorrhizal fungi (ECM) are influenced by several biotic and abiotic factors. To understand their underlying dynamics, ECM communities have been surveyed with ribosomal DNA-based sequencing methods. However, most identification methods are both time-consuming and limited by the number of samples that can be treated in a realistic time frame. As a result of ongoing implementation, the array technique has gained throughput capacity in terms of the number of samples and the capacity for parallel identification of several species. Thus far, although phylochips (microarrays that are used to detect species) have been mostly developed to trace bacterial communities or groups of specific fungi, no phylochip has been developed to carry oligonucleotides for several ectomycorrhizal species that belong to different genera. Results We have constructed a custom ribosomal DNA phylochip to identify ECM fungi. Specific oligonucleotide probes were targeted to the nuclear internal transcribed spacer (ITS) regions from 95 fungal species belonging to 21 ECM fungal genera. The phylochip was first validated using PCR amplicons of reference species. Ninety-nine percent of the tested oligonucleotides generated positive hybridisation signals with their corresponding amplicons. Cross-hybridisation was mainly restricted at the genus level, particularly for Cortinarius and Lactarius species. The phylochip was subsequently tested with environmental samples that were composed of ECM fungal DNA from spruce and beech plantation fungal communities. The results were in concordance with the ITS sequencing of morphotypes and the ITS clone library sequencing results that were obtained using the same PCR products. Conclusion For the first time, we developed a custom phylochip that is specific for several ectomycorrhizal fungi. To overcome cross-hybridisation problems, specific filter and evaluation strategies that used spot signal intensity were applied. Evaluation of the phylochip by hybridising environmental samples confirmed the possible application of this technology for detecting and monitoring ectomycorrhizal fungi at specific sites in a routine and reproducible manner.

Plants shape and interact continuously with their rhizospheric microbiota, which play a key role in plant health and resilience. However, plant-associated microbial community can be shaped by several factors including plant phenotype and cropping system. Thus, understanding the interplay between microbiome assembly during the onset of plant-pathogen interactions and long-lasting resistance traits in ligneous plants remains a major challenge. To date, such attempts were mainly investigated in herbaceous plants, due to their phenotypic characteristics and their short life cycle. However, only few studies have focused on the microbial structure, dynamic and their drivers in perennial ligneous plants. Ligneous plants coevolved in interaction with specific fungal and bacterial communities that differ from those of annual plants. The specificities of such ligneous plants in shaping their own functional microbial communities could be dependent on their high heterozygosis, physiological and molecular status associated to seasonality and their aging processes, root system and above-ground architectures, long-lasting climatic variations, and specific cultural practices. This article provides an overview of the specific characteristics of perennial ligneous plants that are likely to modulate symbiotic interactions in the rhizosphere, thus affecting the plant's fitness and systemic immunity. Plant and microbial traits contributing to the establishment of plant-microbiome interactions and the adaptation of this holobiont are also discussed., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Soil fungi belonging to different functional guilds, such as saprotrophs, pathogens, and mycorrhizal symbionts, play key roles in forest ecosystems. To date, no study has compared the actual gene expression of these guilds in different forest soils. We used metatranscriptomics to study the competition for organic resources by these fungal groups in boreal, temperate, and Mediterranean forest soils. Using a dedicated mRNA annotation pipeline combined with the JGI MycoCosm database, we compared the transcripts of these three fungal guilds, targeting enzymes involved in C- and N mobilization from plant and microbial cell walls. Genes encoding enzymes involved in the degradation of plant cell walls were expressed at a higher level in saprotrophic fungi than in ectomycorrhizal and pathogenic fungi. However, ectomycorrhizal and saprotrophic fungi showed similarly high expression levels of genes encoding enzymes involved in fungal cell wall degradation. Transcripts for N-related transporters were more highly expressed in ectomycorrhizal fungi than in other groups. We showed that ectomycorrhizal and saprotrophic fungi compete for N in soil organic matter, suggesting that their interactions could decelerate C cycling. Metatranscriptomics provides a unique tool to test controversial ecological hypotheses and to better understand the underlying ecological processes involved in soil functioning and carbon stabilization., (© 2023 His Majesty the King in Right of Canada and The Authors. New Phytologist © 2023 New Phytologist Foundation Reproduced with the permission of the Minister of Natural Resources Canada. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Background: Major advances over the past decade in molecular ecology are providing access to soil fungal diversity in forest ecosystems worldwide, but the diverse functions and metabolic capabilities of this microbial community remain largely elusive. We conducted a field survey in montane old-growth broadleaved and conifer forests, to investigate the relationship between soil fungal diversity and functional genetic traits. To assess the extent to which variation in community composition was associated with dominant tree species (oak, spruce, and fir) and environmental variations in the old-growth forests in the Jade Dragon Snow Mountain in Yunnan Province, we applied rDNA metabarcoding. We also assessed fungal gene expression in soil using mRNA sequencing and specifically assessed the expression of genes related to organic matter decomposition and nutrient acquisition in ectomycorrhizal and saprotrophic fungi., Results: Our taxonomic profiling revealed striking shifts in the composition of the saprotrophic and ectomycorrhizal guilds among the oak-, fir-, and spruce-dominated forests. The core fungal microbiome comprised only ~ 20% of the total OTUs across all soil samples, although the overlap between conifer-associated communities was substantial. In contrast, seasonality and soil layer explained only a small proportion of the variation in community structure. However, despite their highly variable taxonomic composition, fungal guilds exhibited remarkably similar functional traits for growth-related and core metabolic pathways across forest associations, suggesting ecological redundancy. However, we found that the expression profiles of genes related to polysaccharide and protein degradation and nutrient transport notably varied between and within the fungal guilds, suggesting niche adaptation., Conclusions: Overall, our metatranscriptomic analyses revealed the functional potential of soil fungal communities in montane old-growth forests, including a suite of specialized genes and taxa involved in organic matter decomposition. By linking genes to ecological traits, this study provides insights into fungal adaptation strategies to biotic and environmental factors, and sheds light on the importance of understanding functional gene expression patterns in predicting ecosystem functioning. Video Abstract., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation Some of the authors are U.S. Government employees and their work is in the public domain in the USA.)

Global warming is pushing populations outside their range of physiological tolerance. According to the environmental envelope framework, the most vulnerable populations occur near the climatic edge of their species' distributions. In contrast, populations from the climatic center of the species range should be relatively buffered against climate warming. We tested this latter prediction using a combination of linear mixed effects and machine learning algorithms on an extensive, citizen-scientist generated dataset on the fruitbody productivity of the Burgundy (aka summer) truffle (Tuber aestivum Vittad.), a keystone, ectomycorrhizal tree-symbiont occurring on a wide range of temperate climates. T. aestivum's fruitbody productivity was monitored at 3-week resolution over up to 8 continuous years at 20 sites distributed in the climatic center of its European distribution in southwest Germany and Switzerland. We found that T. aestivum fruitbody production is more sensitive to summer drought than would be expected from the breadth of its species' climatic niche. The monitored populations occurring nearly 5°C colder than the edge of their species' climatic distribution. However, interannual fruitbody productivity (truffle mass year -1 ) fell by a median loss of 22% for every 1°C increase in summer temperature over a site's 30-year mean. Among the most productive monitored populations, the temperature sensitivity was even higher, with single summer temperature anomalies of 3°C sufficient to stop fruitbody production altogether. Interannual truffle productivity was also related to the phenology of host trees, with ~22 g less truffle mass for each 1-day reduction in the length of the tree growing season. Increasing summer drought extremes are therefore likely to reduce fruiting among summer truffle populations throughout Central Europe. Our results suggest that European T. aestivum may be a mosaic of vulnerable populations, sensitive to climate-driven declines at lower thresholds than implied by its species distribution model., (© 2022 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

Replacement of primary old-growth forests by secondary woodlands in threatened subtropical biomes drives important changes at the level of the overstory, understory and forest floor, but the impact on belowground microbial biodiversity is yet poorly documented. In the present study, we surveyed by metabarcoding sequencing, the diversity and composition of soil bacteria and fungi in the old-growth forest, dominated by stone oaks ( Lithocarpus spp.) and in the secondary Yunnan pine woodland of an iconic site for biodiversity research, the Ailaoshan National Nature Reserve (Ailao Mountains, Yunnan province, China). We assessed the effect of forest replacement and other environmental factors, including soil horizons, soil physicochemical characteristics and seasonality (monsoon vs. dry seasons). We showed that tree composition and variation in soil properties were major drivers for both bacterial and fungal communities, with a significant influence from seasonality. Ectomycorrhizal Operational Taxonomic Units (OTUs) dominated the functional fungal guilds. Species richness and diversity of the bacterial and fungal communities were higher in the pine woodland compared to the primary Lithocarpus forest, although prominent OTUs were different. The slightly lower complexity of the microbiome in the primary forest stands likely resulted from environmental filtering under relatively stable conditions over centuries, when compared to the secondary pine woodlands. In the old-growth forest, we found a higher number of species, but that communities were homogeneously distributed, whereas in the pine woodlands, there is a slightly lower number of species present but the communities are heterogeneously distributed. The present surveys of the bacterial and fungal diversity will serve as references in future studies aiming to assess the impact of the climate change on soil microbial diversity in both old-growth forests and secondary woodlands in Ailaoshan., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zeng, Lebreton, Man, Jia, Wang, Gong, Buée, Wu, Dai, Yang and Martin.)

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., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Environmental DNA contains information on the species interaction networks that support ecosystem functions and services. Next-generation biomonitoring proposes the use of this data to reconstruct ecological networks in real time and then compute network-level properties to assess ecosystem change. We investigated the relevance of this proposal by assessing: (i) the replicability of DNA-based networks in the absence of ecosystem change, and (ii) the benefits and shortcomings of community- and network-level properties for monitoring change. We selected crop-associated microbial networks as a case study because they support disease regulation services in agroecosystems and analysed their response to change in agricultural practice between organic and conventional systems. Using two statistical methods of network inference, we showed that network-level properties, especially β-properties, could detect change. Moreover, consensus networks revealed robust signals of interactions between the most abundant species, which differed between agricultural systems. These findings complemented those obtained with community-level data that showed, in particular, a greater microbial diversity in the organic system. The limitations of network-level data included (i) the very high variability of network replicates within each system; (ii) the low number of network replicates per system, due to the large number of samples needed to build each network; and (iii) the difficulty in interpreting links of inferred networks. Tools and frameworks developed over the last decade to infer and compare microbial networks are therefore relevant to biomonitoring, provided that the DNA metabarcoding data sets are large enough to build many network replicates and progress is made to increase network replicability and interpretation., (© 2020 John Wiley & Sons Ltd.)

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 β-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., (© 2021 John Wiley & Sons Ltd.)

The genus Phytophthora represents a group of plant pathogens with broad global distribution. The majority of them cause the collar and root-rot of diverse plant species. Little is known about Phytophthora communities in forest ecosystems, especially in the Neotropical forests where natural enemies could maintain the huge plant diversity via negative density dependence. We characterized the diversity of soil-borne Phytophthora communities in the North French Guiana rainforest and investigated how they are structured by host identity and environmental factors. In this little-explored habitat, 250 soil cores were sampled from 10 plots hosting 10 different plant families across three forest environments (Terra Firme, Seasonally Flooded and White Sand). Phytophthora diversity was studied using a baiting approach and metabarcoding (High-Throughput Sequencing) on environmental DNA extracted from both soil samples and baiting-leaves. These three approaches revealed very similar communities, characterized by an unexpected low diversity of Phytophthora species, with the dominance of two cryptic species close to Phytophthora heveae. As expected, the Phytophthora community composition of the French Guiana rainforest was significantly impacted by the host plant family and environment. However, these plant pathogen communities are very small and are dominated by generalist species, questioning their potential roles as drivers of plant diversity in these Amazonian forests., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Mycorrhizal fungi are mutualists that play crucial roles in nutrient acquisition in terrestrial ecosystems. Mycorrhizal symbioses arose repeatedly across multiple lineages of Mucoromycotina, Ascomycota, and Basidiomycota. Considerable variation exists in the capacity of mycorrhizal fungi to acquire carbon from soil organic matter. Here, we present a combined analysis of 135 fungal genomes from 73 saprotrophic, endophytic and pathogenic species, and 62 mycorrhizal species, including 29 new mycorrhizal genomes. This study samples ecologically dominant fungal guilds for which there were previously no symbiotic genomes available, including ectomycorrhizal Russulales, Thelephorales and Cantharellales. Our analyses show that transitions from saprotrophy to symbiosis involve (1) widespread losses of degrading enzymes acting on lignin and cellulose, (2) co-option of genes present in saprotrophic ancestors to fulfill new symbiotic functions, (3) diversification of novel, lineage-specific symbiosis-induced genes, (4) proliferation of transposable elements and (5) divergent genetic innovations underlying the convergent origins of the ectomycorrhizal guild.

Fire is a major disturbance linked to the evolutionary history and climate of Mediterranean ecosystems, where the vegetation has evolved fire-adaptive traits (e.g., serotiny in pines). In Mediterranean forests, mutualistic feedbacks between trees and ectomycorrhizal (ECM) fungi, essential for ecosystem dynamics, might be shaped by recurrent fires. We tested how the structure and function of ECM fungal communities of Pinus pinaster and Pinus halepensis vary among populations subjected to high and low fire recurrence in Mediterranean ecosystems, and analysed the relative contribution of environmental (climate, soil properties) and tree-mediated (serotiny) factors. For both pines, local and regional ECM fungal diversity were lower in areas of high than low fire recurrence, although certain fungal species were favoured in the former. A general decline of ECM root-tip enzymatic activity for P. pinaster was associated with high fire recurrence, but not for P. halepensis. Fire recurrence and fire-related factors such as climate, soil properties or tree phenotype explained these results. In addition to the main influence of climate, the tree fire-adaptive trait serotiny recovered a great portion of the variation in structure and function of ECM fungal communities associated with fire recurrence. Edaphic conditions (especially pH, tightly linked to bedrock type) were an important driver shaping ECM fungal communities, but mainly at the local scale and probably independently of the fire recurrence. Our results show that ECM fungal community shifts are associated with fire recurrence in fire-prone dry Mediterranean forests, and reveal complex feedbacks among trees, mutualistic fungi and the surrounding environment in these ecosystems., (© 2020 John Wiley & Sons Ltd.)

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.

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., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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.

Sequencing of a high number of fungal genomes has become possible due to the development of next generation sequencing techniques (NGS). The most recent developments aim to sequence single-molecule long-reads in order to improve genome assemblies, but consequently needs higher quality (minimum >20 kbp) DNA as starting material. However, environmental-derived samples from soil, wood, or litter often contain phenolic compounds, pigments, and other molecules that can be inhibitors for reactions during sequencing library construction. In this chapter, we propose an optimized protocol allowing the preparation of high quality and long fragment DNA from different samples (mycelium, fruiting body, soil) compatible with the current sequencing requirements.

Fungi provide relevant ecosystem services contributing to primary productivity and the cycling of nutrients in forests. These fungal inputs can be decisive for the resilience of Mediterranean forests under global change scenarios, making necessary an in-deep knowledge about how fungal communities operate in these ecosystems. By using high-throughput sequencing and enzymatic approaches, we studied the fungal communities associated with three genotypic variants of Pinus pinaster trees, in 45-year-old common garden plantations. We aimed to determine the impact of biotic (i.e., tree genotype) and abiotic (i.e., season, site) factors on the fungal community structure, and to explore whether structural shifts triggered functional responses affecting relevant ecosystem processes. Tree genotype and spatial-temporal factors were pivotal structuring fungal communities, mainly by influencing their assemblage and selecting certain fungi. Diversity variations of total fungal community and of that of specific fungal guilds, together with edaphic properties and tree's productivity, explained relevant ecosystem services such as processes involved in carbon turnover and phosphorous mobilization. A mechanistic model integrating relations of these variables and ecosystem functional outcomes is provided. Our results highlight the importance of structural shifts in fungal communities because they may have functional consequences for key ecosystem processes in Mediterranean forests., (© 2017 Society for Applied Microbiology and John Wiley and Sons Ltd.)