A growing demand for renewable carbon (C) has led to intensified forest management resulting in the use of forest residues (e.g. canopy, bark or litter layer) as energy sources with potential ...modifications of soil properties and tree productivity. Because microbes mediate the recycling of C and nutrients sequestered in organic matter, we investigated the effects of organic matter (OM) removal on soil properties, root surfaces, microbial functions and abundance using a new observational forest network. We compared leaf litter and logging residue removal plots to reference plots in six beech forests located in the northern half of France. After three consecutive years of OM removal, C and nitrogen (N) pools were not affected, but OM exportation decreased the cation exchange capacity and available phosphorus (P) pool by respectively 12% and 30% in the topsoil (0–5 cm depth). Fine root surface area significantly increased by 21% in the subsoil (5–10 cm depth) in response to OM removal. Enzymatic activities involved in N and P mobilisation decreased from 12 to 38% with the manipulation of OM. Community-level profiling (CLPP) based on BIOLOG approach revealed that the metabolic potential of the microbial community strongly decreased in response to OM removal. Our results indicated that intensive forestry could affect microbial functions implicated in nutrients mobilisation. We demonstrated that soil organic matter (SOM) content is a predictor of microbial functions resistance to forest residue removal. We recommend that intensive forestry should be reduced or limited in beech forests characterised by low soil OM contents.
•Soil microbial functions change in the short term in response to organic matter removal.•Organic matter removal leads to a decrease in the microbial activities involved in nitrogen and phosphorus mobilisation.•The initial soil organic matter content is a predictor of microbial function sensitivity to organic matter removal.
The screening of a metagenomic library of 250,000 clones generated from a hypersaline soil (Spain) allowed us to identify a single positive clone which confers the ability to degrade N-acyl ...homoserine lactones (AHLs). The sequencing of the fosmid revealed a 42,318 bp environmental insert characterized by 46 ORFs. The subcloning of these ORFs demonstrated that a single gene (hqiA) allowed AHL degradation. Enzymatic analysis using purified HqiA and HPLC/MS revealed that this protein has lactonase activity on a broad range of AHLs. The introduction of hqiA in the plant pathogen Pectobacterium carotovorum efficiently interfered with both the synthesis of AHLs and quorum-sensing regulated functions, such as swarming motility and the production of maceration enzymes. Bioinformatic analyses highlighted that HqiA showed no sequence homology with the known prototypic AHL lactonases or acylases, thus expanding the AHL-degrading enzymes with a new family related to the cysteine hydrolase (CHase) group. The complete sequence analysis of the fosmid showed that 31 ORFs out of the 46 identified were related to Deltaproteobacteria, whilst many intercalated ORFs presented high homology with other taxa. In this sense, hqiA appeared to be assigned to the Hyphomonas genus (Alphaproteobacteria), suggesting that horizontal gene transfer had occurred.
Fungal communities associated with plants and soil influence plant fitness and ecosystem functioning. They are frequently studied by metabarcoding approaches targeting the ribosomal internal ...transcribed spacer (ITS), but there is no consensus concerning the most appropriate bioinformatic approach for the analysis of these data. We sequenced an artificial fungal community composed of 189 strains covering a wide range of Ascomycota and Basidiomycota, to compare the performance of 360 software and parameter combinations. The most sensitive approaches, based on the USEARCH and VSEARCH clustering algorithms, detected almost all fungal strains but greatly overestimated the total number of strains. By contrast, approaches using DADA2 to detect amplicon sequence variants were the most effective for recovering the richness and composition of the fungal community. Our results suggest that analyzing single forward (R1) sequences with DADA2 and no filter other than the removal of low-quality and chimeric sequences is a good option for fungal community characterization.
•A mock community of 189 fungal strains was used to compare 360 pipelines.•Observed fungal richness depended strongly on the bioinformatic approach.•Community richness and composition were well recovered by DADA2.•Single forward (R1) reads often gave better results than assembled reads.•Bioinformatic approach selection should be based on study goals.
The access and recycling of the base cations are essential processes for the long-lasting functioning of forest ecosystems. While the role of soil bacterial communities has been demonstrated in ...mineral weathering and tree nutrition, our understanding of the link between the availability of base cations and the functioning of these communities remains limited. To fill this gap, we developed a microcosm approach to investigate how an increase in key base cations (potassium or magnesium) impacted the taxonomic and functional structures of the bacterial communities. During a 2-month period after fertilization with available potassium or magnesium, soil properties, global functions (metabolic potentials and respiration) as well as mineral weathering bioassays and 16S rRNA amplicon pyrosequencing were monitored. Our analyses showed no or small variations in the taxonomic structure, total densities and global functions between the treatments. In contrast, a decrease in the frequency and effectiveness of mineral weathering bacteria was observed in the fertilized treatments. Notably, quantitative PCR targeting specific genera known for their mineral weathering ability (i.e., Burkholderia and Collimonas) confirmed this decrease. These new results suggest that K and Mg cation availability drives the distribution of the mineral weathering bacterial communities in forest soil.
Extraction of high-quality, high molecular weight DNA is a critical step for sequencing an organism's genome. For fungi, DNA extraction is often complicated by co-precipitation of secondary ...metabolites, the most destructive being polysaccharides, polyphenols, and melanin. Different DNA extraction protocols and clean-up methods have been developed to address challenging materials and contaminants; however, the method of fungal cultivation and tissue preparation also plays a critical role to limit the production of inhibitory compounds prior to extraction. Here, we provide protocols and guidelines for (i) fungal tissue cultivation and processing with solid media containing a cellophane overlay or in liquid media, (ii) DNA extraction with customized recommendations for taxonomically and ecologically diverse plant-associated fungi, and (iii) assessing DNA quantity and quality for downstream genome sequencing with single-molecule technology such as PacBio.
In soils characterized by nutrient-poor conditions trees have developed strategies to maximize the exploration of the environment through their root system. Notably, in shallow soils, trees adopt a ...deep-rooting strategy to access appropriate levels of water and nutrients from the bedrock. Despite the critical importance of microorganisms in nutrient access in topsoil, understanding their involvement in subsoil was rarely addressed. Our study provides the first comprehensive picture of the bacterial communities colonizing deep roots at the bedrock interface. Particularly, we aimed at deciphering if the subsoil edaphic conditions allowed the enrichment of specific bacterial communities in the rhizosphere. To answer such questioning, we focused on a shallow soil dominated by deep-rooting beech trees (Fagus sylvatica). The taxonomic and functional structures of bacterial communities were investigated through 16S rRNA-pyrosequencing analyses and in vitro bioassays on culturable representatives isolated from the saprolite, the limestone rocks and the roots penetrating those two compartments at the bedrock interface. Our taxonomic analyses revealed a rhizosphere effect, with no difference between the limestone- and saprolite-rhizosphere bacterial communities. Notably, our functional assays highlighted a significant enrichment of bacteria effective at mineral weathering in the limestone-rhizosphere compared to the surrounding environment, whereas organic matter decomposing bacteria were exclusively enriched in the saprolite-rhizosphere. Altogether our results suggest that tree roots select specific bacterial communities in subsoil as potential allies to improve nutrient availability and tree nutrition.
•Bacterial communities from the subsurface critical zone were studied.•Saprolite, limestone bedrock and root interfaces were collected at 2 m depth.•Deep roots were colonized by specific bacterial communities.•Limestone bedrock and saprolite roots supported different bacterial functions.•Deep environmental conditions can affect the rhizosphere bacterial selection.
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.
Abstract
The mutualistic ectomycorrhizal (ECM) fungal genus Pisolithus comprises 19 species defined to date which colonize the roots of >50 hosts worldwide suggesting that substantial genomic and ...functional evolution occurred during speciation. To better understand this intra-genus variation, we undertook a comparative multi-omic study of nine Pisolithus species sampled from North America, South America, Asia, and Australasia. We found that there was a small core set of genes common to all species (13%), and that these genes were more likely to be significantly regulated during symbiosis with a host than accessory or species-specific genes. Thus, the genetic “toolbox” foundational to the symbiotic lifestyle in this genus is small. Transposable elements were located significantly closer to gene classes including effector-like small secreted proteins (SSPs). Poorly conserved SSPs were more likely to be induced by symbiosis, suggesting that they may be a class of protein that tune host specificity. The Pisolithus gene repertoire is characterized by divergent CAZyme profiles when compared with other fungi, both symbiotic and saprotrophic. This was driven by differences in enzymes associated with symbiotic sugar processing, although metabolomic analysis suggest that neither copy number nor expression of these genes is sufficient to predict sugar capture from a host plant or its metabolism in fungal hyphae. Our results demonstrate that intra-genus genomic and functional diversity within ECM fungi is greater than previously thought, underlining the importance of continued comparative studies within the fungal tree of life to refine our focus on pathways and evolutionary processes foundational to this symbiotic lifestyle.
Summary
Ectomycorrhizas are an intrinsic component of tree nutrition and responses to environmental variations. How epigenetic mechanisms might regulate these mutualistic interactions is unknown.
By ...manipulating the level of expression of the chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1) and two demethylases DEMETER‐LIKE (DML) in Populus tremula × Populus alba lines, we examined how host DNA methylation modulates multiple parameters of the responses to root colonization with the mutualistic fungus Laccaria bicolor. We compared the ectomycorrhizas formed between transgenic and wild‐type (WT) trees and analyzed their methylomes and transcriptomes.
The poplar lines displaying lower mycorrhiza formation rate corresponded to hypomethylated overexpressing DML or RNAi‐ddm1 lines. We found 86 genes and 288 transposable elements (TEs) differentially methylated between WT and hypomethylated lines (common to both OX‐dml and RNAi‐ddm1) and 120 genes/1441 TEs in the fungal genome suggesting a host‐induced remodeling of the fungal methylome. Hypomethylated poplar lines displayed 205 differentially expressed genes (cis and trans effects) in common with 17 being differentially methylated (cis).
Our findings suggest a central role of host and fungal DNA methylation in the ability to form ectomycorrhizas including not only poplar genes involved in root initiation, ethylene and jasmonate‐mediated pathways, and immune response but also terpenoid metabolism.
See also the Commentary on this article by Zimmermann & Gaillard, 238: 2259–2260.
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