Impact of soil pedogenesis on the diversity and composition of fungal communities across the California soil chronosequence of Mendocino.

Understanding how soil pedogenesis affects microbial communities and their in situ activities according to ecosystem functioning is a central issue in soil microbial ecology, as soils represent essential nutrient reservoirs and habitats for the biosphere. To address this question, soil chronosequences developed from a single, shared mineralogical parent material and having the same climate conditions are particularly useful, as they isolate the factor of time from other factors controlling the character of soils. In our study, we considered a natural succession of uplifted marine terraces in Mendocino, CA, ranging from highly fertile in the younger terrace (about 100,000 years old) to infertile in the older terraces (about 300,000 years old). Using ITS amplicon pyrosequencing, we analysed and compared the diversity and composition of the soil fungal communities across the first terraces (T1 to T3), with a specific focus in the forested terraces (T2 and T3) on soil samples collected below trees of the same species (Pinus muricata) and of the same age. While diversity and richness indices were highest in the grassland (youngest) terrace (T1), they were higher in the older forested terrace (T3) compared to the younger forested terrace (T2). Interestingly, the most abundant ectomycorrhizal (ECM) taxa that we found within these fungal communities showed high homology with ITS Sanger sequences obtained previously directly from ECM root tips from trees in the same study site, revealing a relative conservation of ECM diversity over time. Altogether, our results provide new information about the diversity and composition of the fungal communities as well as on the dominant ECM species in the soil chronosequence of Mendocino in relation to soil age and ecosystem development.

A new promising phylogenetic marker to study the diversity of fungal communities: The Glycoside Hydrolase 63 gene.

In molecular ecology, the development of efficient molecular markers for fungi remains an important research domain. Nuclear ribosomal internal transcribed spacer (ITS) region was proposed as universal DNA barcode marker for fungi, but this marker was criticized for Indel-induced alignment problems and its potential lack of phylogenetic resolution. Our main aim was to develop a new phylogenetic gene and a putative functional marker, from single-copy gene, to describe fungal diversity. Thus, we developed a series of primers to amplify a polymorphic region of the Glycoside Hydrolase GH63 gene, encoding exo-acting α-glucosidases, in basidiomycetes. These primers were validated on 125 different fungal genomic DNAs, and GH63 amplification yield was compared with that of already published functional markers targeting genes coding for laccases, N-acetylhexosaminidases, cellobiohydrolases and class II peroxidases. Specific amplicons were recovered for 95% of the fungal species tested, and GH63 amplification success was strikingly higher than rates obtained with other functional genes. We downloaded the GH63 sequences from 483 fungal genomes publicly available at the JGI mycocosm database. GH63 was present in 461 fungal genomes belonging to all phyla, except Microsporidia and Neocallimastigomycota divisions. Moreover, the phylogenetic trees built with both GH63 and Rpb1 protein sequences revealed that GH63 is also a promising phylogenetic marker. Finally, a very high proportion of GH63 proteins was predicted to be secreted. This molecular tool could be a new phylogenetic marker of fungal species as well as potential indicator of functional diversity of basidiomycetes fungal communities in term of secretory capacities.

Specific impacts of beech and Norway spruce on the structure and diversity of the rhizosphere and soil microbial communities.

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.

Soil Parameters Drive the Structure, Diversity and Metabolic Potentials of the Bacterial Communities Across Temperate Beech Forest Soil Sequences.

Soil and climatic conditions as well as land cover and land management have been shown to strongly impact the structure and diversity of the soil bacterial communities. Here, we addressed under a same land cover the potential effect of the edaphic parameters on the soil bacterial communities, excluding potential confounding factors as climate. To do this, we characterized two natural soil sequences occurring in the Montiers experimental site. Spatially distant soil samples were collected below Fagus sylvatica tree stands to assess the effect of soil sequences on the edaphic parameters, as well as the structure and diversity of the bacterial communities. Soil analyses revealed that the two soil sequences were characterized by higher pH and calcium and magnesium contents in the lower plots. Metabolic assays based on Biolog Ecoplates highlighted higher intensity and richness in usable carbon substrates in the lower plots than in the middle and upper plots, although no significant differences occurred in the abundance of bacterial and fungal communities along the soil sequences as assessed using quantitative PCR. Pyrosequencing analysis of 16S ribosomal RNA (rRNA) gene amplicons revealed that Proteobacteria, Acidobacteria and Bacteroidetes were the most abundantly represented phyla. Acidobacteria, Proteobacteria and Chlamydiae were significantly enriched in the most acidic and nutrient-poor soils compared to the Bacteroidetes, which were significantly enriched in the soils presenting the higher pH and nutrient contents. Interestingly, aluminium, nitrogen, calcium, nutrient availability and pH appeared to be the best predictors of the bacterial community structures along the soil sequences.

Functional profiling and distribution of the forest soil bacterial communities along the soil mycorrhizosphere continuum.

An ectomycorrhiza is a multitrophic association between a tree root, an ectomycorrhizal fungus, free-living fungi and the associated bacterial communities. Enzymatic activities of ectomycorrhizal root tips are therefore result of the contribution from different partners of the symbiotic organ. However, the functional potential of the fungus-associated bacterial communities remains unknown. In this study, a collection of 80 bacterial strains randomly selected and isolated from a soil-ectomycorrhiza continuum (oak-Scleroderma citrinum ectomycorrhizas, the ectomycorrhizosphere and the surrounding bulk soil) were characterized. All the bacterial isolates were identified by partial 16S rRNA gene sequences as members of the genera Burkholderia, Collimonas, Dyella, Mesorhizobium, Pseudomonas, Rhizobium and Sphingomonas. The bacterial strains were then assayed for ß-xylosidase, ß-glucosidase, N-acetyl-hexosaminidase, ß-glucuronidase, cellobiohydrolase, phosphomonoesterase, leucine-aminopeptidase and laccase activities, chitin solubilization and auxin production. Using these bioassays, we demonstrated significant differences in the functional distribution of the bacterial communities living in the different compartments of the soil-ectomycorrhiza continuum. The surrounding bulk soil was significantly enriched in bacterial isolates capable of hydrolysing cellobiose and N-acetylglucosamine. In contrast, the ectomycorrhizosphere appeared significantly enriched in bacterial isolates capable of hydrolysing glucopyranoside and chitin. Notably, chitinase and laccase activities were found only in bacterial isolates belonging to the Collimonas and Pseudomonas genera. Overall, the results suggest that the ectomycorrhizal fungi favour specific bacterial communities with contrasting functional characteristics from the surrounding soil.

Fungal soil communities in a young transgenic poplar plantation form a rich reservoir for fungal root communities.

Fungal communities play a key role in ecosystem functioning. However, only little is known about their composition in plant roots and the soil of biomass plantations. The goal of this study was to analyze fungal biodiversity in their belowground habitats and to gain information on the strategies by which ectomycorrhizal (ECM) fungi form colonies. In a 2-year-old plantation, fungal communities in the soil and roots of three different poplar genotypes (Populus × canescens, wildtype and two transgenic lines with suppressed cinnamyl alcohol dehydrogenase activity) were analyzed by 454 pyrosequencing targeting the rDNA internal transcribed spacer 1 (ITS) region. The results were compared with the dynamics of the root-associated ECM community studied by morphotyping/Sanger sequencing in two subsequent years. Fungal species and family richness in the soil were surprisingly high in this simple plantation ecosystem, with 5944 operational taxonomic units (OTUs) and 186 described fungal families. These findings indicate the importance that fungal species are already available for colonization of plant roots (2399 OTUs and 115 families). The transgenic modification of poplar plants had no influence on fungal root or soil communities. Fungal families and OTUs were more evenly distributed in the soil than in roots, probably as a result of soil plowing before the establishment of the plantation. Saprophytic, pathogenic, and endophytic fungi were the dominating groups in soil, whereas ECMs were dominant in roots (87%). Arbuscular mycorrhizal diversity was higher in soil than in roots. Species richness of the root-associated ECM community, which was low compared with ECM fungi detected by 454 analyses, increased after 1 year. This increase was mainly caused by ECM fungal species already traced in the preceding year in roots. This result supports the priority concept that ECMs present on roots have a competitive advantage over soil-localized ECM fungi.

Beech roots are simultaneously colonized by multiple genets of the ectomycorrhizal fungus Laccaria amethystina clustered in two genetic groups.

In this study, we characterize and compare the genetic structure of aboveground and belowground populations of the ectomycorrhizal fungus Laccaria amethystina in an unmanaged mixed beech forest. Fruiting bodies and mycorrhizas of L. amethystina were mapped and collected in four plots in the Swietokrzyskie Mountains (Poland). A total of 563 fruiting bodies and 394 mycorrhizas were successfully genotyped using the rDNA IGS1 (intergenic spacer) and seven simple sequence repeat markers. We identified two different genetic clusters of L. amethystina in all of the plots, suggesting that a process of sympatric isolation may be occurring at a local scale. The proportion of individuals belonging to each cluster was similar among plots aboveground while it significantly differed belowground. Predominance of a given cluster could be explained by distinct host preferences or by priority effects and competition among genets. Both aboveground and belowground populations consisted of many intermingling small genets. Consequently, host trees were simultaneously colonized by many L. amethystina genets that may show different ecophysiological abilities. Our data showed that several genets may last for at least 1 year belowground and sustain into the next season. Ectomycorrhizal species reproducing by means of spores can form highly diverse and persistent belowground genets that may provide the host tree with higher resilience in a changing environment and enhance ecosystem performance.

454 Pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversity.

* Soil fungi play a major role in ecological and biogeochemical processes in forests. Little is known, however, about the structure and richness of different fungal communities and the distribution of functional ecological groups (pathogens, saprobes and symbionts). * Here, we assessed the fungal diversity in six different forest soils using tag-encoded 454 pyrosequencing of the nuclear ribosomal internal transcribed spacer-1 (ITS-1). No less than 166 350 ITS reads were obtained from all samples. In each forest soil sample (4 g), approximately 30 000 reads were recovered, corresponding to around 1000 molecular operational taxonomic units. * Most operational taxonomic units (81%) belonged to the Dikarya subkingdom (Ascomycota and Basidiomycota). Richness, abundance and taxonomic analyses identified the Agaricomycetes as the dominant fungal class. The ITS-1 sequences (73%) analysed corresponded to only 26 taxa. The most abundant operational taxonomic units showed the highest sequence similarity to Ceratobasidium sp., Cryptococcus podzolicus, Lactarius sp. and Scleroderma sp. * This study validates the effectiveness of high-throughput 454 sequencing technology for the survey of soil fungal diversity. The large proportion of unidentified sequences, however, calls for curated sequence databases. The use of pyrosequencing on soil samples will accelerate the study of the spatiotemporal dynamics of fungal communities in forest ecosystems.

Phylogenetic analysis, genomic organization, and expression analysis of multi-copper oxidases in the ectomycorrhizal basidiomycete Laccaria bicolor.

In forest soils, ectomycorrhizal and saprotrophic Agaricales differ in their strategies for carbon acquisition, but share common gene families encoding multi-copper oxidases (MCOs). These enzymes are involved in the oxidation of a variety of soil organic compounds. The MCO gene family of the ectomycorrhizal fungus Laccaria bicolor is composed of 11 genes divided into two distinct subfamilies corresponding to laccases (lcc) sensu stricto (lcc1 to lcc9), sharing a high sequence homology with the coprophilic Coprinopsis cinerea laccase genes, and to ferroxidases (lcc10 and lcc11) that are not present in C. cinerea. The fet3-like ferroxidase genes lcc10 and lcc11 in L. bicolor are each arranged in a mirrored tandem orientation with an ftr gene coding for an iron permease. Unlike C. cinerea, L. bicolor has no sid1/sidA gene for siderophore biosynthesis. Transcript profiling using whole-genome expression arrays and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) revealed that some transcripts were very abundant in ectomycorrhizas (lcc3 and lcc8), in fruiting bodies (lcc7) or in the free-living mycelium grown on agar medium (lcc9 and lcc10), suggesting a specific function of these MCOs. The amino acid composition of the MCO substrate binding sites suggests that L. bicolor MCOs interact with substrates different from those of saprotrophic fungi.

Gene transcription in Lactarius quietus-Quercus petraea ectomycorrhizas from a forest soil.

Extracting fungal mRNA from ectomycorrhizas (ECMs) and forest soil samples for monitoring in situ metabolic activities is a significant challenge when studying the role of ECMs in biogeochemical cycles. A robust, simple, rapid, and effective method was developed for extracting RNA from rhizospheric soil and ECMs by adapting previous grinding and lysis methods. The quality and yield of the extracted RNA were sufficient to be used for reverse transcription. RNA extracted from ECMs of Lactarius quietus in a 100-year-old oak stand was used to construct a cDNA library and sequence expressed sequence tags. The transcripts of many genes involved in primary metabolism and in the degradation of organic matter were found. The transcription levels of four targeted fungal genes (glutamine synthase, a general amino acid transporter, a tyrosinase, and N-acetylhexosaminidase) were measured by quantitative reverse transcription-PCR in ECMs and in the ectomycorrhizospheric soil (the soil surrounding the ECMs containing the extraradical mycelium) in forest samples. On average, levels of gene expression for the L. quietus ECM root tips were similar to those for the extraradical mycelium, although gene expression varied up to 10-fold among the samples. This study demonstrates that gene expression from ECMs and soil can be analyzed. These results provide new perspectives for investigating the role of ectomycorrhizal fungi in the functioning of forest ecosystems.

The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis.

Mycorrhizal symbioses--the union of roots and soil fungi--are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants. Boreal, temperate and montane forests all depend on ectomycorrhizae. Identification of the primary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored. Here we report the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolor (Fig. 1) and highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-megabase genome assembly contains approximately 20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences. We detected unexpected genomic features, most notably a battery of effector-type small secreted proteins (SSPs) with unknown function, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSPs probably have a decisive role in the establishment of the symbiosis. The unexpected observation that the genome of L. bicolor lacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell wall polysaccharides, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus that enables it to grow within both soil and living plant roots. The predicted gene inventory of the L. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availability of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts interact with plants within their ecosystem to perform vital functions in the carbon and nitrogen cycles that are fundamental to sustainable plant productivity.

The pre-symbiotic growth of arbuscular mycorrhizal fungi is induced by a branching factor partially purified from plant root exudates.

Arbuscular mycorrhizal (AM) symbiosis is an association between obligate biotrophic fungi and more than 80% of land plants. During the pre-symbiotic phase, the host plant releases critical metabolites necessary to trigger fungal growth and root colonization. We describe the isolation of a semipurified fraction from exudates of carrot hairy roots, highly active on germinating spores of Gigaspora gigantea, G. rosea, and G. margarita. This fraction, isolated on the basis of its activity on hyphal branching, contains a root factor (one or several molecules) that stimulates, directly or indirectly, G. gigantea nuclear division. We demonstrate the presence of this active factor in root exudates of all mycotrophic plant species tested (eight species) but not in those of nonhost plant species (four species). We negatively tested the hypothesis that it was a flavonoid or a compound synthesized via the flavonoid pathway. We propose that this root factor, yet to be chemically characterized, is a key plant signal for the development of AM fungi.