Comparative genomics and transcriptomics depict ericoid mycorrhizal fungi as versatile saprotrophs and plant mutualists.

Some soil fungi in the Leotiomycetes form ericoid mycorrhizal (ERM) symbioses with Ericaceae. In the harsh habitats in which they occur, ERM plant survival relies on nutrient mobilization from soil organic matter (SOM) by their fungal partners. The characterization of the fungal genetic machinery underpinning both the symbiotic lifestyle and SOM degradation is needed to understand ERM symbiosis functioning and evolution, and its impact on soil carbon (C) turnover. We sequenced the genomes of the ERM fungi Meliniomyces bicolor, M. variabilis, Oidiodendron maius and Rhizoscyphus ericae, and compared their gene repertoires with those of fungi with different lifestyles (ecto- and orchid mycorrhiza, endophytes, saprotrophs, pathogens). We also identified fungal transcripts induced in symbiosis. The ERM fungal gene contents for polysaccharide-degrading enzymes, lipases, proteases and enzymes involved in secondary metabolism are closer to those of saprotrophs and pathogens than to those of ectomycorrhizal symbionts. The fungal genes most highly upregulated in symbiosis are those coding for fungal and plant cell wall-degrading enzymes (CWDEs), lipases, proteases, transporters and mycorrhiza-induced small secreted proteins (MiSSPs). The ERM fungal gene repertoire reveals a capacity for a dual saprotrophic and biotrophic lifestyle. This may reflect an incomplete transition from saprotrophy to the mycorrhizal habit, or a versatile life strategy similar to fungal endophytes.

A plant growth-promoting symbiosis between Mycena galopus and Vaccinium corymbosum seedlings.

Typically, Mycena species are viewed as saprotrophic fungi. However, numerous detections of Mycena spp. in the roots of green plants suggest that a continuum from saprotrophy to biotrophy could exist. In particular, mycenoid species have repeatedly been found in Ericaceae plant roots. Our study asked whether (1) Mycena species are commonly found in the roots of green Ericaceae plants; (2) Mycena sequences are limited to a single group/lineage within the genus; and (3) a Mycena sp. can behave as a beneficial root associate with a typical ericoid mycorrhizal plant (Vaccinium corymbosum), regardless of how much external labile carbon is available. We detected Mycena sequences in roots of all sampled Ericaceae plants. Our Mycena sequences clustered in four different groups distributed across the Mycena genus. Only one group could be assigned with confidence to a named species (M. galopus). Our Mycena sequences clustered with other Mycena sequences detected in roots of ericoid mycorrhizal plant species collected throughout Europe, America, and Australia. An isolate of M. galopus promoted growth of V. corymbosum seedlings in vitro regardless of external carbon supply in the media. Seedlings inoculated with M. galopus grew as well as those inoculated with the ericoid mycorrhizal fungus Rhizoscyphus ericae. Surprisingly, this M. galopus isolate colonized Vaccinium roots and formed distinctive peg-like structures. Our results suggest that Mycena species might operate along a saprotroph-symbiotic continuum with a range of ericoid mycorrhizal plant species. We discuss our results in terms of fungal partner recruitment by Ericaceae plants.

Metaxa: a software tool for automated detection and discrimination among ribosomal small subunit (12S/16S/18S) sequences of archaea, bacteria, eukaryotes, mitochondria, and chloroplasts in metagenomes and environmental sequencing datasets.

The ribosomal small subunit (SSU) rRNA gene has emerged as an important genetic marker for taxonomic identification in environmental sequencing datasets. In addition to being present in the nucleus of eukaryotes and the core genome of prokaryotes, the gene is also found in the mitochondria of eukaryotes and in the chloroplasts of photosynthetic eukaryotes. These three sets of genes are conceptually paralogous and should in most situations not be aligned and analyzed jointly. To identify the origin of SSU sequences in complex sequence datasets has hitherto been a time-consuming and largely manual undertaking. However, the present study introduces Metaxa ( http://microbiology.se/software/metaxa/ ), an automated software tool to extract full-length and partial SSU sequences from larger sequence datasets and assign them to an archaeal, bacterial, nuclear eukaryote, mitochondrial, or chloroplast origin. Using data from reference databases and from full-length organelle and organism genomes, we show that Metaxa detects and scores SSU sequences for origin with very low proportions of false positives and negatives. We believe that this tool will be useful in microbial and evolutionary ecology as well as in metagenomics.

Depth-structuring of multi-kingdom soil communities in agricultural pastures.

The biodiversity and structure of deep agricultural soil communities are poorly understood, especially for eukaryotes. Using DNA metabarcoding and co-occurrence networks, we tested whether prokaryote, fungal, protist, and nematode biodiversity declines with increasing depth (0-0.1,  0.3-0.5,  and 1.1-1.7m) in pastoral soil; whether deep soil organisms are subsets of those at the surface; and whether multi-kingdom networks become more interconnected with increasing depth. Depth-related richness declines were observed for almost all detected fungal classes, protist phyla, and nematode orders, but only 13 of 25 prokaryote phyla, of which nine had increasing richness with depth. Deep soil communities were not simply subsets of surface communities, with 3.8%-12.2% of eukaryotes and 13.2% of prokaryotes detected only in the deepest samples. Eukaryotes mainly occurred in the upper soil layers whereas prokaryotes were more evenly distributed across depths. Plant-feeding nematodes were most abundant in top soil, whereas bacteria feeders were more abundant in deep soil. Co-occurrence network structure differences suggested that deep soil communities are concentrated around scarce niches of resource availability, in contrast to more spatially homogenous and abundant resources at the surface. Together, these results demonstrate effects of depth on the composition, distribution, and structure of prokaryote and eukaryote soil communities.

Competition and community succession link N transformation and greenhouse gas emissions in urine patches.

Nitrous oxide (N2O) is a strong greenhouse gas produced by biotic/abiotic processes directly linked to both fungal and prokaryotic communities that produce, consume or create conditions leading to its emission. In soils exposed to nitrogen (N) in the form of urea, an ecological succession is triggered resulting in a dynamic turnover of microbial populations. However, knowledge of the mechanisms controlling this succession and the repercussions for N2O emissions remain incomplete. Here, we monitored N2O production and fungal/prokaryotic community changes (via 16S and 18S amplicon sequencing) in soil microcosms exposed to urea. Contributions of microbes to emissions were determined using biological inhibitors. Results confirmed that urea leads to shifts in microbial community assemblages by selecting for certain microbial groups (fast growers) as dictated through life history strategies. Urea reduced overall community diversity by conferring dominance to specific groups at different stages in the succession. The diversity lost under urea was recovered with inhibitor addition through the removal of groups that were actively growing under urea indicating that species replacement is mediated in part by competition. Results also identified fungi as significant contributors to N2O emissions, and demonstrate that dominant fungal populations are consistently replaced at different stages of the succession. These successions were affected by addition of inhibitors which resulted in strong decreases in N2O emissions, suggesting that fungal contributions to N2O emissions are larger than that of prokaryotes.

Soil Nitrogen Treatment Alters Microbiome Networks Across Farm Niches.

Agriculture is fundamental for food production, and microbiomes support agriculture through multiple essential ecosystem services. Despite the importance of individual (i.e., niche specific) agricultural microbiomes, microbiome interactions across niches are not well-understood. To observe the linkages between nearby agricultural microbiomes, multiple approaches (16S, 18S, and ITS) were used to inspect a broad coverage of niche microbiomes. Here we examined agricultural microbiome responses to 3 different nitrogen treatments (0, 150, and 300 kg/ha/yr) in soil and tracked linked responses in other neighbouring farm niches (rumen, faecal, white clover leaf, white clover root, rye grass leaf, and rye grass root). Nitrogen treatment had little impact on microbiome structure or composition across niches, but drastically reduced the microbiome network connectivity in soil. Networks of 16S microbiomes were the most sensitive to nitrogen treatment across amplicons, where ITS microbiome networks were the least responsive. Nitrogen enrichment in soil altered soil and the neighbouring microbiome networks, supporting our hypotheses that nitrogen treatment in soil altered microbiomes in soil and in nearby niches. This suggested that agricultural microbiomes across farm niches are ecologically interactive. Therefore, knock-on effects on neighbouring niches should be considered when management is applied to a single agricultural niche.

New insights into the mycorrhizal Rhizoscyphus ericae aggregate: spatial structure and co-colonization of ectomycorrhizal and ericoid roots.

• Fungi in the Rhizoscyphus ericae aggregate have been recovered from the roots of co-occurring ericaceous shrubs and ectomycorrhizal trees. However, to date, there is no evidence that the same individual genotypes colonize both hosts, and no information on the extent of the mycelial networks that might form. • Using spatially explicit core sampling, we isolated fungi from neighbouring Pinus sylvestris (ectomycorrhizal) and Vaccinium vitis-idaea (ericoid mycorrhizal) roots and applied intersimple sequence repeat (ISSR) typing to assess the occurrence and extent of shared genets. • Most isolates were identified as Meliniomyces variabilis, and isolates with identical ISSR profiles were obtained from neighbouring ericoid and ectomycorrhizal roots on a number of occasions. However, genet sizes were small (< 13 cm), and several genets were found in a single soil core. Genetic relatedness was independent of spatial separation at the scales investigated (< 43 m) and M. variabilis populations from sites 20 km apart were genetically indistinguishable. • We conclude that individual genets of M. variabilis can simultaneously colonize Scots pine and Vaccinium roots, but there is no evidence for the formation of large mycelial networks. Our data also suggest significant genotypic overlap between widely separated populations of this ubiquitous root-associated fungus.

Nitrogen storage and remobilization by trees: ecophysiological relevance in a changing world.

The role of carbon (C) and nitrogen (N) storage by trees will be discussed in terms of uncoupling their growth from resource acquisition. There are profound differences between the physiology of C and N storage. C storage acts as a short-term, temporary buffer when photosynthesis cannot meet current sink demand and remobilization is sink driven. However, the majority of C allocated to non-structural carbohydrates such as starch is not reused so is in fact sequestered, not stored. In contrast, N storage is seasonally programmed, closely linked to tree phenology and operates at temporal scales of months to years, with remobilization being source driven. We examine the ecological significance of N storage and remobilization in terms of regulating plant N use efficiency, allowing trees to uncouple seasonal growth from N uptake by roots and allowing recovery from disturbances such as browsing damage. We also briefly consider the importance of N storage and remobilization in regulating how trees will likely respond to rising atmospheric carbon dioxide concentrations. Most studies of N storage and remobilization have been restricted to small trees growing in a controlled environment where (15)N can be used easily as a tracer for mineral N. We highlight the need to describe and quantify these processes for adult trees in situ where most root N uptake occurs via ectomycorrhizal partners, an approach that now appears feasible for deciduous trees through quantification of the flux of remobilized N in their xylem. This opens new possibilities for studying interactions between N and C allocation in trees and associated mycorrhizal partners, which are likely to be crucial in regulating the response of trees to many aspects of global environmental change.

Reciprocal carbon and nitrogen transfer between an ericaceous dwarf shrub and fungi isolated from Piceirhiza bicolorata ectomycorrhizas.

The overstorey coniferous trees and understorey ericaceous dwarf shrubs of northern temperate and boreal forests have previously been considered to form mycorrhizas with taxonomically and functionally distinct groups of fungi. Here, we tested the hypothesis that Meliniomyces variabilis and Meliniomyces bicolor, isolated from Piceirhiza bicolorata ectomycorrhizas of pine, can function as ericoid mycorrhizal symbionts with Vaccinium vitis-idaea. We used split-compartment microcosms to measure the reciprocal exchange of (13)C and (15)N between V. vitis-idaea and three fungal isolates in the Hymenoscyphus ericae aggregate isolated from Scots pine ectomycorrhizas (M. variabilis and M. bicolor) or Vaccinium roots (M. variabilis). The extramatrical fungal mycelium of labelled mycorrhizal plants was significantly enriched in (13)C, and the leaves were significantly enriched in (15)N, compared with nonmycorrhizal and nonlabelled controls. * These findings show for the first time that fungi in the H. ericae aggregate, isolated from pine ectomycorrhizas, can transfer C and N and can thus form functional ericoid mycorrhizas in an understorey ericaceous shrub.

Small genetic differences between ericoid mycorrhizal fungi affect nitrogen uptake by Vaccinium.

Ericoid mycorrhizal fungi have been shown to differ in their pattern of nitrogen (N) use in pure culture. Here, we investigate whether this functional variation is maintained in symbiosis using three ascomycetes from a clade not previously shown to include ericoid mycorrhizal taxa. Vaccinium macrocarpon and Vaccinium vitis-idaea were inoculated with three fungal strains known to form coils in Vaccinium roots, which differed in their patterns of N use in liquid culture. (15)N was used to trace the uptake of -N, -N and glutamine-N into shoots. (15)N transfer differed among the three fungal strains, including two that had identical internal transcribed spacer (ITS) sequences, and was quantitatively related to fungal growth in liquid culture at low carbon availability. These results demonstrate that functional differences among closely related ericoid mycorrhizal fungi are maintained in symbiosis with their hosts, and suggest that N transfer to plant shoots in ericoid mycorrhizas is under fungal control.

Translocation of nitrogen in the xylem of field-grown cherry and poplar trees during remobilization.

Studies of small trees growing in pots have established that individual amino acids or amides are translocated in the xylem sap of a range of tree species following bud burst, as a consequence of nitrogen (N) remobilization from storage. This paper reports the first study of N translocation in the xylem of large, deciduous, field-grown trees during N remobilization in the spring. We applied 15N fertilizer to the soil around 10-year-old Prunus avium L. and Populus trichocharpa Torr. & Gray ex Hook var. Hastata (Dode) A. Henry x Populus balsamifera L. var. Michauxii (Dode) Farwell trees before bud burst to label N taken up by the roots. Recovery of unlabeled N in xylem sap and leaves was used to demonstrate that P. avium remobilizes N in both glutamine (Gln) and asparagine (Asn). Sap concentrations of both amides rose sharply after bud burst, peaking 14 days after bud burst for Gln, and remaining high some 45 days for Asn. There was no 15N enrichment of either amide until 21 days after bud burst. In the Populus trees, nearly all the N was translocated in the sap as Gln, the concentration of which peaked and then declined before the amide was enriched with 15N, 40 days after bud burst. Xylem sap of clonal P. avium trees was sampled at different positions in the crown to assess if the amino acid and amide composition of the sap varied within the crown. Sap was sampled during remobilization (when the concentration of Gln was maximal), at the end of remobilization and at the end of the experiment (68 days after bud burst). Although the date of sampling had a highly significant effect on sap composition, the effect of position of sampling was marginal. The results are discussed in relation to N translocation in adult trees and the possibility of measuring N remobilization by calculating the flux of N translocation in the xylem.

Archaeorhizomycetes: unearthing an ancient class of ubiquitous soil fungi.

Estimates suggest that only one-tenth of the true fungal diversity has been described. Among numerous fungal lineages known only from environmental DNA sequences, Soil Clone Group 1 is the most ubiquitous. These globally distributed fungi may dominate below-ground fungal communities, but their placement in the fungal tree of life has been uncertain. Here, we report cultures of this group and describe the class, Archaeorhizomycetes, phylogenetically placed within subphylum Taphrinomycotina in the Ascomycota. Archaeorhizomycetes comprises hundreds of cryptically reproducing filamentous species that do not form recognizable mycorrhizal structures and have saprotrophic potential, yet are omnipresent in roots and rhizosphere soil and show ecosystem and host root habitat specificity.

Tidying up international nucleotide sequence databases: ecological, geographical and sequence quality annotation of its sequences of mycorrhizal fungi.

Sequence analysis of the ribosomal RNA operon, particularly the internal transcribed spacer (ITS) region, provides a powerful tool for identification of mycorrhizal fungi. The sequence data deposited in the International Nucleotide Sequence Databases (INSD) are, however, unfiltered for quality and are often poorly annotated with metadata. To detect chimeric and low-quality sequences and assign the ectomycorrhizal fungi to phylogenetic lineages, fungal ITS sequences were downloaded from INSD, aligned within family-level groups, and examined through phylogenetic analyses and BLAST searches. By combining the fungal sequence database UNITE and the annotation and search tool PlutoF, we also added metadata from the literature to these accessions. Altogether 35,632 sequences belonged to mycorrhizal fungi or originated from ericoid and orchid mycorrhizal roots. Of these sequences, 677 were considered chimeric and 2,174 of low read quality. Information detailing country of collection, geographical coordinates, interacting taxon and isolation source were supplemented to cover 78.0%, 33.0%, 41.7% and 96.4% of the sequences, respectively. These annotated sequences are publicly available via UNITE (http://unite.ut.ee/) for downstream biogeographic, ecological and taxonomic analyses. In European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena/), the annotated sequences have a special link-out to UNITE. We intend to expand the data annotation to additional genes and all taxonomic groups and functional guilds of fungi.

Environmental change and carbon limitation in trees: a biochemical, ecophysiological and ecosystem appraisal.

As C(3) photosynthesis is not yet CO(2)-saturated, forests offer the possibility of enhanced growth and carbon (C) sequestration with rising atmospheric CO(2). However, at an ecosystem scale, increased photosynthetic rates are not always translated into faster tree growth, and in free air carbon enrichment (FACE) experiments with trees, the stimulation in above-ground growth often declines with time. So is tree growth C-limited? The evidence is reviewed here at three different scales. First, at the biochemical scale, the role of Rubisco is discussed by considering its evolution and role as a nitrogen (N) storage protein. Second, at the ecophysiological scale, C allocation to gain nutrients from the soil is considered and it is argued that any C limitation is only through a limitation to soil nutrient cycling. Finally, the response of forest ecosystems to rising atmospheric CO(2) concentrations is considered and evidence from FACE experiments is discussed. From the three lines of evidence we conclude that the growth of trees is not C-limited, with the key to understanding future responses to climate change being turnover of soil organic matter and nutrient cycling.

Carbon availability affects nitrogen source utilisation by Hymenoscyphus ericae.

We compared the ability of five strains of the ericoid mycorrhizal fungus Hymenoscyphus ericae to utilise glutamine, ammonium or nitrate at high or low carbon (C) availability. The pattern of intraspecific variation in growth was affected by C availability. When C supply was high, growth differences between strains were explained by the total amount of nitrogen (N) taken up, suggesting variation in uptake kinetics. Under C-limiting conditions, strain differences were linked with their nitrogen use efficiency, implying intraspecific differences in N metabolism. The relationship between growth on glutamine and pH shifts in the media indicated that there was intraspecific variation in glutamine transporters. In addition, the correlation between pH changes and the amount of glutamine-N recovered as ammonium in the media indicated that there were intraspecific variations within the enzymatic pathways involved in glutamine metabolism. Our findings, compared with those of a previous study involving the same ericoid strains, draw attention to the temporal variation in nitrogen source utilisation by ericoid mycorrhizal fungi when maintained in axenic culture.