Pervasive associations between dark septate endophytic fungi with tree root and soil microbiomes across Europe.

Trees interact with a multitude of microbes through their roots and root symbionts such as mycorrhizal fungi and root endophytes. Here, we explore the role of fungal root symbionts as predictors of the soil and root-associated microbiomes of widespread broad-leaved trees across a European latitudinal gradient. Our results suggest that, alongside factors such as climate, soil, and vegetation properties, root colonization by ectomycorrhizal, arbuscular mycorrhizal, and dark septate endophytic fungi also shapes tree-associated microbiomes. Notably, the structure of root and soil microbiomes across our sites is more strongly and consistently associated with dark septate endophyte colonization than with mycorrhizal colonization and many abiotic factors. Root colonization by dark septate endophytes also has a consistent negative association with the relative abundance and diversity of nutrient cycling genes. Our study not only indicates that root-symbiotic interactions are an important factor structuring soil communities and functions in forest ecosystems, but also that the hitherto less studied dark septate endophytes are likely to be central players in these interactions.

Metabarcoding of soil environmental DNA to estimate plant diversity globally.

Introduction: Traditional approaches to collecting large-scale biodiversity data pose huge logistical and technical challenges. We aimed to assess how a comparatively simple method based on sequencing environmental DNA (eDNA) characterises global variation in plant diversity and community composition compared with data derived from traditional plant inventory methods. Methods: We sequenced a short fragment (P6 loop) of the chloroplast trnL intron from from 325 globally distributed soil samples and compared estimates of diversity and composition with those derived from traditional sources based on empirical (GBIF) or extrapolated plant distribution and diversity data. Results: Large-scale plant diversity and community composition patterns revealed by sequencing eDNA were broadly in accordance with those derived from traditional sources. The success of the eDNA taxonomy assignment, and the overlap of taxon lists between eDNA and GBIF, was greatest at moderate to high latitudes of the northern hemisphere. On average, around half (mean: 51.5% SD 17.6) of local GBIF records were represented in eDNA databases at the species level, depending on the geographic region. Discussion: eDNA trnL gene sequencing data accurately represent global patterns in plant diversity and composition and thus can provide a basis for large-scale vegetation studies. Important experimental considerations for plant eDNA studies include using a sampling volume and design to maximise the number of taxa detected and optimising the sequencing depth. However, increasing the coverage of reference sequence databases would yield the most significant improvements in the accuracy of taxonomic assignments made using the P6 loop of the trnL region.

Global diversity and distribution of nitrogen-fixing bacteria in the soil.

Our knowledge of microbial biogeography has advanced in recent years, yet we lack knowledge of the global diversity of some important functional groups. Here, we used environmental DNA from 327 globally collected soil samples to investigate the biodiversity patterns of nitrogen-fixing bacteria by focusing on the nifH gene but also amplifying the general prokaryotic 16S SSU region. Globally, N-fixing prokaryotic communities are driven mainly by climatic conditions, with most groups being positively correlated with stable hot or seasonally humid climates. Among soil parameters, pH, but also soil N content were most often shown to correlate with the diversity of N-fixer groups. However, specific groups of N-fixing prokaryotes show contrasting responses to the same variables, notably in Cyanobacteria that were negatively correlated with stable hot climates, and showed a U-shaped correlation with soil pH, contrary to other N-fixers. Also, the non-N-fixing prokaryotic community composition was differentially correlated with the diversity and abundance of N-fixer groups, showing the often-neglected impact of biotic interactions among bacteria.

Do commercial arbuscular mycorrhizal inoculants contain the species that they claim?

The use of arbuscular mycorrhizal (AM) fungal inoculants as a means to promote plant growth is gaining momentum worldwide. Although there is an increasing number of commercial products available for various applications, the quality of these remains uncertain. We determined the AM fungal species composition in eleven inoculants from four producers by using DNA metabarcoding and compared them to the AM fungal species declared on the product labels. Our DNA metabarcoding of the inoculants revealed a concerning discrepancy between the declared and detected AM fungal species compositions of the products. While nine products contained at least one declared species, two did not contain any matching species and all inoculants but one contained additional species not declared on the product label. These findings highlight the need for better guidelines and industry standards to ensure consumer protection in the AM fungal inoculum market. Additionally, we call for caution when using commercial AM fungal inoculants in scientific experiments without confirmatory information about their species composition.

Dominance, diversity, and niche breadth in arbuscular mycorrhizal fungal communities.

Classical theory identifies resource competition as the major structuring force of biotic communities and predicts that (i) levels of dominance and richness in communities are inversely related, (ii) narrow niches allow dense "packing" in niche space and thus promote diversity, and (iii) dominants are generalists with wide niches, such that locally abundant taxa also exhibit wide distributions. Current empirical support, however, is mixed. We tested these expectations using published data on arbuscular mycorrhizal (AM) fungal community composition worldwide. We recorded the expected negative relationship between dominance and richness and, to a degree, the positive association between local and global dominance. However, contrary to expectations, dominance was pronounced in communities where more specialists were present and, conversely, richness was higher in communities with more generalists. Thus, resource competition and niche packing appear to be of limited importance in AM fungal community assembly; rather, patterns of dominance and diversity seem more consistent with habitat filtering and stochastic processes.

Global taxonomic and phylogenetic assembly of AM fungi.

Arbuscular mycorrhizal (AM) fungi are a ubiquitous group of plant symbionts, yet processes underlying their global assembly - in particular the roles of dispersal limitation and historical drivers - remain poorly understood. Because earlier studies have reported niche conservatism in AM fungi, we hypothesized that variation in taxonomic community composition (i.e., unweighted by taxon relatedness) should resemble variation in phylogenetic community composition (i.e., weighted by taxon relatedness) which reflects ancestral adaptations to historical habitat gradients. Because of the presumed strong dispersal ability of AM fungi, we also anticipated that the large-scale structure of AM fungal communities would track environmental conditions without regional discontinuity. We used recently published AM fungal sequence data (small-subunit ribosomal RNA gene) from soil samples collected worldwide to reconstruct global patterns in taxonomic and phylogenetic community variation. The taxonomic structure of AM fungal communities was primarily driven by habitat conditions, with limited regional differentiation, and there were two well-supported clusters of communities - occurring in cold and warm conditions. Phylogenetic structure was driven by the same factors, though all relationships were markedly weaker. This suggests that niche conservatism with respect to habitat associations is weakly expressed in AM fungal communities. We conclude that the composition of AM fungal communities tracks major climatic and edaphic gradients, with the effects of dispersal limitation and historic factors considerably less apparent than those of climate and soil.

Diversity of arbuscular mycorrhizal fungi and its chemical drivers across dryland habitats.

Qatar is largely characterized by a hyper-arid climate and low soil fertility which create a stressful soil environment for arbuscular mycorrhizal (AM) fungi. In a study of AM fungal communities and their relationship with soil chemical characteristics, we used a high-throughput sequencing technique to explore AM fungal diversity and community composition in different habitats across Qatar. We identified a total of 79 AM fungal taxa, over 77% of which were species from the Glomeraceae family. The lowest AM fungal diversity was observed in saltmarsh and in one rawdha site, while the highest richness, effective number of species, and diversity were observed in rawdha and sabkha communities. NMDS and multiple regression analyses showed that AM fungi were negatively correlated with soil pH and TC, but positively correlated with K and NO3-. AM fungi also were positively correlated with Cd, with the latter suggesting that very low levels of heavy metals may not always be harmful to AM fungi. These findings provide baseline information on AM fungal assemblages and the chemical drivers of diversity across communities in Qatar. This work partly compensates for the current lack of broad-scale studies in the Arabian Peninsula by providing understanding of overall patterns of AM fungi and their drivers in the region.

Impacts of elevated atmospheric CO2 on arbuscular mycorrhizal fungi and their role in moderating plant allometric partitioning.

Elevated atmospheric CO2 concentration (eCO2) effects on plants depend on several factors including plant photosynthetic physiology (e.g. C3, C4), soil nutrient availability and plants' co-evolved soil-dwelling fungal symbionts, namely arbuscular mycorrhizal (AM) fungi. Complicated interactions among these components will determine the outcomes for plants. Therefore, clearer understanding is needed of how plant growth and nutrient uptake, along with root-colonising AM fungal communities, are simultaneously impacted by eCO2. We conducted a factorial growth chamber experiment with a C3 and a C4 grass species (± AM fungi and ± eCO2). We found that eCO2 increased plant biomass allocation towards the roots, but only in plants without AM fungi, potentially associated with an eCO2-driven increase in plant nutrient requirements. Furthermore, our data suggest a difference in the identities of root-colonising fungal taxa between ambient CO2 and eCO2 treatments, particularly in the C4 grass species, although this was not statistically significant. As AM fungi are ubiquitous partners of grasses, their response to increasing atmospheric CO2 is likely to have important consequences for how grassland ecosystems respond to global change.

Arbuscular Mycorrhizal Fungal Communities in the Soils of Desert Habitats.

Deserts cover a significant proportion of the Earth's surface and continue to expand as a consequence of climate change. Mutualistic arbuscular mycorrhizal (AM) fungi are functionally important plant root symbionts, and may be particularly important in drought stressed systems such as deserts. Here we provide a first molecular characterization of the AM fungi occurring in several desert ecosystems worldwide. We sequenced AM fungal DNA from soil samples collected from deserts in six different regions of the globe using the primer pair WANDA-AML2 with Illumina MiSeq. We recorded altogether 50 AM fungal phylotypes. Glomeraceae was the most common family, while Claroideoglomeraceae, Diversisporaceae and Acaulosporaceae were represented with lower frequency and abundance. The most diverse site, with 35 virtual taxa (VT), was in the Israeli Negev desert. Sites representing harsh conditions yielded relatively few reads and low richness estimates, for example, a Saudi Arabian desert site where only three Diversispora VT were recorded. The AM fungal taxa recorded in the desert soils are mostly geographically and ecologically widespread. However, in four sites out of six, communities comprised more desert-affiliated taxa (according to the MaarjAM database) than expected at random. AM fungal VT present in samples were phylogenetically clustered compared with the global taxon pool, suggesting that nonrandom assembly processes, notably habitat filtering, may have shaped desert fungal assemblages.

Temperature and pH define the realised niche space of arbuscular mycorrhizal fungi.

The arbuscular mycorrhizal (AM) fungi are a globally distributed group of soil organisms that play critical roles in ecosystem function. However, the ecological niches of individual AM fungal taxa are poorly understood. We collected > 300 soil samples from natural ecosystems worldwide and modelled the realised niches of AM fungal virtual taxa (VT; approximately species-level phylogroups). We found that environmental and spatial variables jointly explained VT distribution worldwide, with temperature and pH being the most important abiotic drivers, and spatial effects generally occurring at local to regional scales. While dispersal limitation could explain some variation in VT distribution, VT relative abundance was almost exclusively driven by environmental variables. Several environmental and spatial effects on VT distribution and relative abundance were correlated with phylogeny, indicating that closely related VT exhibit similar niche optima and widths. Major clades within the Glomeraceae exhibited distinct niche optima, Acaulosporaceae generally had niche optima in low pH and low temperature conditions, and Gigasporaceae generally had niche optima in high precipitation conditions. Identification of the realised niche space occupied by individual and phylogenetic groups of soil microbial taxa provides a basis for building detailed hypotheses about how soil communities respond to gradients and manipulation in ecosystems worldwide.

Different wheat cultivars exhibit variable responses to inoculation with arbuscular mycorrhizal fungi from organic and conventional farms.

The present study aimed to investigate the effects of arbuscular mycorrhizal (AM) fungal communities originating from organic and conventional agriculture on wheat growth and yield. Six different spring wheat cultivars released in different years in north and central European countries were considered. We hypothesised that AM fungal inoculum collected from organic agricultural fields would elicit a greater positive growth response than inoculum collected from conventional agricultural fields; and that older cultivars, which were developed under conditions of low fertilizer input, would exhibit overall greater growth responses to the presence of AM fungi, compared with more recent cultivars, and that AM fungal inoculum from conventional fields might have the most beneficial effect on the growth and yield of recent cultivars. The results showed that the overall effects on the growth and yield of spring wheat grown with organic and conventional AM fungal inocula did not differ greatly. However, the inoculation growth response, showing the difference of the effects of organic and conventional inocula, varied between particular wheat cultivars. Inoculation growth response of the cultivar Pikker (released in 1959) was the most positive, while that of the cultivar Arabella (released in 2012) was the most negative. The use of AM fungal inoculum from organic fields resulted in slightly taller plant individuals. Pikker showed relatively higher yield and stronger growth when the organic AM fungal inoculum was used. Arabella exhibited relatively lower yield and weaker growth when the organic inoculum was used. Whether the positive response of Pikker to Estonian organic inoculation reflects adaptation to the locally occurring AM fungal community needs to be established by further studies of the communities of AM fungi colonizing wheat roots.

Host preference and network properties in biotrophic plant-fungal associations.

Analytical methods can offer insights into the structure of biological networks, but mechanisms that determine the structure of these networks remain unclear. We conducted a synthesis based on 111 previously published datasets to assess a range of ecological and evolutionary mechanisms that may influence the plant-associated fungal interaction networks. We calculated the relative host effect on fungal community composition and compared nestedness and modularity among different mycorrhizal types and endophytic fungal guilds. We also assessed how plant-fungal network structure was related to host phylogeny, environmental and sampling properties. Orchid mycorrhizal fungal communities responded most strongly to host identity, but the effect of host was similar among all other fungal guilds. Community nestedness, which did not differ among fungal guilds, declined significantly with increasing mean annual precipitation on a global scale. Orchid and ericoid mycorrhizal fungal communities were more modular than ectomycorrhizal and root endophytic communities, with arbuscular mycorrhizal fungi in an intermediate position. Network properties among a broad suite of plant-associated fungi were largely comparable and generally unrelated to phylogenetic distance among hosts. Instead, network metrics were predominantly affected by sampling and matrix properties, indicating the importance of study design in properly inferring ecological patterns.

Local-scale spatial structure and community composition of orchid mycorrhizal fungi in semi-natural grasslands.

Orchid mycorrhizal (OrM) fungi play a crucial role in the ontogeny of orchids, yet little is known about how the structure of OrM fungal communities varies with space and environmental factors. Previous studies suggest that within orchid patches, the distance to adult orchids may affect the abundance of OrM fungi. Many orchid species grow in species-rich temperate semi-natural grasslands, the persistence of which depends on moderate physical disturbances, such as grazing and mowing. The aim of this study was to test whether the diversity, structure and composition of OrM fungal community are influenced by the orchid patches and management intensity in semi-natural grasslands. We detected putative OrM fungi from 0 to 32 m away from the patches of host orchid species (Orchis militaris and Platanthera chlorantha) in 21 semi-natural calcareous grasslands using pyrosequencing. In addition, we assessed different ecological conditions in semi-natural grasslands but primarily focused on the effect of grazing intensity on OrM fungal communities in soil. We found that investigated orchid species were mostly associated with Ceratobasidiaceae and Tulasnellaceae and, to a lesser extent, with Sebacinales. Of all the examined factors, the intensity of grazing explained the largest proportion of variation in OrM fungal as well as total fungal community composition in soil. Spatial analyses showed limited evidence for spatial clustering of OrM fungi and their dependence on host orchids. Our results indicate that habitat management can shape OrM fungal communities, and the spatial distribution of these fungi appears to be weakly structured outside the orchid patches.

Temporal patterns of orchid mycorrhizal fungi in meadows and forests as revealed by 454 pyrosequencing.

Orchid mycorrhizal (OrM) symbionts play a key role in the growth of orchids, but the temporal variation and habitat partitioning of these fungi in roots and soil remain unclear. Temporal changes in root and rhizosphere fungal communities of Cypripedium calceolus, Neottia ovata and Orchis militaris were studied in meadow and forest habitats over the vegetation period by using 454 pyrosequencing of the full internal transcribed spacer (ITS) region. The community of typical OrM symbionts differed by plant species and habitats. The root fungal community of N. ovata changed significantly in time, but this was not observed in C. calceolus and O. militaris. The rhizosphere community included a low proportion of OrM symbionts that exhibited a slight temporal turnover in meadow habitats but not in forests. Habitat differences in OrM and all fungal associates are largely attributable to the greater proportion of ectomycorrhizal fungi in forests. Temporal changes in OrM fungal communities in roots of certain species indicate selection of suitable fungal species by plants. It remains to be elucidated whether these shifts depend on functional differences inside roots, seasonality, climate or succession.

Towards a unified paradigm for sequence-based identification of fungi.

The nuclear ribosomal internal transcribed spacer (ITS) region is the formal fungal barcode and in most cases the marker of choice for the exploration of fungal diversity in environmental samples. Two problems are particularly acute in the pursuit of satisfactory taxonomic assignment of newly generated ITS sequences: (i) the lack of an inclusive, reliable public reference data set and (ii) the lack of means to refer to fungal species, for which no Latin name is available in a standardized stable way. Here, we report on progress in these regards through further development of the UNITE database (http://unite.ut.ee) for molecular identification of fungi. All fungal species represented by at least two ITS sequences in the international nucleotide sequence databases are now given a unique, stable name of the accession number type (e.g. Hymenoscyphus pseudoalbidus|GU586904|SH133781.05FU), and their taxonomic and ecological annotations were corrected as far as possible through a distributed, third-party annotation effort. We introduce the term 'species hypothesis' (SH) for the taxa discovered in clustering on different similarity thresholds (97-99%). An automatically or manually designated sequence is chosen to represent each such SH. These reference sequences are released (http://unite.ut.ee/repository.php) for use by the scientific community in, for example, local sequence similarity searches and in the QIIME pipeline. The system and the data will be updated automatically as the number of public fungal ITS sequences grows. We invite everybody in the position to improve the annotation or metadata associated with their particular fungal lineages of expertise to do so through the new Web-based sequence management system in UNITE.

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.