Search

Control of the potato late blight pathogen Phytophthora infestans relies heavily on chemicals. The fungicide metalaxyl-M (Mefenoxam) has played an important role in controlling the disease, but insensitivity to the fungicide in certain isolates is now of major concern. A genetic basis for resistance to metalaxyl suggests the possibility for linking resistance phenotypes to specific population genetic markers, but in order to do this, the population genetic structure and mode of reproduction in a population must first be well described. The dynamics of metalaxyl-M resistance in the Danish population of P. infestans was characterized over the course of the 2013 growing season, as was the population genetic structure, using simple sequence repeat (SSR) genotypes and single nucleotide polymorphism (SNP)-based mitochondrial haplotyping of over 80 isolates. Both mating types A1 and A2 were present in most fields, but tests for recombination showed that clonal reproduction dominates in Danish populations. Genotype was not linked to haplotype and no differentiation was observed at the haplotype level, but rather between fields. Resistance phenotypes were linked to specific SSR alleles, demonstrating the potential for a more precise SNP-based marker system for predicting resistance to metalaxyl-M. [ABSTRACT FROM AUTHOR]

Abstract The mitochondrial ribosomal large subunit (Ls) DNA was used to identify the orchid mycorrhizal fungi found in roots of Dactylorhiza majalis. The gene was amplified using DNA extracted from single pelotons obtained from fresh and silica gel dried roots. Furthermore, sequencing a variety of well-characterized orchid isolates expanded the fungal database of the mitochondrial ribosomal LsDNA. Polymerase chain reaction product length variants present in D. majalis were sequenced and identified using the expanded database. These analyses revealed two different peloton-forming fungi in samples from D. majalis, which sometimes occurred together as a single two-taxa peloton within the same cortex cell. The first taxon belonged to the genus Tulasnella and the second taxon was distantly related to Laccaria. [ABSTRACT FROM AUTHOR]

Arbuscular mycorrhizal (AM) symbioses are known to play a role in increased resistance of plants against soilborne pathogens. Mechanisms involved in this phenomenon are not yet well understood. This work investigates possible roles of endoproteolytic activities in bioprotection of Pisum sativum roots by Glomus mosseae against Aphanomyces euteiches. First, it is demonstrated that bioprotection occurs only in pre-mycorrhizal plants. Second, endoproteolytic activities were analysed qualitatively and quantitatively during AM symbiosis, in plants infected with either zoospores or mycelium of A. euteiches, and in mycorrhizal plants infected with the pathogen. In mycorrhizal symbiosis a progressive increase in endoproteolytic activities was observed following root colonization by G. mosseae. By contrast, in roots inoculated with A. euteiches, a drastic increase in endoproteolytic activities was observed which was correlated with the amount of pathogen occurring in roots. Qualitative differences were seen among the endoproteolytic activities detected in roots inoculated with zoospores or mycelium. The constitutive as well as mycorrhizal and pathogen-induced activities were further characterized as 'trypsin-like' serine endoproteases. Interestingly, in a situation of bioprotection, only low levels of the activities normally associated with the infection by A. euteiches were detected, suggesting that the synthesis of these proteins is directly linked to the growth or virulence of the pathogen. [ABSTRACT FROM AUTHOR]

A new method to measure enzyme activity of the fungal root pathogen Aphanomyces euteiches in pea roots is described. The specific enzymes of the fungus and the host were separated by polyacrylamide gel electrophoresis (PAGE) and the activity of fungal Glucose-6-phosphate dehydrogenase and Phosphoglucomutase were quantified by densitometry. Fungal activity could be correlated to the percentage infected root length and to the disease symptoms of the plants. The activity of A. euteiches was studied in a time course experiment with increasing levels of zoospore inoculum. The results indicated that an increase in inoculum level resulted in a faster disease development in the plants. The relation between fungal enzyme activity and infection level is discussed. [ABSTRACT FROM AUTHOR]

Soil microbial communities perform vital ecosystem functions, such as the decomposition of organic matter to provide plant nutrition. However, despite the functional importance of soil microorganisms, attribution of ecosystem function to particular constituents of the microbial community has been impeded by a lack of information linking microbial function to community composition and structure. Here, we propose a function-first framework to predict how microbial communities influence ecosystem functions. We first view the microbial community associated with a specific function as a whole and describe the dependence of microbial functions on environmental factors (e.g., the intrinsic temperature dependence of bacterial growth rates). This step defines the aggregate functional response curve of the community. Second, the contribution of the whole community to ecosystem function can be predicted, by combining the functional response curve with current environmental conditions. Functional response curves can then be linked with taxonomic data in order to identify sets of "biomarker" taxa that signal how microbial communities regulate ecosystem functions. Ultimately, such indicator taxa may be used as a diagnostic tool, enabling predictions of ecosystem function from community composition. In this paper, we provide three examples to illustrate the proposed framework, whereby the dependence of bacterial growth on environmental factors, including temperature, pH, and salinity, is defined as the functional response curve used to interlink soil bacterial community structure and function. Applying this framework will make it possible to predict ecosystem functions directly from microbial community composition., (© 2021 The Authors. Ecology published by Wiley Periodicals LLC on behalf of Ecological Society of America.)

The foliar microbiome can extend the host plant phenotype by expanding its genomic and metabolic capabilities. Despite increasing recognition of the importance of the foliar microbiome for plant fitness, stress physiology, and yield, the diversity, function, and contribution of foliar microbiomes to plant phenotypic traits remain largely elusive. The recent adoption of high-throughput technologies is helping to unravel the diversityand spatiotemporal dynamics of foliar microbiomes, but we have yet to resolve their functional importance for plant growth, development, and ecology. Here, we focus on the processes that govern the assembly of the foliar microbiome and the potential mechanisms involved in extended plant phenotypes. We highlight knowledge gaps and provide suggestions for new research directions that can propel the field forward. These efforts will be instrumental in maximizing the functional potential of the foliar microbiome for sustainable crop production.

Wood ash is alkaline and contains base-cations. Application of wood ash to forests therefore counteracts soil acidification and recycle nutrients removed during harvest. Wood ash application to soil leads to strong vertical gradients in physicochemical parameters. Consequently, we designed an experimental system where small-scale vertical changes in soil properties and prokaryotic community structure could be followed after wood ash application. A mixed fly and bottom ash was applied in dosages of 3 and 9 t ha -1 to the surface of soil mesocosms, simulating a typical coniferous podzol. Soil pH, exchangeable cations and 16S prokaryotic community was subsequently assessed at small depth intervals to 5 cm depth at regular intervals for one year. Wood ash significantly changed the prokaryotic community in the top of the soil column. Also, the largest increases in pH and concentrations of exchangeable cations was found here. The relative abundance of prokaryotic groups directionally changed, suggesting that wood ash favors copiotrophic prokaryotes at the expense of oligotrophic and acidophilic taxa. The effect of wood ash were negligible both in terms of pH- and biological changes in lower soil layers. Consequently, by micro-vertical profiling we showed that wood ash causes a steep gradient of abiotic factors driving biotic changes but only in the top-most soil layers.

Background and Aims: There are a number of disparate models predicting variation in plant chemical defences between species, and within a single species over space and time. These can give conflicting predictions. Here we review a number of these theories, before assessing their power to predict the spatial-temporal variation of thapsigargins between and within populations of the deadly carrot (Thapsia garganica). By utilizing multiple models simultaneously (optimum defence theory, growth rate hypothesis, growth-differentiation balance hypothesis, intra-specific framework and resource exchange model of plant defence), we will highlight gaps in their predictions and evaluate the performance of each., Methods: Thapsigargins are potent anti-herbivore compounds that occur in limited richness across the different plant tissues of T. garganica, and therefore represent an ideal system for exploring these models. Thapsia garganica plants were collected from six locations on the island of Ibiza, Spain, and the thapsigargins quantified within reproductive, vegetative and below-ground tissues. The effects of sampling time, location, mammalian herbivory, soil nutrition and changing root-associated fungal communities on the concentrations of thapsigargins within these in situ observations were analysed, and the results were compared with our model predictions., Key Results: The models performed well in predicting the general defence strategy of T. garganica and the above-ground distribution of thapsigargins, but failed to predict the considerable proportion of defences found below ground. Models predicting variation over environmental gradients gave conflicting and less specific predictions, with intraspecific variation remaining less understood., Conclusion: Here we found that multiple models predicting the general defence strategy of plant species could likely be integrated into a single model, while also finding a clear need to better incorporate below-ground defences into models of plant chemical defences. We found that constitutive and induced thapsigargins differed in their regulation, and suggest that models predicting intraspecific defences should consider them separately. Finally, we suggest that in situ studies be supplemented with experiments in controlled environments to identify specific environmental parameters that regulate variation in defences within species., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Recycling of wood ash from energy production may counteract soil acidification and return essential nutrients to soils. However, wood ash amendment affects soil physicochemical parameters that control composition and functional expression of the soil microbial community. Here, we applied total RNA sequencing to simultaneously assess the impact of wood ash amendment on the active soil microbial communities and the expression of functional genes from all microbial taxa. Wood ash significantly affected the taxonomic (rRNA) as well as functional (mRNA) profiles of both agricultural and forest soil. Increase in pH, electrical conductivity, dissolved organic carbon and phosphate were the most important physicochemical drivers for the observed changes. Wood ash amendment increased the relative abundance of the copiotrophic groups Chitinonophagaceae (Bacteroidetes) and Rhizobiales (Alphaproteobacteria) and resulted in higher expression of genes involved in metabolism and cell growth. Finally, total RNA sequencing allowed us to show that some groups of bacterial feeding protozoa increased concomitantly to the enhanced bacterial growth, which shows their pivotal role in the regulation of bacterial abundance in soil., (© FEMS 2020.)

Plant traits-the morphological, anatomical, physiological, biochemical and phenological characteristics of plants-determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait-based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits-almost complete coverage for 'plant growth form'. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait-environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives., (© 2019 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

Dispersal of mycorrhizal fungi via animals and the importance for the interacting partners' life history as well as for ecosystems is an understudied topic. In this review, we describe the available evidence and the most important knowledge gaps and finally suggest ways to gain the missing information. So far, 33 articles have been published proving a successful transfer of mycorrhizal propagules by animals. The vast majority of research on invertebrates was focused on arbuscular mycorrhizal (AM) fungi, whereas papers on vertebrates (mainly rodents and artiodactyls) equally addressed ectomycorrhizal (ECM) and AM fungi. Effective dispersal has been mostly shown by the successful inoculation of bait plants and less commonly by spore staining or germination tests. Based on the available data and general knowledge on animal lifestyles, collembolans and oribatid mites may be important in transporting ECM fungal propagules by ectozoochory, whereas earthworms, isopods, and millipedes could mainly transfer AM fungal spores in their gut systems. ECM fungal distribution may be affected by mycophagous dipterans and their hymenopteran parasitoids, while slugs, snails, and beetles could transport both mycorrhizal groups. Vertebrates feeding on fruit bodies were shown to disperse mainly ECM fungi, while AM fungi are transported mostly accidentally by herbivores. The important knowledge gaps include insufficient information on dispersal of fungal propagules other than spores, the role of invertebrates in the dispersal of mycorrhizal fungi, the way in which propagules pass through food webs, and the spatial distances reached by different dispersal mechanisms both horizontally and vertically.

An outbreak of the potato late blight pathogen Phytophthora infestans in Denmark was characterized in order to resolve the population structure and determine to what extent sexual reproduction was occurring. A standard set of microsatellite simple sequence repeats (SSRs) and single nucleotide polymorphism (SNP) markers generated using restriction site-associated DNA sequencing (RAD-seq) were employed in parallel. A total of 83 individuals, isolated from seven different potato fields in 2014, were analysed together with five Danish whole-genome sequenced isolates, as well as two Mexican individuals used as an outgroup. From a filtered dataset of 55 288 SNPs, population genomics analyses revealed no sign of recombination, implying clonality. In spite of this, multilocus genotypes were unique to individual potato fields, with little evidence of gene flow between fields. Ploidy analysis performed on the SNPs dataset indicated that the majority of isolates were diploid. These contradictory results with clonality and high genotypic diversity may suggest that rare sexual events likely still contribute to the population. Comparison of the results generated by SSRs vs SNPs data indicated that large marker sets, generated by RAD-seq, may be advised going forward, as it provides a higher level of genetic discrimination than SSRs., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Wood ash recycling to forests is beneficial because it regains nutrients and prevents acidification, but wood ash application is restricted due to its cadmium (Cd) content. We question if Cd in wood ash represents a problem, since decreases in Cd bioavailability due to ash-induced pH changes may counteract increased total Cd concentration. We studied effects of wood ash (0, 3, 9 and 30 t ha -1 ) and lime (pH increase equivalent to the wood ash treatments) on growth and Cd uptake in Deschampsia flexuosa. After four months, we measured plant biomass and Cd accumulation, and extracted Cd from the soil using three different methods; HNO 3 (total), EDTA (chelator-based) and NH 4 NO 3 (salt-based). Wood ash and lime strongly stimulated plant growth. Cd concentration in the plant tissue decreased with wood ash and lime addition, and correlated positively with the NH 4 NO 3 extractable fraction of Cd in the soil. In contrast, HNO 3 and EDTA extracted more Cd with increased wood ash application. We conclude that wood ash amendment increases soil pH, total Cd concentration, nutrient levels and stimulates plant growth. However, it does not increase Cd accumulation in D. flexuosa, as pH-driven decreases in Cd bioavailability leads to reduced plant Cd uptake. Finally, soil bioavailable Cd is best determined using NH 4 NO 3 -extraction., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Most plants form symbiotic associations with arbuscular mycorrhizal fungi (AMF). AMF increase the uptake of plant nutrients by extending their extra-radical mycelium (ERM) in the soil where other groups of microorganisms may suppress the activity of the ERM. However, little is known about such suppression in natural soils. This work aimed to investigate the incidence of AMF suppression among soils sampled from highly variable natural ecosystems, and used 33P uptake by the ERM to evaluate AMF activity. A second aim was to identify factors behind the observed AMF-suppression. We found that AMF-suppressiveness varied markedly among natural soils and occurred more frequently in low pH than in high pH soils. A previous study for cultivated soils revealed a strong biological component of suppressiveness against AMF, and in accordance we found that the composition of both fungal and bacterial communities differed significantly between AMF-suppressive and non-suppressive natural soils. Acidobacteria, Acidothermus, Xanthomonadaceae, Archaeorhizomyces sp., Mortierella humilis and some Mycena spp. were significantly more abundant in AMF-suppressive soils and may therefore be direct antagonists of AMF. This implies that the functioning of AMF in natural ecosystems is strongly modulated by specific soil microbes., (© FEMS 2019.)

There is an increasing consensus that microbial communities have an important role in mediating ecosystem processes. Trait-based ecology predicts that the impact of the microbial communities on ecosystem functions will be mediated by the expression of their traits at community level. The link between the response of microbial community traits to environmental conditions and its effect on plant functioning is a gap in most current microbial ecology studies. In this study, we analyzed functional traits of ectomycorrhizal fungal species in order to understand the importance of their community assembly for the soil-plant relationships in holm oak trees ( Quercus ilex subsp. ballota ) growing in a gradient of exposure to anthropogenic trace element (TE) contamination after a metalliferous tailings spill. Particularly, we addressed how the ectomycorrhizal composition and morphological traits at community level mediate plant response to TE contamination and its capacity for phytoremediation. Ectomycorrhizal fungal taxonomy and functional diversity explained a high proportion of variance of tree functional traits, both in roots and leaves. Trees where ectomycorrhizal fungal communities were dominated by the abundant taxa Hebeloma cavipes and Thelephora terrestris showed a conservative root economics spectrum, while trees colonized by rare taxa presented a resource acquisition strategy. Conservative roots presented ectomycorrhizal functional traits characterized by high rhizomorphs formation and low melanization which may be driven by resource limitation. Soil-to-root transfer of TEs was explained substantially by the ectomycorrhizal fungal species composition, with the highest transfer found in trees whose roots were colonized by Hebeloma cavipes . Leaf phosphorus was related to ectomycorrhizal species composition, specifically higher leaf phosphorus was related to the root colonization by Thelephora terrestris . These findings support that ectomycorrhizal fungal community composition and their functional traits mediate plant performance in metal-contaminated soils, and have a high influence on plant capacity for phytoremediation of contaminants. The study also corroborates the overall effects of ectomycorrhizal fungi on ecosystem functioning through their mediation over the plant economics spectrum.

DNA metabarcoding is promising for cost-effective biodiversity monitoring, but reliable diversity estimates are difficult to achieve and validate. Here we present and validate a method, called LULU, for removing erroneous molecular operational taxonomic units (OTUs) from community data derived by high-throughput sequencing of amplified marker genes. LULU identifies errors by combining sequence similarity and co-occurrence patterns. To validate the LULU method, we use a unique data set of high quality survey data of vascular plants paired with plant ITS2 metabarcoding data of DNA extracted from soil from 130 sites in Denmark spanning major environmental gradients. OTU tables are produced with several different OTU definition algorithms and subsequently curated with LULU, and validated against field survey data. LULU curation consistently improves α-diversity estimates and other biodiversity metrics, and does not require a sequence reference database; thus, it represents a promising method for reliable biodiversity estimation.

Recirculation of wood ash from energy production to forest soil improves the sustainability of this energy production form as recycled wood ash contains nutrients that otherwise would be lost at harvest. In addition, wood-ash is beneficial to many soils due to its inherent acid-neutralizing capabilities. However, wood ash has several ecosystem-perturbing effects like increased soil pH and pore water electrical conductivity both known to strongly impact soil bacterial numbers and community composition. Studies investigating soil bacterial community responses to wood ash application remain sparse and the available results are ambiguous and remain at a general taxonomic level. Here we investigate the response of bacterial communities in a spruce forest soil to wood ash addition corresponding to 0, 5, 22, and 167 t wood ash ha -1 . We used culture-based enumerations of general bacteria, Pseudomonas and sporeforming bacteria combined with 16S rRNA gene amplicon sequencing to valuate soil bacterial responses to wood ash application. Results showed that wood ash addition strongly increased soil pH and electrical conductivity. Soil pH increased from acidic through neutral at 22 t ha -1 to alkaline at 167 t ha -1 . Bacterial numbers significantly increased up to a wood ash dose of 22 t ha -1 followed by significant decrease at 167 t ha -1 wood ash. The soil bacterial community composition changed after wood ash application with copiotrophic bacteria responding positively up to a wood ash dose of 22 t ha -1 while the adverse effect was seen for oligotrophic bacteria. Marked changes in bacterial community composition occurred at a wood ash dose of 167 t ha -1 with a single alkaliphilic genus dominating. Additionally, spore-formers became abundant at an ash dose of 167 t ha -1 whereas this was not the case at lower ash doses. Lastly, bacterial richness and diversity strongly decreased with increasing amount of wood ash applied. All of the observed bacterial responses can be directly explained by the wood ash induced changes in pH, electrical conductivity and the addition of wood ash inherent nutrients.

Based on molecular and morphological data we investigated the taxonomy and phylogeny of the ectomycorrhizal genus Tricholoma in northern Europe. Our phylogenetic tree confirmed the presence of at least 72 well circumscribed species within the region. Of these, three species, viz. T. boreosulphurescens , T. bryogenum and T. ilkkae are described as new to science, based on morphological, distributional, ecological and molecular data. Several other terminal branches represent putative cryptic taxa nested within classical species or species groups. Molecular type studies and/or designation of sequenced neotypes are needed in these groups, before the taxonomy can be settled. In general our phylogenetic analysis supported previous suprageneric classification systems, but with some substantial changes. Most notably, T. virgatum and allies were found to belong to sect. Tricholoma rather than sect. Atrosquamosa , while T. focale was found to be clearly nested in sect. Genuina rather than in sect. Caligata . In total, ten sections are accepted, with five species remaining unassigned. The combination of morphological and molecular data showed pileus colour, pileipellis structure, presence of clamp connections and spore size to be rather conservative characters within accepted sections, while the presence of a distinct ring, and especially host selection were highly variable within these.

Reutilizing biomass ashes in agriculture can substitute inputs of P from finite primary sources. However, recycling of ashes is disputed due to their content of toxic substances such as heavy metals. This study evaluates the potential risk of replacing easily soluble inorganic P fertilizer with P in biomass ashes in a barley crop grown on soil with adequate P status. Two contrasting doses of three different types of ashes were applied to an agricultural field with spring barley and compared to similar doses of triple-superphosphate fertilizer. In the second growing season after biomass ash application, grain, straw and root dry matter yield, and P and Cd uptake were determined. Resin-extractable P was measured in soil and the symbiotic arbuscular mycorrhizal fungal activity, colonization, and community composition were assessed. Crop yield was not affected by ash application, while P-uptake and mycorrhizal status were slightly enhanced with high ash applications. Changes to the mycorrhizal community composition were evident with high ash doses. Cadmium uptake in aboveground plant tissue was unaffected by ash treatments, but increased in roots with increasing doses. Consequently, we conclude that fertilization with biomass ashes can replace conventional fertilizers without risk to barley crops in the short term., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In 1998, a toxic mine spill polluted a 55-km(2) area in a basin southward to Doñana National Park (Spain). Subsequent attempts to restore those trace element-contaminated soils have involved physical, chemical, or biological methodologies. In this study, the restoration approach included application of different types and doses of organic amendments: biosolid compost (BC) and leonardite (LEO). Twelve years after the last addition, molecular analyses of arbuscular mycorrhizal (AM) fungal communities associated with target plants (Lamarckia aurea and Chrysanthemum coronarium) as well as analyses of trace element concentrations both in soil and in plants were performed. The results showed an improved soil quality reflected by an increase in soil pH and a decrease in trace element availability as a result of the amendments and dosages. Additionally, the phylogenetic diversity of the AM fungal community increased, reaching the maximum diversity at the highest dose of BC. Trace element concentration was considered the predominant soil factor determining the AM fungal community composition. Thereby, the studied AM fungal community reflects a community adapted to different levels of contamination as a result of the amendments. The study highlights the long-term effect of the amendments in stabilizing the soil system.

Phylogenetic analyses of Mycena sect. Calodontes using ITS previously suggested ten cryptic monophyletic ITS lineages within the Mycena pura morphospecies. Here, we compare ITS data (645 bp incl. gaps) from 46 different fruit bodies that represent the previously described ITS diversity with partial tEF-1-α (423 bp) and RNA polymerase II (RPB1) (492 bp) sequence data to test the genealogical concordance. While neither of the markers were in complete topological agreement, the branches differing between the tEF and RPB1 trees had a low bootstrap (<50) support, and the partition homogeneity incongruence length difference (ILD) tests were not significant. ILD tests revealed significant discordances between ITS and the tEF and RPB1 markers in several lineages. And our analyses suggested recombination between ITS1 and ITS2, most pronounced in one phylospecies that was identical in tEF and RPB1. Based on the agreement between tEF and RPB1, we defined 11 mutually concordant terminal clades as phylospecies inside the M. pura morphospecies; most of them cryptic. While neither of the markers showed an unequivocal barcoding gap between inter- and intraspecific diversity, the overlap was most pronounced for ITS (intraspecific diversity 0-3.5 %, interspecific diversity 0.4 %-8.8 %). A clustering analysis on tEF separated at a 1.5 % level returned all phylogenetic species as Operational Taxonomic Units (OTUs), while ITS at both a 1.5 % level and at a 3 % threshold level not only underestimated diversity as found by the tEF and RPB1, but also identified an OTU which was not a phylogenetic species. Thus, our investigation does not support the universal suitability of ITS for species recognition in particular, and emphasises the general limitation of single gene analyses combined with single percentage separation values., (Copyright © 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Novel high-throughput sequencing methods outperform earlier approaches in terms of resolution and magnitude. They enable identification and relative quantification of community members and offer new insights into fungal community ecology. These methods are currently taking over as the primary tool to assess fungal communities of plant-associated endophytes, pathogens, and mycorrhizal symbionts, as well as free-living saprotrophs. Taking advantage of the collective experience of six research groups, we here review the different stages involved in fungal community analysis, from field sampling via laboratory procedures to bioinformatics and data interpretation. We discuss potential pitfalls, alternatives, and solutions. Highlighted topics are challenges involved in: obtaining representative DNA/RNA samples and replicates that encompass the targeted variation in community composition, selection of marker regions and primers, options for amplification and multiplexing, handling of sequencing errors, and taxonomic identification. Without awareness of methodological biases, limitations of markers, and bioinformatics challenges, large-scale sequencing projects risk yielding artificial results and misleading conclusions., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Global species richness patterns of soil micro-organisms remain poorly understood compared to macro-organisms. We use a global analysis to disentangle the global determinants of diversity and community composition for ectomycorrhizal (EcM) fungi-microbial symbionts that play key roles in plant nutrition in most temperate and many tropical forest ecosystems. Host plant family has the strongest effect on the phylogenetic community composition of fungi, whereas temperature and precipitation mostly affect EcM fungal richness that peaks in the temperate and boreal forest biomes, contrasting with latitudinal patterns of macro-organisms. Tropical ecosystems experience rapid turnover of organic material and have weak soil stratification, suggesting that poor habitat conditions may contribute to the relatively low richness of EcM fungi, and perhaps other soil biota, in most tropical ecosystems. For EcM fungi, greater evolutionary age and larger total area of EcM host vegetation may also contribute to the higher diversity in temperate ecosystems. Our results provide useful biogeographic and ecological hypotheses for explaining the distribution of fungi that remain to be tested by involving next-generation sequencing techniques and relevant soil metadata., (© 2012 Blackwell Publishing Ltd.)

• Nitrogen (N) availability is known to influence ectomycorrhizal fungal components, such as fungal community composition, biomass of root tips and production of mycelia, but effects have never been demonstrated within the same forest. • We measured concurrently the abundance of ectomycorrhizal root tips and the production of external mycelia, and explored the changes in the ectomycorrhizal community composition, across a stand-scale N deposition gradient (from 27 to 43 kg N ha⁻¹ yr⁻¹) at the edge of a spruce forest. The N status was affected along the gradient as shown by a range of N availability indices. • Ectomycorrhizal root tip abundance and mycelial production decreased five and 10-fold, respectively, with increasing N deposition. In addition, the ectomycorrhizal fungal community changed and the species richness decreased. The changes were correlated with the measured indices of N status, in particular N deposition and N leaching. • The relationship between the altered ectomycorrhizal community, root tip abundance and mycelial production is discussed in the context of the N parameters. We suggest that increased N deposition to forests will cause large changes in ectomycorrhizal fungal community structure and functioning, which, in turn, may result in reduced N uptake by roots and fungi, and increased losses of N by leaching., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

Cortinarius is the most species rich genus of mushroom forming fungi with an estimated 2000 spp. worldwide. However, species delimitation within the genus is often controversial. This is particularly true in the section Calochroi (incl. section Fulvi), where the number of accepted taxa in Europe ranges between c.60 and c.170 according to different taxonomic schools. Here, we evaluated species delimitation within this taxonomically difficult group of species and estimated their phylogenetic relationships. Species were delimited by phylogenetic inference and by comparison of ITS sequence data in combination with morphological characters. A total of 421 ITS sequences were analyzed, including data from 53 type specimens. The phylogenetic relationships of the identified species were estimated by analyzing ITS data in combination with sequence data from the two largest subunits of RNA polymerase II (RPB1 and RPB2). Seventy-nine species were identified, which are believed to constitute the bulk of the diversity of this group in Europe. The delimitation of species based on ITS sequences is more consistent with a conservative morphological species concept for most groups. ITS sequence data from 30 of the 53 types were identical to other taxa, and most of these can be readily treated as synonyms. This emphasizes the importance of critical analysis of collections before describing new taxa. The phylogenetic separation of species was, in general, unambiguous and there is considerable potential for using ITS sequence data as a barcode for the group. A high level of homoplasy and phenotypic plasticity was observed for morphological and ecological characters. Whereas most species and several minor lineages can be recognized by morphological and ecological character states, these same states are poor indicators at higher levels.

Extensive knowledge of various ectomycorrhizal fungal communities has been obtained over the past 10 years based on molecular identification of the fungi colonizing fine roots. In contrast, only limited information exists about the species composition of ectomycorrhizal hyphae in soil. This study compared the ectomycorrhizal external mycelial community with the adjacent root-tip community in a Danish beech forest. Sand-filled in-growth mesh bags were used to trap external mycelia by incubating the mesh bags in the soil for 70 days. The adjacent ectomycorrhizal root-tip communities were recorded at the times of insertion and retrieval of the mesh bags. Ectomycorrhizal fungi were identified by sequencing the internal transcribed spacer region. In total, 20, 31 and 24 ectomycorrhizal species were recorded from the two root-tip harvests and from the mesh bags, respectively. Boletoid species were significantly more frequent as mycelia than as root tips, while russuloid and Cortinarius species appeared to be less dominant as mycelia than as root tips. Tomentella species were equally frequent as root tips and as mycelia. These discrepancies between the root-tip and the mycelial view of the ectomycorrhizal fungal community are discussed within the framework of ectomycorrrhizal exploration types.

A growing body of molecular research is discovering a high diversity of pezizalean ectomycorrhiza (EcM), yet most remain unidentified at the genus or species level. This study describes EcM-forming taxa within the Pezizales. EcM-forming Pezizales were revealed by morphotyping and sequencing of EcM root tips from forests in Estonia and Denmark. The taxa on EcM root tips were identified using phylogenetic analyses of large-subunit rDNA sequences derived from sporocarps of 301 pezizalean species, and comparisons with internal transcribed spacer rDNA sequences. Thirty-three species are suggested as EcM symbionts, representing all three major clades of Pezizales, the genera Genea, Geopora, Humaria, Tarzetta, Trichophaea, Wilcoxina, Helvella, Hydnotrya, Tuber, Pachyphloeus, Peziza and Sarcosphaera, and two Pezizaceae anamorphs. EcM of Pezizales species are easily distinguished by their anatomy, particularly thick cell walls and stout hyphae. This study demonstrates that Pezizales species constitute a considerable proportion of the mycobionts in EcM fungal communities in mature boreal deciduous and coniferous forests, in several soil types. Fruit-body sequences and EcM descriptions will facilitate identification of pezizalean EcM in future studies.

Identification of ectomycorrhizal (ECM) fungi is often achieved through comparisons of ribosomal DNA internal transcribed spacer (ITS) sequences with accessioned sequences deposited in public databases. A major problem encountered is that annotation of the sequences in these databases is not always complete or trustworthy. In order to overcome this deficiency, we report on UNITE, an open-access database. UNITE comprises well annotated fungal ITS sequences from well defined herbarium specimens that include full herbarium reference identification data, collector/source and ecological data. At present UNITE contains 758 ITS sequences from 455 species and 67 genera of ECM fungi. UNITE can be searched by taxon name, via sequence similarity using blastn, and via phylogenetic sequence identification using galaxie. Following implementation, galaxie performs a phylogenetic analysis of the query sequence after alignment either to pre-existing generic alignments, or to matches retrieved from a blast search on the UNITE data. It should be noted that the current version of UNITE is dedicated to the reliable identification of ECM fungi. The UNITE database is accessible through the URL http://unite.zbi.ee

•  Species of arbuscular mycorrhizal fungi (AMF) differ markedly in their improvement of plant nutrition and health. However, it is not yet possible to relate the diversity of an AMF community to its functional properties due to the lack of information on the functional diversity at each taxonomic level. This study investigates the inter- and intraspecific functional diversity of four Glomus species in relation to a phylogenetic analysis of large ribosomal subunit (LSU) sequences. •  Growth and P nutrition of cucumber (Cucumis sativus) associated with 24 different isolates of AMF were measured in a two-compartment system with a 33 P-labelled root-free soil compartment. •  Intraspecific differences were found in plant growth response and the extension of the fungal mycelium into the root-free soil patch whereas length-specific P uptake of the hyphae remained rather constant within each AMF species. Hence, the length-specific P uptake differed according to species, whereas lower phylogenetic levels were required to match functional characteristics such as fungal growth pattern and plant growth promotion. •  The large intraspecific diversity observed for mycelium growth and improvement of P uptake means that AMF communities of low species diversity may still contain considerable functional heterogeneity.

The colonisation intensity and composition of the mycorrhizal community in the aquatic plants Lobelia dortmanna and Littorella uniflora were studied. The mycorrhizal fungi were characterised by fungal specific nested PCR and sequencing using the 5'-end of the LSU rDNA as target. For this, primers for the clade of Acaulospora, the clade including Glomus mosseae and G. intraradices and the clade containing G. etunicatum and G. claroideum were used. The nested PCR products were screened for different sequence types using single stranded conformation polymorphism (SSCP) and representatives for each type were sequenced. A phylogenetic analysis of the sequences showed two phylotypes of Acaulospora, one phylotype within the clade of G. etunicatum/G. claroideum and five within the G. mosseae/ G. intraradices clade. The colonisation intensity was comparable to that seen in typical grassland vegetation. The neutral lipid fatty acid 16: 1omega5 was seen to be indicative of mycorrhizal colonisation with concentrations up to 35 nmol mg(-1) root DW, which indicates that the fungi are active.

In this study we examine the distribution of Rhizopogon species in spore banks from five California pine forests. Four of the forest sites were discontinuous populations of Pinus muricata and a fifth was a Pinus ponderosa stand in Sierra National Forest. Rhizopogon species were retrieved by bioassaying the soils with pine seedlings followed by isolation of axenic cultures from individual root tips with typical Rhizopogon ectomycorrhizal morphology. The cultures were screened by ITS-RFLP and all unique patterns were sequenced. These sequences then were compared with those derived from identified sporocarp material. Bioassaying proved to be an efficient way to bring Rhizopogon species into culture. Approximately 50% of the pots contained ectomycorrhizal tips with Rhizopogon-like morphology, and axenic Rhizopogon cultures were obtained from half these pots. Our results showed that Rhizopogon spores usually are well distributed within local forest areas, while there is significant structuring of species at the regional scale. Spore longevity and homogenization by soil and water movement might explain their distribution within local forest areas, while the regional pattern might be explained by limited long distance dispersal or climatic and edaphic differences.

This is the first reported study of the interactions between indigenous arbuscular mycorrhizal fungi (AMF) and Aphanomyces euteiches in pea under field conditions. A. euteiches was applied to the soil by adding oospores produced in vitro. Attempts were made to create a non-mycorrhizal control by incorporating carbendazim (Derosal Fl) in the topsoil before sowing. However, all carbendazim-treated plants showed approximately 20% root colonisation with AMF. Pea plants not treated with carbendazim showed a wide variation in AMF colonisation of 35-70% at the full flowering stage. In these control plots, root length infected with oospores of A. euteiches and colonisation by AMF were negatively correlated. Application of carbendazim increased the percent root length infected with oospores by 50-70%, depending on inoculum density of A. euteiches. Despite the lower levels of AMF colonisation in these treated plots, a negative correlation with oospore-containing root length was still observed. No correlation was found between AMF colonisation and disease severity, disease incidence or pathogen enzymatic activity (glucose-6-phosphate dehydrogenase). Thus, AMF do not seem to influence the vegetative stage of pathogen development during which cortical root rotting takes place, but rather the reproductive stage when oospores are produced. The results of this study underline the importance of field experiments for validating the significance of mycorrhizal fungi for plant health.

ABSTRACT To describe the disease cycle of the root pathogen Aphanomyces euteiches, enzymatic activity in the mycelium was compared with the development of oospores in pea roots. Plants were inoculated with two zoospore concentrations to achieve different disease levels. Hyphae were stained for fungal alkaline phosphatase activity in the roots. Additionally, enzyme activity was measured after electrophoresis of an A. euteiches-specific glucose-6-phosphate isozyme. Development of oospores in the roots was measured after staining the oospores with trypan blue. In plants inoculated with the higher zoospore concentration, the enzymatic activity of the pathogen mycelium peaked 10 to 14 days after inoculation, when oospore formation was initiated. Oospore formation was associated with a gradual increase in disease symptoms. At the last harvest, plants inoculated with the higher zoospore concentration had died. In these plants, oospores were found in 90% of the root length, while the enzymatic activity of the mycelium was low. This suggests that the pathogen mycelium is only active on living plants and does not grow saprophytically on dead plant material.