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Fungal research is experiencing a new wave of methodological improvements that most probably will boost mycology as profoundly as molecular phylogeny has done during the last 15 years. Especially the next generation sequencing technologies can be expected to have a tremendous effect on fungal biodiversity and ecology research. In order to realise the full potential of these exciting techniques by accelerating biodiversity assessments, identification procedures of fungi need to be adapted to the emerging demands of modern large-scale ecological studies. But how should fungal species be identified in the near future? While the answer might seem trivial to most microbiologists, taxonomists working with fungi may have other views. In the present review, we will analyse the state of the art of the so-called barcoding initiatives in the light of fungi, and we will seek to evaluate emerging trends in the field. We will furthermore demonstrate that the usability of DNA barcoding as a major tool for identification of fungi largely depends on the development of high-quality sequence databases that are thoroughly curated by taxonomists and systematists.

With high-throughput sequencing (HTS), we are able to explore the hidden world of microscopic organisms to an unpre-cedented level. The fast development of molecular technology and statistical methods means that microbial ecologists must keep their toolkits updated. Here, we review and evaluate some of the more widely adopted and emerging techniques for analysis of diversity and community composition, and the inference of species interactions from co-occurrence data generated by HTS of marker genes. We emphasize the importance of observational biases and statistical properties of the data and methods. The aim of the review is to critically discuss the advantages and disadvantages of established and emerging statistical methods, and to contribute to the integration of HTS-based marker gene data into community ecology. This is an overview of the more widely adopted and emerging techniques for analysis of diversity and community composition, and the inference of species interactions from co-occurrence data generated by high-throughput sequencing of marker genes. Graphical Abstract Figure. This is an overview of the more widely adopted and emerging techniques for analysis of diversity and community composition, and the inference of species interactions from co-occurrence data generated by high-throughput sequencing of marker genes.

Comparative investigations of plant-associated fungal communities (mycobiomes) in distinct habitats and under distinct climate regimes have been rarely conducted in the past. Nowadays, high-throughput sequencing allows routine examination of mycobiome responses to environmental changes and results at an unprecedented level of detail. In the present study, we analysed Illumina-generated fungal ITS1 sequences from European beech (Fagus sylvatica) originating from natural habitats at two different altitudes in the German Alps and from a managed tree nursery in northern Germany. In general, leaf-inhabiting mycobiome diversity and composition correlated significantly with the origin of the trees. Under natural condition the mycobiome was more diverse at lower than at higher elevation, whereas fungal diversity was lowest in the artificial habitat of the tree nursery. We further identified significant correlation of leaf chlorophylls and flavonoids with both habitat parameters and mycobiome biodiversity. The present results clearly point towards a pronounced importance of local stand conditions for the structure of beech leaf mycobiomes and for a close interrelation of phyllosphere fungi and leaf physiology.

This paper introduces a new approach-the Principal Component Gradient Analysis (PCGA)-to detect ecological gradients in time-series populations, i.e. several time-series originating from different individuals of a population. Detection of ecological gradients is of particular importance when dealing with time-series from heterogeneous populations which express differing trends. PCGA makes use of polar coordinates of loadings from the first two axes obtained by principal component analysis (PCA) to define groups of similar trends. Based on the mean inter-series correlation (rbar) the gain of increasing a common underlying signal by PCGA groups is quantified using Monte Carlo Simulations. In terms of validation PCGA is compared to three other existing approaches. Focusing on dendrochronological examples, PCGA is shown to correctly determine population gradients and in particular cases to be advantageous over other considered methods. Furthermore, PCGA groups in each example allowed for enhancing the strength of a common underlying signal and comparably well as hierarchical cluster analysis. Our results indicate that PCGA potentially allows for a better understanding of mechanisms causing time-series population gradients as well as objectively enhancing the performance of climate transfer functions in dendroclimatology. While our examples highlight the relevance of PCGA to the field of dendrochronology, we believe that also other disciplines working with data of comparable structure may benefit from PCGA.

Plant-associated mycobiomes in extreme habitats are understudied and poorly understood. We analysed Illumina-generated ITS1 sequences from the needle mycobiome of white spruce (Picea glauca) at the northern treeline in Alaska (USA). Sequences were obtained from the same DNA that was used for tree genotyping. In the present study, fungal metabarcoding and tree microsatellite data were compared for the first time. In general, neighbouring trees shared more fungal taxa with each other than trees growing in further distance. Mycobiomes correlated strongly with phenological host traits and local habitat characteristics contrasting a dense forest stand with an open treeline site. Genetic similarity between trees did not influence fungal composition and no significant correlation existed between needle mycobiome and tree genotype. Our results suggest the pronounced influence of local habitat conditions and phenotypic tree traits on needle-inhabiting fungi. By contrast, the tree genetic identity cannot be bench-marked as a dominant driver for needle-inhabiting mycobiomes, at least not for white spruce in this extreme environment.

Comparative simultaneous studies of environmental high-throughput sequencing (HTS) and cultivation of plant-associated fungi have rarely been conducted in the past years. For the present contribution, HTS and extinction culturing were applied for the same leaf samples of European beech ( Fagus sylvatica ) in order to trace both “real” environmental drivers as well as method-dependent signals of the observed mycobiomes. Both approaches resulted in non-overlapping community composition and pronounced differences in taxonomic classification and trophic stages. However, both methods revealed similar correlations of the fungal communities with local environmental conditions. Our results indicate undeniable advantages of HTS over cultivation in terms of revealing a good representation of the major functional guilds, rare taxa and biodiversity signals of leaf-inhabiting fungi. On the other hand our results demonstrate that the immense body of literature about cultivable endophytic fungi can and should be used for the interpretation of community signals and environmental correlations obtained from HTS studies and that cultivation studies should be continued at the highest standards, e.g. when sequencing facilities are not available or if such surveys are expanded into functional aspects with experiments on living isolates.

In 2005, researchers at the Leipzig Canopy Crane Research Facility collected living leaves of four temperate tree species at heights of between 15 and 33 m above the ground. Following surface sterilisation of the leaves, leaf-fragments were cultured on malt extract agar which allowed the growth of endophytic fungi into the surrounding medium. Isolated cultures were identified by morphology and sequence analysis of the D1/D2 region of the large subunit rDNA. Phylogenetic analysis established the taxonomic positions of the fungi. A total of 49 different taxa were identified, representing 20 families and ten orders. With the exception of one basidiomycetous yeast, all taxa belonged to filamentous ascomycetes. Species richness was highest on Tilia cordata and lowest on Quercus robur. Species-accumulation curves showed that the sampling effort was not sufficient to cover the majority of the likely species at the investigation site. Most endophytes proved to be ubiquitous within the canopy of the investigation site, but habitat preferences in terms of different tree species, different light regimes and season (sampling times) were obvious for some abundant endophytes. Apiognomonia errabunda and Aspergillus niger occurred predominantly on Q. robur, Diplodina acerina on Acer pseudoplatanus, one species of Phoma significantly prefered shaded leaves from the lower canopy layer whereas Sordaria fimicola prefered sun-exposed leaves from the upper tree crowns. Seasonal patterns were observed, for example, for A. errabunda, which was abundant in young leaves in the spring and almost completely absent in aged autumn-leaves, thus suggesting the accumulation of antifungal secondary plant metabolites during the growing season.

ABSTRACT The drainage of peatlands for their agricultural use leads to huge emissions of greenhouse gases. One sustainable alternative is the cultivation of peat mosses after rewetting (‘Sphagnum farming’). Environmental parameters of such artificial systems may differ from those of natural Sphagnum ecosystems which host a rich fungal community. We studied the fungal community at a 4 ha Sphagnum farming field site in Northwestern Germany and compared it with that of natural Sphagnum ecosystems. Additionally, we asked if any fungi occur with potentially negative consequences for the commercial production and/or use of Sphagnum biomass. Samples were collected every 3 months within 1 year. High-throughput sequencing of the fungal ITS2 barcode was used to obtain a comprehensive community profile of the fungi. The dominant taxa in the fungal community of the Sphagnum farming field site were all commonly reported from natural Sphagnum ecosystems. While the taxonomic composition showed clear differences between seasons, a stable functional community profile was identified across seasons. Additionally, nutrient supply seems to affect composition of fungal community. Despite a rather high abundance of bryophyte parasites, and the occurrence of both Sphagnum-species-specific and general plant pathogens, their impact on the productivity and usage of Sphagnum biomass as raw material for growing media was considered to be low. Artificial Sphagnum farming sites are surrogate habitats for bog and Sphagnum-specific fungi.

Salicinoid phenolic glycosides are common defence substances in salicaceous trees and specialist leaf beetles use these compounds for their own defence against predators. Salicinoids vary qualitatively and qualitatively in aspen (Populus tremula) and this variation has a genetic basis. The foliar endophyte mycobiome is plentiful and we hypothesised that it is related to plant genotype, potentially mediated by salicinoid composition, and that interactions with the leaf beetle Chrysomela tremula may alter this relationship. We studied these three-way interactions in controlled greenhouse experiments. Endophytic fungi were isolated from sterilised leaf tissues with and without beetle damage, and from beetles. We confirmed that endophyte composition was influenced by host genotype. Beetle activity added generalist morphs to the mycobiome that overrode the initial host association. Yeast-like genera (Cryptococcus and Rhodotorula) were isolated only from beetle-damaged tissues and from beetles, whereas fast-growing filamentous fungi dominated beetle-free control plants. Competition experiments between filamentous fungi of plant origin and beetle-related yeasts suggested interaction of both stimulating and inhibiting modes of action amongst the fungi. As a result, we detected examples of amensalism, commensalism, parasitism and competition between the morphs tested, but we found no evidence of mutualism, and consequently no co-evolutionary relationship could be demonstrated, between yeasts carried by beetles, host genotype and associated filamentous morphs. Endophyte studies are method-dependent and high-throughput sequencing technology best define the fungal mycobiome, culturing however continues to be a cheap way to provide fundamental ecological insights and it is also required for experimental studies.

• Premise of the study: To understand the early evolution of mycorrhizal symbioses, it is important to know the fungal partners of gametophytes and sporophytes for basal lineages of vascular plants. Subterranean mycotrophic gametophytes of the clubmoss Diphasiastrum alpinum found at three localities gave an opportunity to study their morphology and anatomy and to identify and describe their hitherto unknown fungal endophytes. In addition, sporophytes were screened for fungal partners.• Methods: Gametophytes with attached young sporophytes were excavated, and their anatomy and their associated fungi were studied by light microscopy. DNA was isolated and amplified with both universal and group-specific fungal primers for the ITS region, the large subunit and small subunit of the nuclear rDNA, respectively, to identify the fungal partner.• Key results: Gametophytes were uniformly colonized by a fungus with septate hyphae forming coils and vesicles. Its morphology resembles that of the sebacinoid genus Piriformospora. Both ITS and LSU sequences were identified as Sebacinales group B, a basal clade of the Agaricomycetes (Basidiomycota). This fungus was detected in 11 gametophytes from two localities and in rootlets of adjacent Calluna vulgaris (Ericaceae) plants, but was absent in roots of sporophytes. In addition, several ascomycetes and glomeromycetes were found by DNA sequencing.• Conclusions: Our study suggests a fungus belonging to Sebacinales group B as the main fungal host of the D. alpinum gametophytes. However, Sebacinales group B fungi occur as well in adjacent Ericaceae plants; therefore, we assume the mycoheterotrophic gametophyte to be epiparasitic on Ericaceae, which would explain the steady association of these plants.