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The thallus structure of the lichen symbiosis provides a fungal shelter for the growth of algal partners. The long-living thallus also provides a habitat for other fungi, but experimental studies, which could inform us about the details of their interactions have hardly been conducted. We present a new approach by embedding axenically cultured strains of fungi together with algae in alginate spheres, which allows easy transfer of co-cultures on solid media. As the growth rates of the organisms are differentially triggered by the underlying medium, alginate embedding can help to adjust optimal parameters for stable culture of the combined symbionts. In our experiments, direct contacts between hyphae and algae and the formation of layered structures were observed in a fungus that is living as a commensal in the host lichen without visible symbiotic structures. The growth of primary lichen symbionts cannot be accelerated by alginate embedding so far, but our approach could artificially enforce symbiotic interactions that are not normally observed in nature.

Lichens are frequently colonized by specialized, lichenicolous fungi. Symptomatic lichenicolous fungi usually display typical phenotypes and reproductive structures on the lichen hosts. The classification based on these structures revealed different host specificity patterns. Other fungi occur asymptomatically in the lichen thalli and are much less known. We aimed at studying the diversity of lichen-associated fungi in specific, lichen-rich communities on rocks in the Alps. We tested whether lichenicolous fungi developing symptomatically on their known hosts also occur asymptomatically in other thalli of the same or of different host species. We collected lichen thalli according to a uniform sampling design comprising individuals adjacent to thalli that showed symptoms of lichenicolous fungal infections. The total fungal communities in the selected lichen thalli were further studied by single-strand conformation polymorphism (SSCP) fingerprinting analyses and sequencing of internal transcribed spacer (ITS) fragments. The systematic, stratified sampling strategy helped to recover 17 previously undocumented lichenicolous fungi and almost exhaustively the species diversity of symptomatic lichenicolous fungi in the studied region. The results from SSCP and the sequencing analyses did not reveal asymptomatic occurrence of normally symptomatic lichenicolous fungi in thalli of both the same and different lichen host species. The fungal diversity did not correlate with the species diversity of the symptomatic lichenicolous fungus-lichen host associations. The complex fingerprint patterns recovered here for fungal communities, in associations of well-delimited lichen thalli, suggest lichen symbiosis as suitable subjects for fungal metacommunity studies.

DNA phylogenetic comparisons have shown that morphology-based species recognition often underestimates fungal diversity. Therefore, the need for accurate DNA sequence data, tied to both correct taxonomic names and clearly annotated specimen data, has never been greater. Furthermore, the growing number of molecular ecology and microbiome projects using high-throughput sequencing require fast and effective methods for en masse species assignments. In this article, we focus on selecting and re-annotating a set of marker reference sequences that represent each currently accepted order of Fungi. The particular focus is on sequences from the internal transcribed spacer region in the nuclear ribosomal cistron, derived from type specimens and/or ex-type cultures. Re-annotated and verified sequences were deposited in a curated public database at the National Center for Biotechnology Information (NCBI), namely the RefSeq Targeted Loci (RTL) database, and will be visible during routine sequence similarity searches with NR_prefixed accession numbers. A set of standards and protocols is proposed to improve the data quality of new sequences, and we suggest how type and other reference sequences can be used to improve identification of Fungi. Database URL: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA177353., (Published by Oxford University Press 2013. This work is written by US Government employees and is in the public domain in the US.)

The lichen symbiosis allows a self-sustained life under harsh environmental conditions, yet symbiotic integrity can be affected by fungal parasites. Nothing is known about the impact of these biologically diverse and often specific infections on the recently detected bacterial community in lichens. To address this question, we studied the arctic-alpine 'chocolate chip lichen' Solorina crocea, which is frequently infected by Rhagadostoma lichenicola. We sampled healthy and infected lichens at two different sites in the Eastern Alps. High abundances of Acidobacteria, Planctomycetes, and Proteobacteria were identified analyzing 16S rRNA gene regions obtained by barcoded pyrosequencing. At the phylum and genus level, no significant alterations were present among infected and healthy individuals. Yet, evidence for a differentiation of communities emerged, when data were analyzed at the strain level by detrended correspondence analysis. Further, a profile clustering network revealed strain-specific abundance shifts among Acidobacteria and other bacteria. Study of stability and change in host-associated bacterial communities requires a fine-grained analysis at strain level. No correlation with the infection was found by analysis of nifH genes responsible for nitrogen fixation., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Lichens are unique among fungal symbioses in that their mycelial structures are compact and exposed to the light as thallus structures. The myriad intersections of unique fungal species with photosynthetic partner organisms (green algae in 90% of lichens) produce a wide variety of diverse shapes and colours of the fully synthesized lichen thallus when growing in nature. This characteristic complex morphology is, however, not achieved in the fungal axenic state. Even under ideal environmental conditions, the lichen life cycle faces considerable odds: first, meiotic spores are only produced on well-established thalli and often only after achieving considerable age in a stable environment, and second, even then in vivo resynthesis requires the presence of compatible algal strains where fungal spores germinate. Many lichen species have evolved a way around the resynthesis bottleneck by producing asexual propagules for joint propagation of symbionts. These different dispersal strategies ostensibly shape the population genetic structure of lichen symbioses, but the relative contributions of vertical (joint) and horizontal (independent) symbiont transmission have long eluded lichen evolutionary biologists. In this issue of Molecular Ecology, Dal Grande et al. (2012) close in on this question with the lung lichen, Lobaria pulmonaria, a flagship species in the conservation of old growth forests. By capitalizing on available microsatellite markers for both fungal and algal symbionts, they show that while vertical transmission is the predominant mode of reproduction, horizontal transmission is demonstrable and actively shapes population genetic structure. The resulting mixed propagation system is a highly successful balance of safe recruitment of symbiotic clones and endless possibilities for fungal recombination and symbiont shuffling.

Recent and substantial yield losses of Styrian oil pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) are primarily caused by the ascomycetous fungus Didymella bryoniae but bacterial pathogens are frequently involved as well. The diversity of endophytic microbial communities from seeds (spermosphere), roots (endorhiza), flowers (anthosphere), and fruits (carposphere) of three different pumpkin cultivars was studied to develop a biocontrol strategy. A multiphasic approach combining molecular, microscopic, and cultivation techniques was applied to select a consortium of endophytes for biocontrol. Specific community structures for Pseudomonas and Bacillus, two important plant-associated genera, were found for each microenvironment by fingerprinting of 16S ribosomal RNA genes. All microenvironments were dominated by bacteria; fungi were less abundant. Of the 2,320 microbial isolates analyzed in dual culture assays, 165 (7%) were tested positively for in vitro antagonism against D. bryoniae. Out of these, 43 isolates inhibited the growth of bacterial pumpkin pathogens (Pectobacterium carotovorum, Pseudomonas viridiflava, Xanthomonas cucurbitae); here only bacteria were selected. Microenvironment-specific antagonists were found, and the spermosphere and anthosphere were revealed as underexplored reservoirs for antagonists. In the latter, a potential role of pollen grains as bacterial vectors between flowers was recognized. Six broad spectrum antagonists selected according to their activity, genotypic diversity, and occurrence were evaluated under greenhouse conditions. Disease severity on pumpkins of D. bryoniae was significantly reduced by Pseudomonas chlororaphis treatment and by a combined treatment of strains (Lysobacter gummosus, P. chlororaphis, Paenibacillus polymyxa, and Serratia plymuthica). This result provides a promising prospect to biologically control pumpkin diseases.

In this study we investigate bacterial communities in association with an enriched black-fungal community in the plant phyllosphere to test whether these fungi create an environment for specific bacteria. Under organic conditions of agriculture, grapevine plants (Vitis vinifera) display an increased occurrence of the black fungi Aureobasidium pullulans and Epicoccum nigrum. Their enrichment agrees with the tolerance of these fungi to copper and sulphate, both used as main fungicides in organic viticulture. Both fungi also intrude the plant material to grow endophytically. Bacterial communities associated with black fungi of the plant surface and endosphere showed no differences compared to those found in conventionally managed V. vinifera plants. This suggests that despite an increase of these black fungi in organic practice, they do not shape bacterial diversity in grapevine plants. Nevertheless, dual cultures revealed a negative effect of Aureobasidium on the growth of certain bacilli, whereas growth of Aureobasidium was impeded by one Pseudomonas strain. Such singular effects are either not apparent in the natural black-fungal--bacterial community of the grape phyllosphere or are of rather localized effect., (Copyright © 2011. Published by Elsevier Ltd.)

Specific indoor environments select for certain stress-tolerant fungi and can drive their evolution towards acquiring medically important traits. Here we review the current knowledge in this area of research, focussing on the so-called black yeasts. Many of these melanised stress-tolerant organisms originate in unusual ecological niches in nature, and they have a number of preadaptations that make them particularly suited for growth on human-made surfaces and substrates. Several pathogenic species have been isolated recently from various domestic habitats. We argue that in addition to enriching for - potentially - pathogenic species, the selection pressure and stress acting on microorganisms in indoor environments are driving their evolution towards acquiring the missing virulence factors and further enhancing their stress tolerance and pathogenic potential. Some of the polyextremotolerant fungi are particularly problematic: they can grow at elevated temperatures, and so they have a higher potential to colonise warm-blooded organisms. As several species of black fungi are already implicated in health problems of various kinds, their selection and possible evolution in human environments are of concern., (Copyright © 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Environmental proteomics, also referred to as metaproteomics, is an emerging technology to study the structure and function of microbial communities. Here, we applied semi-quantitative label-free proteomics using one-dimensional gel electrophoresis combined with LC-MS/MS and normalized spectral counting together with fluorescence in situ hybridization and confocal laser scanning microscopy to characterize the metaproteome of the lung lichen symbiosis Lobaria pulmonaria. In addition to the myco- and photobiont, L. pulmonaria harbors proteins from a highly diverse prokaryotic community, which is dominated by Proteobacteria and including also Archaea. While fungal proteins are most dominant (75.4% of all assigned spectra), about the same amount of spectra were assigned to prokaryotic proteins (10%) and to the green algal photobiont (9%). While the latter proteins were found to be mainly associated with energy and carbohydrate metabolism, a major proportion of fungal and bacterial proteins appeared to be involved in PTMs and protein turnover and other diverse functions., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lichenized and non-lichenized fungi produce a wide range of secondary metabolites. So far, type I polyketide synthases (PKSs) are the suggested catalysts for the biosynthesis of lichen compounds. We were interested whether lichen mycobionts also contain type III PKSs, representing a class that was only recently discovered in fungi. With an alignment of known type III CHS-like genes we applied the CODEHOP strategy to design degenerate PCR primers. We further screened available fungal genomes for type III PKS genes and aligned these sequences for a phylogenetic analysis. Type III-like genes from lichen mycobionts are closely related to those known from non-lichenized fungi, but not to those of bacteria and/or plants. We conclude that type III PKS genes are ubiquitous in fungi. They are present in diverse unrelated lichen mycobionts, but their function in lichens is so far unclear., (Copyright © 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Our planet offers many opportunities for life on the edge: high and low temperatures, high salt concentrations, acidic and basic conditions and toxic environments, to name but a few extremes. Recent studies have revealed the diversity of fungi that can occur in stressful environments that are hostile to most eukaryotes. We review these studies here, with the additional purpose of proposing some mechanisms that would allow for the evolutionary adaptation of eukaryotic microbial life under extreme conditions. We focus, in particular, on life in ice and life at high salt concentrations, as there is a surprising similarity between the fungal populations in these two kinds of environments, both of which are characterized by low water activity. We propose steps of evolution of generalist species towards the development of specialists in extreme habitats. We argue that traits present in some fungal groups, such as asexuality, synthesis of melanin-like pigments and a flexible morphology, are preadaptations that facilitate persistence and eventual adaptation to conditions on the ecological edge, as well as biotope switches. These processes are important for understanding the evolution of extremophiles; moreover, they have implications for the emergence of novel fungal pathogens.

Ecological samples of fungal associations pose particular challenges for nucleic acid extraction due to the presence of several genomes. Thorough examination of the samples prior to extraction is important to assess the risks of contamination. If manual separation of symbionts or their axenic cultivation is not feasible, symbiont-specific primers can be applied in PCR experiments. A basic protocol is suggested here which can be optimized for specific applications.

We present a method for rapid genetic analysis of small amounts of fungal material. Sterile glass slides, sufficiently small to fit in a standard PCR tube, were placed on agar inside a Petri dish. After a few days, fungal cultures start to overgrow the glass slides. Glass slides with attached mycelium were harvested, analysed microscopically, and placed into a standard PCR tube. Conserved primers flanking the ITS regions of rDNA repeat were used in a direct PCR with the fungal material. Sequence data were generated to be included in phylogenetic analyses to investigate the relationships of the studied mycorrhizal fungi from orchids. The mycelium attached to glass slides was also used for an in situ hybridization experiment using fluorescent labelled oligonucleotides as probes. Fluorescent signal was found throughout the cytoplasm when a probe specific to a site in the nuclear small subunit rRNA is used.