Photochemical defense as trait of fungi from Cortinarius subgenus Dermocybe.

The photobiological activity of ten colorful species belonging to subgenus Dermocybe of the basidiomycete genus Cortinarius was investigated. Extracts of all species produced singlet oxygen and are thus photoactive. Pigment analysis was performed and showed similarities of the anthraquinone pigments across the species in dependency to their respective pigmentation types. Detailed content analysis of the pigments in the whole agaricoid fruiting body compared to the three different tissue types (pileus, stipe, and lamellae) revealed that the pigments emodin, dermocybin, and dermorubin, as well as their respective glycosides, are enhanced in the gills. In an independent experiment, the gills were shown to be the most photoactive tissues of the fruiting body. Photobiological experiments with invertebrates (i.e., glassworm Chaoborus crystallinus) proved a phototoxic effect of the methanolic extract of the red blood webcap (Cortinarius sanguineus var. aurantiovaginatus). This work adds further evidence to a common photobiological trait in Cortinarius subgenus Dermocybe and underpins the possibility of a photochemical defense mechanism in fungi.

Growth, morphology, and formation of cinnabarin in Pycnoporus cinnabarinus in relation to different irradiation spectra.

BACKGROUND: The demand for natural pigments in general, and for fungi-derived pigments in particular, is constantly rising. Wood-decomposing fungi represent a promising source for natural pigments and they are usually easy to cultivate in pure culture. One of them, i.e., Pycnoporus cinnabarinus, offers a highly interesting spectrum of bioactivity, partly due to the formation of the orange-red pigment cinnabarin. However, apart from a few studies addressing its diverse potential biotechnological applications, there is still a large gap of knowledge concerning the influence of light on the formation of cinnabarin. The aim of this work was to investigate the effect of different irradiations on the cinnabarin content, the growth, and the morphology of three different P. cinnabarinus strains. We used highly standardized irradiation conditions and cultivation techniques in combination with newly developed methods for the extraction and direct quantification of cinnabarin. RESULTS: Red, green, blue, and UV-A irradiation (mean irradiance Ee = 1.5 ± 0.18 W m-2) had considerable effects on the growth and colony appearance of all three P. cinnabarinus strains tested. The cinnabarin content determined was, thus, dependent on the irradiation wavelength applied, allowing strain-specific thresholds to be defined. Irradiation with wavelengths below this strain-specific threshold corresponded to a lower cinnabarin content, at least at the intensity applied. The orange-red pigment appeared by light microscopy as incrusted extracellular plaques present on the hyphal walls. Highly efficient vegetative propagation occurred by arthroconidia, and we observed the tendency that this asexual reproduction was (i) most frequent in the dark but (ii) never occurred under UV-A exposure. CONCLUSION:  This study highlights a differential photo-dependence of growth, morphology, and cinnabarin formation in P. cinnabarinus. This confirms that it is advisable to consider the wavelength of the light used in future biotechnological productions of natural pigments.

Highlighting the Phototherapeutical Potential of Fungal Pigments in Various Fruiting Body Extracts with Informed Feature-Based Molecular Networking.

Fungal pigments are characterized by a diverse set of chemical backbones, some of which present photosensitizer-like structures. From the genus Cortinarius, for example, several biologically active photosensitizers have been identified leading to the hypothesis that photoactivity might be a more general phenomenon in the kingdom Fungi. This paper aims at testing the hypothesis. Forty-eight fruiting body-forming species producing pigments from all four major biosynthetic pathways (i.e., shikimate-chorismate, acetate-malonate, mevalonate, and nitrogen heterocycles) were selected and submitted to a workflow combining in vitro chemical and biological experiments with state-of-the-art metabolomics. Fungal extracts were profiled by high-resolution mass spectrometry and subsequently explored by spectral organization through feature-based molecular networking (FBMN), including advanced metabolite dereplication techniques. Additionally, the photochemical properties (i.e., light-dependent production of singlet oxygen), the phenolic content, and the (photo)cytotoxic activity of the extracts were studied. Different levels of photoactivity were found in species from all four metabolic groups, indicating that light-dependent effects are common among fungal pigments. In particular, extracts containing pigments from the acetate-malonate pathway, e.g., extracts from Bulgaria inquinans, Daldinia concentrica, and Cortinarius spp., were not only efficient producers of singlet oxygen but also exhibited photocytotoxicity against three different cancer cell lines. This study explores the distribution of photobiological traits in fruiting body forming fungi and highlights new sources for phototherapeutics.

Fungal Anthraquinone Photoantimicrobials Challenge the Dogma of Cationic Photosensitizers.

The photoantimicrobial potential of four mushroom species (i.e., Cortinarius cinnabarinus, C. holoxanthus, C. malicorius, and C. sanguineus) was explored by studying the minimal inhibitory concentrations (MIC) via a light-modified broth microdilution assay based on the recommended protocols of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The extracts were tested against Candida albicans, Escherichia coli, and Staphylococcus aureus under blue (λ = 428 and 478 nm, H = 30 J/cm2) and green light (λ = 528 nm, H = 30 J/cm2) irradiation. Three extracts showed significant photoantimicrobial effects at concentrations below 25 µg/mL. Targeted isolation of the major pigments from C. sanguineus led to the identification of two new potent photoantimicrobials, one of them (i.e., dermocybin) being active against S. aureus and C. albicans under green light irradiation [PhotoMIC530 = 39.5 µM (12.5 µg/mL) and 2.4 µM (0.75 µg/mL), respectively] and the other one (i.e., emodin) being in addition active against E. coli in a low micromolar range [PhotoMIC428 = 11.1 µM (3 µg/mL)]. Intriguingly, dermocybin was not (photo)cytotoxic against the three tested cell lines, adding an additional level of selectivity. Since both photoantimicrobials are not charged, this discovery shifts the paradigm of cationic photosensitizers.

Optimized isolation of 7,7'-biphyscion starting from Cortinarius rubrophyllus, a chemically unexplored fungal species rich in photosensitizers.

Mushrooms such as the dermocyboid Cortinarius rubrophyllus are characterized by strikingly colorful fruiting bodies. The molecular dyes responsible for such colors recently experienced a comeback as photoactive compounds with remarkable photophysical and photobiological properties. One of them-7,7'-biphyscion-is a dimeric anthraquinone that showed promising anticancer effects in the low nanomolar range under blue-light irradiation. Compared to acidic anthraquinones, 7,7'-biphyscion was more efficiently taken up by cells and induced apoptosis after photoactivation. However, seasonal collection of mushrooms producing this compound, low extraction yields, and tricky fungal identification hamper further developments to the clinics. To bypass these limitations, we demonstrate here an alternative approach utilizing a precursor of 7,7'-biphyscion, i.e., the pre-anthraquinone flavomannin-6,6'-dimethyl ether, which is abundant in many species of the subgenus Dermocybe. Controlled oxidation of the crude extract significantly increased the yield of 7,7'-biphyscion by 100%, which eased the isolation process. We also present the mycochemical and photobiological characterization of the yet chemically undescribed species, i.e. C. rubrophyllus. In total, eight pigments (1-8) were isolated, including two new glycosylated anthraquinones (1 and 2). Light-dependent generation of singlet oxygen was detected for the first time for emodin-1-O-ß-D-glucopyranoside (3) [photophysical measurement: Φ∆ = 0.11 (CD3OD)]. Furthermore, emodin (7) was characterized as promising compound in the photocytotoxicity assay with EC50-values in the low micromolar range under irradiation against cells of the cancer cell lines AGS, A549, and T24.

A convenient separation strategy for fungal anthraquinones by centrifugal partition chromatography.

As recently shown, some fungal pigments exhibit significant photoactivity turning them into promising agents for the photodynamic treatment of microbial infections or malignant diseases. In the present study, a separation strategy for fungal anthraquinones was developed based on centrifugal partition chromatography. A suitable method was explored employing a methanolic extract of the fruiting bodies of Cortinarius sanguineus (Agaricales, Basidiomycota). An excellent fractionation was achieved using a biphasic solvent system comprising chloroform/ethyl acetate/methanol/water/acetic acid (3:1:3:2:1, v/v/v/v/v) operating in ascending mode. Experiments on an analytical scale with extracts of closely related Cortinarius species exhibited broad applicability of the devised system. Up to six pigments could be purified directly from the crude extract. Preparative-scale fractionation of the methanol extracts of C. malicorius and C. sanguineus demonstrated that up-scaling was possible without compromising selectivity.

Habitat specialisation controls ectomycorrhizal fungi above the treeline in the European Alps.

Alpine habitats are one of the most vulnerable ecosystems to environmental change, however, little information is known about the drivers of plant-fungal interactions in these ecosystems and their resilience to climate change. We investigated the influence of the main drivers of ectomycorrhizal (EM) fungal communities along elevation and environmental gradients in the alpine zone of the European Alps and measured their degree of specialisation using network analysis. We sampled ectomycorrhizas of Dryas octopetala, Bistorta vivipara and Salix herbacea, and soil fungal communities at 28 locations across five countries, from the treeline to the nival zone. We found that: (1) EM fungal community composition, but not richness, changes along elevation, (2) there is no strong evidence of host specialisation, however, EM fungal networks in the alpine zone and within these, EM fungi associated with snowbed communities, are more specialised than in other alpine habitats, (3) plant host population structure does not influence EM fungal communities, and (4) most variability in EM fungal communities is explained by fine-scale changes in edaphic properties, like soil pH and total nitrogen. The higher specialisation and narrower ecological niches of these plant-fungal interactions in snowbed habitats make these habitats particularly vulnerable to environmental change in alpine ecosystems.

Emerging from the ice-fungal communities are diverse and dynamic in earliest soil developmental stages of a receding glacier.

We used amplicon sequencing and isolation of fungi from in-growth mesh bags to identify active fungi in three earliest stages of soil development (SSD) at a glacier forefield (0-3, 9-14, 18-25 years after retreat of glacial ice). Soil organic matter and nutrient concentrations were extremely low, but the fungal diversity was high [220 operational taxonomic units (OTUs)/138 cultivated OTUs]. A clear successional trend was observed along SSDs, and species richness increased with time. Distinct changes in fungal community composition occurred with the advent of vascular plants. Fungal communities of recently deglaciated soil are most distinctive and rather similar to communities typical for cryoconite or ice. This indicates melting water as an important inoculum for native soil. Moreover, distinct seasonal differences were detected in fungal communities. Some fungal taxa, especially of the class Microbotryomycetes, showed a clear preference for winter and early SSD. Our results provide insight into new facets regarding the ecology of fungal taxa, for example, by showing that many fungal taxa might have an alternative, saprobial lifestyle in snow-covered, as supposed for a few biotrophic plant pathogens of class Pucciniomycetes. The isolated fungi include a high proportion of unknown species, which can be formally described and used for experimental approaches.

Lanostane Triterpenes from Gloeophyllum odoratum and Their Anti-Influenza Effects.

In an in vitro screening for anti-influenza agents from European polypores, the fruit body extract of Gloeophyllum odoratum dose-dependently inhibited the cytopathic effect of the H3N2 influenza virus A/Hong Kong/68 (HK/68) in Madin Darby canine kidney cells with a 50% inhibitory concentration (IC50) of 15 µg/mL, a noncytotoxic concentration. After a chromatographic work-up, eight lanostane triterpenes (1: -8: ) were isolated and their structures were elucidated based on high-resolution electrospray ionization mass spectrometry analyses, and one- and two-dimensional nuclear magnetic resonance experiments. Constituents 1: (gloeophyllin K) and 2: (gloeophyllin L) are reported here for the first time, and compounds 5: , 7: , and 8: have not been described for the investigated fungal material so far. The highest activity was determined for trametenolic acid B (3: ) against HK/68 and the 2009 pandemic H1N1 strain A/Jena/8178/09 with IC50 values of 14 and 11 µM, respectively. In a plaque reduction assay, this compound was able to bind to cell-free viruses and to neutralize their infectivity.

Spring and early summer species of Cortinarius, subgenus Telamonia, section Colymbadini and /Flavobasilis, in the mountains of western North America.

Seven species of Cortinarius, subgenus Telamonia, section Colymbadini and /Flavobasilis, are reported from conifer forests in the mountains of western North America. They typically produce basidiomes in the spring and summer. Only one species, C. colymbadinus, is widespread, occurring in Europe and western North America, but to date not reported from California. Cortinarius bridgei, C. flavobasilis, C. rumoribrunsi, C. vernalishastensis, and C. vernalisierraensis are new species. The first two are found throughout the western mountains, whereas the latter three thus far are known only from California. Cortinarius ahsii, a common species in the Rocky Mountains and Pacific Northwest, also has not been recorded from California.

Myrmecridium hiemale sp. nov. from snow-covered alpine soil is the first eurypsychrophile in this genus of anamorphic fungi.

Myrmecridium hiemale sp. nov. was isolated from snow-covered alpine bare soil and is described as the first eurypsychrophilic species of this genus of filamentous fungi. Colony growth temperature experiments were carried out in the range 4-37 °C. Morphological characteristics and colony appearance were in accordance with characteristics typical for Myrmecridium, but M. hiemale does not grow at temperatures of 25 °C and above. Sequence analyses of the internal transcribed spacer and LSU rRNA D1/D2 regions indicated that the strain in question represents a distinct taxon within the genus Myrmecridium (Myrmecridiaceae, Sordariomycetes, Ascomycota). The type strain of M. hiemale is CBS 141017T(=JMRC 12083T). A morphological description is provided, and a key is presented for the currently known taxa of Myrmecridium, a group of interesting fungi that are either saprobes or plant endophytes.

Finding a robust strain for biomethanation: anaerobic fungi (Neocallimastigomycota) from the Alpine ibex (Capra ibex) and their associated methanogens.

Anaerobic fungi occupy the rumen and digestive tract of herbivores, where they play an important role in enzymatic digestion of lignocellulosic and cellulosic substrates, i.e. organic material that their hosts are unable to decompose on their own. In this study we isolated anaerobic fungi from a typical alpine herbivore, the Alpine ibex (C. ibex). Three fungal strains, either as pure culture (ST2) or syntrophic co-culture with methanogens (ST3, ST4) were successfully obtained and morphologically characterised by different microscopy- and staining-techniques and by rDNA ITS gene sequencing. The isolated fungi were identified as Neocallimastix frontalis (ST2) and Caecomyces communis (ST3 and ST4). We introduce a novel field of application for lactofuchsin-staining, combined with confocal laser scanning microscopy. This approach proved as an effective method to visualize fungal structures, especially in the presence of plant biomass, generally exhibiting high autofluorescence. Moreover, we could demonstrate that fungal morphology is subject to changes depending on the carbon source used for cultivation. Oxygen tolerance was confirmed for both, C. communis-cultures for up to three, and for the N. frontalis-isolate for up to 12 h, respectively. With PCR, FISH and an oligonucleotide microarray we found associated methanogens (mainly Methanobacteriales) for C. communis, but not for N. frontalis.

Fungal and bacterial communities and their associations in snow-free and snow covered (sub-)alpine Pinus cembra forest soils.

BACKGROUND: In Europe, Pinus cembra forests cover subalpine and alpine areas and they are of high conservational and ecological relevance. These forests experience strong seasonality with alternating snow-free and snow covered periods. Although P. cembra is known for mycorrhization and mycorrhizae usually involve fungi, plants and bacteria, the community compositions of fungi and bacteria and their associations in (sub-)alpine P. cembra forests remain vastly understudied. Here, we studied the fungal and bacterial community compositions in three independent (sub-)alpine P. cembra forests and inferred their microbial associations using marker gene sequencing and network analysis. We asked about the effect of snow cover on microbial compositions and associations. In addition, we propose inferring microbial associations across a range of filtering criteria, based on which we infer well justified, concrete microbial associations with high potential for ecological relevance that are typical for P. cembra forests and depending on snow cover. RESULTS: The overall fungal and bacterial community structure was comparable with regards to both forest locations and snow cover. However, occurrence, abundance, and diversity patterns of several microbial taxa typical for P. cembra forests differed among snow-free and snow covered soils, e.g. Russula, Tetracladium and Phenoliphera. Moreover, network properties and microbial associations were influenced by snow cover. Here, we present concrete microbial associations on genus and species level that were repeatedly found across microbial networks, thereby confirming their ecological relevance. Most importantly, ectomycorrhizal fungi, such as Basidioascus, Pseudotomentella and Rhizopogon, as well as saprobic Mortierella changed their bacterial association partners depending on snow cover. CONCLUSION: This is the first study researching fungal-bacterial associations across several (sub-)alpine P. cembra forests. The poorly investigated influence of snow cover on soil fungi and bacteria, especially those mycorrhizing P. cembra roots, but also saprobic soil organisms, underlines the relevance of forest seasonality. Our findings highlight that the seasonal impact of snow cover has significant consequences for the ecology of the ecosystem, particularly in relation to mycorrhization and nutrient cycling. It is imperative to consider such effects for a comprehensive understanding of the functioning resilience and responsiveness of an ecosystem.

Revised taxon definition in European Cortinarius subgenus Dermocybe based on phylogeny, chemotaxonomy, and morphology.

Cortinarius (Fr.) Fr. is one of the most species-rich genera in the Agaricales (Basidiomycota). Cortinarius subgen. Dermocybe (Fr.) Trog includes brightly coloured Cortinarii with anthraquinone pigments. The chemotaxonomic approach has always been as important as classical methods for species definition of Dermocybe and helped to improve overall species concepts. However, some species concepts within this group remain unclear. We therefore address this topic based on a combined phylogenetic, morphological, and pigment-chemical approach. For this, sequence data, HPLC-MS pigment profiles and spore sizes were included were included to obtain a better resolution of taxa. The study was based on 173 recent collections and 12 type specimens. A total of 117 rDNA ITS sequences were produced from the collections in this study, 102 sequences were retrieved from databases. We could detect and clearly delimit 19 Dermocybe species occurring in central European habitats, from which 16 are discussed in detail. Additionally, we grouped the detected anthraquinone pigments into four groups. This detailed analysis of dermocyboid Cortinarius species occurring in a restricted number of habitat types confirmed our hypothesis that species diversity is much higher than currently assumed. This high diversity is blurred by too wide and incorrect species concepts of several classical species like C. croceus and C. cinnamomeus. Molecular and chemotaxonomical studies carried out together with careful phenotypical analyses resulted in a good differentiation of species. A key is presented for these taxa to allow a better identification of Cortinarius subgenus Dermocybe spp. occurring in Central Europe mainly in the alpine range. Supplementary Information: The online version contains supplementary material available at 10.1007/s11557-024-01959-z.

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.

Co-cultivation of Mortierellaceae with Pseudomonas helmanticensis affects both their growth and volatilome.

Volatile organic compounds (VOCs) might mediate microbial interactions, especially in spatially structured environments, such as soil. However, the variety and specificity of VOC production are poorly understood. Here, we studied 25 Mortierellaceae strains belonging to the genera Linnemannia and Entomortierella in both pure and co-culture with Pseudomonas helmanticensis under laboratory conditions. We analysed both the fungal growth depending on co-cultivation and the cultures' volatilomes applying proton-transfer-reaction time-of-flight and gas chromatography-mass spectrometry (PTR-ToF-MS and GC-MS). In a strain-specific manner, we found the fungi's radial growth rate and colony morphology affected by the presence of P. helmanticensis. The fungus seemed to generally reduce the bacterial growth. The volatilomes of the fungal and bacterial pure and co-cultures were diverse. While the fungi frequently consumed VOCs, P. helmanticensis produced a higher diversity and amount of VOCs than any fungal strain. Our results support that both the pure and co-culture volatilomes are taxonomically conserved. Taken together, our data supports the relevance of VOCs in Mortierellaceae-P. helmanticensis interaction. We also discuss individual VOCs that appear relevant in the interaction.

Fungal growth and biomass development is boosted by plants in snow-covered soil.

Soil microbial communities follow distinct seasonal cycles which result in drastic changes in processes involving soil nutrient availability. The biomass of fungi has been reported to be highest during winter, but is fungal growth really occurring in frozen soil? And what is the effect of plant cover on biomass formation and on the composition of fungal communities? To answer these questions, we monitored microbial biomass N, ergosterol, and the amount of fungal hyphae during summer and winter in vegetated and unvegetated soils of an alpine primary successional habitat. The winter fungal communities were identified by rDNA ITS clone libraries. Winter soil temperatures ranged between -0.6°C and -0.1°C in snow-covered soil. We found distinct seasonal patterns for all biomass parameters, with highest biomass concentrations during winter in snow-covered soil. The presence of plant cover had a significant positive effect on the amount of biomass in the soil, but the type of plant cover (plant species) was not a significant factor. A mean hyphal ingrowth of 5.6 m g(-1) soil was detected in snow-covered soil during winter, thus clearly proving fungal growth during winter in snow-covered soil. Winter fungal communities had a typical species composition: saprobial fungi were dominating, among them many basidiomycete yeasts. Plant cover had no influence on the composition of winter fungal communities.

The photosensitizer emodin is concentrated in the gills of the fungus Cortinarius rubrophyllus.

The colorful agaricoid fruiting bodies of dermocyboid Cortinarii owe their magnificent hue to a mixture of anthraquinone (AQ) pigments. Recently, it was discovered that some of these fungal anthraquinones have an impressive photopharmacological effect. The question, therefore, arises as to whether these pigments are also of ecological or functional significance. According to the optimal defense hypothesis, toxic molecules should be enriched in spore-producing structures, such as the gills of agarics. To test this hypothesis, we studied the distribution of fungal AQs in the fruiting body of Cortinarius rubrophyllus. The fungus belongs to the well-studied Cortinarius subgenus Dermocybe but has not been chemically characterized. Here, we report on the pigment profile of this beautiful fungus and focus on the distribution of anthraquinone pigments in the fruiting body for the first time. Here it is statistically confirmed that the potent photosensitizer emodin is significantly enriched in the gills. Furthermore, we show that the extract is photoactive against cancer cells and bacteria.

Targeted isolation of photoactive pigments from mushrooms yielded a highly potent new photosensitizer: 7,7'-biphyscion.

Pigments of fungi are a fertile ground of inspiration: they spread across various chemical backbones, absorption ranges, and bioactivities. However, basidiomycetes with strikingly colored fruiting bodies have never been explored as agents for photodynamic therapy (PDT), even though known photoactive compound classes (e.g., anthraquinones or alkaloids) are used as chemotaxonomic markers. In this study, we tested the hypothesis that the dyes of skin-heads (dermocyboid Cortinarii) can produce singlet oxygen under irradiation and thus are natural photosensitizers. Three photosensitizers based on anthraquinone structures were isolated and photopharmaceutical tests were conducted. For one of the three, i.e., (-)-7,7'-biphyscion (1), a promising photoyield and photocytotoxicity of EC50 = 0.064 µM against cancer cells (A549) was found under blue light irradiation (λexc = 468 nm, 9.3 J/cm2). The results of molecular biological methods, e.g., a viability assay and a cell cycle analysis, demonstrated the harmlessness of 1 in the dark and highlighted the apoptosis-inducing PDT potential under blue light irradiation. These results demonstrate for the first time that pigments of dermocyboid Cortinarii possess a so far undescribed activity, i.e., photoactivity, with significant potential for the field of PDT. The dimeric anthraquinone (-)-7,7'-biphyscion (1) was identified as a promising natural photosensitizer.