Fungal-fungal and fungal-bacterial interactions in aquatic decomposer communities: bacteria promote fungal diversity

Fungi produce a variety of extracellular enzymes, making recalcitrant substrates bioavailable. Thus, fungi are central for the decomposition of dead organic matter such as leaf litter. Despite their ecological importance, our understanding of relationships between fungal species diversity and ecosystem functioning is limited, especially with regard to aquatic habitats. Moreover, fungal interactions with other groups of microorganisms such as bacteria are rarely investigated. This lack of information may be attributed to methodological limitations in tracking the biomass of individual fungal species in communities, impeding a detailed assessment of deviations from the overall performance expected from the sum of individual species' performances, so-called net diversity effects (NDEs). We used fungal species-specific biomolecular tools to target fungal-fungal and fungal-bacterial interactions on submerged leaves using four cosmopolitan aquatic fungal species and a stream microbial community dominated by bacteria. In microcosms, we experimentally manipulated fungal diversity and bacterial absence/presence and assessed functional performances and fungal community composition after 14 d of incubation. Fungal community data were used to evaluate NDEs on leaf colonization. The individual fungal species were functionally distinct and fungal cultures were on average more efficient than the bacterial culture. In absence of bacteria, NDEs correlated with growth rate (negatively) and genetic divergence (positively), but were predominantly negative, suggesting that higher fungal diversity led to a lower colonization success (niche overlap). In both absence and presence of bacteria, the overall functional performances of the communities were largely defined by their composition (i.e., no interactions at the functional level). In the presence of bacteria, NDEs correlated with genetic divergence (positively) and were largely positive, suggesting higher fungal diversity stimulated colonization (niche complementarity). This stimulation may be driven by a bacteria-induced inhibition of fungal growth, alleviating competition among fungi. Resulting feedback loops eventually promote fungal coexistence and synergistic interactions. Nonetheless, overall functional performances are reduced compared to bacteria-free cultures. These findings highlight the necessity to conduct future studies, investigating biodiversity-ecosystem functioning relationships using artificial systems, without exclusion of key organisms naturally co-occurring in the compartment of interest. Otherwise, study outcomes might not reflect true ecological relationships and ultimately misguide conservation strategies

The Evolution of the Satratoxin and Atranone Gene Clusters of Stachybotrys chartarum

Stachybotrys chartarum is frequently isolated from damp building materials or improperly stored animal forage. Human and animal exposure to the secondary metabolites of this mold is linked to severe health effects. The mutually exclusive production of either satratoxins or atranones defines the chemotypes A and S. Based upon the genes (satratoxin cluster, SC1-3, sat or atranone cluster, AC1, atr) that are supposed to be essential for satratoxin and atranone production, S. chartarum can furthermore be divided into three genotypes: the S-type possessing all sat- but no atr-genes, the A-type lacking the sat- but harboring all atr-genes, and the H-type having only certain sat- and all atr-genes. We analyzed the above-mentioned gene clusters and their flanking regions to shed light on the evolutionary relationship. Furthermore, we performed a deep re-sequencing and LC-MS/MS (Liquid chromatography–mass spectrometry) analysis. We propose a first model for the evolution of the S. chartarum genotypes. We assume that genotype H represents the most ancient form. A loss of the AC1 and the concomitant acquisition of the SC2 led to the emergence of the genotype S. According to our model, the genotype H also developed towards genotype A, a process that was accompanied by a loss of SC1 and SC3

Environmentally relevant fungicide levels modify fungal community composition and interactions but not functioning

Aquatic hyphomycetes (AHs), a group of saprotrophic fungi adapted to submerged leaf litter, play key functional roles in stream ecosystems as decomposers and food source for higher trophic levels. Fungicides, controlling fungal pathogens, target evolutionary conserved molecular processes in fungi and contaminate streams via their use in agricultural and urban landscapes. Thus fungicides pose a risk to AHs and the functions they provide. To investigate the impacts of fungicide exposure on the composition and functioning of AH communities, we exposed four AH species in monocultures and mixed cultures to increasing fungicide concentrations (0, 5, 50, 500, and 2500 mg/L). We assessed the biomass of each species via quantitative real-time PCR. Moreover, leaf decomposition was investigated. In monocultures, none of the species was affected at environmentally relevant fungicide levels (5 and 50 mg/L). The two most tolerant species were able to colonize and decompose leaves even at very high fungicide levels (>= 500 mg/L), although less efficiently. In mixed cultures, changes in leaf decomposition reflected the response pattern of the species most tolerant in monocultures. Accordingly, the decomposition process may be safeguarded by tolerant species in combination with functional redundancy. In all fungicide treatments, however, sensitive species were displaced and interactions between fungi changed from complementarity to competition. As AH community composition determines leaves' nutritional quality for consumers, the data suggest that fungicide exposures rather induce bottom-up effects in food webs than impairments in leaf decomposition. (C) 2021 The Author(s). Published by Elsevier Ltd

A multigene phylogeny toward a new phylogenetic classification of Leotiomycetes

Fungi in the class Leotiomycetes are ecologically diverse, including mycorrhizas, endophytes of roots and leaves, plant pathogens, aquatic and aero-aquatic hyphomycetes, mammalian pathogens, and saprobes. These fungi are commonly detected in cultures from diseased tissue and from environmental DNA extracts. The identification of specimens from such character-poor samples increasingly relies on DNA sequencing. However, the current classification of Leotiomycetes is still largely based on morphologically defined taxa, especially at higher taxonomic levels. Consequently, the formal Leotiomycetes classification is frequently poorly congruent with the relationships suggested by DNA sequencing studies. Previous class-wide phylogenies of Leotiomycetes have been based on ribosomal DNA markers, with most of the published multi-gene studies being focussed on particular genera or families. In this paper we collate data available from specimens representing both sexual and asexual morphs from across the genetic breadth of the class, with a focus on generic type species, to present a phylogeny based on up to 15 concatenated genes across 279 specimens. Included in the dataset are genes that were extracted from 72 of the genomes available for the class, including 10 new genomes released with this study. To test the statistical support for the deepest branches in the phylogeny, an additional phylogeny based on 3156 genes from 51 selected genomes is also presented. To fill some of the taxonomic gaps in the 15-gene phylogeny, we further present an ITS gene tree, particularly targeting ex-type specimens of generic type species. A small number of novel taxa are proposed: Marthamycetales ord. nov., and Drepanopezizaceae and Mniaeciaceae fams. nov. The formal taxonomic changes are limited in part because of the ad hoc nature of taxon and specimen selection, based purely on the availability of data. The phylogeny constitutes a framework for enabling future taxonomically targeted studies using deliberate specimen selection. Such studies will ideally include designation of epitypes for the type species of those genera for which DNA is not able to be extracted from the original type specimen, and consideration of morphological characters whenever genetically defined clades are recognized as formal taxa within a classification

Taxonomic annotation of public fungal ITS sequences from the built environment - A report from an April 10-11, 2017 workshop (Aberdeen, UK)

The UNITE database community gratefully acknowledges support from the Alfred P. Sloan Foundation. HN and CW gratefully acknowledges financial support from Stiftelsen Olle Engkvist Byggmästare, Stiftelsen Lars Hiertas Minne, Kapten Carl Stenholms Donationsfond, and Birgit och Birger Wålhströms Minnesfond. CW gratefully acknowledges a Marie Skłodowska-Curie post doctoral grant from the ERC. Leho Tedersoo is gratefully acknowledged for providing helpful feedback on an earlier draft of this manuscript.Peer reviewedPublisher PD

Entwicklung und Anwendung ribosomal gerichteter Hybridisierungssonden und Phylogenie aquatischer Pilze

Das Ziel der Arbeit war die molekulare phylogenetische Charakterisierung aquatischer Pilze, die Evaluierung und Optimierung der Fluoreszenz in situ Methode (FISH) für filamentöse Pilze sowie die Entwicklung taxonspezifischer Oligonukleotidsonden für aquatische Pilze und deren Anwendung in dem Tieflandfluß Elbe und dem österreichischen Gebirgsbach Oberer Seebach. In der Elbe war innerhalb dreier Jahre mit elf verschiedenen Spezies nur eine geringe Diversität an aquatischen Pilzen nachweisbar. Die Mehrzahl der aus der Elbe isolierten Pilze waren den Bodenpilzen oder durch die Luft verbreiteten Arten zuzuordnen. Fixierte Schaumproben aus der Elbe wiesen mit durchschnittlich 3,75 Konidien pro 10 ml sehr niedrige Konidienzahlen auf, während in Schaumproben aus dem Oberen Seebach 5635 (Herbst) und 2654 Konidien (Sommer) pro 10 ml gefunden wurden. Während zwei dreiwöchigen Probenahmen am Oberen Seebach wurden 36 verschiedene Spezies aquatischer Pilze nachgewiesen. Die phylogenetische Analyse unterschiedlicher ribosomaler DNS-Regionen von elf Spezies bestätigt einen polyphyletischen Ursprung aquatischer Pilze. Auf der Basis von 18S rRNS-Sequenzdaten wurden neun Spezies (Tetracladium marchalianum, Lemonniera terrestris, L. aquatica, Varicosporium elodeae, Tricladium angulatum, T. splendens, Alatospora acuminata, Anguillospora crassa und A. furtiva) in die Klasse der Leotiomycetes eingeordnet. Zwei Spezies wurden in die Klasse der Sordariomycetes (Heliscus lugdunensis) bzw. der Dothideomycetes (Anguillospora longissima) eingeordnet. Die Analysen der ITS und LSU-Sequenzbereiche stellten T. marchalianum in die Nähe der Ordnung Helotiales. Auf der Basis von ITS-Sequenzdaten wurden die sensu stricto und sensu lato Morphotypen von A. acuminata separiert. Die Ergebnisse der ITS-Sequenzanalysen von T. splendens und A. crassa weisen auf eine enge Verwandtschaft beider Arten untereinander und zu dem marinen Pilz Zalerion varium (Helotiales) hin. T. angulatum wurde in ITS-Analysen den Helotiales zugeordnet und zeigt enge Beziehungen zur Familie Hyaloscyphaceae. Während A. crassa der Ordnung Helotiales näher steht, wurde A. longissima in die Ordnung Pleosporales eingeordnet. Hier zeigte A. longissima eine nähere Verwandtschaft zu Arten der Gattung Lophiostoma als zu Arten der vermuteten Teleomorph-Gattung Massarina. Folglich muß die Gattung Anguillospora auch aufgrund der Ergebnisse der Sequenzanalysen überarbeitet werden. Für die phylogenetischen Analysen und die Entwicklung taxonspezifischer Oligonukleotidsonden wurde das ARB Softwareprogramm an die Bearbeitung von pilzlichen Sequenzdaten angepaßt. Die bestehende Datenbank wurde um umfangreiche Datensätze von 18S, ITS und 28S rRNS- Pilzsequenzen erweitert. Die neu entwickelten Sonden FUN1429 und MY1574, spezifisch für einen breiten phylogenetischen Bereich der Eumycota, die gattungspezifische Sonde TCLAD1395 (Tetracladium) sowie die artspezifischen Sonden ALacumi1698 (A. acuminata), TRIang322 (T. angulatum), Alongi340 (A. longissima) und Hlug1698 (H. lugdunensis) sind gegen die 18S rRNS gerichtet. Die artspezifischen Sonden für T. marchalianum (TmarchB10, TmarchC1_1, TmarchC1_2) und A. longissima (AlongiB16) sind 28S rRNS gerichtet. Die Spezifität aller neu entwickelten Sonden konnte durch computergestützte Sequenzanalyse und in situ Hybridisierungen gegen Zielorganismen und Vertreter anderer phylogenetischer Gruppen bestätigt werden. Umfangreiche Testserien ergaben, daß die durch die Sonden vermittelten Fluoreszenzsignale sowohl qualitativ als auch quantitativ durch die Fixierungsmethode, das Myzelalter, unterschiedliche Stoffwechselaktivität verschiedener Strukturen im Myzel, Autofluoreszenzen, die Mikroskopiertechnik sowie die Permeabilität der Pilzzellwände wesentlich beeinflußt werden. Durch Markierung der Sonden mit roten Fluoreszenzfarbstoffen konnte die natürliche Autofluoreszenz der Pilze im Bereich des grünen Lichts umgangen werden. Mit steigendem Alter des Myzels erhöhte sich in der Regel auch die Intensität der Autofluoreszenz. Die Autofluoreszenz von Organismen und Substraten konnte durch Verwendung von Natriumborhydrid reduziert werden. Bei Verwendung von konfokaler Laser-Scanning-Mikroskopie wurden Fluoreszenz-Emissionen außerhalb der Fokusebene minimiert und die dreidimensionale Darstellung von Pilzen auf Blättern ermöglicht. Die Intensität der Sondensignale war positiv korreliert mit dem metabolischen Status unterschiedlicher Strukturen im Myzelkörper. Die Durchlässigkeit der Pilzzellwände für Oligonukleotidsonden konnte durch eine Behandlung mit Chitinase verbessert werden. Erheblich konnte die Intensität sowie die Reproduzierbarkeit der Sondensignale durch den Einsatz der neu entwickelten Electroporation-FISH Methode gesteigert werden. Durch Einsatz der Sonden MY1574 und Hlug1698 konnten stoffwechselaktive Pilze in der Elbe nachgewiesen werden. Ebenso wurden Pilze aus dem Oberen Seebach auf Blättern und Polyethylen-Objektträgern mit den neu entwickelten, pilzspezifischen Gensonden nachgewiesen.The aim of the study was the molecular phylogenetic characterisation of aquatic hyphomycetes, the evaluation of the factors influencing their accessibility for fluorescence in situ hybridisation and the application of newly developed taxon specific rRNA-targeted oligonucleotide probes in the German lowland river Elbe and the Austrian alpine 2nd order stream Oberer Seebach. During 24 sampling campaigns in three years performed in the Elbe river, with only eleven different species a remarkable low diversity of aquatic hyphomycetes could be distinguished while the majority of fungi observed were terrestrial and airborne fungi. Conidial numbers in fixed foam samples of 3.75 10 ml -1 (autumn) were extremely low in the Elbe river compared to conidial numbers of 5635 10 ml -1 (autumn) and 2654 10 ml -1 (summer) in foam samples from the Oberer Seebach. During two short term, three week campaigns 36 different species of aquatic hyphomycetes could be observed and isolated from different substrates in the Oberer Seebach. Phylogenetic analyses of sequences of different rDNA regions of 11 species of aquatic hyphomycetes confirmed the assumption of a polyphyletic origin of aquatic hyphomycetes. On the basis of 18S rDNA data, nine species (Tetracladium marchalianum, Lemonniera terrestris, L. aquatica, Varicosporium elodeae, Tricladium angulatum, T. splendens, Alatospora acuminata, Anguillospora crassa, and A. furtiva) were placed in the Ascomycota class Leotiomycetes. The species Heliscus lugdunensis and Anguillospora longissima were placed in the classes Sordariomycetes and Dothideomycetes, respectively. In ITS and LSU sequence analyses T. marchalianum showed most likely phylogenetic affinities to the Helotiales. On the basis of ITS sequence data, two morphotypes of A. acuminata were separated into two clades containing the sensu lato and the sensu stricto type, respectively. T. splendens showed close relationships to A. crassa and to the marine hyphomycete Zalerion varium (Helotiales) in ITS analyses. T. angulatum is related to the Helotiales and showed close relation to members of the family Hyaloscyphaceae as revealed from ITS analyses. A. crassa was placed most likely within the Helotiales. In contrast A. longissima is a member of the Pleosporales with closer relation to the genus Lophiostoma than to the suspected teleomorph genus Massarina. Thus the genus Anguillospora has to be revised. For the phylogenetic analyses and subsequent probe design, the ARB software environment for sequence data was adapted to properly work with fungi by incorporating 18S rRNA and 28S rRNA secondary structure information and extending and generating 18S rRNA, ITS and 28S rRNA sequence databases. Newly developed 18S rRNA-targeted taxon-specific probes termed FUN1429, MY1574 are specific for a wide range of Eumycota. The genus specific probe TCLAD1395 (Tetracladium) as well as the species specific probes ALacumi1698 (A. acuminata), TRIang322 (T. angulatum), Alongi340 (A. longissima) and Hlug1698 (H. lugdunensis) are targeted against the 18S rRNA. The probes TmarchB10, TmarchC1_1, TmarchC1_2 and AlongiB16, specific for T. marchalianum and A. longissima, respectively, are targeted on the 28S rRNA. The specificity of all newly designed probes was successfully tested in whole fungal hybridisations against target and non-target organisms. The fixation method, the fungal inherent and substrate mediated autofluorescence, the imaging technique, the age of the mycelium, the different mycelial parts, and the permeability of fungal cell walls were shown to be the most obvious influencing factors for the detection of fungal FISH signals. Autofluorescence scans revealed that many freshwater fungi showed inherent fluorescence in the green visible light spectrum. This could be successfully overcome by use of probe labels emitting red fluorescence. Generally, autofluorescence intensities increased with the age of the fungal mycelium. Bleaching of autofluorescing organisms and substrates with sodiumborohydrid was useful for enhanced probe signal detection. Employing epifluorescence microscopy, fungal FISH signals were often insufficient to detect due to high background noise. In contrast, confocal laser scanning microscopy significantly enhanced the detection of fungal FISH signals. Additionally, the spatial distribution of freshwater fungi on leaves could be documented more clearly by CLSM. The FISH signal intensity and rRNA content resembling the metabolical status of different structures of the mycelium were positively correlated. The cell wall permeability could be enhanced by chitinase treatment. However, the most reliable results for FISH signal detection were yielded by employing the Electroporation-FISH method newly developed in this study. Application of the probes MY1574 and Hlug1698 yielded FISH signals of metabolically active hyphae in the Elbe river. In the Oberer Seebach aquatic hyphomycetes were successfully detected on PE slides, leaves and in germination experiments using the set of newly developed fungal FISH probes

In Situ Detection of Freshwater Fungi in an Alpine Stream by New Taxon-Specific Fluorescence In Situ Hybridization Probes▿

New rRNA-targeting oligonucleotide probes permitted the fluorescence in situ hybridization (FISH) identification of freshwater fungi in an Austrian second-order alpine stream. Based on computer-assisted comparative sequence analysis, nine taxon-specific probes were designed and evaluated by whole-fungus hybridizations. Oligonucleotide probe MY1574, specific for a wide range of Eumycota, and the genus (Tetracladium)-specific probe TCLAD1395, as well as the species-specific probes ALacumi1698 (Alatospora acuminata), TRIang322 (Tricladium angulatum), and Alongi340 (Anguillospora longissima), are targeted against 18S rRNA, whereas probes TmarchB10, TmarchC1_1, TmarchC1_2, and AlongiB16 are targeted against the 28S rRNA of Tetracladium marchalianum and Anguillospora longissima, respectively. After 2 weeks and 3 months of exposure of polyethylene slides in the stream, attached germinating conidia and growing hyphae of freshwater fungi were accessible for FISH. Growing hyphae and germinating conidia on leaves and in membrane cages were also visualized by the new FISH probes

Production of Satratoxin G and H Is Tightly Linked to Sporulation in Stachybotrys chartarum

Stachybotrys chartarum is a toxigenic fungus that is frequently isolated from damp building materials or improperly stored forage. Macrocyclic trichothecenes and in particular satratoxins are the most potent mycotoxins known to be produced by this fungus. Exposure of humans or animals to these secondary metabolites can be associated with severe health problems. To assess the pathogenic potential of S. chartarum isolates, it is essential to cultivate them under conditions that reliably promote toxin production. Potato dextrose agar (PDA) was reported to be the optimal nutrition medium for satratoxin production. In this study, the growth of S. chartarum genotype S strains on PDA from two manufacturers led to divergent results, namely, well-grown and sporulating cultures with high satratoxin concentrations (20.8 ± 0.4 µg/cm2) versus cultures with sparse sporulation and low satratoxin production (0.3 ± 0.1 µg/cm2). This finding is important for any attempt to identify toxigenic S. chartarum isolates. Further experiments performed with the two media provided strong evidence for a link between satratoxin production and sporulation. A comparison of three-point and one-point cultures grown on the two types of PDA, furthermore, demonstrated an inter-colony communication that influences both sporulation and mycotoxin production of S. chartarum genotype S strains

Potentials and limitations of quantification of fungi in freshwater environments based on PLFA profiles

Aquatic fungi are increasingly recognized for their contribution to carbon cycling in aquatic ecosystems, both as saprotrophs and parasites. Their quantification in mixed communities is crucial to assess their ecological significance but remains challenging. We characterized the phospholipid-derived fatty acid (PLFA) composition of fifteen aquatic fungal isolates from Chytridiomycota (chytrids) and Dikarya. Additionally, we identified PLFA patterns of chytrids infecting phytoplankton and their zoospores. PLFA composition of zoospores was highly similar among different taxa, but were distinct from their respective sporangial life-stage. Finally, we applied a fatty acid-based Bayesian mixed model (FASTAR) and tested its potential to quantify fungi in complex mixtures with bacteria and phytoplankton using PLFA profiles. While the quantification of chytrid biomass in low quantities was rather imprecise, the model predicted the contribution of filamentous fungi and other components with fair accuracy, supporting the suitability of this approach to quantify fungal biomass in aquatic environments