Elevated Fungicide and Nutrient Concentrations Change Structure but not Function of Aquatic Microbial Communities.

Leaf decomposition is a key process in stream ecosystems within forested catchments; it is driven by microbial communities, particularly fungi and bacteria. These microorganisms make nutrients and energy bound in leaves available for wider parts of the food web. Leaf-associated microorganisms are subjected to anthropogenic pressures, such as the increased exposure to nutrients and fungicides associated with land-use change. We assessed the sensitivity of leaf-associated microbial communities with differing exposure histories, namely, from pristine (P) streams, and streams impacted by wastewater (W) and agricultural run-off (vineyards; V). In the laboratory, microbial communities were exposed to elevated nutrient (NO3-N: 0.2-18.0 mg/L, PO4-P: 0.02-1.8 mg/L) and fungicide concentrations (sum concentration 0-300 µg/L) in a fully crossed 3 × 4 × 4-factorial design over 21 days. Leaf decomposition and exoenzyme activity were measured as functional endpoints, and fungal community composition and microbial abundance served as structural variables. Overall, leaf decomposition did not differ between fungicide treatments or exposure histories. Nonetheless, substantial changes in the fungal community composition were observed after exposure to environmentally relevant fungicide concentrations. Elevated nutrient concentrations assisted leaf decomposition, and the effect size depended on the exposure history. The observed changes in the fungal community composition support the principle of functional redundancy, with highly efficient decomposers maintaining leaf decomposition. Environ Toxicol Chem 2024;43:1300-1311. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Comparative genome analysis of the freshwater fungus Filosporella fistucella indicates potential for plant-litter degradation at cold temperatures.

Freshwater fungi play an important role in the decomposition of organic matter of leaf litter in rivers and streams. They also possess the necessary mechanisms to endure lower temperatures caused by habitat and weather variations. This includes the production of cold-active enzymes and antifreeze proteins. To better understand the physiological activities of freshwater fungi in their natural environment, different methods are being applied, and genome sequencing is one in the spotlight. In our study, we sequenced the first genome of the freshwater fungus Filosporella fistucella (45.7 Mb) and compared the genome with the evolutionary close-related species Tricladium varicosporioides (48.2 Mb). The genomes were annotated using the carbohydrate-active enzyme database where we then filtered for leaf-litter degradation-related enzymes (cellulase, hemicellulase, laccase, pectinase, cutinase, amylase, xylanase, and xyloglucanase). Those enzymes were analyzed for antifreeze properties using a machine-learning approach. We discovered that F. fistucella has more enzymes to participate in the breakdown of sugar, leaf, and wood than T. varicosporioides (855 and 719, respectively). Filosporella fistucella shows a larger set of enzymes capable of resisting cold temperatures than T. varicosporioides (75 and 66, respectively). Our findings indicate that in comparison with T. varicosporioides, F. fistucella has a greater capacity for aquatic growth, adaptability to freshwater environments, and resistance to low temperatures.

MediaDive: the expert-curated cultivation media database.

We present MediaDive (https://mediadive.dsmz.de), a comprehensive and expert-curated cultivation media database, which comprises recipes, instructions and molecular compositions of >3200 standardized cultivation media for >40 000 microbial strains from all domains of life. MediaDive is designed to enable broad range applications from every-day-use in research and diagnostic laboratories to knowledge-driven support of new media design and artificial intelligence-driven data mining. It offers a number of intuitive search functions and comparison tools, for example to identify media for related taxonomic groups and to integrate strain-specific modifications. Besides classical PDF archiving and printing, the state-of-the-art website allows paperless use of media recipes on mobile devices for convenient wet-lab use. In addition, data can be retrieved using a RESTful web service for large-scale data analyses. An internal editor interface ensures continuous extension and curation of media by cultivation experts from the Leibniz Institute DSMZ, which is interlinked with the growing microbial collections at DSMZ. External user engagement is covered by a dedicated media builder tool. The standardized and programmatically accessible data will foster new approaches for the design of cultivation media to target the vast majority of uncultured microorganisms.

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.

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.

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.

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 µg/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 µg/L). The two most tolerant species were able to colonize and decompose leaves even at very high fungicide levels (≥500 µg/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.

The Fungicide Tebuconazole Confounds Concentrations of Molecular Biomarkers Estimating Fungal Biomass.

Due to their ecological importance, fungi are suitable indicator organisms for anthropogenic stress. To estimate fungal biomass, the fungal membrane molecule ergosterol is often quantified as a proxy. Estimates based on ergosterol may, however, be distorted by exposure to demethylase inhibiting (DMI) fungicides, interfering with sterol synthesis. To test this hypothesis, we exposed ten fungal species to the DMI fungicide tebuconazole and measured concentrations of ergosterol and DNA per unit dry mass of the fungal hyphae. The latter served as alternative biomass proxy that is not specifically targeted by tebuconazole. Effects of tebuconazole on ergosterol concentrations were species-specific, while concentrations were on average reduced by 13%. In contrast, DNA concentrations were on average increased by 13%. We demonstrate that DMI fungicides - at close to field relevant levels - can distort fungal biomass estimation, complicating the use of this endpoint for environmental management.

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.

Long-read DNA metabarcoding of ribosomal RNA in the analysis of fungi from aquatic environments.

DNA metabarcoding is widely used to study prokaryotic and eukaryotic microbial diversity. Technological constraints limit most studies to marker lengths below 600 base pairs (bp). Longer sequencing reads of several thousand bp are now possible with third-generation sequencing. Increased marker lengths provide greater taxonomic resolution and allow for phylogenetic methods of classification, but longer reads may be subject to higher rates of sequencing error and chimera formation. In addition, most bioinformatics tools for DNA metabarcoding were designed for short reads and are therefore unsuitable. Here, we used Pacific Biosciences circular consensus sequencing (CCS) to DNA-metabarcode environmental samples using a ca. 4,500 bp marker that included most of the eukaryote SSU and LSU rRNA genes and the complete ITS region. We developed an analysis pipeline that reduced error rates to levels comparable to short-read platforms. Validation using a mock community indicated that our pipeline detected 98% of chimeras de novo. We recovered 947 OTUs from water and sediment samples from a natural lake, 848 of which could be classified to phylum, 397 to genus and 330 to species. By allowing for the simultaneous use of three databases (Unite, SILVA and RDP LSU), long-read DNA metabarcoding provided better taxonomic resolution than any single marker. We foresee the use of long reads enabling the cross-validation of reference sequences and the synthesis of ribosomal rRNA gene databases. The universal nature of the rRNA operon and our recovery of >100 nonfungal OTUs indicate that long-read DNA metabarcoding holds promise for studies of eukaryotic diversity more broadly.

A unique fungal strain collection from Vietnam characterized for high performance degraders of bioecological important biopolymers and lipids.

Fungal strains are abundantly used throughout all areas of biotechnology and many of them are adapted to degrade complex biopolymers like chitin or lignocellulose. We therefore assembled a collection of 295 fungi from nine different habitats in Vietnam, known for its rich biodiversity, and investigated their cellulase, chitinase, xylanase and lipase activity. The collection consists of 70 isolates from wood, 55 from soil, 44 from rice straw, 3 found on fruits, 24 from oil environments (butchery), 12 from hot springs, 47 from insects as well as 27 from shrimp shells and 13 strains from crab shells. These strains were cultivated and selected by growth differences to enrich phenotypes, resulting in 211 visually different fungi. DNA isolation of 183 isolates and phylogenetic analysis was performed and 164 species were identified. All were subjected to enzyme activity assays, yielding high activities for every investigated enzyme set. In general, enzyme activity corresponded with the environment of which the strain was isolated from. Therefore, highest cellulase activity strains were isolated from wood substrates, rice straw and soil and similar substrate effects were observed for chitinase and lipase activity. Xylanase activity was similarly distributed as cellulase activity, but substantial activity was also found from fungi isolated from insects and shrimp shells. Seven strains displayed significant activities against three of the four tested substrates, while three degraded all four investigated carbon sources. The collection will be an important source for further studies. Therefore representative strains were made available to the scientific community and deposited in the public collection of the Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig.

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

Recent DNA-based studies have shown that the built environment is surprisingly rich in fungi. These indoor fungi - whether transient visitors or more persistent residents - may hold clues to the rising levels of human allergies and other medical and building-related health problems observed globally. The taxonomic identity of these fungi is crucial in such pursuits. Molecular identification of the built mycobiome is no trivial undertaking, however, given the large number of unidentified, misidentified, and technically compromised fungal sequences in public sequence databases. In addition, the sequence metadata required to make informed taxonomic decisions - such as country and host/substrate of collection - are often lacking even from reference and ex-type sequences. Here we report on a taxonomic annotation workshop (April 10-11, 2017) organized at the James Hutton Institute/University of Aberdeen (UK) to facilitate reproducible studies of the built mycobiome. The 32 participants went through public fungal ITS barcode sequences related to the built mycobiome for taxonomic and nomenclatural correctness, technical quality, and metadata availability. A total of 19,508 changes - including 4,783 name changes, 14,121 metadata annotations, and the removal of 99 technically compromised sequences - were implemented in the UNITE database for molecular identification of fungi (https://unite.ut.ee/) and shared with a range of other databases and downstream resources. Among the genera that saw the largest number of changes were Penicillium, Talaromyces, Cladosporium, Acremonium, and Alternaria, all of them of significant importance in both culture-based and culture-independent surveys of the built environment.

The molecular phylogeny of aquatic hyphomycetes with affinity to the Leotiomycetes.

Aquatic hyphomycetes play a key role in decomposition of submerged organic matter and stream ecosystem functioning. We examined the phylogenetic relationships among various genera of aquatic hyphomycetes belonging to the Leotiomycetes (Ascomycota) using sequences of internal transcribed spacer (ITS) and large subunit (LSU) regions of rDNA generated from 42 pure cultures including 19 ex-types. These new sequence data were analyzed together with additional sequences from 36 aquatic hyphomycetes and 60 related fungi obtained from GenBank. Aquatic hyphomycetes, characterized by their tetraradiate or sigmoid conidia, were scattered in nine supported clades within the Helotiales (Leotiomycetes). Tricladium, Lemonniera, Articulospora, Anguillospora, Varicosporium, Filosporella, and Flagellospora are not monophyletic, with species from the same genus distributed among several major clades. The Gyoerffyella clade and the Hymenoscyphus clade accommodated species from eight and six different genera, respectively. Thirteen aquatic hyphomycete taxa were grouped in the Leotia-Bulgaria clade while twelve species clustered within the Hymenoscyphus clade along with several amphibious ascomycetes. Species of Filosporella and some species from four other aquatic genera were placed in the Ascocoryne-Hydrocina clade. It is evident that many aquatic hyphomycetes have relatives of terrestrial origin. Adaptation to colonize the aquatic environment has evolved independently in multiple phylogenetic lineages within the Leotiomycetes.

Two new Tricladium species from streams in Alaska.

Two new species of aquatic hyphomycetes in the genus Tricladium are described from streams in Alaska, USA. Both species were isolated from submerged decaying sedges. Tricladium kelleri has blackish colonies and typical tricladioid conidia formed on sympodial conidiogenous cells. Tricladium alaskense has conidia with fine elements and 0-4 lateral branches; conidia are formed on sympodial conidiogenous cells. The two species are compared to other species in the genus and related genera using morphological characters and/or rDNA sequencing data (ITS and 28S). Molecular phylogenetic analysis placed both species in the Helotiales.

Molecular techniques for pathogen identification and fungus detection in the environment.

Many species of fungi can cause disease in plants, animals and humans. Accurate and robust detection and quantification of fungi is essential for diagnosis, modeling and surveillance. Also direct detection of fungi enables a deeper understanding of natural microbial communities, particularly as a great many fungi are difficult or impossible to cultivate. In the last decade, effective amplification platforms, probe development and various quantitative PCR technologies have revolutionized research on fungal detection and identification. Examples of the latest technology in fungal detection and differentiation are discussed here.

Interactions between hyphosphere-associated bacteria and the fungus Cladosporium herbarum on aquatic leaf litter.

We investigated microbial interactions of aquatic bacteria associated with hyphae (the hyphosphere) of freshwater fungi on leaf litter. Bacteria were isolated directly from the hyphae of fungi from sedimented leaves of a small stream in the National Park "Lower Oder," Germany. To investigate interactions, bacteria and fungi were pairwise co-cultivated on leaf-extract medium and in microcosms loaded with leaves. The performance of fungi and bacteria was monitored by measuring growth, enzyme production, and respiration of mono- and co-cultures. Growth inhibition of the fungus Cladosporium herbarum by Ralstonia pickettii was detected on leaf extract agar plates. In microcosms, the presence of Chryseobacterium sp. lowered the exocellulase, endocellulase, and cellobiase activity of the fungus. Additionally, the conversion of leaf material into microbial biomass was retarded in co-cultures. The respiration of the fungus was uninfluenced by the presence of the bacterium.

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.