Christian Wurzbacher

Importance of Saprotrophic Freshwater Fungi for Pollen Degradation

Fungi and bacteria are the major organic matter (OM) decomposers in aquatic ecosystems. While bacteria are regarded as primary mineralizers in the pelagic zone of lakes and oceans, fungi dominate OM decomposition in streams and wetlands. Recent findings indicate that fungal communities are also active in lakes, but little is known about their diversity and interactions with bacteria. Therefore, the decomposer niche overlap of saprotrophic fungi and bacteria was studied on pollen (as a seasonally recurring source of fine particulate OM) by performing microcosm experiments with three different lake types. Special emphasis was placed on analysis of fungal community composition and diversity. We hypothesized that (I) pollen select for small saprotrophic fungi and at the same time for typical particle-associated bacteria; (II) fungal communities form specific free-living and attached sub-communities in each lake type; (III) the ratio between fungi or bacteria on pollen is controlled by the lakes chemistry. Bacteria-to-fungi ratios were determined by quantitative PCR (qPCR), and bacterial and fungal diversity were studied by clone libraries and denaturing gradient gel electrophoresis (DGGE) fingerprints. A protease assay was used to identify functional differences between treatments. For generalization, systematic differences in bacteria-to-fungi ratios were analyzed with a dataset from the nearby Baltic Sea rivers. High abundances of Chytridiomycota as well as occurrences of Cryptomycota and yeast-like fungi confirm the decomposer niche overlap of saprotrophic fungi and bacteria on pollen. As hypothesized, microbial communities consistently differed between the lake types and exhibited functional differences. Bacteria-to-fungi ratios correlated well with parameters such as organic carbon and pH. The importance of dissolved organic carbon and nitrogen for bacteria-to-fungi ratios was supported by the Baltic Sea river dataset. Our findings highlight the fact that carbon-to-nitrogen ratios may also control fungal contributions to OM decomposition in aquatic ecosystems.

Microbial diversity and community respiration in freshwater sediments influenced by artificial light at night

An increasing proportion of the Earths surface is illuminated at night. In aquatic ecosystems, artificial light at night (ALAN) may influence microbial communities living in the sediments. These communities are highly diverse and play an important role in the global carbon cycle. We combined field and laboratory experiments using sediments from an agricultural drainage system to examine how ALAN affects communities and alters carbon mineralization. Two identical light infrastructures were installed parallel to a drainage ditch before the start of the experiment. DNA metabarcoding indicated that both sediment communities were similar. After one was lit for five months (July–December 2012) we observed an increase in photoautotroph abundance (diatoms, Cyanobacteria) in ALAN-exposed sediments. In laboratory incubations mimicking summer and winter (six weeks each), communities in sediments that were exposed to ALAN for 1 year (July 2012–June 2013) showed less overall seasonal change compared with ALAN-naive sediments. Nocturnal community respiration was reduced in ALAN-exposed sediments. In long-term exposed summer-sediments, we observed a shift from negative to positive net ecosystem production. Our results indicate ALAN may alter sediment microbial communities over time, with implications for ecosystem-level functions. It may thus have the potential to transform inland waters to nocturnal carbon sinks.

Integrating chytrid fungal parasites into plankton ecology: research gaps and needs

Chytridiomycota, often referred to as chytrids, can be virulent parasites with the potential to inflict mass mortalities on hosts, causing e.g. changes in phytoplankton size distributions and succession, and the delay or suppression of bloom events. Molecular environmental surveys have revealed an unexpectedly large diversity of chytrids across a wide range of aquatic ecosystems worldwide. As a result, scientific interest towards fungal parasites of phytoplankton has been gaining momentum in the past few years. Yet, we still know little about the ecology of chytrids, their life cycles, phylogeny, host specificity and range. Information on the contribution of chytrids to trophic interactions, as well as co-evolutionary feedbacks of fungal parasitism on host populations is also limited. This paper synthesizes ideas stressing the multifaceted biological relevance of phytoplankton chytridiomycosis, resulting from discussions among an international team of chytrid researchers. It presents our view on the most pressing research needs for promoting the integration of chytrid fungi into aquatic ecology.

Distribution of acI-Actinorhodopsin genes in Baltic Sea salinity gradients indicates adaptation of facultative freshwater photoheterotrophs to brackish waters

Knowledge on Actinobacteria rhodopsin gene (actR) diversity and spatial distribution is scarce. The Baltic Sea is characterized by strong salinity gradients leading to the coexistence of marine and freshwater bacteria and hence is an ideal study area to elucidate the dispersion and phylogenetic affiliation of actR in dependence on salinity. ActR DGGE fingerprints in summer 2008 revealed between 3 and 19 distinct bands within a salinity range of 2.4-27 PSU. Environmental actR clone sequences were obtained from stations distributed along the whole salinity gradient. Overall, 20 different actR sequence groups (operational taxonomic units) were found, with up to 11 different ones per station. Phylogenetically, the actR sequences were predominantly (80%) affiliated with freshwater acI-Actinobacteria whose 16S rRNA gene accounted for 2-33% of total 16S rRNA genes in both the Bothnian Sea and central Baltic Sea. However, at salinities above 14 PSU, acI-16S rRNA gene accounted for less than 1%. In contrast, the diversity of actR remained high. Changes in actR gene diversity were significantly correlated with salinity, oxygen, silica or abundance of Synechococcus sp. Our results demonstrate a wide distribution of freshwater actR along the Baltic Sea salinity gradient indicating that some freshwater Actinobacteria might have adapted to higher salinities.

Environmental actinorhodopsin expression revealed by a new in situ filtration and fixation sampler

Freshwater Actinobacteria are an important and dominant group of bacterioplankton in most temperate freshwater systems. Recently, metagenomic studies discovered rhodopsin-like protein-coding sequences present in Actinobacteria which could be a decisive hint for their success in freshwater ecosystems. We analysed the diversity of actinorhodopsin (ActR) in Lake Stechlin (northern Germany) and assessed the actR expression profile during a diurnal cycle. We obtained 85 positive actR clones which could be subsequently grouped to 17 operational taxonomic units assuming a 90% sequence similarity. The phylogenetic analysis points to a close relationship of all obtained sequences to the acI lineage of Actinobacteria, forming six independent clusters. For the first time, we followed in situ transcription of actR in Lake Stechlin revealing a rather constitutive circadian gene expression. For analysing in situ expression patterns of functional genes in aquatic ecosystems, such as actR, we invented a new in situ filtration and fixation sampler (IFFS). The IFFS enables the representative investigation of microbial transcriptomes in any aquatic ecosystem at all water depths. The IFFS sampler is simple and inexpensive, and we provide all engineering plans for an easy rebuild. Consequently, our IFFS is suitable to reliably study expression of any known functional gene of any aquatic microorganism.

Effects of Light and Autochthonous Carbon Additions on Microbial Turnover of Allochthonous Organic Carbon and Community Composition

The fate of allochthonous dissolved organic carbon (DOC) in aquatic systems is primarily controlled by the turnover of heterotrophic bacteria. However, the roles that abiotic and biotic factors such as light and DOC release by aquatic primary producers play in the microbial decomposition of allochthonous DOC is not well understood. We therefore tested if light and autochthonous DOC additions would increase allochthonous DOC decomposition rates and change bacterial growth efficiencies and community composition (BCC). We established continuous growth cultures with different inocula of natural bacterial communities and alder leaf leachates (DOCleaf) with and without light exposure before amendment. Furthermore, we incubated DOCleaf together with autochthonous DOC from lysed phytoplankton cultures (DOCphyto). Our results revealed that pretreatments of DOCleaf with light resulted in a doubling of bacterial growth efficiency (BGE), whereas additions of DOCphyto or combined additions of DOCphyto and light had no effect on BGE. The change in BGE was not accompanied by shifts in the phylogenetic structure of the BCC, but BCC was influenced by the DOC source. Our results highlight that a doubling of BGE is not necessarily accompanied by a shift in BCC and that BCC is more strongly affected by resource properties.

Metaxa2 Diversity Tools: Easing microbial community analysis with Metaxa2

DNA sequencing has become an integrated part of microbial ecology, and taxonomic marker genes such as the SSU and LSU rRNA are frequently used to assess community structure. One solution for taxonomic community analysis based on shotgun metagenomic data is the Metaxa2 software, which can extract and classify sequence fragments belonging to the rRNA genes. This paper describes the Metaxa2 Diversity Tools, a set of new open-source software programs that extends the capabilities of the Metaxa2 software. These tools allow for better handling of data from multiple samples, improved species classifications, rarefaction analysis accounting for unclassified entries, and determination of significant differences in community composition of different samples. We demonstrate the performance of the software tools on rRNA data extracted from different shotgun metagenomes, and find the tools to streamline and improve the assessments of community diversity, particularly for samples from environments for which few reference genomes are available. Finally, we establish that our resampling algorithm for determining community dissimilarity is robust to differences in coverage depth, suggesting that it forms a complement to multidimensional visualization approaches for finding differences between communities. The Metaxa2 Diversity Tools are included in recent versions (2.1 and later) of Metaxa2 (http://microbiology.se/software/metaxa2/) and facilitate implementation of Metaxa2 within software pipelines for taxonomic analysis of environmental communities.

Improved detection and identification of aquatic fungi and chitin in aquatic environments

Chitin is a polymer of major importance in aquatic environments. We report on a new chitin-staining method for environmental samples. Major advantages of this method are (i) its high specificity and (ii) its suitability for combination with general stains, such as 4′,6-diamidino-2-phenylindole (DAPI), or taxa-specific methods, such as fluorescence in situ hybridization (FISH). This method lets aquatic mycologists explore basal fungal lineages directly in situ, identify their hosts, occurrence and evolutionary traits.

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

Abstract 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.

Metabarcoding-based fungal diversity on coarse and fine particulate organic matter in a first-order stream in Nova Scotia, Canada

Most streams receive substantial inputs of allochthonous organic material in the form of leaves and twigs (CPOM , coarse particulate organic matter). Mechanical and biological processing converts this into fine particulate organic matter (FPOM). Other sources of particles include flocculated dissolved matter and soil particles. Fungi are known to play a role in the CPOM conversion process, but the taxonomic affiliations of these fungi remain poorly studied. The present study seeks to shed light on the composition of fungal communities on FPOM and CPOM as assessed in a natural stream in Nova Scotia, Canada. Maple leaves were exposed in a stream for four weeks and their fungal community evaluated through pyrosequencing. Over the same period, four FPOM size fractions were collected by filtration and assessed. Particles had much lower ergosterol contents than leaves, suggesting major differences in the extent of fungal colonization. Pyrosequencing documented a total of 821 fungal operational taxonomic units (OTU), of which 726 were exclusive to particles and 47 to leaf samples. Most fungal phyla were represented, including yeast lineages (e.g., Taphrinaceae and Saccharomycotina), Basidiomycota, Chytridiomycota and Cryptomycota, but several classes of Pezizomycontina (Ascomycota) dominated. Cluster dendrograms clearly separated fungal communities from leaves and from particles. Characterizing fungal communities may shed some light on the processing pathways of fine particles in streams and broadens our view of the phylogenetic composition of fungi in freshwater ecosystems.