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70 records · Page 4

The endohyphal microbiome: current progress and challenges for scaling down integrative multi-omic microbiome research

Abstract As microbiome research has progressed, it has become clear that most, if not all, eukaryotic organisms are hosts to microbiomes composed of prokaryotes, other eukaryotes, and viruses. Fungi have only recently been considered holobionts with their own microbiomes, as filamentous fungi have been found to harbor bacteria (including cyanobacteria), mycoviruses, other fungi, and whole algal cells within their hyphae. Constituents of this complex endohyphal microbiome have been interrogated using multi-omic approaches. However, a lack of tools, techniques, and standardization for integrative multi-omics for small-scale microbiomes (e.g., intracellular microbiomes) has limited progress towards investigating and understanding the total diversity of the endohyphal microbiome and its functional impacts on fungal hosts. Understanding microbiome impacts on fungal hosts will advance explorations of how “microbiomes within microbiomes” affect broader microbial community dynamics and ecological functions. Progress to date as well as ongoing challenges of performing integrative multi-omics on the endohyphal microbiome is discussed herein. Addressing the challenges associated with the sample extraction, sample preparation, multi-omic data generation, and multi-omic data analysis and integration will help advance current knowledge of the endohyphal microbiome and provide a road map for shrinking microbiome investigations to smaller scales.

59 BASIC BIOLOGICAL SCIENCES↗

Quantitative stable isotope probing (qSIP) and cross-domain networks reveal bacterial-fungal interactions in the hyphosphere

Interactions between fungi and bacteria have the potential to substantially influence soil carbon dynamics in soil, but we have yet to fully identify these interactions and partners in their natural environment. In this study, we stacked two powerful methods, 13 C quantitative stable isotope probing (qSIP) and cross-domain co-occurrence network, to identify interacting fungi and bacteria in a California grassland soil. We used in-field whole plant 13 CO 2 labeling along with sand-filled ingrowth bags (that trap fungi and hyphae-associated bacteria) to amplify the signal of fungal-bacterial interactions, separate from the bulk soil background. We found a total of 54 bacterial ASVs and 9 fungal OTUs that were significantly 13 C-enriched. These were saprotrophic and biotrophic fungi, and motile, sometimes predatory bacteria. Among these, 70% of all 13 C-enriched bacteria identified were motile. Notably, we detected fungal-bacterial network links between a fungal OTU of the genus Alternaria and several bacterial ASVs of the genera Bacteriovorax, Mucilaginibacter, and Flavobacterium, providing empirical evidence of their direct interactions through C exchange. We observed a strong positive co-occurrence pattern between predatory bacteria of the phylum Bdellovibrionota and fungal OTUs, suggesting the transfer of C across the soil food web. To date, our ability to associate microbial co-occurrence network patterns with biological interactions is limited, but the incorporation of qSIP allowed us to more precisely detect interacting partners by narrowing in on the taxa that were actively incorporating plant-fixed, fungal-transported labeled substrates. Together, these approaches can help build a mechanistic understanding of the complex nature of fungal-bacterial interactions in soil.

59 BASIC BIOLOGICAL SCIENCES↗

Monitoring the impact of confinement on hyphal penetration and fungal behavior

Through their expansive mycelium network, soil fungi alter the physical arrangement and chemical composition of their local environment. This can significantly impact bacterial distribution and nutrient transport and can play a dramatic role in shaping the rhizosphere around a developing plant. However, direct observation and quantitation of such behaviors is extremely difficult due to the opacity and complex porosity of the soil microenvironment. In this study, we demonstrate the development and use of an engineered microhabitat to visualize fungal growth in response to varied levels of confinement. Microfluidics were fabricated using photolithography and conventional soft lithography, assembled onto glass slides, and prepared to accommodate fungal cultures. Selected fungal strains across three phyla (Ascomycota: Morchella sextalata , Fusarium falciforme ; Mucoromycota: Linnemannia elongata , Podila minutissima , Benniella ; Basidiomycota: Laccaria bicolor , and Serendipita sp.) were cultured within microhabitats and imaged using time-lapse microscopy to visualize development at the mycelial level. Fungal hyphae of each strain were imaged as they penetrated through microchannels with well-defined pore dimensions. The hyphal penetration rates through the microchannels were quantified via image analysis. Other behaviors, including differences in the degree of branching, peer movement, and tip strength were also recorded for each strain. Our results provide a repeatable and easy-to-use approach for culturing fungi within a microfluidics platform and for visualizing the impact of confinement on hyphal growth and other fungal behaviors pertinent to their remodeling of the underground environment.

Guo, Yi-Syuan (ORCID:0000000340142225)↗

Testing mechanisms of how mycorrhizal associations affect forest soil carbon and nitrogen cycling (Final Technical Report)

Trees are in a symbiotic partnership with mycorrhizal fungi in which they provide the fungi with carbon from photosynthesis and the fungi provide the trees with nutrients and water. In temperate forests, the vast majority of trees form symbioses with one of two types of mycorrhizal fungi—arbuscular mycorrhizal (AM) fungi or ectomycorrhizal (EcM) fungi. These fungi differ in their morphology, hyphal length, and nutrient acquisition strategies. Many studies have found systematic differences in soil organic matter and nitrogen availability between forest stands dominated by AM-associating trees versus EcM-associating trees. For instance, there is a larger proportion of organic matter that is mineral-associated, more available nitrogen, and lower soil carbon to nitrogen ratios in AM forest stands relative to EcM forests stands. However, the mechanisms driving these patterns are not known, which complicates our ability to model soil organic matter dynamics in forested ecosystems. The main objective of this research was to understand the degree to which the observed differences in soil C and N dynamics between AM and EcM dominated forests are driven by tree traits like litter decomposability and root exudation versus mycorrhizal fungal nutrient acquisition strategies. We investigated these mechanisms using observations and targeted experiments and incorporated this knowledge into a process-based soil organic matter model. The observational studies compared the importance of leaf litter decomposability versus fungal identity on soil organic matter processes. We found that often fungal identity and traits were more important drivers of soil organic matter patterns than leaf litter decomposability. We ran two novel experiments: 1) a growth chamber experiment across four EcM and four AM tree species using a 13 C-labeled atmosphere to trace seedling-derived C into hyphae, the rhizosphere, and soil; and 2) an in situ decomposition experiment of six different 13 C and 15 N labeled litters that ranged in decomposability incubated across a gradient of EcM dominance at three sites that capture important variation in climate, soils, and forest species composition. In the first experiment, we found no significant differences of seedling mycorrhizal association on soil carbon sequestration over a growing season, but we did find that mycorrhizal association affected rhizodeposition with EcM-associating seedlings depositing more carbon in response to increased nitrogen availability. The decomposition experiment is still ongoing, but thus far, we have found slower litter decomposition in only one of three EcM-dominated forests which suggests that differences between AM- and EcM-dominated forests depend on the environmental context and identity of the EcM fungi. Lastly, we explicitly incorporated mycorrhizal processes into the Carbon, Organisms, Rhizosphere, and Protection in the Soil Environment (CORPSE) model creating Myco-CORPSE. By including the different nutrient acquisition strategies of AM and EcM fungi, we explored the conditions under which EcM fungi can slow decomposition rates and lead to greater soil organic carbon accumulation compared to AM fungi. We found that the effect of EcM fungi was highly context dependent and that EcM fungi decreased decomposition in colder forests with recalcitrant litter inputs and when they produced oxidases and necromass-degrading enzymes. Our research highlights the importance of fungal nutrient acquisition in driving soil organic matter patterns and the need to move beyond the AM-EcM dichotomy to consider the identity and traits of the specific fungi participating in the symbiosis. Overall, this research has resulted in six, peer-reviewed published papers in journals such as Global Change Biology, Ecology (2), Soil Biology and Biochemistry, and Ecosystems (2). There are at least two more papers in progress on this research including one that was recently submitted to Global Change Biology.

54 ENVIRONMENTAL SCIENCES↗

KS4A-Omics1.0_FspDS682

Soil fungi facilitate the translocation of inorganic nutrients from soil minerals to other microorganisms and plants. This ability is particularly advantageous in impoverished soils, because fungal mycelial networks can bridge otherwise spatially disconnected and inaccessible nutrient hotspots. However, the molecular mechanisms underlying fungal mineral weathering and transport through soil remains poorly understood. Here, we addressed this knowledge gap by directly visualizing nutrient acquisition and transport through fungal hyphae in a mineral doped soil micromodel using a multimodal imaging approach. Here, we observed how a representative of common saprotrophic soil fungi, Fusarium sp. DS 682, exhibited a mechanosensory response (thigmotropism) around obstacles and through pore spaces (~12 μm) in the presence of minerals.This study establishes the significance of fungal biology and nutrient translocation mechanisms in maintaining fungal growth under water and nutrient limitations in a soil-like microenvironment, using a high-throughput multi-omic analysis approach. Data package KS4A-Omics.1.0_FspDS682 (Publication: Fungal Mineral Weathering Mechanisms Revealed Through Direct Molecular Visualization) contents reported here are the first version (1.0) and contain pre- and post-processed data using high throughput data capture technologies for multi-omic analysis and integration, this data package contains raw and post-processed experimental data for X-Ray Absorption Near Edge Structure Spectroscopy (XANES/XRF), Optical Microscopy, Proteomics, Scanning Electron Microscope (SEM), Time-of-Flight Secondary Ion Mass Spectroscopy (ToF-SIMS), X-Ray Diffraction Spectroscopy (XRD) files, and X- Ray Photoelectron Spectroscopy (XPS) using EMSL capabilities. This data package DOI contains a comprehensive collection of high-throughput multi-omics data and process metadata catalog. Support files include additional data download contents “Read Me” with dataset descriptor information and data source method application ontologies (see data dictionary section). Reported data download content is structured for compliance with reported guidelines provided by community standard initiatives and publisher stakeholder policies supporting FAIR data principles.

47 OTHER INSTRUMENTATION↗

Hyaloscypha finlandica Metabolome Repository

This repository provides the curated data tables, manuscript figure and table exports, dependency records, and workflow scripts supporting an integrated comparative genomics and untargeted LC-MS/MS metabolomics analysis of Hyaloscypha finlandica strain PMI 746, a root-associated dark septate endophyte of poplar. The repository includes genome-mining summaries from antiSMASH, FunBGCeX, BGC-Prophet, and BiG-SCAPE; processed metabolomics inputs; metabolite annotation evidence; statistical outputs; and publication-facing figures and tables. Raw LC-MS/MS spectra, full genome/protein downloads, and large generated tool outputs are referenced through public archive/accession records and are not stored in Git.

59 BASIC BIOLOGICAL SCIENCES↗

Phylogeny and character evolution in the Dacrymycetes , and systematics of Unilacrymaceae and Dacryonaemataceae fam. nov.

We present a multilocus phylogeny of the class Dacrymycetes, based on data from the 18S, ITS, 28S, RPB1,RPB2,TEF-1α, 12S, andATP6DNA regions, with c. 90 species including the types of most currently accepted genera. A variety of methodological approaches was used to infer phylogenetic relationships among the Dacrymycetes, from a supermatrix strategy using maximum likelihood and Bayesian inference on a concatenated dataset, to coalescence-based calculations, such as quartet-based summary methods of independent single-locus trees, and Bayesian integration of single-locus trees into a species tree under the multispecies coalescent. We evaluate for the first time the taxonomic usefulness of some cytological phenotypic characters, i.e., vacuolar contents (vacuolar bodies and lipid bodies), number of nuclei of recently discharged basidiospores, and pigments, with especial emphasis on carotenoids. These characters, along with several others traditionally used for the taxonomy of this group (basidium shape, presence and morphology of clamp connections, morphology of the terminal cells of cortical/marginal hyphae, presence and degree of ramification of the hyphidia), are mapped on the resulting phylogenies and their evolution through the class Dacrymycetes discussed. Our analyses reveal five lineages that putatively represent five different families, four of which are accepted and named. Three out of these four lineages correspond to previously circumscribed and published families (Cerinomycetaceae, Dacrymycetaceae, and Unilacrymaceae), and one is proposed as the new family Dacryonaemataceae. Provisionally, only a single order, Dacrymycetales, is accepted with in the class. Furthermore, the systematics of the two smallest families, Dacryonaemataceae and Unilacrymaceae, are investigated to the species level, using coalescence-based species delimitation on multilocus DNA data, and a detailed morphological study including morphometric analyses of the basidiospores. Three species are accepted in Dacryonaema, the type, Da. rufum, the newly combined Da. Macnabbii (basionym Dacrymyces macnabbii), and a new species named Da. macrosporum. Two species are accepted in Unilacryma, the new U. bispora, and the type, U. unispora, the latter treated in a broad sense pending improved sampling across the Holarctic.

59 BASIC BIOLOGICAL SCIENCES↗

The use of models to predict potential contamination aboard orbital vehicles

A model of fungal growth on air-exposed, nonnutritive solid surfaces, developed for utilization aboard orbital vehicles is presented. A unique feature of this testable model is that the development of a fungal mycelium can facilitate its own growth by condensation of water vapor from its environment directly onto fungal hyphae. The fungal growth rate is limited by the rate of supply of volatile nutrients and fungal biomass is limited by either the supply of nonvolatile nutrients or by metabolic loss processes. The model discussed is structurally simple, but its dynamics can be quite complex. Biofilm accumulation can vary from a simple linear increase to sustained exponential growth, depending on the values of the environmental variable and model parameters. The results of the model are consistent with data from aquatic biofilm studies, insofar as the two types of systems are comparable. It is shown that the model presented is experimentally testable and provides a platform for the interpretation of observational data that may be directly relevant to the question of growth of organisms aboard the proposed Space Station.

Boraas, Martin E.↗

Distribution of inorganic species in two Antarctic cryptoendolithic microbial communities

Chemical differences were noted between two Antarctic cryptoendolithic (hidden within rock) microenvironments colonized by different microbial communities. Microenvironments dominated by cyanobacteria (BPC) had a higher pH (pH 7-8) than those dominated by lichen (LTL) (pH 4.5-5.5). In order to understand the interactions between the microbiota and the inorganic environment, the inorganic environment was characterized. Water-soluble, carbonate-bound, metal-oxide, organically bound, and residual inorganic species were sequentially extracted from rock samples by chemical means. Each fraction was then quantified using inductively coupled plasma atomic emission spectrometry. BPC contained much more water-soluble and carbonate-bound Ca and Mg than LTL. Metal-oxide species of Al, Fe, and Mn were more abundant in LTL than BPC. Metal oxides appeared to be mobilized (in the order Mn > Fe > Al) from the LTL lichen zone but remained immobile in BPC sandstone. The distribution of K and P bound to metal oxide reflected the distribution of iron oxide in LTL, an indication of the importance of iron in controlling the availability of nutrients in this ecosystem. Metal oxides in turn were likely controlled or influenced by organic matter associated with the lichen community. Despite overall depletion of Fe, Al, and K in the lichen zone, SEM X-ray analysis showed that they were enriched in fungal hyphae. Water-soluble P was present despite the presence of metal oxides, which sequester phosphate. This has biological relevance since P is an essential nutrient.

NASA Program Exobiology↗

Colonization by Cladosporium spp. of painted metal surfaces associated with heating and air conditioning systems

Cladosporium cladosporioides and C. hebarum colonized painted metal surfaces of covering panels and register vents of heating, air conditioning and ventilation systems. Hyphae penetrated the paint film and developed characteristic conidiophores and conidia. The colonies were tightly appressed to the metal surface and conidia were not readily detectable via standard air sampling procedures.

NASA Program Environmental Health↗

Mutualism in a Reduced Gravity Environment (MuRGE)

Mutualism in a Reduced Gravity Environment (MuRGE) is a ground research study to determine the feasibility of assessing fungi-plant (Piriformospora indica-Arabidopsis thaliana) interactions in microgravity. Seeds from the plant Arabiddospsis thaliana (At) will be grown in the presence of Piriformospora indica (Pi) an endophytic Sebacinacae family fungus. Pi is capable of colonizing the roots of a wide variety of plant species, including non-mycorrhizal hosts like At, and promoting plant growth similarly to AMF (arbusuclar mychorrizal fungi) unlike most AMF, Pi is not an obligate plant symbiont and can be grown in the absence of a host. In the presence of a suitable plant host, Pi can attach to and colonize root tips. Interaction visualization is accomplished with strong autofluorescence in the roots, followed by root colonization via fungal hyphae, and chlamydospore production. Increased root growth can be observed even before root colonization is detectable. In addition, Pi chlamydospores generated from axenic culture in microgravity will be used to inoculate roots of At grown in 1g to determine the effect of microgravity upon the inherent virulence or beneficial effects. Based on recent reports of increased virulence of S. typhimurium, P. aeruginosa, and S. Pneumoniae in reduced gravity, differences in microbial pathogenic responses and host plant systemic acquired resistance are expected. The focus of this project within MuRGE involved the development P. indica culture media evaluation and microscopy protocol development. High, clean spore harvest yields for the detection of fungi-plant interactions microscopically was the immediate goal of this experiment.

Haire, Timothy C.↗

Lipid biosynthesis and abiotic stress resilience in photosynthetic organisms

This application describes consortium between fungi and algae, where the algae are incorporated within hyphae of the fungi. The fungi, the algae, or both can be modified to express heterologous lipid synthesizing enzymes. Incorporation of algae into fungi facilitates harvesting of the algae and products produced by the consortia. Such consortia are robust. For example, the fungi and algae can symbiotically provide nutrients to each other and are tolerant of environmental stresses.

Bonito, Gregory↗

Fine-scale cation dynamics and control by Rhodonia placenta and Pleurotus ostreatus during wood decay

Fungi are the primary decomposers of wood across the globe. They redistribute the vast pool of wood carbon to other parts of the carbon cycle using mechanisms that also have intriguing potential in bioconversion and biotechnology applications. To make a living in the relatively sparse microenvironment of wood, these filamentous fungi must import many of their needs, including cations. Understanding the timing of cation import can help us validate the functions of cations in wood decay and create a clearer understanding of these complex wood degradation mechanisms. In this study, we resolved cation timing dynamics across space for two fungi (brown rot fungus Rhodonia placenta and white rot fungus Pleurotus ostreatus) using a wood wafer system. We found some expected patterns of the cation dynamics for both fungi, and a clear role for iron at the early stages of brown rot decay. On the other hand, the lack of an increase in manganese during initial white rot decay was surprising. Unexpectedly, we also saw a spike in copper during early brown rot decay that demands more investigation as a potential player in the brown rot mechanism.

59 BASIC BIOLOGICAL SCIENCES↗

Substrate quality drives fungal necromass decay and decomposer community structure under contrasting vegetation types

Fungal mycelium is increasingly recognized as a central component of soil biogeochemical cycling, yet our current understanding of the ecological controls on fungal necromass decomposition is limited to single sites and vegetation types. By deploying common fungal necromass substrates in a temperate oak savanna and hardwood forest in the midwestern USA, we assessed the generality of the rate at which high- and low-quality fungal necromass decomposes; further, we investigated how the decomposer ‘necrobiome’ varies both across and within sites under vegetation types dominated by either arbuscular or ectomycorrhizal plants. In this work, the effects of necromass quality on decay rate were robust to site and vegetation type differences, with high-quality fungal necromass decomposing, on average, 2.5 times faster during the initial stages of decay. Across vegetation types, bacterial and fungal communities present on decaying necromass differed from bulk soil microbial communities and were influenced by necromass quality. Moulds, yeasts and copiotrophic bacteria consistently dominated the necrobiome of high-quality fungal substrates. Synthesis. We show that regardless of differences in decay environments, high-quality fungal substrates decompose faster and support different types of decomposer micro-organisms when compared with low-quality fungal tissues. These findings help to refine our theoretical understanding of the dominant factors affecting fast cycling components of soil organic matter and the microbial communities associated with rapid decay.

59 BASIC BIOLOGICAL SCIENCES↗

Dataset for "A Microfluidic Spore Chamber for Long-Term Imaging of Single-Spore Hyphal Development"

Understanding the life cycle of fungal spores is essential for elucidating their roles in pathogenesis, dispersal, and survival. However, studying spore development under controlled, spatially defined conditions remains challenging. Here, we present the Spore Chamber, a custom-built microfluidic platform engineered for parallel trapping and long-term imaging of individual spores under defined media conditions, enabling real-time visualization of hyphal development. Using Aspergillus fumigatus as a model organism, we demonstrate that sparse trapping of individual spores within size-matched trap geometries enables long-term time-lapse imaging of key developmental stages, including germination, polarized hyphal elongation, branching, and conidiophore formation. To assess the device’s capacity to resolve morphogenetic responses to exogenous signals, we introduced lipochitooligosaccharides (LCOs) and short-chain chitooligosaccharides (COs). Rhizobium-derived, non-sulfated LCO (nsLCO) mixtures induced enhanced secondary branching (hyperbranching), a response not previously reported in A. fumigatus under these signal conditions, to our knowledge, whereas sulfated LCOs and CO4 did not significantly alter branching patterns. In addition, long-term confinement and imaging revealed rare developmental morphologies previously described primarily in mutant strains, including split conidiophore formation, elongated phialides, microcyclic conidiation, and chlamydospore development. Together, these results establish the Spore Chamber as a targeted microfluidic platform for single-spore phenotyping and long-term developmental analysis, with applications in fungal biology, chemical signaling studies, and host–microbe interaction research. Videos of the observed phenomena are included in this data set.

59 BASIC BIOLOGICAL SCIENCES↗