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At least 181 records · Page 10

HT-SIP: a semi-automated stable isotope probing pipeline identifies cross-kingdom interactions in the hyphosphere of arbuscular mycorrhizal fungi

Abstract Background Linking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive for this goal, Stable-isotope probing—SIP—remains among the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, actively growing microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated, high-throughput SIP (HT-SIP) pipeline to support well-replicated, temporally resolved amplicon and metagenomics experiments. We applied this pipeline to a soil microhabitat with significant ecological importance—the hyphosphere zone surrounding arbuscular mycorrhizal fungal (AMF) hyphae. AMF form symbiotic relationships with most plant species and play key roles in terrestrial nutrient and carbon cycling. Results Our HT-SIP pipeline for fractionation, cleanup, and nucleic acid quantification of density gradients requires one-sixth of the hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We applied HT-SIP to 13 C-AMF hyphosphere DNA from a 13 CO 2 plant labeling study and created metagenome-assembled genomes (MAGs) using high-resolution SIP metagenomics (14 metagenomes per gradient). SIP confirmed the AMF Rhizophagus intraradices and associated MAGs were highly enriched (10–33 atom% 13 C), even though the soils’ overall enrichment was low (1.8 atom% 13 C). We assembled 212 13 C-hyphosphere MAGs; the hyphosphere taxa that assimilated the most AMF-derived 13 C were from the phyla Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia-oxidizing archaeon genus Nitrososphaera . Conclusions Our semi-automated HT-SIP approach decreases operator time and improves reproducibility by targeting the most labor-intensive steps of SIP—fraction collection and cleanup. We illustrate this approach in a unique and understudied soil microhabitat—generating MAGs of actively growing microbes living in the AMF hyphosphere (without plant roots). The MAGs’ phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling.

59 BASIC BIOLOGICAL SCIENCES↗

Widespread bacterial diversity within the bacteriome of fungi

Abstract Knowledge of associations between fungal hosts and their bacterial associates has steadily grown in recent years as the number and diversity of examinations have increased, but current knowledge is predominantly limited to a small number of fungal taxa and bacterial partners. Here, we screened for potential bacterial associates in over 700 phylogenetically diverse fungal isolates, representing 366 genera, or a tenfold increase compared with previously examined fungal genera, including isolates from several previously unexplored phyla. Both a 16 S rDNA-based exploration of fungal isolates from four distinct culture collections spanning North America, South America and Europe, and a bioinformatic screen for bacterial-specific sequences within fungal genome sequencing projects, revealed that a surprisingly diverse array of bacterial associates are frequently found in otherwise axenic fungal cultures. We demonstrate that bacterial associations with diverse fungal hosts appear to be the rule, rather than the exception, and deserve increased consideration in microbiome studies and in examinations of microbial interactions.

59 BASIC BIOLOGICAL SCIENCES↗

Whole-Genome Comparisons of Ergot Fungi Reveals the Divergence and Evolution of Species within the Genus Claviceps Are the Result of Varying Mechanisms Driving Genome Evolution and Host Range Expansion

The genus Claviceps has been known for centuries as an economically important fungal genus for pharmacology and agricultural research. Only recently have researchers begun to unravel the evolutionary history of the genus, with origins in South America and classification of four distinct sections through ecological, morphological, and metabolic features (Claviceps sects. Citrinae, Paspalorum, Pusillae, and Claviceps). The first three sections are additionally characterized by narrow host range, whereas section Claviceps is considered evolutionarily more successful and adaptable as it has the largest host range and biogeographical distribution. However, the reasons for this success and adaptability remain unclear. Our study elucidates factors influencing adaptability by sequencing and annotating 50 Claviceps genomes, representing 21 species, for a comprehensive comparison of genome architecture and plasticity in relation to host range potential. Our results show the trajectory from specialized genomes (sects. Citrinae and Paspalorum) toward adaptive genomes (sects. Pusillae and Claviceps) through colocalization of transposable elements around predicted effectors and a putative loss of repeat-induced point mutation resulting in unconstrained tandem gene duplication coinciding with increased host range potential and speciation. Alterations of genomic architecture and plasticity can substantially influence and shape the evolutionary trajectory of fungal pathogens and their adaptability. Furthermore, our study provides a large increase in available genomic resources to propel future studies of Claviceps in pharmacology and agricultural research, as well as, research into deeper understanding of the evolution of adaptable plant pathogens.

59 BASIC BIOLOGICAL SCIENCES↗

The ModelSEED Biochemistry Database for the integration of metabolic annotations and the reconstruction, comparison and analysis of metabolic models for plants, fungi and microbes

Abstract For over 10 years, ModelSEED has been a primary resource for the construction of draft genome-scale metabolic models based on annotated microbial or plant genomes. Now being released, the biochemistry database serves as the foundation of biochemical data underlying ModelSEED and KBase. The biochemistry database embodies several properties that, taken together, distinguish it from other published biochemistry resources by: (i) including compartmentalization, transport reactions, charged molecules and proton balancing on reactions; (ii) being extensible by the user community, with all data stored in GitHub; and (iii) design as a biochemical ‘Rosetta Stone’ to facilitate comparison and integration of annotations from many different tools and databases. The database was constructed by combining chemical data from many resources, applying standard transformations, identifying redundancies and computing thermodynamic properties. The ModelSEED biochemistry is continually tested using flux balance analysis to ensure the biochemical network is modeling-ready and capable of simulating diverse phenotypes. Ontologies can be designed to aid in comparing and reconciling metabolic reconstructions that differ in how they represent various metabolic pathways. ModelSEED now includes 33,978 compounds and 36,645 reactions, available as a set of extensible files on GitHub, and available to search at https://modelseed.org and KBase.

59 BASIC BIOLOGICAL SCIENCES↗

Ant handling changes myrmecochore seed coat microbiomes and alters diversity of seed-borne plant pathogenic fungi

1. The putative benefits to seeds in myrmecochory (ant- mediated seed dispersal) are often cast in a reward context. However, microbes have been mostly overlooked as seed mortality agents in myrmecochory, as have potential treatments provided by ant-handling. 2. We investigated the effects of ant handling on the diversity of seed coat fungal communities of three myrmecochorous plant species. 3. Ant-handling altered measures of both alpha and beta diversity of fungal communities. Ant-handled seeds harboured different overall fungal communities and plant pathogen communities than non-ant-handled seeds. The myrmecochore pathogenic fungal community showed high dissimilarity (high pairwise community turnover) between ant-handled and control seeds, while beta diversity measures for ant- handled seeds and seeds with manually removed elaiosomes were less dissimilar. 4. Ant handling may offer an additional benefit to myrmecochorous seeds via the reduction in the seed coat pathogenic community, which may be driven by elaiosome removal or as a by-product of ant cleaning behaviours and chemical secretions.

59 BASIC BIOLOGICAL SCIENCES↗

Climate, soil mineralogy and mycorrhizal fungi influence soil organic matter fractions in eastern US temperate forests

Identifying the primary controls of particulate (POM) and mineral-associated organic matter (MAOM) content in soils is critical for determining future stocks of soil carbon (C) and nitrogen (N) across the globe. However, drivers of these soil organic matter fractions are likely to vary among ecosystems in response to climate, soil type and the composition of local biological communities. We tested how soil factors, climate and plant–fungal associations influenced the distribution and concentrations of C and N in MAOM and POM in seven temperate forests in the National Ecological Observatory Network (NEON) across the eastern United States. Samples of upper mineral horizon soil within each forest were collected in plots representing a gradient of dominant tree–mycorrhizal association, allowing us to test how plant and microbial communities influenced POM and MAOM across sites differing in climate and soil conditions. We found that concentrations of C and N in soil organic matter were primarily driven by soil mineralogy, but the relative abundance of MAOM versus POM C was strongly linked to plot-level mycorrhizal dominance. Furthermore, the effect of dominant tree mycorrhizal type on the distribution of N among POM and MAOM fractions was sensitive to local climate: in cooler sites, an increasing proportion of ectomycorrhizal-associated trees was associated with lower proportions of N in MAOM, but in warmer sites, we found the reverse. As an indicator of soil carbon age, we measured radiocarbon in the MAOM fraction but found that within and across sites, Δ 14 C was unrelated to mycorrhizal dominance, climate, or soil factors, suggesting that additional site-specific factors may be primary determinants of long-term SOM persistence. Our results indicate that while soil mineralogy primarily controls SOM C and N concentrations, the distribution of SOM among density fractions depends on the composition of vegetation and microbial communities, with these effects varying across sites with distinct climates. Here we also suggest that within biomes, the age of mineral-associated soil carbon is not clearly linked to the factors that control concentrations of MAOM C and N.

54 ENVIRONMENTAL SCIENCES↗