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All sources NASA NTRS DOE OSTI

DOE OSTI2020

Fungal Community, Not Substrate Quality, Drives Soil Microbial Function in Northeastern U.S. Temperate Forests

Mycorrhizal fungi can affect soil organic matter cycling through several mechanisms including priming, nutrient competition, and direct enzyme production. Differences in nutrient foraging strategies between ectomycorrhizal (EcM) and arbuscular mycorrhizal (AM) fungi produce divergent belowground dynamics: where EcM can take up organic nitrogen and directly break down soil organic matter (SOM) by producing enzymes, AM fungi are limited to scavenging mineral N. EcM-associated tree species also have leaf litter with relatively higher ratios of carbon to nitrogen (C:N), and belowground saprotrophic communities more dominated by fungi. Consequently, free-living microbes in EcM-dominated soils should experience nitrogen limitation, with subsequent increases in enzyme production and decreased carbon use efficiency (CUE). However, the relative importance of the effects of substrate quality and fungal community composition on enzyme production and CUE are unclear. To assess this distinction, we sampled the organic horizon and 10 cm of the mineral horizon in northern temperate forest soils along a gradient of EcM dominance. We characterized fungal community composition by measuring EcM relative abundances from extracted fungal DNA and the fungal to bacterial (F:B) ratios from phospholipid fatty acid (PLFA) analysis. We assessed soil substrate quality as the soil C:N ratio. Soil microbial functions were measured as potential activities of five hydrolytic and two oxidative enzymes, and microbial CUE. We found that the fungal community, represented by either the F:B ratio, EcM relative abundance, or both, affected CUE and six measured enzyme activities, while the C:N ratio affected only oxidative and chitin-targeting extracellular enzyme activities. Our results highlight the use of EcM relative dominance as a predictor of soil microbial community composition and function independent of substrate quality.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2020

Ectomycorrhizal fungal diversity interacts with soil nutrients to predict plant growth despite weak plant-soil feedbacks

Background and aims Plant-soil feedbacks are the result of multiple abiotic and biotic mechanisms. However, few studies have addressed how feedbacks vary based on abiotic context or attempted to identify microbiota responsible for feedbacks. In this work, we investigated whether plant-soil feedbacks of an ectomycorrhizal tree (Quercus macrocarpa) varied based on soil nutrient status and whether fungal community composition and diversity could explain feedback patterns. Furthermore, we inoculated Q. macrocarpa seedlings with field-sampled soils taken from five soil origins – including heterospecific and conspecific trees and an old field – which were profiled using fungal DNA metabarcoding.Results Despite finding that soils associated with different hosts harbored distinct fungal communities and that fungal communities were predictive of plant growth, we did not find any significant plant-soil feedbacks regardless of fertilization status. We did find that the growth promotive effect associated with ectomycorrhizal OTU diversity was weakened with fertilization, suggesting context-dependent relations between plant growth and a guild of fungal mutualists. Our results demonstrate that the host-specific accumulation of functionally important soil microbes is not always sufficient to drive observable plant-soil feedbacks. Our data provide support for a role for ECM fungal diversity in mediating plant growth responses, though it is unclear whether this effect is direct or indirect.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2001

Microbial Contamination of Allende and Murchison Carbonaceous Chondrites; Developing a Protocol for Life Detection in Extraterrestrial Materials Using Biotechnology

The arguments used to refute the McKay et al., (1996) hypothesis of possible Martian life in ALH84001 failed to use contamination of the meteorite as a source. This has worrying implications for our ability to detect terrestrial microbiota in meteorites and therefore any potential extraterrestrial biosignatures in both meteorites and possible returned samples. We report on imaging and microbial culturing of both Allende and Murchison carbonaceous chondrites and on the use of molecular biology techniques on a sample of Allende. Contaminating fungi and bacteria were observed (in the case of Murchison) and cultured from both meteorites. DNA was successfully extracted and subsequent PCR showed the presence of both bacterial and fungal DNA although no Archaea were detected. These results show that it is possible to use molecular biological techniques on very small quantities (300 mg) of extraterrestrial material.

Steele, A.↗

DOE OSTI2023

Mycorrhiza Better Predict Soil Fungal Community Composition and Function than Aboveground Traits in Temperate Forest Ecosystems

Forests in the northeastern US are experiencing shifts in community composition due to the northward migration of warm-adapted tree species and certain species’ declines (for example, white ash and eastern hemlock) due to invasive insects. Changes in belowground fungal communities and associated functions will inevitably follow. Therefore, we sought to investigate the relative importance of two important tree characteristics—mycorrhizal type [ectomycorrhizal (EcM) or arbuscular mycorrhizal (AM)] and leaf habit (deciduous or evergreen) on soil fungal community composition and organic matter cycling. We sampled soil in the organic and mineral horizons beneath two AM-associated (Fraxinus americana and Thuja occidentalis) and two ECM-associated tree species (Betula alleghaniensis and Tsuga canadensis), with an evergreen and deciduous species in each mycorrhizal group. To characterize fungal communities and organic matter decomposition beneath each tree species, we sequenced the ITS1 region of fungal DNA and measured the potential activity of carbon- and nitrogen-targeting extracellular enzymes. Each tree species harbored distinct fungal communities, supporting the need to consider both mycorrhizal type and leaf habit. However, between tree characteristics, mycorrhizal type better predicted fungal communities. Across fungal guilds, saprotrophic fungi were the most important group in shaping fungal community differences in soils beneath all tree species. The effect of leaf habit on carbon- and nitrogen-targeting hydrolytic enzymes depended on tree mycorrhizal association in the organic horizon, while oxidative enzyme activities were higher beneath EcM-associated trees across both soil horizons and leaf habits.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Mycorrhiza better predict soil fungal community composition and function than aboveground traits in temperate forest ecosystems

We sampled soil in the organic and mineral horizons beneath two AM-associated (Fraxinus americana, Thuja occidentalis) and two ECM-associated tree species (Betula alleghaniensis, and Tsuga canadensis), with an evergreen and deciduous species in each mycorrhizal group. To characterize fungal communities and organic matter decomposition beneath each tree species, we sequenced the ITS1 region of fungal DNA and measured the potential activity of carbon and nitrogen-targeting extracellular enzymes. Each tree species harbored distinct fungal communities, supporting the need to consider both mycorrhizal type and leaf habit. However, between tree characteristics, mycorrhizal type better predicted fungal communities. Across fungal guilds, saprotrophic fungi were the most important group in shaping fungal community differences in soils beneath all tree species. The effect of leaf habit on carbon and nitrogen-targeting hydrolytic enzymes depended on tree mycorrhizal association in the organic horizon, while oxidative enzyme activities were higher beneath EcM-associated trees across both soil horizons and leaf habits. These data include extracellular hydrolytic and oxidative enzyme activities, ITS sequencing fungal community data, soil carbon to nitrogen, and soil pH. Site level data include climate (mean annual temperature, precipitation), elevation, and soil series and order information.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2020

Soil microbial function and fungal community data along an ectomycorrhizal basal area gradient, Vermont, May 2019

Mycorrhizal fungi can affect soil organic matter cycling through several mechanisms including priming, nutrient competition, and direct enzyme production. Differences in nutrient foraging strategies between ectomycorrhizal (EcM) and arbuscular mycorrhizal (AM) fungi produce divergent belowground dynamics: where EcM can take up organic nitrogen and directly break down SOM by producing enzymes, AM fungi are limited to scavenging mineral N. EcM-associated tree species also have leaf litter with relatively higher ratios of carbon to nitrogen (C:N), and belowground saprotrophic communities more dominated by fungi. Consequently, free-living microbes in EcM-dominated soils should experience nitrogen limitation, with subsequent increases in enzyme production and decreased carbon use efficiency (CUE). However, the relative importance of the effects of substrate quality and fungal community composition on enzyme production and CUE are unclear. To assess this distinction, we sampled the organic horizon and 10 cm of the mineral horizon in northern temperate forest soils along a gradient of EcM dominance (see SCxTS_DBH.csv for full tree species information). We characterized fungal community composition by measuring EcM relative abundances from extracted fungal DNA (To run raw ASV data through R code, use SCxTS_ASV_taxonomy.csv, and R code ITS_processing.Rmd; to see processed data with EcM relative abundance, use file SCxTS_sum_afitch_Feb18.20.csv) and the fungal to bacterial ratios (see data file SCxTS_plfa.csv) from phospholipid fatty acid analysis. Soil microbial functions were measured as potential activities of five hydrolytic and two oxidative enzymes ( To run raw data through R code us SCxTS_metadata.csv, hydrolytic_oxidative_raw.csv, MUBraw.csv, Enzyme_soilweight.csv, and incubationtime.csv, and R code SCxTS_enzymes.Rmd; to use Enzyme data ready to run through statistics code, use file Enzyme.data.csv) and microbial carbon use efficiency (see file SCxTS_CUE.csv and for full calculations file 18O_CUEcalcs_SCxTS_2019.xlsx). We assessed soil substrate quality as the soil carbon:nitrogen ratio (See file Organic_CN.csv). Statistical analysis can be run using R code from file SCxTS_analysis.Rmd.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2025

Tropical Forest Soil Microbiome Modulates Leaf Heat Tolerance More Strongly Under Warming Than Ambient Conditions

ABSTRACT It is unclear how plants respond to increasing temperatures. Leaf heat tolerance (LHT) is often at its upper limit in tropical forests, suggesting that climate change might negatively impact these forests. We hypothesized that intraspecific variation in LHT might be associated with changes in the soil microbiome, which might also respond to climate. We hypothesized that warming would increase LHT through changes in the soil microbiome: we combined an in situ tropical warming experiment with a shade house experiment in Puerto Rico. The shade house experiment consisted of growing seedlings of Guarea guidonia , a dominant forest species, under different soil microbiome treatments (reduced arbuscular mycorrhizal fungi, reduced plant pathogens, reduced microbes, and unaltered) and soil inoculum from the field experiment. Heat tolerance was determined using chlorophyll fluorescence ( F V /F m ) on individual seedlings in the field and on groups of seedlings (per pot) in the shade house. We sequenced soil fungal DNA to analyze the impacts of the treatments on the soil microbiome. In the field, seedlings from ambient temperature plots showed higher F V /F m values under high temperatures (0.648 at 46°C and 0.067 at 52°C) than seedlings from the warming plots (0.535 at 46°C and 0.031 at 52°C). In the shade house, the soil microbiome treatments significantly influenced the fungal community composition and LHT ( T crit and F V /F m ). Reduction in fungal pathogen abundance and diversity altered F V /F m before T 50 for seedlings grown with soil inoculum from the warming plots but after T 50 for seedlings grown with soil inoculum from the ambient plots. Our findings emphasize that the soil microbiome plays an important role in modulating the impacts of climate change on plants. Understanding and harnessing this relationship might be vital for mitigating the effects of warming on forests, emphasizing the need for further research on microbial responses to climate change.

Hernandes Villani, Gabriela [Department of Plant B↗

DOE OSTI2023

DNA hypomethylation of the host tree impairs interaction with mutualistic ectomycorrhizal fungus

Summary Ectomycorrhizas are an intrinsic component of tree nutrition and responses to environmental variations. How epigenetic mechanisms might regulate these mutualistic interactions is unknown. By manipulating the level of expression of the chromatin remodeler DECREASE IN DNA METHYLATION 1 ( DDM1 ) and two demethylases DEMETER‐LIKE ( DML ) in Populus tremula × Populus alba lines, we examined how host DNA methylation modulates multiple parameters of the responses to root colonization with the mutualistic fungus Laccaria bicolor . We compared the ectomycorrhizas formed between transgenic and wild‐type (WT) trees and analyzed their methylomes and transcriptomes. The poplar lines displaying lower mycorrhiza formation rate corresponded to hypomethylated overexpressing DML or RNAi‐ddm1 lines. We found 86 genes and 288 transposable elements (TEs) differentially methylated between WT and hypomethylated lines (common to both OX‐ dml and RNAi‐ ddm1 ) and 120 genes/1441 TEs in the fungal genome suggesting a host‐induced remodeling of the fungal methylome. Hypomethylated poplar lines displayed 205 differentially expressed genes ( cis and trans effects) in common with 17 being differentially methylated ( cis ). Our findings suggest a central role of host and fungal DNA methylation in the ability to form ectomycorrhizas including not only poplar genes involved in root initiation, ethylene and jasmonate‐mediated pathways, and immune response but also terpenoid metabolism.

Vigneaud, Julien↗

DOE OSTI2023

Dynamic Reconfiguration of Switchgrass Proteomes in Response to Rust ( Puccinia novopanici ) Infection

Switchgrass (Panicum virgatum L.) can be infected by the rust pathogen (Puccinia novopanici) and results in lowering biomass yields and quality. Label-free quantitative proteomics was conducted on leaf extracts harvested from non-infected and infected plants from a susceptible cultivar (Summer) at 7, 11, and 18 days after inoculation (DAI) to follow the progression of disease and evaluate any plant compensatory mechanisms to infection. Some pustules were evident at 7 DAI, and their numbers increased with time. However, fungal DNA loads did not appreciably change over the course of this experiment in the infected plants. In total, 3830 proteins were identified at 1% false discovery rate, with 3632 mapped to the switchgrass proteome and 198 proteins mapped to different Puccinia proteomes. Across all comparisons, 1825 differentially accumulated switchgrass proteins were identified and subjected to a STRING analysis using Arabidopsis (A. thaliana L.) orthologs to deduce switchgrass cellular pathways impacted by rust infection. Proteins associated with plastid functions and primary metabolism were diminished in infected Summer plants at all harvest dates, whereas proteins associated with immunity, chaperone functions, and phenylpropanoid biosynthesis were significantly enriched. At 18 DAI, 1105 and 151 proteins were significantly enriched or diminished, respectively. Many of the enriched proteins were associated with mitigation of cellular stress and defense.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Rapid and robust squashed spore/colony PCR of industrially important fungi

Fungi have been utilized for centuries in medical, agricultural, and industrial applications. Development of systems biology techniques has enabled the design and metabolic engineering of these fungi to produce novel fuels, chemicals, and enzymes from renewable feedstocks. Many genetic tools have been developed for manipulating the genome and creating mutants rapidly. However, screening and confirmation of transformants remain an inefficient step within the design, build, test, and learn cycle in many industrial fungi because extracting fungal genomic DNA is laborious, time-consuming, and involves toxic chemicals. In this study we developed a rapid and robust technique called “Squash-PCR” to break open the spores and release fungal genomic DNA as a template for PCR. The efficacy of Squash-PCR was investigated in eleven different filamentous fungal strains. Clean PCR products with high yields were achieved in all tested fungi. Spore age and type of DNA polymerase did not affect the efficiency of Squash-PCR. However, spore concentration was found to be the crucial factor for Squash-PCR in Aspergillus niger, with the dilution of starting material often resulting in higher PCR product yield. We then further evaluated the applicability of the squashing procedure for nine different yeast strains. We found that Squash-PCR can be used to improve the quality and yield of colony PCR in comparison to direct colony PCR in the tested yeast strains. The developed technique will enhance the efficiency of screening transformants and accelerate genetic engineering in filamentous fungi and yeast.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2020

Prokaryotic and Fungal Characterization of the Facilities Used to Assemble, Test, and Launch the OSIRIS-REx Spacecraft

To characterize the ATLO (Assembly, Test, and Launch Operations) environment of the OSIRIS-REx spacecraft, we analyzed 17 aluminum witness foils and two blanks for bacterial, archaeal, fungal, and arthropod DNA. Under NASA’s Planetary Protection guidelines, OSIRIS-REx is a Category II outbound, Category V unrestricted sample return mission. As a result, it has no bioburden restrictions. However, the mission does have strict organic contamination requirements to achieve its primary objective of returning pristine carbonaceous asteroid regolith to Earth. Its target, near-Earth asteroid (101955) Bennu, is likely to contain organic compounds that are biologically available. Therefore, it is useful to understand what organisms were present during ATLO as part of the larger contamination knowledge effort—even though it is unlikely that any of the organisms will survive the multi-year deep space journey. Even though these samples of opportunity were not collected or preserved for DNA analysis, we successfully amplified bacterial and archaeal DNA (16S rRNA gene) from 16 of the 17 witness foils containing as few as 7 ± 3 cells per sample. Fungal DNA (ITS1) was detected in 12 of the 17 witness foils. Despite observing arthropods in some of the ATLO facilities, arthropod DNA (COI gene) was not detected. We observed 1,009 bacterial and archaeal sOTUs (sub-operational taxonomic units, 100% unique) and 167 fungal sOTUs across all of our samples (25–84 sOTUs per sample). The most abundant bacterial sOTU belonged to the genus Bacillus. This sOTU was present in blanks and may represent contamination during sample handling or storage. The sample collected from inside the fairing just prior to launch contained several unique bacterial and fungal sOTUs that describe previously uncharacterized potential for contamination during the final phase of ATLO. Additionally, fungal richness (number of sOTUs) negatively correlates with the number of carbon-bearing particles detected on samples. The total number of fungal sequences positively correlates with total amino acid concentration. These results demonstrate that it is possible to use samples of opportunity to characterize the microbiology of low-biomass environments while also revealing the limitations imposed by sample collection and preservation methods not specifically designed with biology in mind.

Aaron B. Regberg↗

NASA NTRS2017

Field Exploration and Life Detection Sampling Through Planetary Analogue Sampling (FELDSPAR).

Exploration missions to Mars rely on rovers to perform analyses over small sampling areas; however, landing sites for these missions are selected based on large-scale, low-resolution remote data. The use of Earth analogue environments to estimate the multi-scale spatial distributions of key signatures of habitability can help ensure mission science goals are met. A main goal of FELDSPAR is to conduct field operations analogous to Mars sample return in its science, operations, and technology from landing site selection, to in-field sampling location selection, remote or stand-off analysis, in situ analysis, and home laboratory analysis. Lava fields and volcanic regions are relevant analogues to Martian landscapes due to desiccation, low nutrient availability, and temperature extremes. Operationally, many Icelandic lava fields are remote enough to require that field expeditions address several sampling constraints that are experienced in robotic exploration, including in situ and sample return missions. The Fimmvruhls lava field was formed by a basaltic effusive eruption associated with the 2010 Eyjafjallajkull eruption. Mlifellssandur is a recently deglaciated plain to the north of the Myrdalsjkull glacier. Holuhraun was formed by a 2014 fissure eruptions just north of the large Vatnajkull glacier. Dyngjusandur is an alluvial plain apparently kept barren by repeated mechanical weathering. Informed by our 2013 expedition, we collected samples in nested triangular grids every decade from the 10 cm scale to the 1 km scale (as permitted by the size of the site). Satellite imagery is available for older sites, and for Mlifellssandur, Holuhraun, and Dyngjusandur we obtained overhead imagery at 1 m to 200 m elevation. PanCam-style photographs were taken in the field by sampling personnel. In-field reflectance spectroscopy was also obtained with an ASD spectrometer in Dyngjusandur. All sites chosen were 'homogeneous' in apparent color, morphology, moisture, grain size, and reflectance spectra at all scales greater than 10 cm. Field lab assays were conducted to monitor microbial habitation, including ATP quantification, qPCR for fungal, bacterial, and archaeal DNA, and direct cell imaging using fluorescence microscopy. Home laboratory analyses include Raman spectroscopy and community sequencing. ATP appeared to be significantly more sensitive to small changes in sampling location than qPCR or fluorescence microscopy. Bacterial and archaeal DNA content were more consistent at the smaller scales, but similarly variable across more distant sites. Conversely, cell counts and fungal DNA content have significant local variation but appear relatively homogeneous over scales of 1 km. ATP, bacterial DNA, and archaeal DNA content were relatively well correlated at many spatial scales. While we have observed spatial variation at various scales and are beginning to observe how that variation fluctuates over time as biodiversity recovers after an eruption, we do not yet fully understand what parameters lead to the observed spatial variation. Home laboratory analyses will help us further understand the elemental and structural composition of the basaltic matrices, but further field analyses are vital for the understanding how temperature, moisture, incident radiation, and so forth influence the habitability of a microclimate.

Field↗

DOE OSTI2021

Agricultural Management Affects the Active Rhizosphere Bacterial Community Composition and Nitrification

Cropping system diversity provides yield benefits that may result from shifts in the composition of root-associated bacterial and fungal communities, which either enhance nutrient availability or limit nutrient loss. We investigated whether temporal diversity of annual cropping systems (four versus two crops in rotation) influences the composition and metabolic activities of root-associated microbial communities in maize at a developmental stage when the peak rate of nitrogen uptake occurs. We monitored total (DNA-based) and potentially active (RNA-based) bacterial communities and total (DNA-based) fungal communities in the soil, rhizosphere, and endosphere. Cropping system diversity strongly influenced the composition of the soil microbial communities, which influenced the recruitment of the resident microbial communities and, in particular, the potentially active rhizosphere and endosphere bacterial communities. The diversified cropping system rhizosphere recruited a more diverse bacterial community (species richness), even though there was little difference in soil species richness between the two cropping systems. In contrast, fungal species richness was greater in the conventional rhizosphere, which was enriched in fungal pathogens; the diversified rhizosphere, however, was enriched in Glomeromycetes. While cropping system influenced endosphere community composition, greater correspondence between DNA- and RNA-based profiles suggests a higher representation of active bacterial populations. Cropping system diversity influenced the composition of ammonia oxidizers, which coincided with diminished potential nitrification activity and gross nitrate production rates, particularly in the rhizosphere. The results of our study suggest that diversified cropping systems shift the composition of the rhizosphere’s active bacterial and total fungal communities, resulting in tighter coupling between plants and microbial processes that influence nitrogen acquisition and retention.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

Horizontal gene transfer in Fungi

Horizontal gene transfer (HGT), the sharing of genes between species outside of inheritance, is a common feature among prokaryotes, but a growing body of evidence supports HGT in eukaryotic genomes. The driving forces behind horizontal gene transfer occur within a shared environment, and are selective—meaning they improve survivability of stress, increase nutrient access, or provide an evolutionary advantage. The development of toxic and pathogenic properties was one of the first indications of phenotypic transfer among fungal species. An growing species diversity of genome sequences has brought a new set of tools to tracking HGT. The detection of HGT based on sequence similarity utilizes species tree phylogenies, which compare similarity of core conserved genes to determine lineage. Divergence of an HGT candidate gene from a phylogenetic background tree can show inter-species DNA transfer. It is by following phenotypes and analysis of DNA sequence context and content that we can uncover previous transfer events, and track lab fostered and recorded HGT. Evidence of HGT is found heavily in parasitic and pathogenic fungi; both of these lifestyles rely on phagocytosis and assimilation of host parts and pieces, suggesting phagocytosis is implicated in DNA uptake mechanisms. Our understanding of the molecular mechanisms of DNA uptake and incorporation of foreign DNA into fungal genomes, i.e. natural competence, remain based on a set of laboratory conditions used in molecular transformation. The mechanism of DNA uptake in the natural world and new DNA incorporation remain under investigation.

Bredeweg, Erin L.↗

NASA NTRS2026

Does the International Space Station Leak DNA? Preliminary Results from the ISS External Microorganisms Payload

Existing crewed spacecraft like the ISS (International Space Station) leak by design. The ISS routinely releases gas to maintain life support systems and when astronauts exit the station to perform space walks. The chemical component of this leakage is well characterized, but the biological components are not. The ISS is not subject to planetary protection requirements, but planned missions to Mars will use similar systems and will be subject to planetary protection requirements. If detectable microorganisms are escaping through vents and or airlocks we may need to redesign our crewed habitats to minimize this type of contamination. To test the hypothesis that microorganisms from inside ISS are detectable on exterior surfaces an astronaut used the ISS External Microorganisms sampling kit (Rucker et al. 2018) to sample exterior surfaces of the ISS during an EVA (Extra Vehicular Activity) in January of 2025. These samples were returned to Earth for DNA extraction and sequencing. We successfully, extracted and sequenced bacterial, fungal and viral DNA from these samples that was not present in the negative controls. These results should help NASA refine the planetary protection requirements for crewed missions. Methods: The samples were collected using sterile, DNA free, buccal swabs (23 mm. diameter) housed in custom canisters. Each canister uses a 0.2 μm Teflon filter to maintain sterility as the caddy, holding 8 swabs moves in and out of vacuum. The astronaut sampled the: 1) airlock vestibule, 2) airlock thermal cover, 3) a gap in the micrometeorite shielding near the airlock, 4) a handrail near the airlock, 5) the Carbon Dioxide Removal Assembly vent, and 6) the Vacuum Exhaust System vent. The seventh swab was exposed to vacuum during the EVA without touching it to a surface. The eighth swab, a negative control, was not opened until the caddy returned to Earth. DNA was extracted from the swabs using a QIamp UCP Pathogen kit and prepared for sequencing on an Aviti (Element Biosciences) sequencer (Arslan et al. 2024). The resulting sequences were analyzed using the EDGE Bioinformatics platform (Li et al. 2017). The sequences were analyzed individually using tools like BLAST, GOTTCHA2, Kraken2, and PanGIA. The data were also assembled into metagenome assembled genomes) using tools like CONCOCT, MaxBin2 and MetaBAT2. Results: We successfully extracted and sequenced bacterial, archaeal, fungal and viral DNA from all seven samples. The handrail swab had the lowest number of reads (768,651) and the airlock thermal cover had the highest number of reads (8,819,230). These samples contain DNA from human associated bacteria (e.g. Crynebacterium riegelii ), fungi (.e.g. Penicillium rubens ), and viruses (e.g Alphapapillomavirus ). Conclusion: Preliminary interpretation suggest that the airlock and the space suits themselves are the largest sources of contaminant DNA. Most if not all of the DNA is from organisms known to be present inside the ISS. Vents attached to life support systems may be a lesser source of biological contamination. Further analysis should help NASA address planetary protection knowledge gaps for crewed missions.

Aaron B Regberg↗

DOE OSTI2021

Colonization of Naive Roots from Populus tremula × alba Involves Successive Waves of Fungi and Bacteria with Different Trophic Abilities

ABSTRACT Through their roots, trees interact with a highly complex community of microorganisms belonging to various trophic guilds and contributing to tree nutrition, development, and protection against stresses. Tree roots select for specific microbial species from the bulk soil communities. The root microbiome formation is a dynamic process, but little is known on how the different microorganisms colonize the roots and how the selection occurs. To decipher whether the final composition of the root microbiome is the product of several waves of colonization by different guilds of microorganisms, we planted sterile rooted cuttings of gray poplar obtained from plantlets propagated in axenic conditions in natural poplar stand soil. We analyzed the root microbiome at different time points between 2 and 50 days of culture by combining high-throughput Illumina MiSeq sequencing of the fungal ribosomal DNA internal transcribed spacer and bacterial 16S rRNA amplicons with confocal laser scanning microscopy observations. The microbial colonization of poplar roots took place in three stages, but bacteria and fungi had different dynamics. Root bacterial communities were clearly different from those in the soil after 2 days of culture. In contrast, if fungi were also already colonizing roots after 2 days, the initial communities were very close to that in the soil and were dominated by saprotrophs. They were slowly replaced by endophytes and ectomycorhizal fungi. The replacement of the most abundant fungal and bacterial community members observed in poplar roots over time suggest potential competition effect between microorganisms and/or a selection by the host. IMPORTANCE The tree root microbiome is composed of a very diverse set of bacterial and fungal communities. These microorganisms have a profound impact on tree growth, development, and protection against different types of stress. They mainly originate from the bulk soil and colonize the root system, which provides a unique nutrient-rich environment for a diverse assemblage of microbial communities. In order to better understand how the tree root microbiome is shaped over time, we observed the composition of root-associated microbial communities of naive plantlets of poplar transferred in natural soil. The composition of the final root microbiome relies on a series of colonization stages characterized by the dominance of different fungal guilds and bacterial community members over time. Our observations suggest an early stabilization of bacterial communities, whereas fungal communities are established following a more gradual pattern.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Host genotype controls ecological change in the leaf fungal microbiome

Leaf fungal microbiomes can be fundamental drivers of host plant success, as they contain pathogens that devastate crop plants and taxa that enhance nutrient uptake, discourage herbivory, and antagonize pathogens. We measured leaf fungal diversity with amplicon sequencing across an entire growing season in a diversity panel of switchgrass ( Panicum virgatum ). We also sampled a replicated subset of genotypes across 3 additional sites to compare the importance of time, space, ecology, and genetics. We found a strong successional pattern in the microbiome shaped both by host genetics and environmental factors. Further, we used genome-wide association (GWA) mapping and RNA sequencing to show that 3 cysteine-rich receptor-like kinases (crRLKs) were linked to a genetic locus associated with microbiome structure. We confirmed GWAS results in an independent set of genotypes for both the internal transcribed spacer (ITS) and large subunit (LSU) ribosomal DNA markers. Fungal pathogens were central to microbial covariance networks, and genotypes susceptible to pathogens differed in their expression of the 3 crRLKs, suggesting that host immune genes are a principal means of controlling the entire leaf microbiome.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1999

Scaling features of noncoding DNA

We review evidence supporting the idea that the DNA sequence in genes containing noncoding regions is correlated, and that the correlation is remarkably long range--indeed, base pairs thousands of base pairs distant are correlated. We do not find such a long-range correlation in the coding regions of the gene, and utilize this fact to build a Coding Sequence Finder Algorithm, which uses statistical ideas to locate the coding regions of an unknown DNA sequence. Finally, we describe briefly some recent work adapting to DNA the Zipf approach to analyzing linguistic texts, and the Shannon approach to quantifying the "redundancy" of a linguistic text in terms of a measurable entropy function, and reporting that noncoding regions in eukaryotes display a larger redundancy than coding regions. Specifically, we consider the possibility that this result is solely a consequence of nucleotide concentration differences as first noted by Bonhoeffer and his collaborators. We find that cytosine-guanine (CG) concentration does have a strong "background" effect on redundancy. However, we find that for the purine-pyrimidine binary mapping rule, which is not affected by the difference in CG concentration, the Shannon redundancy for the set of analyzed sequences is larger for noncoding regions compared to coding regions.

Non-NASA Center↗