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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Optimized CRISPR Interference System for Investigating Pseudomonas alloputida Genes Involved in Rhizosphere Microbiome Assembly

Pseudomonas alloputida KT2440 (formerly P. putida) has become both a well-known chassis organism for synthetic biology and a model organism for rhizosphere colonization. Here, we describe a CRISPR interference (CRISPRi) system in KT2440 for exploring microbe–microbe interactions in the rhizosphere and for use in industrial systems. Our CRISPRi system features three different promoter systems (XylS/P m , LacI/P lac , and AraC/P BAD ) and a dCas9 codon-optimized for Pseudomonads, all located on a mini-Tn7-based transposon that inserts into a neutral site in the genome. It also includes a suite of pSEVA-derived sgRNA expression vectors, where the expression is driven by synthetic promoters varying in strength. We compare the three promoter systems in terms of how well they can precisely modulate gene expression, and we discuss the impact of environmental factors, such as media choice, on the success of CRISPRi. We demonstrate that CRISPRi is functional in bacteria colonizing the rhizosphere, with repression of essential genes leading to a 10–100-fold reduction in P. alloputida cells per root. Finally, we show that CRISPRi can be used to modulate microbe–microbe interactions. When the gene pvdH is repressed and P. alloputida is unable to produce pyoverdine, it loses its ability to inhibit other microbes in vitro. Furthermore, our design is amendable for future CRISPRi-seq studies and in multispecies microbial communities, with the different promoter systems providing a means to control the level of gene expression in many different environments.

Bacteria↗
DOE OSTI2022

Assessment of rhizosphere bacterial diversity and composition in a metal hyperaccumulator (Boehmeria nivea) and a nonaccumulator (Artemisia annua) in an antimony mine

Abstract Aims Heavy metal hyperaccumulators are widely used in mining restoration due to their ability to accumulate and transport heavy metals, compared to nonaccumulators. Rhizosphere bacteria in metal hyperaccumulators play a key role in the uptake of heavy metals from soil; however, assessments of the differences of rhizosphere bacteria between metal hyperaccumulators and nonaccumulator are scarce. Methods and results To understand the difference of bacterial composition between hyperaccumulator and nonaccumulator in rhizosphere, the diversity and composition of rhizosphere bacteria in a metal hyperaccumulator (Boehmeria nivea) and a nonaccumulator (Artemisia annua) grown in the same field in Xikuangshan were evaluated using Illumina Miseq high-throughput sequencing technology. Boehmeria nivea and A. annua had 3926 overlapping OTUs, 19,736 and 17,579 unique OTUs, respectively. Boehmeria nivea had lower Chao1 index, Shannon index and Pielou index than A. annua. The dominant phyla and genera of rhizosphere bacteria in B. nivea and A. annua were similar, but some rhizosphere bacterial communities with heavy metal remediation ability mainly appeared in the rhizosphere of the hyperaccumulator. Compared to A. annua, B. nivea showed a significantly higher relative abundance of rhizosphere bacteria, such as Acidobacteria and Bacteroidete at the phylum level and RB41 at the genus level. Some specific rhizosphere bacteria with the ability to bind metal, such as Leifsonia and Kibdelosporangium, were only found in the rhizosphere of B. nivea. Conclusion Results indicated that B. nivea, as a metal hyperaccumulator, has a key function in governing metal-resistant rhizosphere bacteria in response to antimony compound pollution stress. Significance and Impact of Study Understanding the diversity of rhizosphere bacteria between hyperaccumulators and nonaccumulators is beneficial to formulate strategies to improve metal uptake efficiency by selecting specific plant species and rhizosphere bacteria grown on polluted soil.

Lin, Yuxiang↗
DOE OSTI2024

Assessing horizontal gene transfer in the rhizosphere of Brachypodium distachyon using fabricated ecosystems (EcoFABs)

ABSTRACT Horizontal gene transfer (HGT) is a major process by which genes are transferred between microbes in the rhizosphere. However, examining HGT remains challenging due to the complexity of mimicking conditions within the rhizosphere. Fabricated ecosystems (EcoFABs) have been used to investigate several complex processes in plant-associated environments. Here we show that EcoFABs are efficient tools to examine and measure HGT frequency in the rhizosphere. We provide the first demonstration of gene transfer via a triparental conjugation system in the Brachypodium distachyon rhizosphere in an EcoFAB using Pseudomonas putida KT2440 as both donor and recipient bacterial strain with the donor containing a mobilizable and non-self-transmissible plasmid. We observed that the frequency of plasmid transfer in the rhizosphere is potentially dependent on the plant developmental stage and the composition and amount of root exudates. The frequency of plasmid transfer also increased with higher numbers of donor cells. We demonstrate the transfer of plasmid from P. putida to another B. distachyon root colonizer, Burkholderia sp. OAS925, showing HGT within a rhizosphere microbial community. Environmental stresses also influenced the rate and efficiency of HGT in the rhizosphere between different species and genera. This study provides a robust workflow to evaluate transfer of engineered plasmids in the rhizosphere when such plasmids are potentially introduced in a field or other plant-associated environments. IMPORTANCE We report the use of EcoFABs to investigate the HGT process in a rhizosphere environment. It highlights the potential of EcoFABs in recapitulating the dynamic rhizosphere conditions as well as their versatility in studying plant-microbe interactions. This study also emphasizes the importance of studying the parameters impacting the HGT frequency. Several factors such as plant developmental stages, nutrient conditions, number of donor cells, and environmental stresses influence gene transfer within the rhizosphere microbial community. This study paves the way for future investigations into understanding the fate and movement of engineered plasmids in a field environment.

Priya, Shweta↗
DOE OSTI2023

Uncovering the dominant role of root metabolism in shaping rhizosphere metabolome under drought in tropical rainforest plants

Plant-soil-microbe interactions are crucial for driving rhizosphere processes that contribute to metabolite turnover and nutrient cycling. With the increasing frequency and severity of water scarcity due to climate warming, understanding how plant-mediated processes, such as root exudation, influence soil organic matter turnover in the rhizosphere is essential. In this study, we used 16S rRNA gene amplicon sequencing, rhizosphere metabolomics, and position-specific 13 C-pyruvate labeling to examine the effects of three different plant species (Piper auritum, Hibiscus rosa sinensis, and Clitoria fairchildiana) and their associated microbial communities on soil organic carbon turnover in the rhizosphere. Here, our findings indicate that in these tropical plants, the rhizosphere metabolome is primarily shaped by the response of roots to drought rather than direct shifts in the rhizosphere bacterial community composition. Specifically, the reduced exudation of plant roots had a notable effect on the metabolome of the rhizosphere of P. auritum, with less reliance on neighboring microbes. Contrary to P. auritum, H. rosa sinensis and C. fairchildiana experienced changes in their exudate composition during drought, causing alterations to the bacterial communities in the rhizosphere. This, in turn, had a collective impact on the rhizosphere's metabolome. Furthermore, the exclusion of phylogenetically distant microbes from the rhizosphere led to shifts in its metabolome. Additionally, C. fairchildiana appeared to be associated with only a subset of symbiotic bacteria under drought conditions. These results indicate that plant species-specific microbial interactions systematically change with the root metabolome. As roots respond to drought, their associated microbial communities adapt, potentially reinforcing the drought tolerance strategies of plant roots. These findings have significant implications for maintaining plant health and preference during drought stress and improving plant performance under climate change.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Nutrient and moisture limitations reveal keystone metabolites linking rhizosphere metabolomes and microbiomes

Plants release a wealth of metabolites into the rhizosphere that can shape the composition and activity of microbial communities in response to environmental stress. The connection between rhizodeposition and rhizosphere microbiome succession has been suggested, particularly under environmental stress conditions, yet definitive evidence is scarce. In this study, we investigated the relationship between rhizosphere chemistry, microbiome dynamics, and abiotic stress in the bioenergy crop switchgrass grown in a marginal soil under nutrient-limited, moisture-limited, and nitrogen (N)-replete, phosphorus (P)-replete, and NP-replete conditions. We combined 16S rRNA amplicon sequencing and LC-MS/MS-based metabolomics to link rhizosphere microbial communities and metabolites. We identified significant changes in rhizosphere metabolite profiles in response to abiotic stress and linked them to changes in microbial communities using network analysis. N-limitation amplified the abundance of aromatic acids, pentoses, and their derivatives in the rhizosphere, and their enhanced availability was linked to the abundance of bacterial lineages from Acidobacteria, Verrucomicrobia, Planctomycetes, and Alphaproteobacteria. Conversely, N-amended conditions increased the availability of N-rich rhizosphere compounds, which coincided with proliferation of Actinobacteria. Treatments with contrasting N availability differed greatly in the abundance of potential keystone metabolites; serotonin and ectoine were particularly abundant in N-replete soils, while chlorogenic, cinnamic, and glucuronic acids were enriched in N-limited soils. Serotonin, the keystone metabolite we identified with the largest number of links to microbial taxa, significantly affected root architecture and growth of rhizosphere microorganisms, highlighting its potential to shape microbial community and mediate rhizosphere plant–microbe interactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Root‐Pore Interactions, the Underestimated Driver for Rhizosphere Structure and Rhizosheath Development

Physical characteristics of rhizosphere and rhizosheath, that is, root-adhering soil, are crucial for plant performance. Yet, the drivers of the rhizosphere's structural properties and their relationships with rhizosheath development remain unclear. We used X-ray computed micro-tomography (i) to explore two drivers of rhizosphere porosity: root-induced changes vs. preferential root growth into soil with certain pore characteristics and (ii) to estimate their contributions to rhizosphere macroporosity gradients and rhizosheath formation. Rhizosheath development was assessed in relation to rhizosphere macroporosity and rhizodeposition after ¹⁴C labeling. Our results confirmed that both root-induced changes and growth preferences shape rhizosphere structure, with their relative significance depending on the inherent macropore availability. In intact soils, growth preferences were the dominant factor, while in sieved soils the root-induced changes became equally important. Rhizosheath formation was associated with roots compacting their surrounding and releasing carbon. However, no correlation was found between rhizosheath formation and the actual rhizosphere, that is, the volume of soil adjacent to the roots. The study offers new process-level understanding of rhizosphere porosity gradients, while emphasizing caution in interpreting root growth data from sieved soil studies. Similarly, traditional destructively sampled rhizosheath may not fully capture the true characteristics of the actual rhizosphere, underscoring importance of intact-soil analyses.

macroporosity gradients↗
DOE OSTI2026

Shrub and sedge rhizosphere communities display distinct affinities toward exudates and soil organic matter degradation: a quantitative stable isotope probing analysis

Warming temperatures are accelerating permafrost thaw and changing tundra vegetation, where woody shrubs are displacing sedges. Shrubs, such as Betula nana, and sedges, such as Eriophorum vaginatum, exhibit distinct life strategies including unique root-associated, or rhizosphere microbial communities. As permafrost thaws it unlocks previously unavailable carbon and nutrient sources resulting in deeper roots and a translocation of rhizosphere communities. Because permafrost microbial communities contain lower diversity and biomass than rhizosphere communities, the coalescence of rhizosphere and permafrost microbial communities could alter soil organic matter (SOM) degradation rates and increase greenhouse gas emissions. To identify metabolic strategies across distinct rhizosphere and permafrost microbial communities we conducted an isotope tracing incubation experiment. We inoculated thawed permafrost with shrub and sedge rhizosphere communities while adding exudates or water daily and compared this to an uninoculated control. After 46 days, we spiked samples with 18O enriched water or 13C enriched exudates and measured isotope incorporation into microbial DNA with quantitative stable isotope probing (qSIP). Our results indicate that exudate additions had little effect on uninoculated permafrost communities but the addition of exudates and rhizosphere inoculants had a compounding effect on respiration rates. We found that soils inoculated with shrub rhizosphere communities contained a mixture of exudate and SOM degraders while soils inoculated with sedge rhizosphere communities contained mainly SOM degraders. Finally, we found that individual microbial taxa exhibited maximum growth rates under specific combinations of microbial inoculant communities and exudate addition treatments. Our results reveal that microbial niches are strongly influenced by substrate preferences and community context, and suggest that a reduction in sedges and an expansion of shrubs may provide a mechanism by which permafrost carbon losses are mitigated through corresponding shifts in microbial communities and their substrate preferences.

Schaefer, Sean R [Natural Resources and Earth Syst↗
DOE OSTI2025

Root size and soil physicochemical properties drive microscale spatial patterns of Fe and As retention in the rice rhizosphere

Background and Aims: Radial oxygen loss from rice roots in flooded soils oxidizes and precipitates dissolved Fe(II), Mn(II), and As(III) into mixed Fe(III), Mn(III/IV), and As(V) as root plaque and in the rhizosphere soil. It is unknown how different soils and root sizes impact the spatial extent of Fe and As retention outside the root. Methods: We imaged cross-sections of 90 roots from 6 different soils using synchrotron μXRF imaging followed by k-means clustering and elliptical averaging to distinguish bulk soil, rhizosphere, plaque, and roots based on As and Fe patterns. Results: We found preferential As retention in the plaque and rhizospheres of most roots except small (< 0.45 mm) roots in silty soils with low P or high As. In contrast, clayey soils had similar As-Fe correlations across plaque, rhizosphere, and bulk soil. Large (> 0.45 mm) roots often had no oxidized rhizosphere region. We obtained an extensive dataset of 256 As and 155 Mn synchrotron μXANES measurements, which revealed that rhizosphere and plaque As was mainly inorganic As(V) and As(III), and Mn oxidation state varied between soils but not between belowground locations. Conclusion: Small roots in coarse-textured soils were less likely to have As retention in the plaque or rhizosphere compared to large roots and fine-textured soils. Furthermore, the unique and extensive data in this study provides new insight into soil and root size impacts on As retention in the rhizosphere. It is essential to investigate a representative number of samples to draw conclusions from XRF imaging.

36 MATERIALS SCIENCE↗
DOE OSTI2022

Spectroscopy-based isotopic (δ 13 C) analysis for high spatial resolution of carbon exchange in the rhizosphere

The rhizosphere is a highly dynamic zone bridging plant roots with needed nutrient resources in soil. While the rhizosphere may be small, it has a disproportionally large impact on plant success and biomass production. A suite of rhizosphere-hosted microbial and geochemical interactions facilitate nutrient acquisition by plant roots, and, in turn, the roots stimulate these processes by supplying organic carbon into the rhizosphere. The small physical dimensions of the rhizosphere, however, can constrain efforts to elucidate key carbon exchange processes and their spatial extent and localization. We present a method for spatially resolved δ 13 C analysis of rhizosphere samples by coupling laser ablation (LA) sampling with isotopic analysis using capillary absorption spectroscopy (CAS) which differs from conventional mass spectrometer (MS) approaches. The CAS system has high sensitivity (requires fewer nanomoles of CO 2 per analysis) than comparable MS systems, which enables reduced sample size requirements to thereby improve spatial resolution (from 25 μm to as low as a projected 5 μm spatial resolution). We demonstrate the utility of CAS using rhizosphere samples from switchgrass plants exposed to 13 CO 2 . As a result, this technique will provide a capability for tracking the extent and spatial distribution of root exudate into the rhizosphere at highly detailed spatial scales.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Root exudate composition from different plant species influences the growth of rhizosphere bacteria

Plant roots release exudates that fuel microbial activities and can structure rhizosphere microbial communities, but how different plant species use their root exudate to potentially select for different soil microbes in the rhizosphere is not well understood. Here, we investigated how root exudate from plants of three diverging lineages, Lactuca sativa (lettuce), Brassica juncea (mustard cabbage), and Zea mays (maize) influence the growth of their own rhizosphere bacteria (host) and those from other plant species (non-host) in growth bioassays. We found that on the community level, lettuce rhizosphere bacteria grew better in non-host exudate, but mustard cabbage and maize rhizosphere bacteria grew similarly well in both host and non-host exudate. However, individual bacteria taxa showed strong preferences for exudate from different plant species. The bacterial growth patterns were independent of C and N quantity, suggesting that certain exometabolic compounds may drive the growth patterns. Our results demonstrate that root exudate from a given plant species have the potential to stimulate or suppress soil bacteria and hint at a mechanism that different plant species use to select for their specific suite of rhizosphere bacteria. Furthermore, these findings contribute to our broader understanding of how root exudate quality could be a mechanism that plants use to select for distinct microbial communities in the rhizosphere.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Plant root exudates and rhizosphere bacterial communities shift with neighbor context

A plant's neighborhood context can alter its interactions with other organisms, but little is known about how these dynamics occur belowground, especially with soil microbes. Microbial communities in rhizosphere soil are influenced by many factors, including abiotic conditions and root-derived signals. In particular, root exudates have strong effects on rhizosphere assembly, respond to changes in abiotic conditions, and help plants interact with neighbors. Therefore, we predicted that root exudates likely play a central role in neighbor-induced shifts in rhizosphere communities. We conducted a greenhouse experiment to test this and determine how the rhizosphere bacterial community of a focal plant, Panicum virgatum, changed when beside different neighbors, and whether these shifts were mediated by neighbor-induced changes in root exudation. We found that neighbor altered both focal plant exudates and rhizosphere community, and that changes were largest when the focal plant was beside the most competitive neighbor, Rudbeckia hirta, which reduced both focal plant growth and nitrogen uptake. Several factors contributed to neighbor impacts on rhizosphere assembly, including neighbor-induced changes in root exudates during nitrogen-limitation and microbial spillover from roots of larger neighbors. Using an additional soil incubation, we also found that these changes in exudates can have even greater effects on soil nutrients than on microbial assembly. Overall, we show that neighbors influence one another's microbiomes, and highlight neighbor-induced changes in root exudates as one mechanism through which this may occur. Furthermore, this work suggests that rhizosphere assembly may differ in mixed-species communities and thus emphasizes a need for microbiome studies that consider neighborhood context.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Rhizosphere Soil Biogeochemical Data and Photosynthetic Data of Vicia Faba in a Rhizobox

Here we share the data in column format via csv files for pH, redox, and dissolved oxygen collected at hourly resolution from microelectrodes. Dissolved organic carbon concentrations collected from TOC are also provided in a similar format but are composited samples from hourly microdialysis collection. This provided resolution of diel rhizosphere dynamics belowground. Plant physiological data was also collected at every 5 min for 24 hr cycles in order to capture diel dynamics aboveground. This data was used to parameterize the reaction transport model eSTOMP-ROOTS, which examines the rhizosphere biogeochemistry of a growing Vicia faba plant. The aim was to investigate plant activity and belowground biogeochemical processes, particularly their impact on mineral-organic associations in the rhizosphere. We combined in-situ rhizosphere microsensor and plant physiological measurements with a 3-D plant-soil reactive transport model to explore the behavior of dissolved organic carbon (DOC) in the rhizosphere. Over several days, microdialysis probes placed at the root-soil interface in live soil showed distinct daily patterns of DOC concentration in the pore water. Spikes in DOC concentrations during the day aligned with peaks in leaf-level photosynthesis, accompanied by decreasing redox potential and dissolved oxygen levels, and increasing pH in the rhizosphere. This new mechanistic modeling framework, which integrates aboveground plant physiological data with non-destructive, high-resolution monitoring of rhizosphere processes, offers significant potential for studying the factors that control carbon storage in soils.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Enzymatically hydrolyzed fluorescence-based chemical probe enables in situ mapping of chitinase activity in the rhizosphere

Rhizosphere microbiomes and root exudates play a pivotal biochemical role in helping to catalyze chitin catabolism. Chitin is a recalcitrant and ubiquitous soil biopolymer, estimated to be the second most abundant organic soil biopolymer on Earth. Despite its abundance, role as a source of C and N in soil, and importance to ecosystem function, the biochemical mechanisms controlling chitin fate in the rhizosphere are elusive and poorly understood. To enable spatial mapping of chitinase activity in the rhizosphere, we designed and synthesized an enzymatically activated fluorogenic substrate, chitotriose-TokyoGreen (chitotriose-TG), by incorporating a fluorescein derivative (TG) onto the trimeric unit of chitin. This non-fluorescent substrate is selectively hydrolyzed by chitinase to release TG and yield a high fluorescence signal, which can be used to spatially image and measure chitinase activity in the rhizosphere. To demonstrate the application of this technique, we grew switchgrass (Panicum virgatum) in rhizoboxes amended with a horizontal layer of chitin. We extracted mobile proteins from the rhizobox using a nitrocellulose membrane blotting technique which offer non-destructive enzyme extraction while preserving the 2D spatial position of the enzymes. We then subjected these membranes to the synthesized chitotriose-TG stain to spatially visualize the distribution of chitinase activity within the rhizosphere. Furthermore, we observed increased chitinase activity near plant roots and higher activity within the soil zone enriched in chitin, showing an adaptive response of chitinase activity with spatial focusing in areas of higher chitin abundance. Thus, the enzyme extraction and visualization strategy we describe here can help enlighten efforts to better understand spatial controls on chitin breakdown in rhizosphere, further elucidating the role of chitin as a C and N source in these systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Resolving dynamic mineral-organic interactions in the rhizosphere by combining in-situ microsensors with plant-soil reactive transport modeling

Associations between minerals and organic matter represent one of the most important carbon storage mechanisms in soils. Plant roots are major sources of soil carbon, and resolving the dynamics and dominance of microbial consumption versus mineral sorption of root-derived carbon is critical to understanding soil carbon storage. Here we integrate in-situ rhizosphere microsensor and plant physiological measurements with a 3-D plant-soil reactive transport model to explore the fate of dissolved organic carbon (DOC) in the rhizosphere, particularly its microbial consumption and interaction with Fe oxide minerals. Over several days, a microdialysis probe sampling pore water at the root-soil interface of growing Vicia faba roots in live soil, revealed clear diel patterns of DOC concentration. Daytime DOC spikes coincided with peaks in leaf-level photosynthesis rates and were accompanied by declining redox potential and dissolved oxygen as well as increasing pH in the rhizosphere. Incorporating microsensor data into our modeling framework showed that the measured rapid loss of DOC after each mid-day spike could not be explained by consumption via aerobic respiration, nor via anaerobic respiration dominated by Fe oxide reduction. Rather, in the model, a large fraction of rhizosphere DOC was rapidly immobilized each day by adsorption to Fe oxides. Further, modeled microbial Fe reduction (fueled by DOC) did not mobilize significant organic carbon from Fe oxides during the day. Instead, the model predicted equilibrium desorption of organic carbon from Fe oxides at night. This new mechanistic modeling framework, which couples aboveground plant physiological measurements with non-destructive high-resolution monitoring of rhizosphere processes, has great potential for exploring the dynamics and balance of the various microbial reactions and mineral interactions controlling carbon transformations and storage in soils.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Rhizosphere and detritusphere habitats modulate expression of soil N-cycling genes during plant development

Interactions between plant roots and rhizosphere bacteria modulate nitrogen (N)-cycling processes and create habitats rich in low molecular weight compounds (exudates) and complex organic molecules (decaying root litter) compared to those of bulk soil. Microbial N-cycling is regulated by edaphic conditions and genes from many interconnected metabolic pathways, but most studies of soil N-cycling gene expression have focused on single pathways. Currently, we lack a comprehensive understanding of the interplay between soil N-cycling gene regulation, spatial habitat, and time. We present results from a replicated time series of soil metatranscriptomes; we followed gene expression of multiple N transformations in four soil habitats (rhizosphere, detritusphere, rhizo-detritusphere, and bulk soil) during active root growth for the annual grass, Avena fatua. The presence of root litter and living roots significantly altered the trajectories of N-cycling gene expression. Upregulation of assimilatory nitrate reduction in the rhizosphere suggests that rhizosphere bacteria were actively competing with roots for nitrate. Simultaneously, ammonium assimilatory pathways were upregulated in both rhizosphere and detritusphere soil, which could have limited N availability to plants. The detritusphere supported dissimilatory processes DNRA and denitrification. Expression of nitrification genes was dominated by three phylotypes of Thaumarch aeota and was upregulated in bulk soil. Unidirectional ammonium assimilation and its regulatory genes (GS/GOGAT) were upregulated near relatively young roots and highly decayed root litter, suggesting N may have been limiting in these habitats (GS/GOGAT is typically activated under N limitation). In conclusion, our comprehensive analysis indicates that differences in carbon and inorganic N availability control contemporaneous transcription of N-cycling pathways in soil habitats.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Developing stable, simplified, functional consortia from Brachypodium rhizosphere for microbial application in sustainable agriculture

The rhizosphere microbiome plays a crucial role in supporting plant productivity and ecosystem functioning by regulating nutrient cycling, soil integrity, and carbon storage. However, deciphering the intricate interplay between microbial relationships within the rhizosphere is challenging due to the overwhelming taxonomic and functional diversity. Here we present our systematic design framework built on microbial colocalization and microbial interaction, toward successful assembly of multiple rhizosphere-derived Reduced Complexity Consortia (RCC). We enriched co-localized microbes from Brachypodium roots grown in field soil with carbon substrates mimicking Brachypodium root exudates, generating 768 enrichments. By transferring the enrichments every 3 or 7 days for 10 generations, we developed both fast and slow-growing reduced complexity microbial communities. Most carbon substrates led to highly stable RCC just after a few transfers. 16S rRNA gene amplicon analysis revealed distinct community compositions based on inoculum and carbon source, with complex carbon enriching slow growing yet functionally important soil taxa like Acidobacteria and Verrucomicrobia. Network analysis showed that microbial consortia, whether differentiated by growth rate (fast vs. slow) or by succession (across generations), had significantly different network centralities. Besides, the keystone taxa identified within these networks belong to genera with plant growth-promoting traits, underscoring their critical function in shaping rhizospheric microbiome networks. Furthermore, tested consortia demonstrated high stability and reproducibility, assuring successful revival from glycerol stocks for long-term viability and use. Our study represents a significant step toward developing a framework for assembling rhizosphere consortia based on microbial colocalization and interaction, with future implications for sustainable agriculture and environmental management.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Rhizosphere Microbiome Diversity Potentially Supports Robust Nature of Field Pennycress ( Thlaspi arvense L.) in Dryland Cropping Systems of Eastern Washington

ABSTRACT Field pennycress ( Thlaspi arvense L.) is an annual in the Brassicaceae family and is currently being developed as an oilseed intermediate crop suitable for renewable biodiesel and jet fuel. It displays many desirable characteristics for this role including cold tolerance, a rapid life cycle, and a seed fatty acid profile conducive to bioenergy generation. These traits make field pennycress favorable for winter oilseed cultivation in the inland Pacific Northwest (iPNW). Simultaneously, intermediate crops are an increasingly recognized component of both agronomic sustainability and soil health management. Intermediate crops enhance soil microbial diversity, which benefits both soil and plant health. To understand the impact of field pennycress on soil microbial diversity, two natural accessions and seven experimental accessions were grown at three sites in Eastern Washington. Aboveground biomass and rhizosphere soil were then collected. Soil genomic DNA was extracted from rhizosphere samples and used to generate an amplicon library for bacterial (16S) and fungal (ITS) rRNA sequences. The resulting libraries were analyzed in QIIME2, which revealed that not only did the fad2 deficient line from the Spring32‐10 background have significantly increased aboveground biomass production compared to other pennycress genotypes, but also displayed significantly higher β‐diversity in the rhizosphere community specifically at the site experiencing the driest conditions. ANCOM analysis showed that multiple sequences similar to beneficial plant and soil health enhancing organisms such as Trichoderma spirale , Pseudomonas spp., and Methylobacterium goesingense were found to be enriched in the microbiome of the fad2 Spring32‐10 background also at that site. To add additional context to rhizosphere community data, root exudates from two pennycress genotypes were captured in magenta boxes and analyzed using HPLC. Future work will expand our understanding of the mechanisms by which field pennycress creates diversity in the rhizosphere, thus expanding our ability to cultivate this crop in the iPNW.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Long-term compost amendment modulates wheat genotype differences in belowground carbon allocation, microbial rhizosphere recruitment and nitrogen acquisition

The implementation of soil health-promoting practices, such as cover cropping and compost application, has important implications for nutrient cycling and management in agroecosystems. At the same time, plant belowground carbon (C) allocation patterns can influence nutrient cycling and availability in soil through changes to the microbial community, but the effects may depend on the crop genotype and management practices in place. We evaluated belowground C allocation patterns using 13 C labeling and root architecture in two genotypes of winter wheat (Triticum aestivum) with different levels of exudation and belowground allocation strategies in soils with contrasting compost amendment legacy (108.7 Mg ha -1 every 2 years over 10 years vs. no compost). We also measured microbial community structure and function in the rhizosphere and quantified uptake of residue-derived N from 15 N-labelled cover crop residues. We found an interactive effect between soil management and genotype, where in the no-compost soil, the high-exudation genotype (Snowmass) increased exudation by over 4-fold, while the low-exudate genotype (Byrd) increased only 2-fold. While we did not observe genotype differences in rhizosphere enzyme activity or dissolved N pools, residue N uptake was 1.8 times greater for Snowmass in the compost-amended soil. There were more rhizosphere microbial taxa associated with the high-exudate genotype (Snowmass); nine bacterial and seven fungal families were indicative of Snowmass, versus one bacterial and four fungal families for Byrd. Our results suggest that the high-exudation strategy can influence the rhizosphere microbial community, and lead to greater short-term residue N uptake in high SOM soil. By directly linking root architecture, exudation, microbial communities, and N mineralization and uptake dynamics, this work demonstrates that plasticity in root C allocation is genotype-specific and influences microbial communities and nutrient cycling depending on the soil health context.

59 BASIC BIOLOGICAL SCIENCES↗