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Arbuscular mycorrhizal fungi (AMF) transport substantial plant carbon (C) that serves as a substrate for soil organisms, a precursor of soil organic matter (SOM), and a driver of soil microbial dynamics. Using two-chamber microcosms where an air gap isolated AMF from roots, we 13CO2-labeled Avena barbata for 6 wk and measured the C Rhizophagus intraradices transferred to SOM and hyphosphere microorganisms. NanoSIMS imaging revealed hyphae and roots had similar 13C enrichment. SOM density fractionation, 13C NMR, and IRMS showed AMF transferred 0.77 mg C g-1 of soil (increasing total C by 2% relative to non-mycorrhizal controls); 33% was found in occluded or mineral-associated pools. In the AMF hyphosphere, there was no overall change in community diversity but 36 bacterial ASVs significantly changed in relative abundance. With stable isotope probing (SIP)-enabled shotgun sequencing, we found taxa from the Solibacterales, Sphingobacteriales, Myxococcales, and Nitrososphaerales (ammonium oxidizing archaea) were highly enriched in AMF-imported 13C (> 20 atom%). Mapping sequences from 13C-SIP metagenomes to total ASVs showed at least 92 bacteria and archaea were significantly 13C-enriched. Our results illustrate the quantitative and ecological impact of hyphal C transport on the formation of potentially protective SOM pools and microbial roles in the AMF hyphosphere soil food web.

Stable isotope probing (SIP) facilitates culture-independent identification of active microbial populations within complex ecosystems through isotopic enrichment of nucleic acids. Many DNA-SIP studies rely on 16S rRNA gene sequences to identify active taxa, but connecting these sequences to specific bacterial genomes is often challenging. Here, we describe a standardized laboratory and analysis framework to quantify isotopic enrichment on a per-genome basis using shotgun metagenomics instead of 16S rRNA gene sequencing. To develop this framework, we explored various sample processing and analysis approaches using a designed microbiome where the identity of labeled genomes and their level of isotopic enrichment were experimentally controlled. With this ground truth dataset, we empirically assessed the accuracy of different analytical models for identifying active taxa and examined how sequencing depth impacts the detection of isotopically labeled genomes. We also demonstrate that using synthetic DNA internal standards to measure absolute genome abundances in SIP density fractions improves estimates of isotopic enrichment. In addition, our study illustrates the utility of internal standards to reveal anomalies in sample handling that could negatively impact SIP metagenomic analyses if left undetected. Finally, we present SIPmg, an R package to facilitate the estimation of absolute abundances and perform statistical analyses for identifying labeled genomes within SIP metagenomic data. This experimentally validated analysis framework strengthens the foundation of DNA-SIP metagenomics as a tool for accurately measuring the in situ activity of environmental microbial populations and assessing their genomic potential. IMPORTANCE Answering the questions, "who is eating what?" and "who is active?" within complex microbial communities is paramount for our ability to model, predict, and modulate microbiomes for improved human and planetary health. These questions can be pursued using stable isotope probing to track the incorporation of labeled compounds into cellular DNA during microbial growth. However, with traditional stable isotope methods, it is challenging to establish links between an active microorganism's taxonomic identity and genome composition while providing quantitative estimates of the microorganism's isotope incorporation rate. Here, we report an experimental and analytical workflow that lays the foundation for improved detection of metabolically active microorganisms and better quantitative estimates of genome-resolved isotope incorporation, which can be used to further refine ecosystem-scale models for carbon and nutrient fluxes within microbiomes.

Nitrogen (N) is frequently limiting to plant growth, in part because most soil N is present as polymeric organic compounds that are not readily taken up by plants. Microbial depolymerization of these large macromolecular N-substrates gradually releases available inorganic N. While many studies have researched and modeled controls on soil organic matter formation and bulk N mineralization, the ecological-spatial, temporal and phylogenetic-patterns underlying organic N degradation remain unclear. We analyzed 48 time-resolved metatranscriptomes and quantified N-depolymerization gene expression to resolve differential expression by soil habitat and time in specific taxonomic groups and gene-based guilds. We observed much higher expression of extracellular serine-type proteases than other extracellular N-degrading enzymes, with protease expression of predatory bacteria declining with time and other taxonomic patterns driven by the presence (Gammaproteobacteria) or absence (Thermoproteota) of live roots and root detritus (Deltaproteobacteria and Fungi). The primary chitinase chit1 gene was more highly expressed by eukaryotes near root detritus, suggesting predation of fungi. In some lineages, increased gene expression over time suggests increased competitiveness with rhizosphere age (Chloroflexi). Phylotypes from some genera had protease expression patterns that could benefit plant N nutrition, for example, we identified a Janthinobacterium phylotype and two Burkholderiales that depolymerize organic N near young roots and a Rhizobacter with elevated protease levels near mature roots. These taxon-resolved gene expression results provide an ecological read-out of microbial interactions and controls on N dynamics in specific soil microhabitats and could be used to target potential plant N bioaugmentation strategies.

The growth and physiology of soil microorganisms, which play vital roles in biogeochemical cycling, are shaped by both current and historical soil environmental conditions. Here, we developed and applied a genome-resolved metagenomic implementation of quantitative stable isotope probing (qSIP) with an H218O labeling experiment to identify actively growing soil microorganisms and their genomic capacities. qSIP enabled measurement of taxon-specific growth because isotopic incorporation into microbial DNA requires production of new genome copies. We studied three Mediterranean grassland soils across a rainfall gradient to evaluate the hypothesis that historic precipitation levels are an important factor controlling trait selection. We used qSIP-informed genome-resolved metagenomics to resolve the active subset of soil community members and identify their characteristic ecophysiological traits. Higher year-round precipitation levels correlated with higher activity and growth rates of flagellar motile microorganisms. In addition to heavily isotopically labeled bacteria, we identified abundant isotope-labeled phages, suggesting phage-induced cell lysis likely contributed to necromass production at all three sites. Further, there was a positive correlation between phage activity and the activity of putative phage hosts. Contrary to our expectations, the capacity to decompose the diverse complex carbohydrates common in soil organic matter or oxidize methanol and carbon monoxide were broadly distributed across active and inactive bacteria in all three soils, implying that these traits are not highly selected for by historical precipitation. IMPORTANCE Soil moisture is a critical factor that strongly shapes the lifestyle of soil organisms by changing access to nutrients, controlling oxygen diffusion, and regulating the potential for mobility. We identified active microorganisms in three grassland soils with similar mineral contexts, yet different historic rainfall inputs, by adding water labeled with a stable isotope and tracking that isotope in DNA of growing microbes. By examining the genomes of active and inactive microorganisms, we identified functions that are enriched in growing organisms, and showed that different functions were selected for in different soils. Wetter soil had higher activity of motile organisms, but activity of pathways for degradation of soil organic carbon compounds, including simple carbon substrates, were comparable for all three soils. We identified many labeled, and thus active bacteriophages (viruses that infect bacteria), implying that the cells they killed contributed to soil organic matter. The activity of these bacteriophages was significantly correlated with activity of their hosts.

Drought disrupts soil microbial activity and many biogeochemical processes. Although plant-associated fungi can support plant performance and nutrient cycling during drought, their effects on nearby drought-exposed soil microbial communities are not well resolved. We used H218O quantitative stable isotope probing (qSIP) and 16S rRNA gene profiling to investigate bacterial community dynamics following water limitation in the hyphospheres of two distinct fungal lineages (Rhizophagus irregularis and Serendipita bescii) grown with the bioenergy model grass Panicum hallii. In uninoculated soil, a history of water limitation resulted in significantly lower bacterial growth potential and growth efficiency, as well as lower diversity in the actively growing bacterial community. In contrast, both fungal lineages had a protective effect on hyphosphere bacterial communities exposed to water limitation: bacterial growth potential, growth efficiency, and the diversity of the actively growing bacterial community were not suppressed by a history of water limitation in soils inoculated with either fungus. Despite their similar effects at the community level, the two fungal lineages did elicit different taxon-specific responses, and bacterial growth potential was greater in R. irregularis compared to S. bescii-inoculated soils. Several of the bacterial taxa that responded positively to fungal inocula belong to lineages that are considered drought susceptible. Overall, H218O qSIP highlighted treatment effects on bacterial community structure that were less pronounced using traditional 16S rRNA gene profiling. Together, these results indicate that fungal-bacterial synergies may support bacterial resilience to moisture limitation.

BackgroundLinking 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.ResultsOur 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 13C-AMF hyphosphere DNA from a 13CO2 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% 13C), even though the soils' overall enrichment was low (1.8 atom% 13C). We assembled 212 13C-hyphosphere MAGs; the hyphosphere taxa that assimilated the most AMF-derived 13C were from the phyla Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia-oxidizing archaeon genus Nitrososphaera.ConclusionsOur 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. Video Abstract.

Switchgrass is a deep-rooted perennial native to the US prairies and an attractive feedstock for bioenergy production; when cultivated on marginal soils it can provide a potential mechanism to sequester and accumulate soil carbon (C). However, the impacts of switchgrass establishment on soil biotic/abiotic properties are poorly understood. Additionally, few studies have reported the effects of switchgrass cultivation on marginal lands that have low soil nutrient quality (N/P) or in areas that have experienced high rates of soil erosion. Here, we report a comparative analyses of soil greenhouse gases (GHG), soil chemistry, and microbial communities in two contrasting soil types (with or without switchgrass) over 17 months (1428 soil samples). These soils are highly eroded, 'Dust Bowl' remnant field sites in southern Oklahoma, USA. Our results revealed that soil C significantly increased at the sandy-loam (SL) site, but not at the clay-loam (CL) site. Significantly higher CO2 flux was observed from the CL switchgrass site, along with reduced microbial diversity (both alpha and beta). Strikingly, methane (CH4) consumption was significantly reduced by an estimated 39 and 47% at the SL and CL switchgrass sites, respectively. Together, our results suggest that soil C stocks and GHG fluxes are distinctly different at highly degraded sites when switchgrass has been cultivated, implying that carbon balance considerations should be accounted for to fully evaluate the sustainability of deep-rooted perennial grass cultivation in marginal lands.

Roots are a primary source of organic carbon input in most soils. The consumption of living and detrital root inputs involves multi-trophic processes and multiple kingdoms of microbial life, but typical microbial ecology studies focus on only one or two major lineages. We used Illumina shotgun RNA sequencing to conduct PCR-independent SSU rRNA community analysis ("community RNA-Seq") and simultaneously assess the bacteria, archaea, fungi, and microfauna surrounding both living and decomposing roots of the annual grass, Avena fatua. Plants were grown in 13CO2-labeled microcosms amended with 15N-root litter to identify the preferences of rhizosphere organisms for root exudates (13C) versus decaying root biomass (15N) using NanoSIMS microarray imaging (Chip-SIP). When litter was available, rhizosphere and bulk soil had significantly more Amoebozoa, which are potentially important yet often overlooked top-down drivers of detritusphere community dynamics and nutrient cycling. Bulk soil containing litter was depleted in Actinobacteria but had significantly more Bacteroidetes and Proteobacteria. While Actinobacteria were abundant in the rhizosphere, Chip-SIP showed Actinobacteria preferentially incorporated litter relative to root exudates, indicating this group's more prominent role in detritus elemental cycling in the rhizosphere. Our results emphasize that decomposition is a multi-trophic process involving complex interactions, and our methodology can be used to track the trajectory of carbon through multi-kingdom soil food webs.

Candidate Phyla Radiation (CPR) bacteria and nanoarchaea populate most ecosystems but are rarely detected in soil. We concentrated particles of less than 0.2 μm in size from grassland soil, enabling targeted metagenomic analysis of these organisms, which are almost totally unexplored in largely oxic environments such as soil. We recovered a diversity of CPR bacterial and some archaeal sequences but no sequences from other cellular organisms. The sampled sequences include Doudnabacteria (SM2F11) and Pacearchaeota, organisms rarely reported in soil, as well as Saccharibacteria, Parcubacteria, and Microgenomates. CPR and archaea of the phyla Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) were enriched 100- to 1,000-fold compared to that in bulk soil, in which we estimate each of these organisms comprises approximately 1 to 100 cells per gram of soil. Like most CPR and DPANN sequenced to date, we predict these microorganisms live symbiotic anaerobic lifestyles. However, Saccharibacteria, Parcubacteria, and Doudnabacteria genomes sampled here also harbor ubiquinol oxidase operons that may have been acquired from other bacteria, likely during adaptation to aerobic soil environments. We conclude that CPR bacteria and DPANN archaea are part of the rare soil biosphere and harbor unique metabolic platforms that potentially evolved to live symbiotically under relatively oxic conditions. IMPORTANCE Here, we investigated overlooked microbes in soil, Candidate Phyla Radiation (CPR) bacteria and Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) archaea, by size fractionating small particles from soil, an approach typically used for the recovery of viral metagenomes. Concentration of these small cells (

Switchgrass is a deep-rooted perennial native to the US prairies and an attractive feedstock for bioenergy production; when cultivated on marginal soils it can provide a potential mechanism to sequester and accumulate soil carbon (C). However, the impacts of switchgrass establishment on soil biotic/abiotic properties are poorly understood. Additionally, few studies have reported the effects of switchgrass cultivation on marginal lands that have low soil nutrient quality (N/P) or in areas that have experienced high rates of soil erosion. Here, we report a comparative analyses of soil greenhouse gases (GHG), soil chemistry, and microbial communities in two contrasting soil types (with or without switchgrass) over 17 months (1428 soil samples). These soils are highly eroded, 'Dust Bowl' remnant field sites in southern Oklahoma, USA. Our results revealed that soil C significantly increased at the sandy-loam (SL) site, but not at the clay-loam (CL) site. Significantly higher CO2 flux was observed from the CL switchgrass site, along with reduced microbial diversity (both alpha and beta). Strikingly, methane (CH4) consumption was significantly reduced by an estimated 39 and 47% at the SL and CL switchgrass sites, respectively. Together, our results suggest that soil C stocks and GHG fluxes are distinctly different at highly degraded sites when switchgrass has been cultivated, implying that carbon balance considerations should be accounted for to fully evaluate the sustainability of deep-rooted perennial grass cultivation in marginal lands.

Microbial activity increases after rewetting dry soil, resulting in a pulse of carbon mineralization and nutrient availability. The biogeochemical responses to wet-up are reasonably well understood and known to be microbially mediated. Yet, the population level dynamics, and the resulting changes in microbial community patterns, are not well understood as ecological phenomena. Here, we used sequencing of 16S rRNA genes coupled with heavy water (H218O) DNA quantitative stable isotope probing to estimate population-specific rates of growth and mortality in response to a simulated wet-up event in a California annual grassland soil. Bacterial growth and mortality responded rapidly to wet-up, within 3 h, and continued throughout the 168 h incubation, with patterns of sequential growth observed at the phylum level. Of the 37 phyla detected in the prewet community, growth was found in 18 phyla while mortality was measured in 26 phyla. Rapid growth and mortality rates were measurable within 3 h of wet-up but had contrasting characteristics; growth at 3 h was dominated by select taxa within the Proteobacteria and Firmicutes, whereas mortality was taxonomically widespread. Furthermore, across the community, mortality exhibited density-independence, consistent with the indiscriminate shock resulting from dry-down and wet-up, whereas growth was density-dependent, consistent with control by competition or predation. Total aggregated growth across the community was highly correlated with total soil CO2 production. Together, these results illustrate how previously "invisible" population responses can translate quantitatively to emergent observations of ecosystem-scale biogeochemistry.

The rhizosphere is a hotspot for microbial carbon transformations, and is the entry point for root polysaccharides and polymeric carbohydrates that are important precursors to soil organic matter (SOM). However, the ecological mechanisms that underpin rhizosphere carbohydrate depolymerization are poorly understood. Using Avena fatua, a common annual grass, we analyzed time-resolved metatranscriptomes to compare microbial functions in rhizosphere, detritusphere, and combined rhizosphere-detritusphere habitats. Transcripts were binned using a unique reference database generated from soil isolate genomes, single-cell amplified genomes, metagenomes, and stable isotope probing metagenomes. While soil habitat significantly affected both community composition and overall gene expression, the succession of microbial functions occurred at a faster time scale than compositional changes. Using hierarchical clustering of upregulated decomposition genes, we identified four distinct microbial guilds populated by taxa whose functional succession patterns suggest specialization for substrates provided by fresh growing roots, decaying root detritus, the combination of live and decaying root biomass, or aging root material. Carbohydrate depolymerization genes were consistently upregulated in the rhizosphere, and both taxonomic and functional diversity were highest in the combined rhizosphere-detritusphere, suggesting coexistence of rhizosphere guilds is facilitated by niche differentiation. Metatranscriptome-defined guilds provide a framework to model rhizosphere succession and its consequences for soil carbon cycling.

Viruses impact nearly all organisms on Earth, with ripples of influence in agriculture, health, and biogeochemical processes. However, very little is known about RNA viruses in an environmental context, and even less is known about their diversity and ecology in soil, 1 of the most complex microbial systems. Here, we assembled 48 individual metatranscriptomes from 4 habitats within a planted soil sampled over a 22-d time series: Rhizosphere alone, detritosphere alone, rhizosphere with added root detritus, and unamended soil (4 time points and 3 biological replicates). We resolved the RNA viral community, uncovering a high diversity of viral sequences. We also investigated possible host organisms by analyzing metatranscriptome marker genes. Based on viral phylogeny, much of the diversity was Narnaviridae that may parasitize fungi or Leviviridae, which may infect Proteobacteria. Both host and viral communities appear to be highly dynamic, and rapidly diverged depending on experimental conditions. The viral and host communities were structured based on the presence of root litter. Clear temporal dynamics by Leviviridae and their hosts indicated that viruses were replicating. With this time-resolved analysis, we show that RNA viruses are diverse, abundant, and active in soil. When viral infection causes host cell death, it may mobilize cell carbon in a process that may represent an overlooked component of soil carbon cycling.

Microbial processing of soil organic matter is a significant driver of C cycling, yet we lack an understanding of what shapes the turnover of this large terrestrial pool. In part, this is due to limited options for accurately identifying the source of C assimilated by microbial communities. Laboratory incubations are the most common method for this; however, they can introduce artifacts due to sample disruption and processing and can take months to produce sufficient CO2 for analysis. We present a biomass extraction method which allows for the direct 14C analysis of microbial biomolecules and compare the results to laboratory incubations. In the upper 50 cm soil depths, the Δ14C from incubations was indistinguishable from that of extracted microbial biomass. Below 50 cm, the Δ14C of the biomass was more depleted than that of the incubations, either due to the stimulation of labile C decomposition in the incubations, the inclusion of biomolecules from non-living cells in the biomass extractions, or differences in C used for assimilation versus respiration. Our results suggest that measurement of Δ14C of microbial biomass extracts can be a useful alternative to soil incubations. [ABSTRACT FROM AUTHOR]

While interactions between roots and microorganisms have been intensively studied, we know little about interactions among root-associated microbes. We used random matrix theory-based network analysis of 16S rRNA genes to identify bacterial networks associated with wild oat (Avena fatua) over two seasons in greenhouse microcosms. Rhizosphere networks were substantially more complex than those in surrounding soils, indicating the rhizosphere has a greater potential for interactions and niche-sharing. Network complexity increased as plants grew, even as diversity decreased, highlighting that community organisation is not captured by univariate diversity. Covariations were predominantly positive (> 80%), suggesting that extensive mutualistic interactions may occur among rhizosphere bacteria; we identified quorum-based signalling as one potential strategy. Putative keystone taxa often had low relative abundances, suggesting low-abundance taxa may significantly contribute to rhizosphere function. Network complexity, a previously undescribed property of the rhizosphere microbiome, appears to be a defining characteristic of this habitat.

The interface between roots and soil, known as the rhizosphere, is a dynamic habitat in the soil ecosystem. Unraveling the factors that control rhizosphere community assembly is a key starting point for understanding the diversity of plant-microbial interactions that occur in soil. The goals of this study were to determine how environmental factors shape rhizosphere microbial communities, such as local soil characteristics and the regional climate, and to determine the relative influence of the rhizosphere on microbial community assembly compared to the pressures imposed by the local and regional environment. We identified the bacteria present in the soil immediately adjacent to the roots of wild oat (A vena spp.) in three California grasslands using deep Illumina 16S sequencing. Rhizosphere communities were more similar to each other than to the surrounding soil communities from which they were derived, despite the fact that the grasslands studied were separated by hundreds of kilometers. The rhizosphere was the dominant factor structuring bacterial community composition (38% variance explained), and was comparable in magnitude to the combined local and regional effects (22% and 21%, respectively). Rhizosphere communities were most influenced by factors related to the regional climate (soil moisture and temperature), while background soil communities were more influenced by soil characteristics (pH, CEC, exchangeable cations, clay content). The Avena core microbiome was strongly phylogenetically clustered according to the metrics NRI and NTI, which indicates that selective processes likely shaped these communities. Furthermore, 17% of these taxa were not detectable in the background soil, even with a robust sequencing depth of approximately 70,000 sequences per sample. These results support the hypothesis that roots select less abundant or possibly rare populations in the soil microbial community, which appear to be lineages of bacteria that have made a physiological tradeoff for rhizosphere competence at the expense of their competitiveness in non-rhizosphere soil.

Changes in soil organic carbon (SOC) storage have the potential to affect global climate; hence identifying environments with a high capacity to gain or lose SOC is of broad interest. Many cross‐site studies have found that SOC‐poor soils tend to gain or retain carbon more readily than SOC‐rich soils. While this pattern may partly reflect reality, here we argue that it can also be created by a pair of statistical artifacts. First, soils that appear SOC‐poor purely due to random variation will tend to yield more moderate SOC estimates upon resampling and hence will appear to accrue or retain more SOC than SOC‐rich soils. This phenomenon is an example of regression to the mean. Second, normalized metrics of SOC change—such as relative rates and response ratios—will by definition show larger changes in SOC at lower initial SOC levels, even when the absolute change in SOC does not depend on initial SOC. These two artifacts create an exaggerated impression that initial SOC stocks are a major control on SOC dynamics. To address this problem, we recommend applying statistical corrections to eliminate the effect of regression to the mean, and avoiding normalized metrics when testing relationships between SOC change and initial SOC. Careful consideration of these issues in future cross‐site studies will support clearer scientific inference that can better inform environmental management. [ABSTRACT FROM AUTHOR]

Perennial bioenergy crops have been shown to increase soil organic carbon (SOC) stocks, potentially offsetting anthropogenic C emissions. The effects of perennial bioenergy crops on SOC are typically assessed at shallow depths (<30 cm), but the deep root systems of these crops may also have substantial effects on SOC stocks at greater depths. We hypothesized that deep (>30 cm) SOC stocks would be greater under bioenergy crops relative to stocks under shallow‐rooted conventional crop cover. To test this, we sampled soils to between 1‐ and 3‐m depth at three sites in Oklahoma with 10‐ to 20‐year‐old switchgrass (Panicum virgatum) stands, and collected paired samples from nearby fields cultivated with shallow rooted annual crops. We measured root biomass, total organic C, 14C, 13C, and other soil properties in three replicate soil cores in each field and used a mixing model to estimate the proportion of recently fixed C under switchgrass based on 14C. The subsoil C stock under switchgrass (defined over 500–1500 kg/m2 equivalent soil mass, approximately 30–100 cm depth) exceeded the subsoil stock in neighboring fields by 1.5 kg C/m2 at a sandy loam site, 0.6 kg C/m2 at a site with loam soils, and showed no significant difference at a third site with clay soils. Using the mixing model, we estimated that additional SOC introduced after switchgrass cultivation comprised 31% of the subsoil C stock at the sandy loam site, 22% at the loam site, and 0% at the clay site. These results suggest that switchgrass can contribute significantly to subsoil organic C—but also indicated that this effect varies across sites. Our analysis shows that agricultural strategies that emphasize deep‐rooted grass cultivars can increase soil C relative to conventional crops while expanding energy biomass production on marginal lands. [ABSTRACT FROM AUTHOR]