Abstract Climate change intensifies grassland drying, which is predicted to decrease soil organic carbon (SOC) storage. Yet how reduced soil water affects the transport and processing of carbon in the rhizosphere — a major source of SOC supporting microbial activities — remains poorly resolved under field conditions. We traced living root-derived carbon (rhizodeposit C) into soils and microbes using ¹³CO₂ labeling in a multi-year precipitation manipulation experiment in a California Mediterranean annual grassland dominated by Avena barbata . Under halved precipitation, season-averaged soil water content was 20% lower and soil matric potential more than threefold lower, without inducing detectable signs of plant stress. Quantitative stable isotope probing (qSIP) of rhizosphere microbial DNA revealed that up to 68% more bacterial and 13% more fungal taxa incorporated rhizodeposit C, and that microbial uptake of rhizodeposit C increased more over time under reduced precipitation, even as total microbial biomass and community composition were unchanged. ¹³C-informed co-occurrence networks of these consumers were larger and more connected under reduced precipitation. The surrounding soils contained less 13 C, and modeled solute transport by soil water was 32-40% lower, implying that rhizodeposit C was retained and intensively processed nearer to roots rather than transported to the wider soil matrix. Our evidence supports that reduced precipitation concentrates rhizodeposit C near roots, increasing the number of distinct consumers and intensifying associations among them. This represents an overlooked mechanism linking precipitation reduction to rhizodeposit C cycling. These findings show that precipitation reduction may reshape rhizosphere C processing before apparent plant stress response, with potential consequences for the fate of rhizodeposit C in grassland soils.

Changes in global patterns can leave a lasting legacy in semiarid grasslands by reshaping microbial growth dynamics and carbon cycling during the first wet-up in the autumn—a period known for intense microbial activity and significant carbon emissions. To study the lasting impacts of decreased winter rain, we implemented two precipitation regimes (100

Widespread bioenergy cropping can transform landscapes, strongly affecting biodiversity. However, the impact of bioenergy cropping on the spatiotemporal scaling of soil biodiversity remains virtually unknown, despite its profound implications for the functioning of the ecological community. Here, we investigated how bioenergy cropping influenced the spatiotemporal scaling of soil bacterial biodiversity in marginal soils (sandy loam and clay loam soils) in Oklahoma, USA. We detected strong, significant species-time-area relationships (STARs) and phylogenetic-time-area relationships (PTARs) in bacterial communities and their lineages, suggesting that STARs and PTARs exist in microbial ecology within the studied system. Also, spatiotemporal scaling rates (the slopes of STAR and PTAR models) varied substantially among bacterial lineages and were positively correlated with their 16S rRNA gene copy numbers, a genomic trait indicative of microbial growth potentials. Strikingly, bioenergy cropping significantly reduced spatiotemporal scaling rates by 6.8%-14.1%, with a more pronounced reduction observed in sandy loam soils, where those rates were significantly lower than in clay loam soils. The heterogeneity of soil phosphorus and carbon resulted in variations in bacterial spatiotemporal scaling rates. Collectively, our findings suggest that bioenergy cropping may alleviate rapid shifts in soil biodiversity across space and time, thereby stabilizing soil biodiversity and supporting its role as part of sustainable land management and climate mitigation strategies.

BackgroundInteractions between fungi and bacteria have the potential to substantially influence soil carbon dynamics in soil, but we have yet to fully identify these interactions and partners in their natural environment. In this study, we stacked two powerful methods, 13C quantitative stable isotope probing (qSIP) and cross-domain co-occurrence network, to identify interacting fungi and bacteria in a California grassland soil. We used in-field whole plant 13CO2 labeling along with sand-filled ingrowth bags (that trap fungi and hyphae-associated bacteria) to amplify the signal of fungal-bacterial interactions, separate from the bulk soil background.ResultsWe found a total of 54 bacterial ASVs and 9 fungal OTUs that were significantly 13C-enriched. These were saprotrophic and biotrophic fungi, and motile, sometimes predatory bacteria. Among these, 70% of all 13C-enriched bacteria identified were motile. Notably, we detected fungal-bacterial network links between a fungal OTU of the genus Alternaria and several bacterial ASVs of the genera Bacteriovorax, Mucilaginibacter, and Flavobacterium, providing empirical evidence of their direct interactions through C exchange. We observed a strong positive co-occurrence pattern between predatory bacteria of the phylum Bdellovibrionota and fungal OTUs, suggesting the transfer of C across the soil food web.ConclusionsTo date, our ability to associate microbial co-occurrence network patterns with biological interactions is limited, but the incorporation of qSIP allowed us to more precisely detect interacting partners by narrowing in on the taxa that were actively incorporating plant-fixed, fungal-transported labeled substrates. Together, these approaches can help build a mechanistic understanding of the complex nature of fungal-bacterial interactions in soil.

In soils, the first rain after a prolonged dry period represents a major pulse event impacting soil microbial community function, yet we lack a full understanding of the genomic traits associated with the microbial response to rewetting. Genomic traits such as codon usage bias and genome size have been linked to bacterial growth in soils—however, often through measurements in culture. Here, we used metagenome-assembled genomes (MAGs) with 18 O-water stable isotope probing and metatranscriptomics to track genomic traits associated with growth and transcription of soil microorganisms over one week following rewetting of a grassland soil. We found that codon bias in ribosomal protein genes was the strongest predictor of growth rate. We also found higher growth rates in bacteria with smaller genomes, suggesting that reduced genome size enables a faster response to pulses in soil bacteria. Faster transcriptional upregulation of ribosomal protein genes was associated with high codon bias and increased nucleotide skew. We found that several of these relationships existed within phyla, indicating that these associations between genomic traits and activity could be generalized characteristics of soil bacteria. Finally, we used publicly available metagenomes to assess the distribution of codon bias across a pH gradient and found that microbial communities in higher pH soils—which are often more water limited and pulse driven—have higher codon usage bias in their ribosomal protein genes. Together, these results provide evidence that genomic characteristics affect soil microbial activity during rewetting and pose a potential fitness advantage for soil bacteria where water and nutrient availability are episodic.

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.

Soil microbial communities perform critical ecosystem services through the collective metabolic activities of numerous individual organisms. Most microbes use corrinoids, a structurally diverse family of cofactors related to vitamin B12. Corrinoid structure influences the growth of individual microbes, yet how these growth responses scale to the community level remains unknown. Analysis of metagenome-assembled genomes suggests that corrinoids are supplied to the community by members of the archaeal and bacterial phyla Thermoproteota, Actinobacteria, and Proteobacteria. Corrinoids were found largely adhered to the soil matrix in a grassland soil, at levels exceeding those required by cultured bacteria. Enrichment cultures and soil microcosms seeded with different corrinoids showed distinct shifts in bacterial community composition, supporting the hypothesis that corrinoid structure can shape communities. Environmental context influenced both community- and taxon-specific responses to specific corrinoids. These results implicate corrinoids as key determinants of soil microbiome structure and suggest that environmental micronutrient reservoirs promote community stability.

Global potent greenhouse gas nitrous oxide (N 2 O) emissions from soil are accelerating, with increases in the proportion of reactive nitrogen emitted as N 2 O, i.e., N 2 O emission factor (EF). Yet, the primary controls and underlying mechanisms of EFs remain unresolved. Based on two independent but complementary global syntheses, and three field studies determining effects of acidity on N 2 O EFs and soil denitrifying microorganisms, we show that soil pH predominantly controls N 2 O EFs and emissions by affecting the denitrifier community composition. Analysis of 5438 paired data points of N 2 O emission fluxes revealed a hump-shaped relationship between soil pH and EFs, with the highest EFs occurring in moderately acidic soils that favored N 2 O-producing over N 2 O-consuming microorganisms, and induced high N 2 O emissions. Our results illustrate that soil pH has a unimodal relationship with soil denitrifiers and EFs, and the net N 2 O emission depends on both the N 2 O/(N 2 O + N 2 ) ratio and overall denitrification rate. These findings can inform strategies to predict and mitigate soil N 2 O emissions under future nitrogen input scenarios.

Unravelling biosphere feedback mechanisms is crucial for predicting the impacts of global warming. Soil priming, an effect of fresh plant-derived carbon (C) on native soil organic carbon (SOC) decomposition, is a key feedback mechanism that could release large amounts of soil C into the atmosphere. However, the impacts of climate warming on soil priming remain elusive. Here, we show that experimental warming accelerates soil priming by 12.7% in a temperate grassland. Warming alters bacterial communities, with 38% of unique active phylotypes detected under warming. The functional genes essential for soil C decomposition are also stimulated, which could be linked to priming effects. We incorporate lab-derived information into an ecosystem model showing that model parameter uncertainty can be reduced by 32–37%. Model simulations from 2010 to 2016 indicate an increase in soil C decomposition under warming, with a 9.1% rise in priming-induced CO 2 emissions. If our findings can be generalized to other ecosystems over an extended period of time, soil priming could play an important role in terrestrial C cycle feedbacks and climate change.

Autotrophic nitrifiers, by catalyzing the oxidation of ammonia to nitrate, play a vital role in the global nitrogen cycle 1–5 . They convert carbon dioxide (CO 2 ) into biomass 1,3,6 and, therefore, are expected to respond positively to increasing atmospheric CO 2 concentrations 1,3,6 . However, in a long-term free-air CO 2 enrichment experiment, we demonstrated that elevated atmospheric CO 2 inhibited the growth of autotrophic nitrifiers, resulting in a reduction in nitrification in a rice ecosystem. By coupling stable isotope probing with metagenomics, we found that the CO 2 inhibition of nitrifiers was mainly a consequence of CO 2 -induced functional loss (that is, incapable of recovering genomes) of dominant but previously uncharacterized autotrophic nitrifying species, especially those of ammonia-oxidizing archaea and nitrite-oxidizing bacteria, which comprised 63% of total dominant members identified from the active nitrifying communities. We further found that the loss of these novel nitrifying species under elevated CO 2 was due largely to the CO 2 -induced aggravation of anoxic stress in the paddy soil. Our results provide insight into the fate of inorganic nitrogen pools in global lowland soil and water systems under future climate change scenarios.

When very dry soil is rewet, rapid stimulation of microbial activity has important implications for ecosystem biogeochemistry, yet associated changes in microbial transcription are poorly known. Here, we present metatranscriptomes of California annual grassland soil microbial communities, collected over 1 week from soils rewet after a summer drought-providing a time series of short-term transcriptional response during rewetting.

Summary 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.

Autotrophic nitrifiers, by catalysing the oxidation of ammonia to nitrate, play a vital role in the global nitrogen cycle. They convert carbon dioxide (CO2) into biomass and, therefore, are expected to respond positively to increasing atmospheric CO2 concentrations. However, in a long-term free-air CO2 enrichment experiment, we demonstrated that elevated atmospheric CO2 inhibited the growth of autotrophic nitrifiers, resulting in a reduction in nitrification in a rice ecosystem. By coupling stable-isotope probing with metagenomics, we found that the CO2 inhibition of nitrifiers was mainly a consequence of CO2-induced functional loss (genomes not recovered from metagenomes) of dominant but previously uncharacterized autotrophic nitrifying species. These species belonged mainly to ammonia-oxidizing archaea and nitrite-oxidizing bacteria and comprised 63% of total dominant members identified from the active nitrifying communities. We further showed that the functional loss of these novel nitrifying species under elevated CO2 was due largely to the CO2-induced aggravation of anoxic stress in the paddy soil. Our results provide insight into the fate of inorganic nitrogen pools in global lowland soil and water systems under climate change. The growth and function of autotrophic nitrifiers are suppressed under elevated CO2 due to the intensification of anoxic stress, according to a long-term free-air CO2 enrichment experiment in nitrogen-rich paddy soil.

As central members of soil trophic networks, viruses have the potential to drive substantial microbial mortality and nutrient turnover. Pinpointing viral contributions to terrestrial ecosystem processes remains a challenge, as temporal dynamics are difficult to unravel in the spatially and physicochemically heterogeneous soil environment. In Mediterranean grasslands, the first rainfall after seasonal drought provides an ecosystem reset, triggering microbial activity during a tractable window for capturing short-term dynamics. Here, we simulated precipitation in microcosms from four distinct dry grassland soils and generated 144 viromes, 84 metagenomes and 84 16S ribosomal RNA gene amplicon datasets to characterize viral, prokaryotic and relic DNA dynamics over 10 days. Vastly different viral communities in each soil followed remarkably similar successional trajectories. Wet-up triggered a significant increase in viral richness, followed by extensive compositional turnover. Temporal succession in prokaryotic communities was much less pronounced, perhaps suggesting differences in the scales of activity captured by viromes (representing recently produced, ephemeral viral particles) and total DNA. Still, differences in the relative abundances of Actinobacteria (enriched in dry soils) and Proteobacteria (enriched in wetted soils) matched those of their predicted phages, indicating viral predation of dominant bacterial taxa. Rewetting also rapidly depleted relic DNA, which subsequently reaccumulated, indicating substantial new microbial mortality in the days after wet-up, particularly of the taxa putatively under phage predation. Production of abundant, diverse viral particles via microbial host cell lysis appears to be a conserved feature of the early response to soil rewetting, and results suggest the potential for 'Cull-the-Winner' dynamics, whereby viruses infect and cull but do not decimate dominant host populations.

Viruses are abundant, ubiquitous members of soil communities that kill microbial cells, but how they respond to perturbation of soil ecosystems is essentially unknown. Here, we investigate lineage-specific virus-host dynamics in grassland soil following “wet-up”, when resident microbes are both resuscitated and lysed after a prolonged dry period. Quantitative isotope tracing, time-resolved metagenomics and viromic analyses indicate that dry soil holds a diverse but low biomass reservoir of virions, of which only a subset thrives following wet-up. Viral richness decreases by 50% within 24 h post wet-up, while viral biomass increases four-fold within one week. Though recent hypotheses suggest lysogeny predominates in soil, our evidence indicates that viruses in lytic cycles dominate the response to wet-up. We estimate that viruses drive a measurable and continuous rate of cell lysis, with up to 46% of microbial death driven by viral lysis one week following wet-up. Thus, viruses contribute to turnover of soil microbial biomass and the widely reported CO 2 efflux following wet-up of seasonally dry soils.

In drought affected ecosystems, a large portion of the annually respired CO2 from soil may occur in the short period following the first rain event after drought. This process, where the rewetting of dry soil results in a pulse of CO­2, is commonly known as the Birch Effect. This pulse of activity influences the stability and persistence of soil carbon which, considering the large and growing extent of dryland and drought-impacted ecosystems, may have far reaching implications. It’s been shown that the consumption of the compounds driving the Birch Effect varies temporally and that different taxa grow over the course of wet-up; however, the transcriptional response of specific taxa during wet-up, and their associated characteristics, has not been fully explored. In this study we map metatranscriptomes against metagenome-assembled genomes (MAGs) in order to assess the transcriptional response of taxa to wet-up at 0, 3, 24, 48, 72, and 168 h post rewetting. We found distinct temporal response patterns that were often conserved on the family-level. Based on response patterns, we grouped genomes into early, mid, and late responders. The average transcriptional profile of MAGs within these different response types did not vary substantially from each other. Instead, for a majority of MAGs, we found shifts in the transcriptional profile of functional genes over time. Together, these findings suggest that much of the temporal dynamics of microbial transcription during the Birch Effect are controlled by differences in within-taxa response time as opposed to stark differences in functional gene transcription between response groups.

ABSTRACT 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 Thaumarchaeota 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). Our comprehensive analysis indicates that differences in carbon and inorganic N availability control contemporaneous transcription of N-cycling pathways in soil habitats. IMPORTANCE Plant roots modulate microbial nitrogen (N) cycling by regulating the supply of root-derived carbon and nitrogen uptake. These differences in resource availability cause distinct micro-habitats to develop: soil near living roots, decaying roots, near both, or outside the direct influence of roots. While many environmental factors and genes control the microbial processes involved in the nitrogen cycle, most research has focused on single genes and pathways, neglecting the interactive effects these pathways have on each other. The processes controlled by these pathways determine consumption and production of N by soil microorganisms. We followed the expression of N-cycling genes in four soil microhabitats over a period of active root growth for an annual grass. We found that the presence of root litter and living roots significantly altered gene expression involved in multiple nitrogen pathways, as well as tradeoffs between pathways, which ultimately regulate N availability to plants.

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.

After the dry season in a Mediterranean climate grassland, the initial soil rewetting event causes a short period of high microbial activity, growth, and mortality. This wet up leads to microbial succession and community reassembly. Climate change in these semiarid environments is projected to cause reduced precipitation, which may affect the structure and function of the microbial community. However, we know little about how microbial functional traits underlie the rewetting succession, and how previous precipitation regimes affect these traits. Using 18O-water stable isotope probing (SIP), we conducted a replicated wet-up experiment in annual grassland soils that had been previously subjected to either average precipitation or 50% of the annual average. We traced microbial succession through 5 time points (0h, 24h, 48h, 72h and 168h) post wet-up. By combining SIP with metagenomics, we identified the actively growing organisms in both precipitation treatments and determined ecophysiological traits that were significantly more represented in growing organisms in each precipitation regime. We observed a legacy effect of average vs. reduced precipitation by comparing the differential abundance of genes observed at time 0h in the two soil treatments. However, this legacy effect was surprisingly short-lived, implying that microbial community function rapidly “restarts itself” before the next growing season, regardless of the precipitation conditions experienced in the previous year. While growing organisms were significantly more abundant than non-growing organisms during the wet-up, the most abundant taxa were slow growers. In contrast, fast growing taxa were less abundant throughout the experiment, suggesting mortality plays a large role in the reformation of the microbial community. We highlight temporal patterns and significant differences based on past precipitation in the abundance of carbohydrate utilization pathways, such as a higher representation of organisms capable of degrading cellulose in the reduced precipitation treatment. There were no temporal patterns in nitrogen cycling pathways; nitrogen acquisition appeared to be based mostly on ammonium assimilation and transport as well as proteases. In conclusion, altering preceding precipitation patterns had a large legacy effect on microbial community assembly and function upon rewetting. However, the functional and compositional changes that resulted from altered precipitation had remarkably short-lived effects after the soils were rewetted.