Jennifer E. Harris

Mixing plant functional groups can boost plant productivity, but the underlying rhizosphere microbial dynamics remain unclear. We tested when to expect divergence from additive expectations by measuring plant traits and active and total rhizosphere microbiomes (via BONCAT-FACS and 16S rRNA gene amplicon sequencing) in monocultures and mixtures of legumes, brassicas, and grasses under varying nitrogen. While shoot biomass was predictable from monocultures, legume-containing mixtures yielded greater-than-expected root biomass and N-fixation. Each plant species supported distinct active and total microbiomes, and nitrogen addition increased microbiome divergence among plant species and their mixtures. In most plant mixtures, microbiomes were additive combinations of those associated with the individual species; legume–grass combinations were a notable exception, showing non-additive shifts in microbial composition, microbial activity, and the abundance of Rhizobium sp. This divergence from additive expectations coincided with non-additive biomass gains in legume-grass mixtures, suggesting that emergent plant-microbe interactions may underlie synergistic belowground responses.

Nitrous oxide (N ₂ O) is a potent greenhouse gas and is largely produced by incomplete denitrification. Although we know many of the microbial species that denitrify, we are still unable to reliably predict N ₂ O production from soils. Recent work in microbial ecology has shown that when key microbes are considered as members of functional ensembles rather than isolated, the predictive power linking their activity to emergent properties increases dramatically. We hypothesized that the active microbial community during high N ₂ O production would be taxonomically distinct from the inactive portion and increases in N₂O production rates would correlate more strongly with increased abundance across multiple active taxa than with dominance by a single active species. We conducted a microcosm experiment where agricultural soil was incubated in anaerobic vials for up to 15 hours while tracking N ₂ O production. Using bioorthogonal non-canonical amino acid tagging paired with fluorescence-activated cell sorting and 16S rRNA amplicon sequencing (BONCAT-FACS-Seq), we probed the active subset of the microbial community throughout the incubation period. Analysis of 16S rRNA gene amplicons revealed that the active and inactive fractions contained distinct taxa, and the taxonomic composition of the active fraction shifted over time. We found that less than 1% of the microbial community was responsible for N ₂ O flux rates as high as 3.84 µg N ₂ O-N g dry soil-1 hr-1. The level of activity (median fluorescent intensity of active cells) correlated well with N ₂ O production rates. The Ensemble Quotient Optimization for Microbiomes (mEQO) tool was used to identify an ensemble of eight organisms whose combined abundance best correlated with N₂O fluxes. Overall, our results reveal that N₂O fluxes are driven not by changes in a single taxon but by shifting ensembles of active microorganisms whose combined functional potential supports consistent emissions. This study applied a novel conceptual and methodological framework with a distinct focus on the active microbial community, rather than the entire community; if our observation that N ₂ O flux rates are correlated with an ensemble of organisms is broadly confirmed, then framing denitrification as a community trait may increase predictability of this key process.

Rhizobia live as free-living microorganisms in the soil and in association with legume hosts. Both environments exert selective pressures on rhizobia, influencing the reproductive success of individual strains (e.g., fitness). The soil, a heterogeneous and fluctuating environment, is often overlooked, and little is known about whether selection in the soil influences the outcomes of the rhizobium-legume mutualism. We exposed a mixture of 68 Sinorhizobium meliloti strains to soil-mediated selection using eight different treatments (temperature, osmotic, and texture perturbations) and host-mediated selection with two Medicago species as hosts. We found that cold (4°C) and warm (32°C) temperatures, as well as salt addition, had the strongest effects on diversity, population composition, or population size. Strain relative fitness was strongly positively correlated among soil treatments, except cold, warm, and salinity, suggesting strains undergo similar selection in the soil. Genome-wide association analysis revealed a complex genetic architecture for soil fitness, characterized by numerous loci of small effect that did not show significant associations. In contrast, when comparing rhizobial fitness between soil and host environments, we found minimal strain fitness correlations, suggesting an independent genetic basis and habitat-specific adaptations. Finally, by examining the relationship between rhizobial fitness in soil and their benefits to the host plant, we found that soil selection influenced the relative abundance of high- and low-quality strains; however, whether these effects were positive or negative for the plant was host-dependent. Our results suggest that rhizobial evolution in soil and host are largely independent, but soil selection can alter mutualism benefits. IMPORTANCE: Rhizobium-legume mutualism is crucial for introducing nitrogen into agricultural and natural ecosystems, and rhizobial persistence in the soil is an important component of agroecosystems. However, we know little about how populations of rhizobia persist and adapt to this environment, especially in the context of the soil's spatial and temporal variations (temperature, moisture, and soil texture). We found that rhizobia are similarly selected across abiotic soil conditions, but their reproductive success in the soil is independent from their reproductive success in the host. Intriguingly, we found that certain soil conditions increase (or decrease) the relative abundance of more beneficial strains. Understanding how rhizobia adapt to diverse environments is crucial for developing effective bioinoculants that persist in the soil while remaining highly competitive for host colonization and beneficial to the plant.

Rhizobia live as free-living microorganisms in the soil and in association with legume hosts. Both environments exert selective pressures on rhizobia, influencing the reproductive success of individual strains (e.g., fitness). The soil, a heterogeneous and fluctuating environment, is often overlooked, and little is known about whether selection in the soil influences the outcomes of the rhizobia-legume mutualism. We exposed a mixture of 68 Sinorhizobium meliloti strains to eight soil treatments (temperature, osmotic, and texture perturbations) and selection by two Medicago plant hosts. We found that cold (4°C) and warm (32°C) temperatures, as well as salt addition, had the strongest effects on diversity, community composition, or population size. Strain relative fitness was strongly positively correlated among soil treatments, except for cold. Genome-wide association analysis revealed a complex genetic architecture for soil fitness. In contrast, when comparing rhizobial fitness between soil and host environments, we found minimal strain fitness correlations, suggesting independent genetic bases and habitat-specific adaptations. Lastly, by examining the relationship between rhizobial fitness in the soil and their nitrogen-fixing plant benefits, we found that soil selection influenced the relative abundance of high- and low-quality strains; However, whether these effects were positive or negative for the plant was host dependent. Our results suggest that rhizobial evolution in soil and host are largely independent, but soil selection can alter mutualism benefits. IMPORTANCE: Rhizobia-legume mutualism is crucial for introducing nitrogen into agricultural and natural ecosystems, and rhizobia persistence in the soil is an important component of agroecosystems. However, we know little about how individual strains of rhizobia persist and adapt to this environment, especially in the context of the soil’s spatial and temporal variations (temperature, moisture, and soil texture). We found that rhizobia similarly adapt to abiotic soil conditions but their reproductive success in the soil is independent from their reproductive success in the host. Intriguingly, we found that certain soil conditions increase (or decrease) the relative abundance of more effective nitrogen-fixing strains. Understanding how rhizobia adapt to diverse environments is crucial for developing effective bioinoculants that maintain high persistence in the soil while are also highly competitive to colonize the host and are beneficial to the plant.

Plant-beneficial microbes have great potential to improve sustainability in agriculture. Still, managing beneficial microbes is challenging because the impact of microbial dormancy on community assembly across the soil, rhizosphere, and endosphere is poorly understood. We address this gap with the first documented use of Biorthogonal Non-Canonical Amino Acid Tagging (BONCAT) to probe active microbes in the soil-to-root gradient. Using nodule-forming legume Trifolium incarnatum, we confirmed that BONCAT is suitable for labeling endospheric microbes with microscopy. Next, we coupled BONCAT to Flow Cytometer Activated Cell Sorting (FACS) and 16S rRNA amplicon sequencing to probe patterns of microbial activity and the structure of the active microbial community across the soil, rhizosphere, root, and nodule with a native soil microbial community. As expected, we found 10 times higher microbial activity in the endosphere than in the rhizosphere or bulk soil, likely due to increased plant resources. Finally, we revealed that microbial activity in the rhizosphere was significantly associated with successful endosphere colonization, more so than microbial abundance alone. This last finding has implications for the development of microbial inoculants, suggesting colonizing plant roots is linked to a microbe’s ability to overcome dormancy once deployed in the soil. IMPORTANCE: Most soil microbes are dormant, so they must exit dormancy to have the potential to carry out plant-beneficial functions. It is unclear if dormant microbes revive in proximity to plant-produced resources and if overcoming dormancy in the soil is important for successful plant colonization. We use a novel microbial activity probing technique for the first time on and in plant roots, and with it, we observe microbes increased in activity 10× inside plant tissues compared to the soil, likely in response to plant-produced resources. In complex, native microbial communities, we observe that microbes that are active and abundant are more likely to colonize plant roots successfully than just abundant microbes. Our research shows that plants could be leveraged to promote a distinct active microbial community from the native soil, a discovery that has the potential to improve sustainability in agriculture.

Plant-beneficial microbes have great potential to improve sustainability in agriculture. Still, managing beneficial microbes is challenging because the impact of microbial dormancy on community assembly across the soil, rhizosphere, and endosphere is poorly understood. We address this gap with the first documented use of Biorthogonal Non-Canonical Amino Acid Tagging (BONCAT) to probe active microbes in the soil-to-root gradient. Using nodule-forming legume Trifolium incarnatum, we confirmed that BONCAT is suitable for labeling endospheric microbes with microscopy. Next, we coupled BONCAT to Flow Cytometer Activated Cell Sorting (FACS) and 16S rRNA amplicon sequencing to probe patterns of microbial activity and the structure of the active microbial community across the soil, rhizosphere, root, and nodule with a native soil microbial community. As expected, we found x10 higher microbial activity in the endosphere than in the rhizosphere or bulk soil, likely due to increased plant resources. Finally, we revealed that microbial activity in the rhizosphere was significantly associated with successful endosphere colonization, more so than microbial abundance alone. This last finding has implications for the development of microbial inoculants, suggesting colonizing plant roots is linked to a microbe’s ability to overcome dormancy once deployed in the soil.