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1 Abstract Mutualisms evolve over time when individuals belonging to different species derive fitness benefits through the exchange of resources and services. Although prevalent in natural and managed ecosystems, mutualisms can be destabilized by environmental fluctuations that alter the costs and benefits of maintaining the symbiosis. In the rhizobia-legume mutualism, bacteria provide reduced nitrogen to the host plant in exchange for photosynthates that support bacterial metabolism. However, this relationship can be disrupted by the addition of external nitrogen sources to the soil, such as fertilizers. While the molecular mechanisms underpinning the rhizobia-legume symbiosis are well-characterized, the genome-wide fitness effects of nitrogen enrichment on symbiotic rhizobia are less clear. Here, we inoculated a randomly barcoded transposon-site sequencing (RB-TnSeq) library of the bacteriumEnsifer(Sinorhizobium)melilotiinto soils containing a host plant, alfalfa (Medicago sativa), under conditions of low and high nitrogen availability. Although plant performance remained robust to fertilization, nitrogen enrichment altered gene fitness for specific traits and functions in the rhizobial partner. Genes involved in carbohydrate metabolism showed increased fitness irrespective of soil nutrient content, whereas fitness gains in quorum-sensing genes were only observed in high-nitrogen environments. We also documented reductions in the fitness of nucleotide metabolism and cell-growth genes, while genes from oxidative phosphorylation and various amino-acid biosynthesis pathways were detrimental to fitness under elevated soil nitrogen, underscoring the complex trade-offs in rhizobial responses to nutrient enrichment. Our experimental functional genomics approach identified gene functions and pathways across allE. melilotireplicons that may be associated with the disruption of an agronomically important mutualism. 2ImportanceUnderstanding the evolutionary dynamics of the rhizobia-legume mutualism is important for elucidating how plant-soil-microbe interactions operate in natural and managed ecosystems. Legumes constitute a significant portion of global food production and generate 25% of all terrestrially fixed nitrogen. The application of chemical fertilizers can disrupt the mutualism by altering the selective pressures experienced by symbiotic rhizobia, potentially affecting gene fitness throughout the microbial genome and leading to the evolution of less productive or cooperative mutualists. To investigate how exogenous nitrogen inputs influence gene fitness during the complex rhizobial lifecycle, we used a barcoded genome-wide mutagenesis screen to quantify gene-level fitness across the rhizobial genome during symbiosis and identify metabolic functions affected by nitrogen enrichment. Our findings provide genomic insight into potential eco-evolutionary mechanisms by which symbioses are maintained or degraded over time in response to changing environmental conditions.
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This content will become publicly available on June 10, 2027
Rhizobia independently adapt to soil and legume host environments, but soil conditions influence the abundance of high-quality partners
ABSTRACT 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 68Sinorhizobium melilotistrains to soil-mediated selection using eight different treatments (temperature, osmotic, and texture perturbations) and host-mediated selection with twoMedicagospecies 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. IMPORTANCERhizobium-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.
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- PAR ID:
- 10703466
- Publisher / Repository:
- American Society for Microbiology
- Date Published:
- Journal Name:
- mBio
- Volume:
- 17
- Issue:
- 6
- ISSN:
- 2150-7511
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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