Jeremy Sutherland

Legumes recruit rhizobial bacteria from the soil to fix nitrogen in root nodules. The legume-rhizobia symbiosis is an early example of humans using microbes to improve crop yields. Rhizobia/microbe-based bioinoculant products often perform inconsistently in the field, across locations, and years. Rather than engineering a one-size-fits-all inoculant, identifying crop varieties that select for and reward beneficial bacteria within each field's diverse microbial community could eliminate the need for repeated inoculant applications as beneficial microbial populations increase over time. Although legume crops have not been explicitly bred to recruit and enrich beneficial rhizobia, the model legume Medicago truncatula can selectively enrich beneficial strains. Here, we investigated whether its perennial relative, Medicago sativa (alfalfa), a globally important forage crop, also has this ability. To test this, we first isolated and sequenced a collection of 117 Sinorhizobium meliloti strains from nodules of three alfalfa varieties in a field trial. Despite being isolated from nodules less than three meters apart, we found high intraspecific genetic diversity in both single-nucleotide polymorphisms and gene presence-absence, with little phylogenetic clustering by source variety. Single-strain inoculations in a nitrogen-free greenhouse experiment revealed substantial variation in nitrogen-fixation ability across strains, with the best-performing strains differing by alfalfa variety. When varieties were inoculated with a mix of all strains, no variety accumulated as much biomass as with the best single strain for that variety, indicating a lack of ability to select only the best strain when given a choice. To confirm, we evaluated relative strain fitness in nodules and found that two of the three varieties weakly enriched for beneficial strains. Our results suggest that alfalfa varieties ability to enrich beneficial rhizobial populations is a variable, breedable trait, opening the door to developing varieties optimized to enrich these populations. Ultimately, breeding hosts to enhance mutualism could improve the reliability and sustainability of field nitrogen fixation.

In the mutualism between leguminous plants and rhizobial bacteria, rhizobia live inside root nodules, creating potential for host genes to shape the rhizobial selective environment. Many host genes that affect symbiosis have been identified; however, the extent to which these genes affect selection acting on rhizobia is unknown. In this study, we inoculated 18 Medicago truncatula symbiotic mutants (including mutants that alter Nodule Cysteine-Rich (NCR) peptide production, plant defence, and nodule number regulation) with a mixture of 86 Sinorhizobium meliloti strains. Most mutations resulted in reduced host benefits, but the effects on rhizobial benefit (i.e. relative strain fitness) varied widely, revealing widespread host-by-strain fitness interactions. Genome-wide association analyses identified variants on rhizobial replicons pSymA and pSymB as important in mediating strain fitness responses to host mutations. Whereas most top variants affected rhizobial fitness with one host mutation (limited effect variants), nine affected fitness across six or more host mutations. These pervasive variants occurred primarily on pSymA, the symbiotic replicon, and include fixL and some metabolic genes. In contrast to the limited effect variants, variants with pervasive positive effects on strain fitness when host genes were mutated tended to adversely affect fitness in wild-type hosts. Competition assays across Medicago genotypes confirmed a pervasive role for one candidate (malonyl-CoA synthase), and AlphaFold multimer modelling suggests that many rhizobial top candidates could interact with host NCR peptides. Our results reveal how host genetic mutations alter strain fitness, setting the stage for improving rhizobial inoculants and breeding legume hosts better adapted to multi-strain environments.

If microbial traits are phylogenetically conserved, then variation in traits of plant hosts may influence rhizosphere microbiomes at higher taxonomic levels. To test this hypothesis and genotype-by-environment-by-microbiome (G × E × M) interactions in switchgrass ( Panicum virgatum), we assessed rhizosphere bacterial composition using 128 host genotypes grown at three distinct field sites. First, we found that growing site was a substantial driver of bacterial composition and that specific bacterial taxa correlated with differences in disease incidence and yield across environments. Second, broad-sense heritability analyses revealed that host genetic effects on rhizosphere composition were strongest at the genus level, suggesting a conserved genetic basis for shaping beneficial or pathogenic taxa. Third, we identified shared host genetic variants associated with bacterial abundance and plant metabolism, indicating possible linkages between key microbial traits and agronomic performance. These findings underscore the importance of incorporating rhizosphere microbiomes into switchgrass breeding efforts and call for further investigation of G × E × M interactions to pinpoint microbial interventions that enhance yield and disease resistance. [ ] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Mutualism breakdown can be prevented if partner species preferentially select and reward partners that provide greater benefit. We examined these two components using the legume Medicago truncatula and its nitrogen-fixing symbiont Sinorhizobium meliloti . First, we reanalyzed data from 202 accessions to show significant genetic variation in the capacity of Medicago to restrict strain diversity, finding that hosts with shorter nodules were more selective. A genome-wide association study on host selectivity identified genes including the hormone leginsulin, pectin degradation, multidrug and toxic compound efflux, Zinc transport, and DNA methylation. Second, we used two well-studied Medicago genotypes with contrasting nodule morphologies to assess the effectiveness of adaptive enrichment mechanisms by sampling the relative frequencies of rhizobial strains in pools of small nodules (indicating a lack of host investment) compared to large nodules (indicating increased host investment) and pairing these results with previous single-strain assessments of strain benefits to hosts. While both hosts enriched beneficial strains in large nodules, the host that formed larger and more variably sized nodules and thus had greater ’potential’ to increase rhizobial populations was less effective. Our findings reveal that host genetic variation affects strain selectivity and suggest that nodule traits warrant attention when exploring mutualism evolution.

In the mutualism between leguminous plants and rhizobia bacteria, rhizobia live inside root nodules, creating potential for host genes to shape the rhizobial selective environment. Many host genes that affect symbiosis have been identified; however, the extent to which these genes affect selection acting on rhizobia is unknown. In this study, we inoculated 18 Medicago truncatula symbiotic mutants (including mutants that alter Nodule Cysteine-Rich (NCR) peptide production, plant defense, and nodule number regulation) with a mixture of 86 Sinorhizobium meliloti strains. Most mutations resulted in reduced host benefits, but the effects on rhizobial benefit (i.e., relative strain fitness) varied widely, revealing widespread host-by-strain fitness interactions. Genome-wide association analyses identified variants on rhizobial replicons pSymA and pSymB as important in mediating strain fitness responses to host mutations. Whereas most top variants affected rhizobial fitness with one host mutant (limited effect variants), nine affected fitness across six or more host mutations. These pervasive variants occurred primarily on pSymA, the symbiotic replicon, and include fixL and some metabolic genes. In contrast to the limited effect variants, variants with pervasive positive effects on strain fitness when host genes were mutated tended to adversely affect fitness in wild-type hosts. Competition assays across Medicago genotypes confirmed a pervasive role for one candidate (malonyl-CoA synthase), and AlphaFold multimer modelling suggests that many rhizobial top candidates could interact with host NCR peptides. Our results reveal how host genetic variation affects rhizobial fitness, setting the stage for improving rhizobial inoculants and breeding legume hosts better adapted to multi-strain environments.

Summary: Since microbial traits are conserved at different taxonomic levels, plant hosts may influence microbiome composition differently at different levels to broadly promote or resist microbiota with traits that impact host fitness. We tested this hypothesis by assessing signals of host genetic influence on bacterial composition in the switchgrass rhizosphere using 128 genotypes in dissimilar growing sites. We employed three common gardens, combined with host genetic mapping, 16S rRNA gene sequence analysis, hierarchical modeling, tests of phylogenetic conservation of host influence, and genome-wide association analyses to determine the contributions of host genetics in shaping rhizosphere bacterial composition at different taxonomic levels. Modeling bacterial assembly showed that growing site was a strong factor shaping bacterial composition in the rhizosphere, though host genetic influence played a significant role. The heritability of bacterial abundance was strongest at the genus level. Phylogenetic signal for heritability was detected within the bacterial phylogeny but conserved clades differed between common gardens. We identified shared host genetic variants associated with bacterial abundance and host traits related to plant metabolism. Our results suggest further investigation is required regarding the genotype-by-environment-by-microbiome relationship to elucidate the factors shaping rhizosphere microbiome composition and the agroecological dynamics shaping plant phenotype.

Host genetic variation can shape the diversity and composition of associated microbiomes, which may reciprocally influence host traits and performance. While the genetic basis of phenotypic diversity of plant populations in nature has been studied, comparatively little research has investigated the genetics of host effects on their associated microbiomes. Switchgrass (Panicum virgatum) is a highly outcrossing, perennial, grass species with substantial locally adaptive diversity across its native North American range. Here, we compared 383 switchgrass accessions in a common garden to determine the host genotypic influence on rhizosphere bacterial composition. We hypothesized that the composition and diversity of rhizosphere bacterial assemblages would differentiate due to genotypic differences between hosts (potentially due to root phenotypes and associated life history variation). We observed higher alpha diversity of bacteria associated with upland ecotypes and tetraploids, compared to lowland ecotypes and octoploids, respectively. Alpha diversity correlated negatively with flowering time and plant height, indicating that bacterial composition varies along switchgrass life history axes. Narrow‐sense heritability (h²) of the relative abundance of 21 core bacterial families was observed. Overall compositional differences among tetraploids, due to genetic variation, supports widespread genotypic influence on the rhizosphere microbiome. Tetraploids were only considered due to complexities associated with the octoploid genomes. Lastly, a genome‐wide association study identified 1861 single‐nucleotide polymorphisms associated with 110 families and genes containing them related to potential regulatory functions. Our findings suggest that switchgrass genomic and life‐history variation influences bacterial composition in the rhizosphere, potentially due to host adaptation to local environments.