Sohini Guha

Sinorhizobium meliloti is a nitrogen-fixing soil bacterium that forms a facultative mutualism with its host alfalfa. S. meliloti strains exhibit diverse colony morphologies on solid culture media that span a continuum from dry to mucoid. Studies on how this trait affects symbiosis rely on mutations in exopolysaccharide II production genes; however, whether natural variation in morphology confers a selective advantage on rhizobia in their natural habitats remains unknown. Here, we used 90 mucoid and 27 dry natural isolates to test whether colony morphology associates with rhizobial fitness in the soil and in the host, or with the nitrogen-fixation benefits they provide to the plant. Colony morphologies were widely distributed across a phylogenetic tree, suggesting that morphological shifts occurred repeatedly and no single genetic variant was strongly associated with this trait. We inoculated soil mesocosms and plants with a mixture of dry and mucoid bacteria. We observed that dry rhizobia have higher fitness, particularly at 22°C and 32°C, but not under cold or drought conditions, independent of the initial rhizobial load. Conversely, mucoid bacteria have a modestly higher relative fitness in host nodules, and single inoculations showed that they tend to confer greater benefits to all three alfalfa varieties. Over time, we also observed rare, repeated conversion from mucoid to dry morphologies in soil and host plants. Taken together, our study reveals that morphological variation within a species in a single agricultural field is important for bacterial fitness in the soil and can have cascading effects on the rhizobia’s plant benefits.

In many host-microbe symbioses, hosts develop specialized organs that house microbial symbionts. In legume-rhizobium symbioses, these organs are called root nodules. While many studies have focused on host traits like plant growth, nodule number, and structural changes, nodule morphology may influence rhizobial benefits (e.g., bacterial population size). Here, we investigated how variation in alfalfa nodule morphology influences the fitness outcome of both partners. We used 117 strains and 3 alfalfa varieties to test how genetic variation influenced (1) nodule branch number, area, and rhizobial fitness at the individual nodule level and (2) the proportion of branched nodules, nodule number, and plant benefits at the whole plant level. In all tested hosts, larger or more branched nodules tended to release more rhizobia regardless of rhizobial strain, although baseline rhizobial fitness values differed across hosts. From the host perspective, the proportion of branched nodules was strongly negatively correlated with total nodule number and positively correlated with host benefit. Both host and strain genetic variation contributed to the proportion of branched nodules on a plant. Together, these results provide insight into the genetic contributions of hosts and rhizobia to nodule morphology and symbiotic outcomes, suggesting a novel direction for research in legume-rhizobium mutualism.

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.

In many host-microbe symbioses, hosts develop specialized organs that house microbial symbionts. In the legume-rhizobium symbiosis, these organs are called root nodules. While many studies have focused on host traits like plant growth, nodule number, and their structural changes, nodule morphology may also influence rhizobial benefits (e.g., bacterial population size). Here, we investigated how variation in alfalfa nodule morphologies influences the fitness outcome of both partners. Specifically, we measured how host and strain genetic variation influenced 1) nodule branch number, area, and rhizobial fitness at the individual nodule level and 2) the proportion of branched nodules, nodule number, and plant benefits at the whole plant level. From the rhizobial perspective, host identity had a stronger effect on rhizobial fitness, and larger or more branched nodules tend to release more rhizobia. From the host perspective, the proportion of branched nodules was strongly negatively correlated with the total nodule number and positively correlated with host benefit across multiple alfalfa genotypes. However, the strength of the relationship differed among hosts. Together, these results provide insight into the host and rhizobial genetic contributions to nodule morphology and symbiotic outcomes, suggesting a novel direction for further research in legume-rhizobium mutualism. Highlight: Plant and bacterial symbiotic effectiveness can be gauged by the number of nodule branches formed in indeterminant legumes-rhizobium systems along with traditional metrics such as nodule count.

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.

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.