Elizabeth L. Paillan

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 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.