Comparative Genomics across Three Ensifer Species Using a New Complete Genome Sequence of the Medicago Symbiont Sinorhizobium (Ensifer) meliloti WSM1022

Here, we report an improved and complete genome sequence of Sinorhizobium (Ensifer) meliloti strain WSM1022, a microsymbiont of Medicago species, revealing its tripartite structure. This improved genome sequence was generated combining Illumina and Oxford nanopore sequencing technologies to better understand the symbiotic properties of the bacterium. The 6.75 Mb WSM1022 genome consists of three scaffolds, corresponding to a chromosome (3.70 Mb) and the pSymA (1.38 Mb) and pSymB (1.66 Mb) megaplasmids. The assembly has an average GC content of 62.2% and a mean coverage of 77X. Genome annotation of WSM1022 predicted 6058 protein coding sequences (CDSs), 202 pseudogenes, 9 rRNAs (3 each of 5S, 16S, and 23S), 55 tRNAs, and 4 ncRNAs. We compared the genome of WSM1022 to two other rhizobial strains, closely related Sinorhizobium (Ensifer) meliloti Sm1021 and Sinorhizobium (Ensifer) medicae WSM419. Both WSM1022 and WSM419 species are high-efficiency rhizobial strains when in symbiosis with Medicago truncatula, whereas Sm1021 is ineffective. Our findings report significant genomic differences across the three strains with some similarities between the meliloti strains and some others between the high efficiency strains WSM1022 and WSM419. The addition of this high-quality rhizobial genome sequence in conjunction with comparative analyses will help to unravel the features that make a rhizobial symbiont highly efficient for nitrogen fixation.

Partners in space: Discordant population structure between legume hosts and rhizobium symbionts in their native range

Coevolution is predicted to depend on how the genetic diversity of interacting species is geographically structured. Plant-microbe symbioses such as the legume-rhizobium mutualism are ecologically and economically important, but distinct life history and dispersal mechanisms for these host and microbial partners, plus dynamic genome composition in bacteria, present challenges for understanding spatial genetic processes in these systems. Here we study the model rhizobium Ensifer meliloti using a hierarchically-structured sample of 191 strains from 21 sites in the native range and compare its population structure to that of its host plant Medicago truncatula. We find high local genomic variation and minimal isolation by distance across the rhizobium genome, particularly at the two symbiosis elements pSymA and pSymB, which have evolutionary histories and population structures that are similar to each other but distinct from both the chromosome and the host. While the chromosome displays weak isolation by distance, it is uncorrelated with hosts. Patterns of discordant population structure among elements with the bacterial genome has implications for bacterial adaptation to life in the soil versus symbiosis, while discordant population genetic structure of hosts and microbes might restrict local adaptation of species to each other and give rise to phenotypic mismatches in coevolutionary traits.

The genetic basis of cooperation and conflict in natural populations of a model symbiont

Although mutualisms are defined as net beneficial interactions among species, whether fitness conflict or alignment drive the evolution of these interactions is unclear. Examining the relationships between host and symbiont fitness proxies at both the organismal and genomic levels can provide new insights. Here, we utilized data from several genome-wide association studies (GWAS) that involved 191 strains of the N-fixing rhizobium symbiont, Ensifer meliloti, collected from natural populations being paired in single or mixed inoculation with two genotypes of the host Medicago truncatula to determine how different proxies of microbial fitness were related to one another, and examine signatures of fitness conflict and alignment between host and symbiont at both the whole-organism and genomic levels. We found little evidence for fitness conflict; instead, loci tended to have concordant effects on both host and symbiont fitness and showed heightened nucleotide diversity and signatures of balancing selection compared to the rest of the genome. We additionally found that single versus competitive measures of rhizobium fitness are distinct, and that the latter should be used given that they better reflect the ecological conditions rhizobia experience in nature. Our results suggest that although conflict appears to be largely resolved in natural populations of rhizobia, mutualistic coevolution between legumes and rhizobia can nonetheless maintain genetic diversity, potentially explaining why variation in symbiotic traits persists in nature.

Partners in space: Discordant population structure between legume hosts and rhizobium symbionts in their native range

Coevolution is predicted to depend on how the genetic diversity of interacting species is geographically structured. Plant-microbe symbioses such as the legume-rhizobium mutualism are ecologically and economically important, but distinct life history and dispersal mechanisms for these macrobial and microbial partners, plus dynamic genome composition in bacteria, present challenges for understanding spatial genetic processes in these systems. Here we study the model rhizobium Ensifer meliloti using a hierarchically-structured sample of 191 strains from 21 sites in the native range and compare its population structure to that of its host plant Medicago truncatula. We find high local genomic variation and minimal isolation by distance across the rhizobium genome, particularly at the two symbiosis elements pSymA and pSymB, which have evolutionary histories and population structures similar to each other and distinct from both the chromosome and the host. While the chromosome displays weak isolation by distance, it is uncorrelated with hosts. Patterns of discordant population structure among elements with the bacterial genome has implications for bacterial adaptation to life in the soil versus symbiosis, while discordant population genetic structure of hosts and microbes might restrict local adaptation of species to each other and give rise to phenotypic mismatches in coevolutionary traits.

Exopolysaccharide Characterization of Rhizobium favelukesii LPU83 and Its Role in the Symbiosis With Alfalfa

One of the greatest inputs of available nitrogen into the biosphere occurs through the biological N2-fixation to ammonium as result of the symbiosis between rhizobia and leguminous plants. These interactions allow increased crop yields on nitrogen-poor soils. Exopolysaccharides (EPS) are key components for the establishment of an effective symbiosis between alfalfa and Ensifer meliloti, as bacteria that lack EPS are unable to infect the host plants. Rhizobium favelukesii LPU83 is an acid-tolerant rhizobia strain capable of nodulating alfalfa but inefficient to fix nitrogen. Aiming to identify the molecular determinants that allow R. favelukesii to infect plants, we studied its EPS biosynthesis. LPU83 produces an EPS I identical to the one present in E. meliloti, but the organization of the genes involved in its synthesis is different. The main gene cluster needed for the synthesis of EPS I in E. meliloti, is split into three different sections in R. favelukesii, which probably arose by a recent event of horizontal gene transfer. A R. favelukesii strain devoided of all the genes needed for the synthesis of EPS I is still able to infect and nodulate alfalfa, suggesting that attention should be directed to other molecules involved in the development of the symbiosis.

Efecto de la inoculación con rizobios y la fertilización fosfatada sobre la nodulación y producción de alfalfa (Medicago sativa L.) en el centro de Santa Fe (Argentina)

La simbiosis entre rizobios y alfalfa (Medicago sativa L.) contribuye a la incorporación de nitrógeno en los agroecosistemas a través de la fijación biológica, que dependerá de la adecuada provisión de nutrientes disponibles en el suelo. El objetivo de este trabajo fue evaluar el efecto de la inoculación con Ensifer meliloti y la fertilización con 0, 20 y 40 kg/ha de fósforo sobre la nodulación y productividad de una pastura de alfalfa en el centro de la provincia de Santa Fe. La inoculación y la aplicación de dosis crecientes de fósforo aumentaron el número de nódulos, biomasa nodular y la producción del forraje de las plantas. La técnica de la inoculación condujo a un incremento de la producción de materia seca entre 23 y 58 % para los tratamientos con el máximo nivel de fertilizante a los 120, 180 y 240 días de la siembra. La adición de la cepa de rizobio utilizada como inoculante y la fertilización fosfatada constituyeron una estrategia favorable para incrementar la producción de alfalfa.

Environment dependence of rhizobial relative fitness in the legume-rhizobia symbiosis

Spatial and temporal variation in resource availability, population density, and composition likely affect the ecology and evolution of symbiotic interactions. We examined how host genotype, Nitrogen addition, rhizobial density, and community complexity affected a legume-rhizobia (Medicago truncatula - Ensifer meliloti) mutualism. Host genotype had the strongest effect on the size, number, and rhizobial composition of root nodules (the symbiotic organ). By contrast, the effect of small changes in N-availability and the complexity of the inoculum community (2, 3, 8, or 68 strains) were minor. Higher inoculum density resulted in a nodule community that was less diverse and more beneficial but only in the more selective host. With the less selective host, higher density resulted in more diverse and less beneficial nodule communities. Density effects on strain composition deserve additional scrutiny as they can create eco-evolutionary feedback and have translational relevance for overcoming establishment barriers in bio-inoculants.Short AbstractThe environmental context of the nitrogen-fixing mutualism between leguminous plants and rhizobial bacteria varies over space and time. The understudied environmental variable of rhizobial density had a larger effect on the relative fitness of 68 rhizobia (Ensifer meliloti) strains in nodules than the addition of low-levels of nitrogen or community complexity.

Experimental evolution makes microbes more cooperative with their local host genotype

Advances in microbiome science require a better understanding of how beneficial microbes adapt to hosts. We tested whether hosts select for more-cooperative microbial strains with a year-long evolution experiment and a cross-inoculation experiment designed to explore how nitrogen-fixing bacteria (rhizobia) adapt to legumes. We paired the bacterium Ensifer meliloti with one of five Medicago truncatula genotypes that vary in how strongly they “choose” bacterial symbionts. Independent of host choice, E. meliloti rapidly adapted to its local host genotype, and derived microbes were more beneficial when they shared evolutionary history with their host. This local adaptation was mostly limited to the symbiosis plasmids, with mutations in putative signaling genes. Thus, cooperation depends on the match between partner genotypes and increases as bacteria adapt to their local host.