Host investment into symbiosis varies among genotypes of the legume Acmispon strigosus, but host sanctions are uniform.

Efficient host control predicts the extirpation of ineffective symbionts, but they are nonetheless widespread in nature. We tested three hypotheses for the maintenance of symbiotic variation in rhizobia that associate with a native legume: partner mismatch between host and symbiont, such that symbiont effectiveness varies with host genotype; resource satiation, whereby extrinsic sources of nutrients relax host control; and variation in host control among host genotypes. We inoculated Acmispon strigosus from six populations with three Bradyrhizobium strains that vary in symbiotic effectiveness on sympatric hosts. We measured proxies of host and symbiont fitness in single- and co-inoculations under fertilization treatments of zero added nitrogen (N) and near-growth-saturating N. We examined two components of host control: 'host investment' into nodule size during single- and co-inoculations, and 'host sanctions' against less effective strains during co-inoculations. The Bradyrhizobium strains displayed conserved growth effects on hosts, and host control did not decline under experimental fertilization. Host sanctions were robust in all hosts, but host lines from different populations varied significantly in measures of host investment in both single- and co-inoculation experiments. Variation in host investment could promote variation in symbiotic effectiveness and prevent the extinction of ineffective Bradyrhizobium from natural populations.

Nonnodulating Bradyrhizobium spp. Modulate the Benefits of Legume-Rhizobium Mutualism.

UNLABELLED: Rhizobia are best known for nodulating legume roots and fixing atmospheric nitrogen for the host in exchange for photosynthates. However, the majority of the diverse strains of rhizobia do not form nodules on legumes, often because they lack key loci that are needed to induce nodulation. Nonnodulating rhizobia are robust heterotrophs that can persist in bulk soil, thrive in the rhizosphere, or colonize roots as endophytes, but their role in the legume-rhizobium mutualism remains unclear. Here, we investigated the effects of nonnodulating strains on the native Acmispon-Bradyrhizobium mutualism. To examine the effects on both host performance and symbiont fitness, we performed clonal inoculations of diverse nonnodulating Bradyrhizobium strains on Acmispon strigosus hosts and also coinoculated hosts with mixtures of sympatric nodulating and nonnodulating strains. In isolation, nonnodulating Bradyrhizobium strains did not affect plant performance. In most cases, coinoculation of nodulating and nonnodulating strains reduced host performance compared to that of hosts inoculated with only a symbiotic strain. However, coinoculation increased host performance only under one extreme experimental treatment. Nearly all estimates of nodulating strain fitness were reduced in the presence of nonnodulating strains. We discovered that nonnodulating strains were consistently capable of coinfecting legume nodules in the presence of nodulating strains but that the fitness effects of coinfection for hosts and symbionts were negligible. Our data suggest that nonnodulating strains most often attenuate the Acmispon-Bradyrhizobium mutualism and that this occurs via competitive interactions at the root-soil interface as opposed to in planta IMPORTANCE: Rhizobia are soil bacteria best known for their capacity to form root nodules on legume plants and enhance plant growth through nitrogen fixation. Yet, most rhizobia in soil do not have this capacity, and their effects on this symbiosis are poorly understood. We investigated the effects of diverse nonnodulating rhizobia on a native legume-rhizobium symbiosis. Nonnodulating strains did not affect plant growth in isolation. However, compared to inoculations with symbiotic rhizobia, coinoculations of symbiotic and nonnodulating strains often reduced plant and symbiont fitness. Coinoculation increased host performance only under one extreme treatment. Nonnodulating strains also invaded nodule interiors in the presence of nodulating strains, but this did not affect the fitness of either partner. Our data suggest that nonnodulating strains may be important competitors at the root-soil interface and that their capacity to attenuate this symbiosis should be considered in efforts to use rhizobia as biofertilizers.

Metapopulation dominance and genomic-island acquisition of Bradyrhizobium with superior catabolic capabilities.

Root nodule-forming rhizobia exhibit a bipartite lifestyle, replicating in soil and also within plant cells where they fix nitrogen for legume hosts. Host control models posit that legume hosts act as a predominant selective force on rhizobia, but few studies have examined rhizobial fitness in natural populations. Here, we genotyped and phenotyped Bradyrhizobium isolates across more than 800 km of the native Acmispon strigosus host range. We sequenced chromosomal genes expressed under free-living conditions and accessory symbiosis loci expressed in planta and encoded on an integrated 'symbiosis island' (SI). We uncovered a massive clonal expansion restricted to the Bradyrhizobium chromosome, with a single chromosomal haplotype dominating populations, ranging more than 700 km, and acquiring 42 divergent SI haplotypes, none of which were spatially widespread. For focal genotypes, we quantified utilization of 190 sole-carbon sources relevant to soil fitness. Chromosomal haplotypes that were both widespread and dominant exhibited superior growth on diverse carbon sources, whereas these patterns were not mirrored among SI haplotypes. Abundance, spatial range and catabolic superiority of chromosomal, but not symbiosis genotypes suggests that fitness in the soil environment, rather than symbiosis with hosts, might be the key driver of Bradyrhizobium dominance.

Native California soils are selective reservoirs for multidrug-resistant bacteria.

Soil bacteria can exhibit extensive antibiotic resistomes and act as reservoirs of important antibiotic resistance traits. However, the geographic sources and evolutionary drivers of resistance traits are poorly understood in these natural settings. We investigated the prevalence, spatial structure and evolutionary drivers of multidrug resistance in natural populations of Bradyrhizobium, a cosmopolitan bacterial lineage that thrives in soil and aquatic systems as well as in plant and human hosts. We genotyped > 400 isolates from plant roots and soils across California and assayed 98 of them for resistance traits against 17 clinically relevant antibiotics. We investigated the geographic and phylogenetic structure of resistance traits, and analysed correlations of resistance with strain abundance, host infection capacity and in vitro fitness. We found: (i) multidrug resistance at all sites, (ii) subsets of resistance traits that are spatially structured and (iii) significant associations between resistance traits and increased strain abundance or host infection capacity. Our results highlight multiple selective factors that can result in the spread of resistance traits in native Bradyrhizobium populations.

Efficiency of partner choice and sanctions in Lotus is not altered by nitrogen fertilization.

Eukaryotic hosts must exhibit control mechanisms to select against ineffective bacterial symbionts. Hosts can minimize infection by less-effective symbionts (partner choice) and can divest of uncooperative bacteria after infection (sanctions). Yet, such host-control traits are predicted to be context dependent, especially if they are costly for hosts to express or maintain. Legumes form symbiosis with rhizobia that vary in symbiotic effectiveness (nitrogen fixation) and can enforce partner choice as well as sanctions. In nature, legumes acquire fixed nitrogen from both rhizobia and soils, and nitrogen deposition is rapidly enriching soils globally. If soil nitrogen is abundant, we predict host control to be downregulated, potentially allowing invasion of ineffective symbionts. We experimentally manipulated soil nitrogen to examine context dependence in host control. We co-inoculated Lotus strigosus from nitrogen depauperate soils with pairs of Bradyrhizobium strains that vary in symbiotic effectiveness and fertilized plants with either zero nitrogen or growth maximizing nitrogen. We found efficient partner choice and sanctions regardless of nitrogen fertilization, symbiotic partner combination or growth season. Strikingly, host control was efficient even when L. strigosus gained no significant benefit from rhizobial infection, suggesting that these traits are resilient to short-term changes in extrinsic nitrogen, whether natural or anthropogenic.

Evolutionary instability of symbiotic function in Bradyrhizobium japonicum.

Bacterial mutualists are often acquired from the environment by eukaryotic hosts. However, both theory and empirical work suggest that this bacterial lifestyle is evolutionarily unstable. Bacterial evolution outside of the host is predicted to favor traits that promote an independent lifestyle in the environment at a cost to symbiotic function. Consistent with these predictions, environmentally-acquired bacterial mutualists often lose symbiotic function over evolutionary time. Here, we investigate the evolutionary erosion of symbiotic traits in Bradyrhizobium japonicum, a nodulating root symbiont of legumes. Building on a previous published phylogeny we infer loss events of nodulation capability in a natural population of Bradyrhizobium, potentially driven by mutation or deletion of symbiosis loci. Subsequently, we experimentally evolved representative strains from the symbiont population under host-free in vitro conditions to examine potential drivers of these loss events. Among Bradyrhizobium genotypes that evolved significant increases in fitness in vitro, two exhibited reduced symbiotic quality, but no experimentally evolved strain lost nodulation capability or evolved any fixed changes at six sequenced loci. Our results are consistent with trade-offs between symbiotic quality and fitness in a host free environment. However, the drivers of loss-of-nodulation events in natural Bradyrhizobium populations remain unknown.