Rhizobial Evolution Research Articles

Diversification of Sinorhizobium populations associated with Medicago polymorpha and Medicago lupulina in purple soil of China.

The double selection of environment adaptation and host specificity forced the diversification of rhizobia in nature. In the tropical region of China, Medicago polymorpha and Medicago lupulina are widely distributed, particularly in purple soil. However, the local distribution and diversity of rhizobia associated with these legumes has not been systematically investigated. To this end, root nodules of M. polymorpha and M. lupulina grown in purple soil at seven locations in Yunnan Province of China were collected for rhizobial isolation. The obtained rhizobia were characterized by RFLP of 16S-23S rRNA intergenic spacer, BOXAIR fingerprinting, and phylogeny of housekeeping and symbiosis genes. As result, a total of 91 rhizobial strains were classified into species Sinorhizobium medicae and S. meliloti, while three nodC gene types were identified among them. S. medicae containing nodC of type I was dominant in farmlands associated with M. polymorpha; while S. meliloti harboring nodC of type III was dominant in wild land nodulated by M. lupulina. For both rhizobial species, greater genetic diversity was detected in the populations isolated from their preferred host plant. A high level of genetic differentiation was observed between the two Sinorhizobium species, and gene flow was evident within the populations of the same species derived from different soil types, indicating that rhizobial evolution is likely associated with the soil features. To examine the effects of environmental features on rhizobial distribution, soil physicochemical traits and rhizobial genotypes were applied for constrained analysis of principle coordinates, which demonstrated that soil features like pH, nitrogen and sodium were the principle factors governing the rhizobial geographical distribution. Altogether, both S. medicae and S. meliloti strains could naturally nodulate with M. polymorpha and M. lupulina, but the rhizobium-legume symbiosis compatibility determined by both the host species and soil factors was also highlighted.

Diversity of rhizobial and non-rhizobial bacteria nodulating wild ancestors of grain legume crop plants.

Chickpeas, lentils, and peas are the oldest grain legume species that spread to other regions after their first domestication in Fertile Crescent, and they could reveal the rhizobial evolution in relation to the microsymbionts of wild species in this region. This study investigated the phenotypic and genotypic diversity of the nodule-forming rhizobial bacteria recovered from Pisum sativum subsp., Cicer pinnatifidum, and Lens culinaris subsp. orientalis exhibiting natural distribution in the Gaziantep province of Turkey. PCA analyses of rhizobial isolates, which were tested to be highly resistant to stress conditions, showed that especially pH and salt concentrations had an important effect on these bacteria. Phylogenetic analysis based on 16S rRNA determined that these wild species were nodulated by at least 7 groups including Rhizobium and non-Rhizobium. The largest group comprised of Rhizobium leguminosarum and Rhizobium sp. while R. pusense, which was previously determined as non-symbiotic species, was found to nodulate C. pinnatifidum and L. culinaris subsp. orientalis. In recent studies, Klebsiella sp., which is stated to be able to nodulate different species, strong evidences have been obtained in present study exhibiting that Klebsiella sp. can nodulate C. pinnatifidum and Pseudomonas sp. was able to nodulate C. pinnatifidum and P. sativum subsp. Additionally, L. culinaris subsp. orientalis unlike other plant species, was nodulated by Burkholderia sp. and Serratia sp. associated isolates. Some isolates could not be characterized at the species level since the 16S rRNA sequence similarity rate was low and the fact that they were in a separate group supported with high bootstrap values in the phylogenetic tree may indicate that these isolates could be new species. The REP-PCR fingerprinting provided results supporting the existence of new species nodulating wild ancestors.

Physiological and symbiotic variation of a long-term evolved Rhizobium strain under alkaline condition

Physiological variation and adaptation of the long-term evolved rhizobia to alkaline environments where no host plant existence and the stability of their symbiotic properties when they are reinoculated to legume host remain unclear. A highly effective N2-fixing Rhizobium yanglingense strain CCBAU 01603 was used as the ancestral strain and was cultured continuously with/without addition of extra alkaline reagent (KOH) in laboratory conditions for approximately 500 generations. Total 60 evolved clones obtained were checked for their adaptation to higher alkaline pH level and inoculated to their host plant Caragana microphylla to evaluate their symbiotic efficiencies. Most of the evolved clones showed increased adaptation to higher alkaline pH but all of them decreased symbiotic efficiencies, resulting in the formation of irregular root nodules with lower nitrogenase activity, production of abnormal bacteroids, and accumulation more starch grains in uninfected nodule cells. Further demonstration of lower symbiotic efficiencies came from the down-regulated expression of genes related to nitrogen fixation in the bacteroids by transcriptome comparison. In addition, genes related to transporters and other diverse functions were up- or down-regulated in the evolved clones in free-living conditions (like yjiS gene) or in symbiotic situations, demonstrating the significant variations in cellular physiology and symbiosis. Our study revealed that the enhancement of alkaline adaptation but loss of symbiotic efficiencies of the evolved clones had happened during the long-term evolution in alkaline environments where no selective pressures from host plant, offering new insight into the molecular mechanism and direction of rhizobial evolution in nature.

Resource acquisition and allocation traits in symbiotic rhizobia with implications for life-history outside of legume hosts.

Resources that microbial symbionts obtain from hosts may enhance fitness during free-living stages when resources are comparatively scarce. For rhizobia in legume root nodules, diverting resources from nitrogen fixation to polyhydroxybutyrate (PHB) has been discussed as a source of host–symbiont conflict. Yet, little is known about natural variation in PHB storage and its implications for rhizobial evolution. We therefore measured phenotypic variation in natural rhizobia populations and investigated how PHB might contribute to fitness in the free-living stage. We found that natural populations of rhizobia from Glycine max and Chamaecrista fasciculata had substantial, heritable variation in PHB acquisition during symbiosis. A model simulating temperature-dependent metabolic activity showed that the observed range of stored PHB per cell could support survival for a few days, for active cells, or over a century for sufficiently dormant cells. Experiments with field-isolated Bradyrhizobium in starvation culture suggest PHB is partitioned asymmetrically in dividing cells, consistent with individual-level bet-hedging previously demonstrated in E. meliloti. High-PHB isolates used more PHB over the first month, yet still retained more PHB for potential long-term survival in a dormant state. These results suggest that stored resources like PHB may support both short-term and long-term functions that contribute to fitness in the free-living stage.

Draft Genome Sequence of Cupriavidus UYMMa02A, a Novel Beta-Rhizobium Species.

We present the draft genome of Cupriavidus UYMMa02A, a rhizobium strain isolated from root nodules of Mimosa magentea. The assembly has approximately 8.1 million bp with an average G+C of 64.1%. Symbiotic and metal-resistance genes were identified. The study of this genome will contribute to the understanding of rhizobial evolution.

Evolution of root nodule bacteria: Reconstruction of the speciation processes resulting from genomic rearrangements in a symbiotic system

The processes of speciation and macroevolution of root nodule bacteria (rhizobia), based on deep rearrangements of their genomes and occurring in the N₂-fixing symbiotic system, are reconstructed. At the first stage of rhizobial evolution, transformation of free-living diazotrophs (related to Rhodopseudomonas) to symbiotic N₂-fixers (Bradyrhizobium) occurred due to the acquisition of the fix gene system, which is responsible for providing nitrogenase with electrons and reducing equivalents, as well as for oxygen-dependent regulation of nitrogenase synthesis in planta, and then of the nod genes responsible for the synthesis of the lipo- chito-oligosaccharide Nod factors, which induce root nodule development. The subsequent rearrangements of bacterial genomes included: (1) increased volume of hereditary information supported by species, genera (pan-genome), and individual strains; (2) transition from the unitary genome to a multicomponent one; and (3) enhanced levels of bacterial genetic plasticity and horizontal gene transfer, resulting in formation of new genera, of which Mesorhizobium, Rhizobium, and Sinorhizobium are the largest, and of over 100 species. Rhizobial evolution caused by development and diversification of the Nod factor synthesizing systems may result in both increased host specificity range (transition of Bradyrhizobium from autotrophic to symbiotrophic carbon metabolism in interaction with a broad spectrum of legumes) and to its contraction (transition of Rhizobium and Sinorhizobium to "altruistic" interaction with legumes of the galegoid clade). Reconstruction of the evolutionary pathway from symbiotic N₂-fixers to their free-living ancestors makes it possible to initiate the studies based on up-to-date genome screening technologies and aimed at the issues of genetic integration of organisms into supracpecies complexes, ratios of the macro- and microevolutionary mechanisms, and developmetn of cooperative adaptations based on altruistic relationship between the symbiotic partners.

Draft Genome Sequence of the Bean-Nodulating Sinorhizobium fredii Strain GR64

Sinorhizobium fredii GR64 is a peculiar strain that is able to effectively nodulate bean but not soybean, the common host of S. fredii. Here we present the draft genome of S. fredii GR64. This information will contribute to a better understanding of the symbiotic rhizobium-plant interaction and of rhizobial evolution.

Controlling the reproductive fate of rhizobia: how universal are legume sanctions?

When a single host plant is infected by more than one strain of rhizobia, they face a tragedy of the commons. Although these rhizobia benefit collectively from nitrogen fixation, which increases host-plant photosynthesis, each strain might nonetheless increase its own reproduction, relative to competing strains, by diverting resources away from nitrogen fixation. Host sanctions can limit the evolutionary success of such rhizobial cheaters (strains that would otherwise benefit by fixing less nitrogen). Host sanctions have been shown in soybean (Glycine max) nodules, where the next generation of symbiotic rhizobia is descended from bacteroids (the differentiated cells that can fix nitrogen). Evidence for sanctions is less clear in legume species that induce rhizobial dimorphism inside their nodules. There, bacteroids are swollen and cannot reproduce regardless of how much nitrogen they fix, but sanctions could reduce reproduction of their undifferentiated clonemates within the same nodule. This rhizobial dimorphism can affect rhizobial evolution, including cheating options, in ways that may affect future generations of legumes. Both the importance of sanctions to hosts and possible physiological mechanisms for sanctions may depend on whether bacteroids are potentially reproductive.

Microevolution of Nodule Bacteria upon Generation of Mutants with Altered Survival in the Plant–Soil System

Simulation of cyclic processes in the plant–soil system was used to analyze the effects of factors responsible for the population dynamics of rhizobia on generation of mutants with changedex planta viability. Rhizobial evolution in a system of ecological niches (soil, rhizosphere, nodules) was described with recurrent equations. Computer experiments were carried out with parameters determining the mutation pressure, selection, and amplitude of the population wave arising in soil on the release of bacteria from nodules and the rhizosphere. Analysis of the model showed that (1) mutants with enhanced ex planta viability do not completely replace the parental strain and (2) mutants with impaired ex planta viability may be fixed in the population. The maintenance of genotypes subject to elimination from the soil and rhizosphere by Darwinian selection was associated with frequency-dependent selection (FDS), which is effective in competition for nodulation. The FDS index was proposed to characterize FDS pressure and was shown to determine the population polymorphism for adaptive traits. An increase in population wave amplitude proved to increase the fixation level (the proportion in the limiting state of the system) of mutants with enhanced viability and to decrease it in mutants with low viability. The results obtained with the model agreed with the data that, in edaphic stress, rhizobial populations remain highly polymorphic, which is associated with the maintenance of sensitive strains. The simulation procedure may be employed in estimating the genetic consequences of introduction of modified rhizobial strains in the environment.

Legume Sanctions and the Evolution of Symbiotic Cooperation by Rhizobia.

The legume-rhizobium symbiosis is an ideal model for studying the factors that limit the evolution of microbial mutualists into parasites. Legumes are unable to consistently recognize parasitic rhizobia that, once established inside plant cells, use plant resources for their own reproduction rather than for N2 fixation. Evolution of parasitism in rhizobia, driven partly by competition among multiple rhizobial strains infecting the same plant, may be countered by postinfection legume sanctions. Both the biochemical options for rhizobial cheating and the evolutionary effect of legume sanctions depend on differences in rhizobial life history associated with nodule type. In legumes with determinate nodule growth, rhizobia typically retain the ability to reproduce after differentiating into N2-fixing bacteroids. Sanctions against individual bacteroids (e.g., acid hydrolases) would therefore select for cooperative rhizobia. In nodules with indeterminate growth, bacteroids generally lose the ability to reproduce, so legume sanctions against bacteroids would have no effect on rhizobial evolution. Whole-nodule sanctions (e.g., decreased nodule O2 permeability) could be effective, via kin selection of undifferentiated rhizobia that persist in indeterminate nodules and replenish soil populations after nodule senescence. Mixed nodules could reduce the effectiveness of whole-nodule sanctions. The frequency of mixed nodules under field conditions is unknown.