Rhizobial Populations Research Articles

Alkalinity of Lanzarote soils is a factor shaping rhizobial populations with Sinorhizobium meliloti being the predominant microsymbiont of Lotus lancerottensis

Lotus lancerottensis is an endemic species that grows widely throughout Lanzarote Island (Canary Is.). Characterization of 48 strains isolated from root nodules of plants growing in soils from eleven locations on the island showed that 38 isolates (79.1%) belonged to the species Sinorhizobium meliloti, whereas only six belonged to Mesorhizobium sp., the more common microsymbionts for the Lotus. Other genotypes containing only one isolate were classified as Pararhizobium sp., Sinorhizobium sp., Phyllobacterium sp. and Bradyrhizobium-like. Strains of S. meliloti were distributed along the island and, in most of the localities they were exclusive or major microsymbionts of L. lancerottensis. Phylogeny of the nodulation nodC gene placed the S. meliloti strains within symbiovar lancerottense and the mesorhizobial strains with the symbiovar loti. Although strains from both symbiovars produced effective N2-fixing nodules, S. meliloti symbiovar lancerottense was clearly the predominant microsymbiont of L. lancerottensis. This fact correlated with the better adaptation of strains of this species to the alkaline soils of Lanzarote, as in vitro characterization showed that while the mesorhizobial strains were inhibited by alkaline pH, S. meliloti strains grew well at pH 9.

Diverse Bacteria Affiliated with the Genera Microvirga, Phyllobacterium, and Bradyrhizobium Nodulate Lupinus micranthus Growing in Soils of Northern Tunisia.

The genetic diversity of bacterial populations nodulating Lupinus micranthus in five geographical sites from northern Tunisia was examined. Phylogenetic analyses of 50 isolates based on partial sequences of recA and gyrB grouped strains into seven clusters, five of which belong to the genus Bradyrhizobium (28 isolates), one to Phyllobacterium (2 isolates), and one, remarkably, to Microvirga (20 isolates). The largest Bradyrhizobium cluster (17 isolates) grouped with the B. lupini species, and the other five clusters were close to different recently defined Bradyrhizobium species. Isolates close to Microvirga were obtained from nodules of plants from four of the five sites sampled. We carried out an in-depth phylogenetic study with representatives of the seven clusters using sequences from housekeeping genes (rrs, recA, glnII, gyrB, and dnaK) and obtained consistent results. A phylogeny based on the sequence of the symbiotic gene nodC identified four groups, three formed by Bradyrhizobium isolates and one by the Microvirga and Phyllobacterium isolates. Symbiotic behaviors of the representative strains were tested, and some congruence between symbiovars and symbiotic performance was observed. These data indicate a remarkable diversity of L. micranthus root nodule symbionts in northern Tunisia, including strains from the Bradyrhizobiaceae, Methylobacteriaceae, and Phyllobacteriaceae families, in contrast with those of the rhizobial populations nodulating lupines in the Old World, including L. micranthus from other Mediterranean areas, which are nodulated mostly by Bradyrhizobium strains.IMPORTANCELupinus micranthus is a legume broadly distributed in the Mediterranean region and plays an important role in soil fertility and vegetation coverage by fixing nitrogen and solubilizing phosphate in semiarid areas. Direct sowing to extend the distribution of this indigenous legume can contribute to the prevention of soil erosion in pre-Saharan lands of Tunisia. However, rhizobial populations associated with L. micranthus are poorly understood. In this context, the diversity of endosymbionts of this legume was investigated. Most Lupinus species are nodulated by Bradyrhizobium strains. This work showed that about half of the isolates from northern Tunisian soils were in fact Bradyrhizobium symbionts, but the other half were found unexpectedly to be bacteria within the genera Microvirga and Phyllobacterium These unusual endosymbionts may have a great ecological relevance. Inoculation with the appropriate selected symbiotic bacterial partners will increase L. micranthus survival with consequent advantages for the environment in semiarid areas of Tunisia.

Natural rhizobial diversity helps to reveal genes and QTLs associated with biological nitrogen fixation in common bean.

Common bean is one of the most important crops for human feed, and the most important legume for direct consumption by millions of people, especially in developing countries. It is a promiscuous host legume in terms of nodulation, able to associate with a broad and diverse range of rhizobia, although the competitiveness for nodulation and the nitrogen fixation capacity of most of these strains is generally low. As a result, common bean is very inefficient for symbiotic nitrogen fixation, and nitrogen has to be supplied with chemical fertilizers. In the last years, symbiotic nitrogen fixation has received increasing attention as a sustainable alternative to nitrogen fertilizers, and also as a more economic and available one in poor countries. Therefore, optimization of nitrogen fixation of bean-rhizobia symbioses and selection of efficient rhizobial strains should be a priority, which begins with the study of the natural diversity of the symbioses and the rhizobial populations associated. Natural rhizobia biodiversity that nodulates common bean may be a source of adaptive alleles acting through phenotypic plasticity. Crosses between accessions differing for nitrogen fixation may combine alleles that never meet in nature. Another way to discover adaptive genes is to use association genetics to identify loci that common bean plants use for enhanced biological nitrogen fixation and, in consequence, for marker assisted selection for genetic improvement of symbiotic nitrogen fixation. In this review, rhizobial biodiversity resources will be discussed, together with what is known about the loci that underlie such genetic variation, and the potential candidate genes that may influence the symbiosis' fitness benefits, thus achieving an optimal nitrogen fixation capacity in order to help reduce reliance on nitrogen fertilizers in common bean.

Growth Behavior, Nodulation and Rhizobium Population, as Affected by Combined Application of Herbicide and Insecticide In Soybean (Glycine max L.)

Growth Behavior, Nodulation and Rhizobium Population, as Affected by Combined Application of Herbicide and Insecticide In Soybean (Glycine max L.)

Ancient Heavy Metal Contamination in Soils as a Driver of Tolerant Anthyllis vulneraria Rhizobial Communities.

Phytostabilization of toxic mine spoils limits heavy metal dispersion and environmental pollution by establishing a sustainable plant cover. This eco-friendly method is facilitated by the use of selected and adapted cover crop legumes living in symbiosis with rhizobia that can stimulate plant growth naturally through biological nitrogen fixation. We studied microsymbiont partners of a metal-tolerant legume, Anthyllis vulneraria, which is tolerant to very highly metal-polluted soils in mining and nonmining sites. Site-specific rhizobial communities were linked to taxonomic composition and metal tolerance capacity. The rhizobial species Mesorhizobium metallidurans was dominant in all Zn-Pb mines but one. It was not detected in unpolluted sites where other distinct Mesorhizobium species occur. Given the different soil conditions at the respective mining sites, including their heavy-metal contamination, revegetation strategies based on rhizobia adapting to local conditions are more likely to succeed over the long term compared to strategies based on introducing less-well-adapted strains.

Pyrosequencing of rpoB uncovers a significant biogeographical pattern of rhizobial species in soybean rhizosphere

AbstractAimMost culture‐independent studies of bacterial biogeography have been at genus or higher taxonomic levels, although many important processes mediated by bacteria are at the strain or species level, such as the competitive nodulation of rhizobia on legumes. Here, at the intra‐species level, we characterized the structural variation in rhizobial populations in soybean rhizosphere under field conditions across 32 sampling sites in three eco‐regions.LocationNorth‐east (Heilongjiang), north (Hebei–Shandong) and south (Jiangxi) China.MethodsThe intra‐species diversity of rhizobia in soybean rhizospheres was investigated by rpoB pyrosequencing. Soybean nodule isolates were characterized using repetitive extragenic palindromic PCR, restriction fragment length polymorphism of 16S‐23S intergenic sequences and rpoB PCR sequencing. Patterns and processes of biogeography of identified rhizobia were studied.ResultsSignificant biogeographical patterns of rhizosphere rhizobia at the species level were found, and the abundant species in the rhizosphere were generally the dominant microsymbionts in soybean nodules. The distribution of rhizobial species in the rhizosphere across eco‐regions correlated with combined effects of spatial, vegetative, edaphic and climatic variables. Gene flow and genetic differentiation analyses revealed that there was no dispersal limitation among geographical populations of the same rhizobial species, and drift or subsequent diversification might be involved in shaping the biogeographical pattern in addition to environmental factors.Main conclusionsThese results constitute the first report of the existence of rhizobial biogeography in legume rhizospheres at the species level, contrasting with most studies, which focused on nodule isolates. The method developed in this study could be used to uncover, at high resolution, the diversity of local rhizobial populations in soils. This information is crucial for choosing suitable strains or species as inoculants for a specific legume host under local field conditions. Moreover, a similar strategy could be used to study other bacteria with important ecological functions.

English

Liquid residue from pig farming contains nutrients that can be used for the fertilization of cultivated soils. The aim of this study was to evaluate chemical and microbiological changes in a sandy soil under pasture with Bermuda Grass (Cynodon spp) that received doses of pig liquid waste (PLW). The experiment was conducted in Cianorte-PR, Brazil, in a Typic Hapludox soil with sandy texture. The treatments consisted of 30, 60 and 90 m3 ha-1 yr-1 of PLW or chemical fertilizer (CF) applied for two years in a randomized block design, with three replications. Soil samples were taken at 0-10 cm, 10-20 cm and 20-40 cm layers, after three months of the second consecutive application of PLW in the second year, before grazing. PLW increased the concentrations of P, C and K at 10-20 and 20-40 cm soil depth, in addition to increasing the microbial biomass carbon and nitrogen and the population of rhizobia at 0-10 cm, in the treatment with 90 m3 ha-1 yr-1. PLW improved the chemical fertility at deeper soil layers and the biological fertility at 0-10 cm of a sandy soil under pasture. Key words: Microbial biomass, organic fertilizer, phosphorus, potassium, rhizobia.

Intrapopulation genomics in a model mutualist: Population structure and candidate symbiosis genes under selection in Medicago truncatula.

Bottom-up evolutionary approaches, including geographically explicit population genomic analyses, have the power to reveal the mechanistic basis of adaptation. Here, we conduct a population genomic analysis in the model legume, Medicago truncatula, to characterize population genetic structure and identify symbiosis-related genes showing evidence of spatially variable selection. Using RAD-seq, we generated over 26,000 SNPs from 191 accessions from within three regions of the native range in Europe. Results from STRUCTURE analysis identify five distinct genetic clusters with divisions that separate east and west regions in the Mediterranean basin. Much of the genetic variation is maintained within sampling sites, and there is evidence for isolation by distance. Extensive linkage disequilibrium was identified, particularly within populations. We conducted genetic outlier analysis with FST -based genome scans and a Bayesian modeling approach (PCAdapt). There were 70 core outlier loci shared between these distinct methods with one clear candidate symbiosis related gene, DMI1. This work sets that stage for functional experiments to determine the important phenotypes that selection has acted upon and complementary efforts in rhizobium populations.

Structural and functional organization of the plasmid regulons of Rhizobium leguminosarum symbiotic genes

The structure of the plasmid locus containing the sym-genes (nod-, nif-, and fix-operons) was investigated in eight Rhizobium leguminosarum strains differing in their origin and host specificity, including five strains of the viciae biovar—symbionts of pea (3), forage beans (1), and Vavilovia (1)—as well as three strains of the biovar trifolii (clover symbionts). Strains of R. leguminosarum bv. viciae, which possess the nodX gene (controlling acetylation of the Nod factor, which is responsible for the ability of rhizobia to form symbioses with a broad spectrum of hosts, including the “Afghan” pea lines, homozygous by the allele sym2A), are characterized by a less compact location of the sym-genes than the strains lacking the nodX gene. The size of the symbiotic cluster in the strains possessing nodX was 94.5 ± 3.5 kb, with the share of the sym-genes of 36.5 ± 1.5%, while for the strains lacking nodX these values were 61.7 ± 3.7 kb and 56.3 ± 1.4%, respectively (significant difference at P0 < 0.01). Syntenic structures were revealed in the symbiotic regions of strains Vaf12, UPM1131, and TOM, as well as syntenic structures of non-symbiotic regions in strains Vaf12, TOM, and WSM1689. The correlation coefficients between the matrices of genetic distances in the analyzed strains for the nodABC, nifHDK, and fixABC operons were on average 0.993 ± 0.002, while their values for the plasmid sites located between the sym-genes were considerably less (0.706 ± 0.010). In these regions, 21 to 27% of the genes were involved in amino acid transport and metabolism, which was substantially higher than the average for the genome of R. leguminosarum bv. viciae (11–12%). These data suggest that the evolution of R. leguminosarum bv. viciae, defined by narrowing of the host specificity (associated with a loss of the nodX gene), was accompanied by reduction of the regions of plasmids located between the sym-genes, as well as by specialization of these areas to perform the functions related to symbiotic nitrogen fixation. The observed increase of density in the cluster of sym-genes may be associated with intensification of their horizontal transfer in the populations of rhizobia, which determines the speed of evolution of the symbiotic system.

English

The growth of tree legumes in degraded areas must be preceded by assessments of nodulation ability of naturally established rhizobia populations since such information contributes to defining the species which can be planted for recovering disturbed areas. The aim of this study was to evaluate the growth and natural nodulation of “sabia” (Mimosa caesalpiniifolia Benth.) and leucaena (Leucaena leucocephala (Lam.) de Wit.) in soils of the Brazilian semiarid area under different systems of land use: native vegetation (locally called caatinga) and areas with different agricultural systems (a monocrop system and an intercropping with various species). For each species, a greenhouse experiment in randomized block design was realized, using soils of different types (Luvisol and Ultisol), with 4 replicates. The results evidentiate significant differences in the evaluated growth characteristics (height, leaflet number and shoot diameter) of M. caesalpiniifolia, that have displayed lower plant growth when cultivated in the Luvisol under conventional system. Plant growth, nodulation and total N accumulation in both seedling tree legumes increased in Ultisol under the different systems of land use. L. leucocephala showed higher potential of biological nitrogen fixation and nodulation effectiveness promoted by indigenous rhizobia. Key words: Biological nitrogen fixation, indigenous rhizobia, symbiosis, sustainable agriculture.

The diversity of bean nodule bacteria in Ukrainian agrocenoses

The aim of our work was to study the diversity of beans nodule bacteria in the natural populations of rhizobia and spend their serological and genetic typing. Beans rhizobia were isolated from bean nodules which were grown in soil samples selected in different regions of Ukraine. Morphological, cultural and physiological properties of rhizobia were studied using standard methods. The diversity of beans nodule bacteria was assessed using a serological method. The restriction analysis of the intergenic region of the ribosomal cluster (ITS) was carried out with the use of restriction endonucleases MspI, HaeIII, NdeII. Fifty strains of beans nodule bacteria were isolated from soils of different regions of Ukraine. Biological diversity of these strains was studied. All the culture of nodule bacteria enters into a symbiotic relationship with the common beans and forms active nodules on the plant roots. These strains were identified as Rhizobium phaseoli. It is established that new strains of R. phaseoli differ in antibiotics sensitivity, serological and genetic properties. On antigenic com- position investigated nodule bacteria were combined into different serological groups. The beans rhizobia, isolated from agrocenosis of Chernihіv region was the most serologically diverse. PCR-RFLP of the ITS- region showed significant genetic polymorphism of common beans microsymbionts, isolated from local populations of rhizobia. On the basis of the restriction profiles of the 16S-23S rDNA these strains were first divided into different IТS-types. It is established that division the strains on ITS-types (NdeII and HaeIII) corresponds to their division into different serological groups.

Nitrogen addition does not influence pre-infection partner choice in the legume-rhizobium symbiosis.

Resource mutualisms such as the symbiosis between legumes and nitrogen-fixing rhizobia are context dependent and are sensitive to various aspects of the environment, including nitrogen (N) addition. Mutualist hosts such as legumes are also thought to use mechanisms such as partner choice to discriminate among potential symbionts that vary in partner quality (fitness benefits conferred to hosts) and thus impose selection on rhizobium populations. Together, context dependency and partner choice might help explain why the legume-rhizobium mutualism responds evolutionarily to N addition, since plant-mediated selection that shifts in response to N might be expected to favor different rhizobium strains in different N environments. We test for the influence of context dependency on partner choice in the model legume, Medicago truncatula, using a factorial experiments with three plant families across three N levels with a mixed inoculation of three rhizobia strains. Neither the relative frequencies of rhizobium strains occupying host nodules, nor the size of those nodules, differed in response to N level. Despite the lack of context dependence, plant genotypes respond very differently to mixed populations of rhizobia, suggesting that these traits are genetically variable and thus could evolve in response to longer-term increases in N.

Inorganic nitrogen application improves the yield and yield traits of common bean (Phaseolus vulgaris L.) irrespective of the indigenous rhizobial population

The effect of number and efficiency of indigenous rhizobia on the need for external inorganic nitrogen (N) application to boost common bean production is unknown. Hence, field experiments were conducted at four locations with soil populations of <100, 100–1 000 and >1 000 rhizobia to evaluate the effect of the indigenous rhizobial population on N requirement of common bean in eastern Ethiopia. The treatments comprised six levels of N application arranged in a randomised complete block design with three replications. The results showed decreases in nodule number (NN) and nodule dry weight (NDW) with increasing rate of inorganic N application in all soil types. Application of 20 kg N ha−1 resulted in a significant increase in the NN and NDW in soil with a low and moderate rhizobial population but not in soils with a high rhizobial population. The highest yield and yield traits in soil containing a high and low rhizobial population was recorded with <40 and >60 kg N ha−1 application, respectively. The association between grain yield and N application was negative in soils with a high and moderate rhizobial population, and positive in soil containing a low rhizobial population. In general, the results revealed that the number of indigenous rhizobia could affect the quantity of N applied.

Native Pseudomonas spp. suppressed the root-knot nematode in in vitro and in vivo, and promoted the nodulation and grain yield in the field grown mungbean

The study describes the effects of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas stutzeri and Pseudomonas striata on root nodulation (Bradyrhizobium sp.), root-knot disease (Meloidogyne incognita), and on the plant growth and yield of field grown mungbean (Vigna radiata). In vitro the Pseudomonas spp. produced hydrogen cyanide (P. fluorescens), ammonia, siderophore and indole acetic acid, and solubilized phosphorus. The bacterial cells and cell free culture filtrate of Pseudomonas spp. suppressed egg hatching and induced mortality to the juveniles of M. incognita with overall greater effects of P. fluorescens and P. striata. The seeds of mungbean cv. T-44 treated with Pseudomonas spp. (1011–12colony formingunits/ml) and Bradyrhizobium (109–10CFU/ml, 5ml/kg seeds) were sown at 45 spots in every field plot (4×2m) with or without the nematode inoculation of 2000juveniles/spot. In the plots without nematodes, grain yield and NPK acquisition of mungbean plants were significantly higher with P. fluorescens or P. striata treatments (P⩽0.05). The Pseudomonas treatments synergized with Bradyrhizobium sp., leading to significant enhancements in the root nodulation, leghemoglobin contents, and the population of rhizobia in the nodule as well as in the soil. The nematode infection caused severe galling on the roots and decreased the yield of mungbean by 21% in the absence of Pseudomonas spp. The nematode infection also suppressed the nodulation, leghemoglobin contents and rhizobia population. However, treatments with Pseudomonas spp. improved the nodulation and other parameters of nematode infected plants. Among the four Pseudomonas spp., P. fluorescens was found more effective against the root-knot nematode, and its application reduced the gall formation, reproduction and soil population of M. incognita (P⩽0.05). The suppressive effect of P. fluorescens on the nematode was relatively less than carbofuran (5g active ingredient/kg seed), but the yield of nematode infected plants with the bacterial treatment was 14% (P⩽0.05) greater than the chemical treatment. Hence, seed treatment with P. fluorescens offers a better substitute of the nematicide in mungbean cultivation.

Pertumbuhan Mucuna bracteata DC. pada Berbagai Waktu Inokulasi dan Dosis Inokulan

ABSTRACT&lt;br /&gt;&lt;br /&gt;Mucuna bracteata DC. is a legume cover crop to prevent erosion and to suppress weed growth. M. bracteata can fix N2 from the air with the help of Rhizobium. M. bracteata get benefit from the symbiosis in the form of increased nodule weight, shoot dry weight, and leaf nitrogen content when the Rhizobium population in the soil is optimal. Application of inoculant is one method to increase the Rhizobium population in the soil. This study aimed to analyze the effects of inoculation times and inoculant rates on M. bracteata growth. The research was conducted in Boyolali, Central Jawa, from February to August 2014. The inoculant contained Bradyrhizobium japonicum and Aeromonas punctata. This experiment was arranged in a randomized block design with two factors and three replications. The first factor was two inoculation times, i.e at the nursery when the seedlings were 2 weeks old and in the field when the seedlings reached 5 weeks old. The second factor was 5 inoculant rates (0, 2, 4, 6, and 8 g per plant). The results showed that inoculation on 5-week-old seedlings was the best result compared to the 2-week-old seedlings. The recommended inoculant rate for Mucuna bracteata was 6.43 g per plant. &lt;br /&gt;&lt;br /&gt;Keywords: legume cover crop, nitrogenase activity, nodule, nutrient content, runner

Symbiont abundance is more important than pre-infection partner choice in a Rhizobium – legume mutualism

It is known that the genetic diversity of conspecific rhizobia present in root nodules differs greatly among populations of a legume species, which has led to the suggestion that both dispersal limitation and the local environment affect rhizobial genotypic composition. However, it remains unclear whether rhizobial genotypes residing in root nodules are representative of the entire population of compatible symbiotic rhizobia. Since symbiotic preferences differ among legume populations, the genetic composition of rhizobia found within nodules may reflect the preferences of the local hosts, rather than the full diversity of potential nodulating rhizobia present in the soil. Here, we assessed whether Vicia cracca legume hosts of different provenances select different Rhizobium leguminosarum genotypes than sympatric V. cracca hosts, when presented a natural soil rhizobial population. Through combining V. cracca plants and rhizobia from adjacent and more distant populations, we found that V. cracca hosts are relatively randomly associated with rhizobial genotypes. This indicates that pre-infection partner choice is relatively weak in certain legume hosts when faced with a natural population of rhizobia.

Selection mosaics differentiate Rhizobium–host plant interactions across different nitrogen environments

The nature and direction of coevolutionary interactions between species is expected to differentiate among distinct environments. Consequently, locally coevolved symbiotic traits would be well matched in similar environments, but mismatched elsewhere. In a classic mutualistic tradeoff, rhizobia provide nitrogen (N) to legume host plants in return for photosynthates. Despite earlier predictions, there is little evidence so far that spatial differences in soil N content mediate the coevolutionary outcome of the legume–Rhizobium mutualism. To test the existence of such selection mosaics, different genotypes of Vicia cracca and Rhizobium leguminosarum originating from spatially and environmentally highly differentiated sites were cross inoculated across different soil N regimes. In accordance with theoretical predictions, we found highly significant effects of genotype by genotype by environment (G× G × E) interactions, on both nodulation and plant growth, even when R. leguminosarum genotypes showed high genetic similarity. Our results show that the trajectory of the coevolutionary interactions between rhizobia and legumes is differentiated across different environments, and that selection mosaics may play an important role in shaping differences in the genetic composition of rhizobial populations.

Evaluation of early maturing cowpea (Vigna unguiculata) germplasm for variation in phosphorus use efficiency and biological nitrogen fixation potential with indigenous rhizobial populations

SUMMARYCowpea genotypes that efficiently utilize phosphorus (P) with high potential for biological nitrogen (N) fixation (BNF) are vital to sustainable cropping systems in West Africa. A total of 175 early maturing cowpea genotypes were evaluated in 2010 and 2011 for P use efficiency (PUE) and BNF with an indigenous rhizobial population at Shika in the Northern Guinea savanna of Nigeria. There were significant genotypic variations for all 11 variables measured. The P utilization index, percentage N derived from the atmosphere and total N fixed ranged between 2·10–4·67, 31·3–61·86% and 11·86–50 kg/ha, respectively. The 175 early maturing cowpea genotypes were divided into five categories using principal component analysis (PCA), whereby total N fixed was associated with N and P uptake and plant biomass yield. Complete linkage cluster analysis revealed a total of three distinctive clusters having remarkable correspondence with the PCA. Some genotypes were identified as potential candidates for further breeding programmes using high PUE genotypes with relatively high capacity for BNF and indigenous rhizobial populations.

Genetic diversity and community structure of rhizobia nodulating Sesbania cannabina in saline–alkaline soils

Sesbania cannabina is a plant that grows naturally along the seashores in Rudong County, China (RDC) and it has been introduced into the Yellow River Delta (YRD) as a pioneer plant to improve the saline–alkaline soils. In order to investigate the diversity of S. cannabina rhizobia in these soils, a total of 198 rhizobial isolates were characterized and phylogenetic trees were constructed based on data from multilocus sequence analysis (MLSA) of the housekeeping genes recA, atpD and glnII, as well as 16S rRNA. Symbiotic features were also studied by establishing the phylogeny of the symbiotic genes nodA and nifH, and by performing nodulation assays. The isolates had highly conserved symbiotic genes and were classified into nine genospecies belonging to the genera Ensifer, Agrobacterium, Neorhizobium and Rhizobium. A unique community structure was detected in the rhizobia associated with S. cannabina in the saline–alkaline soils that was characterized by five novel genospecies and four defined species. In addition, Ensifer sp. I was the predominant rhizobia in YRD, whereas Ensifer meliloti and Neorhizobium huautlense were the dominant species in RDC. Therefore, the study demonstrated for the first time that this plant strongly selected the symbiotic gene background but not the genomic background of its microsymbionts. In addition, biogeographic patterns existed in the rhizobial populations associated with S. cannabina, which were mainly correlated with pH and salinity, as well as the mineral nutrient contents. This study provided novel information concerning the interaction between soil conditions, host plant and rhizobia, in addition to revealing the diversity of S. cannabina rhizobia in saline–alkaline soils.

Ecological genomics of mutualism decline in nitrogen-fixing bacteria.

Anthropogenic changes can influence mutualism evolution; however, the genomic regions underpinning mutualism that are most affected by environmental change are generally unknown, even in well-studied model mutualisms like the interaction between legumes and their nitrogen (N)-fixing rhizobia. Such genomic information can shed light on the agents and targets of selection maintaining cooperation in nature. We recently demonstrated that N-fertilization has caused an evolutionary decline in mutualistic partner quality in the rhizobia that form symbiosis with clover. Here, population genomic analyses of N-fertilized versus control rhizobium populations indicate that evolutionary differentiation at a key symbiosis gene region on the symbiotic plasmid (pSym) contributes to partner quality decline. Moreover, patterns of genetic variation at selected loci were consistent with recent positive selection within N-fertilized environments, suggesting that N-rich environments might select for less beneficial rhizobia. By studying the molecular population genomics of a natural bacterial population within a long-term ecological field experiment, we find that: (i) the N environment is indeed a potent selective force mediating mutualism evolution in this symbiosis, (ii) natural variation in rhizobium partner quality is mediated in part by key symbiosis genes on the symbiotic plasmid, and (iii) differentiation at selected genes occurred in the context of otherwise recombining genomes, resembling eukaryotic models of adaptation.