Horizontal gene transfer and homologous recombination drive the evolution of the nitrogen-fixing symbionts of Medicago species

Using nitrogen-fixing Sinorhizobium species that interact with Medicago plants as a model system, we aimed at clarifying how sex has shaped the diversity of bacteria associated with the genus Medicago on the interspecific and intraspecific scales. To gain insights into the diversification of these symbionts, we inferred a topology that includes the different specificity groups which interact with Medicago species, based on sequences of the nodulation gene cluster. Furthermore, 126 bacterial isolates were obtained from two soil samples, using Medicago truncatula and Medicago laciniata as host plants, to study the differentiation between populations of Sinorhizobium medicae, Sinorhizobium meliloti bv. meliloti, and S. meliloti bv. medicaginis. The former two can be associated with M. truncatula (among other species of Medicago), whereas the last organism is the specific symbiont of M. laciniata. These bacteria were characterized using a multilocus sequence analysis of four loci, located on the chromosome and on the two megaplasmids of S. meliloti. The phylogenetic results reveal that several interspecific horizontal gene transfers occurred during the diversification of Medicago symbionts. Within S. meliloti, the analyses show that nod genes specific to different host plants have spread to different genetic backgrounds through homologous recombination, preventing further divergence of the different ecotypes. Thus, specialization to different host plant species does not prevent the occurrence of gene flow among host-specific biovars of S. meliloti, whereas reproductive isolation between S. meliloti bv. meliloti and S. medicae is maintained even though these bacteria can cooccur in sympatry on the same individual host plants.     

Comparison of rhizobia that nodulate Medicago laciniata and Medicago truncatula present in a single Tunisian arid soil

The rhizobia present in a single arid region Tunisian soil that nodulate Medicago laciniata and Medicago truncatula were compared. All isolates, 40 from each host, were Sinorhizobium meliloti based on 16S rRNA polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) patterns and subsequent confirmation by sequence analysis of the 16S rRNA genes in four representatives from each host species. There was no apparent relationship between Medicago host species of isolation and the nodulating rhizobial genome as determined by repetitive extragenic palandromic PCR. The isolates of M. laciniata were distinguished from those of M. truncatula present in the same soil by variation in PCR-RFLP of nifDK, indicating that this dissimilarity is originally genetic and not geographic. While forming effective symbioses with their own respective isolates, both M. laciniata and M. truncatula formed ineffective true nodules, nodule-like structures, or no nodules at all in cross-inoculation tests, as confirmed by the histological observations.     

Identification and cloning of the bacterial nodulation specificity gene in the Sinorhizobium meliloti--Medicago laciniata symbiosis

Medicago laciniata (cut-leaf medic) is an annual medic that is highly nodulation specific, nodulating only with a restricted range of Sinorhizobium meliloti. e.g., strain 102L4, but not with most strains that nodulate Medicago sativa (alfalfa), e.g., strains RCR2011 and Rm41. Our aim was to identify and clone the S. meliloti 102L4 gene implicated in the specific nodulation of M. laciniata and to characterize the adjacent nodulation (nod) region. An 11-kb EcoRI DNA fragment from S. meliloti 102L4 was shown to complement strain RCR2011 for nodulation of M. laciniata. Nucleotide sequencing revealed that this fragment contained nodABCIJ genes whose overall arrangement was similar to those found in strains RCR2011 and Rm41, which do not nodulate M. laciniata. Data for Tn5 mutagenesis of the nodABCIJ region of strain 102L4 suggested that the nodC gene was involved in the specific nodulation of M. laciniata. Tn5 insertions in the nodIJ genes gave mutants with nodulation delay phenotypes on both M. laciniata and M. sativa. Only subclones of the 11-kb DNA fragment containing a functional nodC gene from strain 102L4 were able to complement strain RCR2011 for nodulation of M. laciniata. The practical implications of these findings are discussed in the context of the development of a specific M. sativa - S. meliloti combination that excludes competition for nodulation by bacterial competitors resident in soil.     

Genetic diversity of Sinorhizobium populations recovered from different medicago varieties cultivated in Tunisian soils

A collection of 468 rhizobial isolates was obtained from different ecological areas of Tunisia by trapping them on Medicago sativa cv. Gabes, Medicago scutelleta cv. Kelson, Medicago truncatula, and Medicago ciliaris. A subsample of 134 rhizobia was chosen to determine their plasmid profile, and 89 isolates were subjected to multilocus enzyme electrophoresis (MLEE) and PCR/RFLP analysis using 16S, IGS (inter genic spacer), and nifKD probes. Twenty-five representatives from these isolates were evaluated for their nodulation and nitrogen fixation capacities. MLEE studies revealed two groups with highly heterogeneous host specificity and geographical origin. The discriminatory power was found to be slightly better with the amplified ribosomal intergenic region, than the nifKD genes. Divisions detected by nifKD amplified DNA analysis matched those established by ribosomal PCR- RFLPs. The comparison between different analyses revealed that MLEE illustrated better phenotypic properties of isolates than PCR-RFLP or plasmid content analysis. Clear distinction between Sinorhizobium meliloti and Sinorhizobium medicae were observed by analysis of the IGS symbiotic regions between nifD and nifK genes. Were able to distinguish three inoculation groups; isolates trapped from M. sativa cv. Gabes and M. scutelleta cv. Kelson formed one inoculation group which was more closely related to isolates trapped from M. truncatula than those trapped from M. ciliaris.     

Stable low molecular weight RNA profiling showed variations within Sinorhizobium meliloti and Sinorhizobium medicae nodulating different legumes from the alfalfa cross-inoculation group

Four different low molecular weight (LMW) RNA profiles, designated I-IV, among 179 isolates from Medicago, Melilotus and Trigonella species growing in a field site in Northern Spain were identified. From sequence analysis of the 16S rRNA, atpD and recA genes as well as DNA-DNA hybridization analysis with representatives of each LMW RNA profile it was evident that isolates with LMW RNA profiles I and II belonged to Sinorhizobium meliloti and those displaying profiles III and IV to Sinorhizobium medicae. Therefore, two distinct LMW RNA electrophoretic mobility profiles were found within each of these two species. Collectively, LMW RNA profiles I and II (identified as S. meliloti) were predominant in Melilotus alba, Melilotus officinalis and Medicago sativa. Profiles III and IV (identified as S. medicae) were predominant in Melilotus parviflora, Medicago sphaerocarpa, Medicago lupulina and Trigonella foenum-graecum. All the four LMW RNA profiles were identified among isolates from Trigonella monspelliaca nodules. These results revealed a different specificity by the hosts of the alfalfa cross-inoculation group towards the two bacterial species found in this study.     

Population genetic structure of Sinorhizobium meliloti and S. medicae isolated from nodules of Medicago spp. in Mexico

We studied the genetic structure of 176 bacterial isolates from nodules of Medicago sativa, M. lupulina and M. polymorpha in fifteen sites distributed in three localities in Mexico. The strains were characterized by multilocus enzyme electrophoresis, plasmid profiles, PCR restriction fragment length polymorphism of 16S rRNA genes and of the intergenic spacer between 16S and 23S rRNA genes, and partial sequences of glnII, recA and nodB. Most of the strains were classified as Sinorhizobium meliloti, and a high genetic diversity was recorded. Six strains were classified as Sinorhizobium medicae, with no genetic variation. Phylogenetic and population genetic analyses revealed evidence of frequent recombination and migration within species.     

Symbiosis between the nodule bacterium Sinorhizobium meliloti and alfalfa (Medicago sativa) under salinization conditions

Two hundred forty-three isolates of alfalfa nodule bacteria (Sinorhizobium meliloti) were obtained from legume nodules and soils sampled in the northern Aral region, experiencing secondary salinization. Isolates obtained from nodules (N isolates) were significantly more salt-tolerant than those from soils (S isolates) when grown in a liquid medium with 3.5% NaCl. It was found that wild species of alfalfa, melilot, and trigonella preferably formed symbioses with salt-tolerant nodule bacteria in both salinized and nonsalinized soils. Only two alfalfa species, Medicago falcata and M. trautvetteri, formed efficient symbioses in soils contrasting in salinity. The formation of efficient symbiosis with alfalfa in the presence of 0.6% NaCl was studied in 36 isolates (N and S) differing in salt tolerance and symbiotic efficiency. Fifteen isolates formed efficient symbioses in the presence of salt. The increase in the dry weight of the plants was 25-68% higher than in the control group. The efficiency of symbiotic interaction under salinization conditions depended on the efficiency of the isolates under standard conditions but did not correlate with the source of nodule bacteria (soil or nodule) or their salt tolerance. The results indicate that nodule bacterium strains forming efficient symbioses under salinization conditions can be found.     

Sugar-binding activity of pea lectin enhances heterologous infection of transgenic alfalfa plants by Rhizobium leguminosarum biovar viciae

Transgenic alfalfa (Medicago sativa L. cv Regen) roots carrying genes encoding soybean lectin or pea (Pisum sativum) seed lectin (PSL) were inoculated with Bradyrhizobium japonicum or Rhizobium leguminosarum bv viciae, respectively, and their responses were compared with those of comparably inoculated control plants. We found that nodule-like structures formed on alfalfa roots only when the rhizobial strains produced Nod factor from the alfalfa-nodulating strain, Sinorhizobium meliloti. Uninfected nodule-like structures developed on the soybean lectin-transgenic plant roots at very low inoculum concentrations, but bona fide infection threads were not detected even when B. japonicum produced the appropriate S. meliloti Nod factor. In contrast, the PSL-transgenic plants were not only well nodulated but also exhibited infection thread formation in response to R. leguminosarum bv viciae, but only when the bacteria expressed the complete set of S. meliloti nod genes. A few nodules from the PSL-transgenic plant roots were even found to be colonized by R. leguminosarum bv viciae expressing S. meliloti nod genes, but the plants were yellow and senescent, indicating that nitrogen fixation did not take place. Exopolysaccharide appears to be absolutely required for both nodule development and infection thread formation because neither occurred in PSL-transgenic plant roots following inoculation with an Exo(-) R. leguminosarum bv viciae strain that produced S. meliloti Nod factor.     

Comparison of nucleotide diversity and symbiotic properties of Rhizobium meliloti populations from annual Medicago species

Abstract: Forty-three isolates of Rhizobium meliloti were trapped from soil with five annual species of Medicago (M. polymorpha, M. truncatula, M. rigidula, M. orbicularis and M. minima ) and one perennial species of Medicago (M. sativa) . The annual species were growing naturally near the soil sampling site, and the commonly studied perennial species was used for comparison. Each R. meliloti was characterized by PCR-RFLP methods applied to two DNA regions nested between 16S rRNA and 23S rRNA genes and between nif D and nif K genes. They fell into two highly divergent groups (groups I and II), separated at a genetic distance of 0.024 by rDNA-amplified pattern analysis (profiles R1 and R2) and at 0.029 by nif -amplified pattern analysis (profiles N1-N2 and N3). These two groups were consistent with some cross-nodulation and -fixation results: rhizobia with the R1 genetic background elicited rudimentary nodules and could not fix nitrogen on M. polymorpha , while they were able to nodulate the five other species of Medicago . In contrast, rhizobia with an R2 profile were highly effective on M. polymorpha and poorly nodulated M. rigidula species, but were able to nodulate efficiently the other species. The striking phenotypic traits on M. polymorpha were also shared by reference strains: strains genetically closed to R2 type triggered typical and efficient nodules on M. polymorpha while those close to R1 type elicited rudimentary and non-efficient ones. Our results suggest that the presence of R. meliloti with R2 genetic backgrounds could be favoured by the distribution of M. polymorpha species.     

Phylogenetic position of Rhizobium sp. strain Or 191, a symbiont of both Medicago sativa and Phaseolus vulgaris, based on partial sequences of the 16S rRNA and nifH genes.

Phenotypic and DNA sequence comparisons are presented for eight Rhizobium isolates that were cultured from field-grown alfalfa (Medicago sativa L.) in Oregon. These isolates were previously shown to nodulate both alfalfa and common bean (Phaseolus vulgaris (L.) Savi.). The objective of the present study was to determine their phylogenetic relationships to the normal symbionts of these plants, Rhizobium meliloti and Rhizobium leguminosarum biovar phaseoli, respectively. Phenotypically, the Oregon isolates more nearly resemble strains from P. vulgaris than those from M. sativa. For example, even though nitrogen fixation levels were low with both host species, the symbiotic efficiency of a representative Rhizobium isolate (Or 191) with common bean was twice that observed with alfalfa. Comparative sequencing of a 260-bp segment of the 16S rRNA gene (directly sequenced after amplification by the polymerase chain reaction) demonstrated that Or 191 is not closely related to the type strain of R. meliloti (ATCC 9930), R. leguminosarum (ATCC 10004), or Rhizobium tropici (CIAT 899). Instead, sequence comparisons of the 16S gene indicated that Or 191 belongs to a distinct and previously unrecognized taxonomic group that includes strains that have previously been called R. leguminosarum bv. phaseoli type I. Unlike type I strains, however, Or 191 has only a single copy of the nifH gene (type I strains have three), and the nucleotide sequence of this gene is substantially different from those of other rhizobial and nonrhizobial nifH genes examined thus far.     

Phylogenetic position of Rhizobium sp. strain Or 191, a symbiont of both Medicago sativa and Phaseolus vulgaris, based on partial sequences of the 16S rRNA and nifH genes.

Phenotypic and DNA sequence comparisons are presented for eight Rhizobium isolates that were cultured from field-grown alfalfa (Medicago sativa L.) in Oregon. These isolates were previously shown to nodulate both alfalfa and common bean (Phaseolus vulgaris (L.) Savi.). The objective of the present study was to determine their phylogenetic relationships to the normal symbionts of these plants, Rhizobium meliloti and Rhizobium leguminosarum biovar phaseoli, respectively. Phenotypically, the Oregon isolates more nearly resemble strains from P. vulgaris than those from M. sativa. For example, even though nitrogen fixation levels were low with both host species, the symbiotic efficiency of a representative Rhizobium isolate (Or 191) with common bean was twice that observed with alfalfa. Comparative sequencing of a 260-bp segment of the 16S rRNA gene (directly sequenced after amplification by the polymerase chain reaction) demonstrated that Or 191 is not closely related to the type strain of R. meliloti (ATCC 9930), R. leguminosarum (ATCC 10004), or Rhizobium tropici (CIAT 899). Instead, sequence comparisons of the 16S gene indicated that Or 191 belongs to a distinct and previously unrecognized taxonomic group that includes strains that have previously been called R. leguminosarum bv. phaseoli type I. Unlike type I strains, however, Or 191 has only a single copy of the nifH gene (type I strains have three), and the nucleotide sequence of this gene is substantially different from those of other rhizobial and nonrhizobial nifH genes examined thus far.     

Genetic relationship of Sinorhizobium meliloti and Sinorhizobium medicae strains isolated from Caucasian region

Sinorhizobium meliloti and Sinorhizobium medicae are two closely related species of the genus Sinorhizobium showing a similar host range, nodulating leguminous species of the genera Medicago, Melilotus and Trigonella, but their phylogenic relationship has not been elucidated yet. In this paper we report the application of three different molecular markers, (i) RFLP of nodD genes, (ii) 16S-23S rDNA intergenic gene spacer fingerprinting and (iii) amplification fragment length polymorphism to S. meliloti and S. medicae strains isolated from the Caucasian area, which is the region of origin of the host plant Medicago. The analysis of data could suggest the origin of S. medicae strains from an ancestral S. meliloti population.     

Salt-tolerant rhizobia isolated from a Tunisian oasis that are highly effective for symbiotic N2-fixation with Phaseolus vulgaris constitute a novel biovar (bv. mediterranense) of Sinorhizobium meliloti

Nodulation of common bean was explored in six oases in the south of Tunisia. Nineteen isolates were characterized by PCR–RFLP of 16S rDNA. Three species of rhizobia were identified, Rhizobium etli, Rhizobium gallicum and Sinorhizobium meliloti. The diversity of the symbiotic genes was then assessed by PCR–RFLP of nodC and nifH genes. The majority of the symbiotic genotypes were conserved between oases and other soils of the north of the country. Sinorhizobia isolated from bean were then compared with isolates from Medicago truncatula plants grown in the oases soils. All the nodC types except for nodC type p that was specific to common bean isolates were shared by both hosts. The four isolates with nodC type p induced N₂-fixing effective nodules on common bean but did not nodulate M. truncatula and Medicago sativa. The phylogenetic analysis of nifH and nodC genes showed that these isolates carry symbiotic genes different from those previously characterized among Medicago and bean symbionts, but closely related to those of S. fredii Spanish and Tunisian isolates effective in symbiosis with common bean but unable to nodulate soybean. The creation of a novel biovar shared by S. meliloti and S. fredii, bv. mediterranense, was proposed.     

Effect of field inoculation with Sinorhizobium meliloti L33 on the composition of bacterial communities in rhizospheres of a target plant (Medicago sativa) and a non-target plant (Chenopodium album)-linking of 16S rRNA gene-based single-strand conformation polymorphism community profiles to the diversity of cultivated bacteria

Fourteen weeks after field release of luciferase gene-tagged Sinorhizobium meliloti L33 in field plots seeded with Medicago sativa, we found that the inoculant also occurred in bulk soil from noninoculated control plots. In rhizospheres of M. sativa plants, S. meliloti L33 could be detected in noninoculated plots 12 weeks after inoculation, indicating that growth in the rhizosphere preceded spread into bulk soil. To determine whether inoculation affected bacterial diversity, 1,119 bacteria were isolated from the rhizospheres of M. sativa and Chenopodium album, which was the dominant weed in the field plots. Amplified ribosomal DNA restriction analysis (ARDRA) revealed plant-specific fragment size frequencies. Dominant ARDRA groups were identified by 16S rRNA gene nucleotide sequencing. Database comparisons indicated that the rhizospheres contained members of the Proteobacteria (alpha, beta, and gamma subgroups), members of the Cytophaga-Flavobacterium group, and gram-positive bacteria with high G+C DNA contents. The levels of many groups were affected by the plant species and, in the case of M. sativa, by inoculation. The most abundant isolates were related to Variovorax sp., Arthrobacter ramosus, and Acinetobacter calcoaceticus. In the rhizosphere of M. sativa, inoculation reduced the numbers of cells of A. calcoaceticus and members of the genus Pseudomonas and increased the number of rhizobia. Cultivation-independent PCR-single-strand conformation polymorphism (SSCP) profiles of a 16S rRNA gene region confirmed the existence of plant-specific rhizosphere communities and the effect of the inoculant. All dominant ARDRA groups except Variovorax species could be detected. On the other hand, the SSCP profiles revealed products which could not be assigned to the dominant cultured isolates, indicating that the bacterial diversity was greater than the diversity suggested by cultivation.     

Ensifer meliloti bv. lancerottense establishes nitrogen-fixing symbiosis with Lotus endemic to the Canary Islands and shows distinctive symbiotic genotypes and host range

Eleven strains were isolated from root nodules of Lotus endemic to the Canary Islands and they belonged to the genus Ensifer, a genus never previously described as a symbiont of Lotus. According to their 16S rRNA and atpD gene sequences, two isolates represented minority genotypes that could belong to previously undescribed Ensifer species, but most of the isolates were classified within the species Ensifer meliloti. These isolates nodulated Lotus lancerottensis, Lotus corniculatus and Lotus japonicus, whereas Lotus tenuis and Lotus uliginosus were more restrictive hosts. However, effective nitrogen fixation only occurred with the endemic L. lancerottensis. The E. meliloti strains did not nodulate Medicago sativa, Medicago laciniata Glycine max or Glycine soja, but induced non-fixing nodules on Phaseolus vulgaris roots. nodC and nifH symbiotic gene phylogenies showed that the E. meliloti symbionts of Lotus markedly diverged from strains of Mesorhizobium loti, the usual symbionts of Lotus, as well as from the three biovars (bv. meliloti, bv. medicaginis, and bv. mediterranense) so far described within E. meliloti. Indeed, the nodC and nifH genes from the E. meliloti isolates from Lotus represented unique symbiotic genotypes. According to their symbiotic gene sequences and host range, the Lotus symbionts would represent a new biovar of E. meliloti for which bv. lancerottense is proposed.     

Development of a cultivation-independent approach for the study of genetic diversity of Sinorhizobium meliloti populations

The development of a species-specific marker for the analysis of the genetic polymorphism of the nitrogen-fixing symbiotic bacterium Sinorhizobium meliloti directly from environmental DNA is reported. The marker is based on terminal-restriction fragment length polymorphism (T-RFLP) methodology targeting specifically the 16S-23S Ribosomal Intergenic Spacer of S. meliloti. Species-specificity and polymorphism of the marker were tested on DNA extracted from soil samples and from a collection of 130 S. meliloti bacterial isolates. These primers and the T-RFLP approach proved useful for the detection and analysis of polymorphism of S. meliloti populations.     

Phenotypical and genotypical characteristics of root-nodulating bacteria isolated from annual Medicago spp. in Soummam Valley (Algeria)

AIMS: In the framework of agro-pastoral system management using local annual medics coupled with their native root-nodulating bacteria to extend pasture zones, increase forage yields and improve ovine and bovine breeding in Algeria, we investigated diversity of rhizobia from annual Medicago spp. (Medicago arabica, Medicago polymorpha, Medicago minima and Medicago orbicularis). METHODS AND RESULTS: Ten nodulating-isolates were characterized by morphological, cultural, physiological and biochemical features, SDS-PAGE analysis and PCR-RFLP of 16S rDNA. The results show some degree of genetic diversity among the isolates; three can be affiliated to Sinorhizobium meliloti, one to Rhizobium galegae and six were separate. CONCLUSIONS: Local annual medics would have a high degree of specificity in their symbiotic interaction. Furthermore, our results support the presence of Rh. galegae in the Mediterranean region. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is a preliminary step towards selection of efficient symbiotic Medicago-rhizobia to develop inoculants for management of agro-pastoral systems using local annual medics in Algeria.     

西北地区天蓝苜蓿根瘤菌16S rDNA RFLP分析

利用RFLP和序列测定方法,对分离自西北地区的67株天蓝苜蓿根瘤菌16S rDNA进行了分析研究。结果表明:所有供试菌株分别归属于中华根瘤菌属(Sinorhizobium)、根瘤菌属(Rhizobium)和土壤杆菌属(Agrobac-terium)。以CCNWNX0042-2为代表的大部分天蓝苜蓿根瘤菌属于草木樨中华根瘤菌(Sinorhizobium meliloti),其余菌株在分群上表现出了较为明显的地域特征。     

Genetic diversity and dynamics of Sinorhizobium meliloti populations nodulating different alfalfa cultivars in Italian soils

We analyzed the genetic diversity of 531 Sinorhizobium meliloti strains isolated from nodules of Medicago sativa cultivars in two different Italian soils during 4 years of plant growth. The isolates were analyzed for DNA polymorphism with the random amplified polymorphic DNA method. The populations showed a high level of genetic polymorphism distributed throughout all the isolates, with 440 different haplotypes. Analysis of molecular variance allowed us to relate the genetic structure of the symbiotic population to various factors, including soil type, alfalfa cultivar, individual plants within a cultivar, and time. Some of these factors significantly affected the genetic structure of the population, and their relative influence changed with time. At the beginning of the experiment, the soil of origin and, even more, the cultivar significantly influenced the distribution of genetic variability of S. meliloti. After 3 years, the rhizobium population was altered; it showed a genetic structure based mainly on differences among plants, while the effects of soil and cultivar were not significant.     

Diversity of Sinorhizobium Meliloti and S. medicae Nodulating Medicago Truncatula According to Host and Soil Origins

Summary The model legume Medicago truncatula was used to trap natural populations of Sinorhizobium meliloti and Sinorhizobium medicae in Tunisian soils to explore their genetic diversity. About 155 Sinorhizobium isolates were trapped from a combination of three soils and four Medicago truncatula populations in order to analyse soil and plant population effects on nodulating Sinorhizobium diversity. The species assignment was done according to the restriction fragment length polymorphism analysis of polymerase chain reaction (PCR/RFLP) of 16S rRNA genes and their infraspecific genetic diversity was assessed with the repetitive extragenic palindromic-polymerase chain reaction (REP-PCR) technique. It appeared that the trapped bacteria were clustered according to the soil of origin, particularly Sinorhizobium medicae isolates. However, regarding the plant population effect, it appeared that no major clustering tendency could be suggested even if the Bulla Regia and Soliman Medicago truncatula populations appeared to nodulate together specific Sinorhizobium medicae genotypes.