Distinctive Mesorhizobium populations associated with Cicer arietinum L. in alkaline soils of Xinjiang, China

Background and aims

Rhizobia associated with chickpea in the main chickpea production zone of Xinjiang, China have never been investigated. Here, we present the first systematic investigation of these rhizobia’s genetic diversity and symbiotic interactions with their host plant.

Methods

Ninety-five isolates obtained from chickpea nodules in eight alkaline-saline (pH?8.24–8.45) sites in Xinjiang were characterized by nodulation test, symbiotic gene analysis, PCR-based restriction fragment length polymorphism (RFLP) of the 16S rRNA gene and 16S–23S rRNA intergenic spacer (IGS), BOX-PCR, phylogenies of 16S rRNA and housekeeping genes (atpD, recA and glnII), multilocus sequence analysis (MLSA) and DNA–DNA hybridization.

Results

All 95 isolates were identified within the genus of Mesorhizobium. Similarities less than 96.5% in MLSA and DNA–DNA hybridization values (<50%) between the new isolates and the defined Mesorhizobium species, and high similarities (>98%) of symbiotic genes (nodC and nifH) with those of the well studied chickpea microsymbioints Mesorhizobium ciceri and Mesorhizobium mediterraneum were found.

Conclusions

Chickpea rhizobia in alkaline-saline soils of Xinjiang, China, form a population distinct from the defined Mesorhizobium species. All these chickpea rhizobia in Xinjiang harbored symbiotic genes highly similar to the type strains of two well-studied chickpea rhizobia, M. ciceri and M. mediterraneum, evidencing the possible lateral transfer of symbiotic genes among these different rhizobial species. On the other hand, chickpea may strongly select rhizobia with a unique symbiotic gene background.     

Genetic Diversity and Host Range of Rhizobia Nodulating Lotus tenuis in Typical Soils of the Salado River Basin (Argentina)

A total of 103 root nodule isolates were used to estimate the diversity of bacteria nodulating Lotus tenuis in typical soils of the Salado River Basin. A high level of genetic diversity was revealed by repetitive extragenic palindromic PCR, and 77 isolates with unique genomic fingerprints were further differentiated into two clusters, clusters A and B, after 16S rRNA restriction fragment length polymorphism analysis. Cluster A strains appeared to be related to the genus Mesorhizobium, whereas cluster B was related to the genus Rhizobium. 16S rRNA sequence and phylogenetic analysis further supported the distribution of most of the symbiotic isolates in either Rhizobium or Mesorhizobium: the only exception was isolate BA135, whose 16S rRNA gene was closely related to the 16S rRNA gene of the genus Aminobacter. Most Mesorhizobium-like isolates were closely related to Mesorhizobium amorphae, Mesorhizobium mediterraneum, Mesorhizobium tianshanense, or the broad-host-range strain NZP2037, but surprisingly few isolates grouped with Mesorhizobium loti type strain NZP2213. Rhizobium-like strains were related to Rhizobium gallicum, Rhizobium etli, or Rhizobium tropici, for which Phaseolus vulgaris is a common host. However, no nodC or nifH genes could be amplified from the L. tenuis isolates, suggesting that they have rather divergent symbiosis genes. In contrast, nodC genes from the Mesorhizobium and Aminobacter strains were closely related to nodC genes from narrow-host-range M. loti strains. Likewise, nifH gene sequences were very highly conserved among the Argentinian isolates and reference Lotus rhizobia. The high levels of conservation of the nodC and nifH genes suggest that there was a common origin of the symbiosis genes in narrow-host-range Lotus symbionts, supporting the hypothesis that both intrageneric horizontal gene transfer and intergeneric horizontal gene transfer are important mechanisms for the spread of symbiotic capacity in the Salado River Basin.     

Phenotypic characterization of Astragalus glycyphyllos symbionts and their phylogeny based on the 16S rDNA sequences and RFLP of 16S rRNA gene

In this study, the nitrogen fixing Astragalus glycyphyllos symbionts were characterized by phenotypic properties, restriction fragment length polymorphism (RFLP), and sequences of 16S rDNA. The generation time of A. glycyphyllos rhizobia in yeast extract mannitol medium was in the range 4–6 h. The studied isolates exhibited a low resistance to antibiotics, a moderate tolerance to NaCl, assimilated di- and trisaccharides, and produced acid in medium containing mannitol as a sole carbon source. In the cluster analysis, based on 86 phenotypic properties of A. glycyphyllos symbionts and the reference rhizobia, examined isolates and the genus Mesorhizobium strains were placed on a single branch, clearly distinct from other lineages of rhizobial genera. By the comparative analysis of 16S rRNA gene sequences and 16S rDNA–RFLP, A. glycyphyllos nodulators were also identified as the members of the genus Mesorhizobium. On the 16S rDNA sequence phylogram, the representatives of A. glycyphyllos nodule isolates formed a robust, monophyletic cluster together with the Mesorhizobium species at 16S rDNA sequence similarity of these bacteria between 95 and 99 %. Similarly, the cluster analysis of the combined RFLP–16S rDNA patterns, obtained with seven restriction endonucleases, showed that A. glycyphyllos rhizobia are closely related to the genus Mesorhizobium bacteria. The taxonomic approaches used in this paper allowed us to classify the studied bacteria into the genus Mesorhizobium.     

<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> in Poland and Japan is nodulated by <Emphasis Type="Italic">Mesorhizobium amorphae</Emphasis> strains

Robinia pseudoacacia microsymbionts from plants growing in Poland and Japan were evaluated for phylogeny and taxonomic position by genomic approach. Based on the comparative analyses of atpD (368 bp) and dnaK (573 bp) gene sequences as well as 16S rDNA restriction analysis (RFLP-16S rDNA), R. pseudoacacia microsymbionts were identified as Mesorhizobium strains. In dnaK and atpD gene phylograms R. pseudoacacia nodulators formed robust, monophyletic clusters with Mesorhizobium species with the nucleotide sequence similarity of 91–98% and 90–98%, respectively. The classification of R. pseudoacacia rhizobia to the genus Mesorhizobium was also supported by amplified 16S rDNA restriction analysis. The studied bacteria formed common clusters with Mesorhizobium species, and their DNA patterns were identical or nearly identical to Mesorhizobium genus strains. When DNA-DNA hybridization was performed, the total DNA of the representative R. pseudoacacia rhizobia exhibited 51–75% relatedness to DNA of Mesorhizobium amorphae ICMP15022 strain and below 41% to DNA of other Mesorhizobium species. These results showed that R. pseudoacacia and M. amorphae belong to the same genomospecies. The G+C content of DNA of R. pseudoacacia two microsymbionts was 59.7 and 60.6 mol% compared to 61–64 mol% across M. amorphae strains.     

Numerical Analysis of Astragalus cicer Microsymbionts

Thirty-seven rhizobium strains, isolated from root nodules of Astragalus cicer (L.) (cicer milkvetch) deriving from different geographic regions, were compared with the representative strains of the known rhizobial species and genera by numerical analysis of phenotypic characteristics. Our results indicated that Astragalus cicer rhizobia were related to the bacteria of Mesorhizobium species and formed two major phena. One phenon, localized on Mesorhizobium loti branch, contained strains from Poland. Another cluster, placed in the vicinity of M. tianshanense, M. mediterraneum, M. ciceri, and M. huakuii, comprised cicer milkvetch nodule isolates from Canada, Ukraine, and one strain from Poland. The relationship of Astragalus cicer microsymbionts to bacteria of the Mesorhizobium species was also supported by phage typing. Received: 10 February 2000 / Accepted: 8 March 2000     

Genetic diversity and biogeography of rhizobia associated with Caragana species in three ecological regions of China

Twenty-two genospecies belonging mainly to Mesorhizobium, and occasionally to Rhizobium and Bradyrhizobium, were defined among the 174 rhizobia strains isolated from Caragana species. Highly similar nodC genes were found in the sole Bradyrhizobium strain and among all the detected Mesorhizobium strains. A clear correlation between rhizobial genospecies and the eco-regions where they were isolated was found using homogeneity analysis. All these results demonstrated that Caragana species had stringently selected the rhizobia symbiotic genotype, but not the genomic background; lateral transfer of symbiotic genes from Mesorhizobium to Bradyrhizobium and among the Mesorhizobium species has happened in the Caragana rhizobia; and biogeography of Caragana rhizobia exists. Furthermore, a combined cluster analysis, based upon the patterns obtained from amplified 16S rRNA gene and 16S–23S intergenic spacer restriction analyses, BOX PCR and SDS-PAGE of proteins, was reported to be an efficient method to define the genospecies.     

Genotypic alteration and competitive nodulation of Mesorhizobium muleiense against exotic chickpea rhizobia in alkaline soils

Mesorhizobium muleiense, Mesorhizobium mediterraneum and Mesorhizobium ciceri are chickpea (Cicer arietinum L.) rhizobia that share a high similarity of the symbiotic genes nodC and nifH, but they have different geographic distributions. M. muleiense has been isolated and found only in alkaline soils of Xinjiang, China, whereas the other two strains have been found in the Mediterranean and India. To investigate the species stability of M. muleiense during natural evolution and its capability of competitive nodulation against the other two exotic species, re-sampling of nodules in the field and competition experiments between the three species were conducted. The results showed that the predominant microsymbiont associated with chickpea grown in Xinjiang was still M. muleiense, but the predominant genotypes of M. muleiense had changed significantly during the four years since a previous survey. The data also showed that M. mediterraneum and M. ciceri were more competitive than the residential strain of M. muleiense CCBAU 83963^T in sterilized vermiculite or soils from Xinjiang. However, in non-sterilized soils, M. muleiense was the predominant nodule occupier. These results indicated that natural or adapting evolution of M. muleiense was occurring in fields subjected to changing environmental factors. In addition, the biogeography and symbiotic associations of rhizobia with their host legumes were also influenced by biological factors in the soil, such as indigenous rhizobia and other organisms.     

Phenotypic and molecular characterization of indigenous rhizobia nodulating chickpea in India

In a combined approach of phenotypic and genotypic characterization, 28 indigenous rhizobial isolates obtained from different chickpea growing regions in peninsular and northern India were analyzed for diversity. The field isolates were compared to two reference strains TAL620 and UPM-Ca142 representing M. ciceri and M. mediterraneum respectively. Phenotypic markers such as resistance to antibiotics, tolerance to salinity, temperature, pH, phosphate solubilization ability, growth rate and also symbiotic efficiency showed considerable diversity among rhizobial isolates. Their phenotypic patterns showed adaptations of rhizobial isolates to abiotic stresses such as heat and salinity. Two salt tolerant strains (1.5% NaCl by T1 and T4) with relatively high symbiotic efficiency and two P-solubilising strains (66.7 and 71 microg/ml by T2 and T5) were identified as potential bioinoculants. Molecular profiling by 16S ribosomal DNA Restriction Fragment Length Polymorphism (RFLP) revealed three clusters at 67% similarity level. Further, the isolates were differentiated at intraspecific level by 16S rRNA gene phylogeny. Results assigned all the chickpea rhizobial field isolates to belong to three different species of Mesorhizobium genus. 46% of the isolates grouped with Mesorhizobium loti and the rest were identified as M. ciceri and M. mediterraneum, the two species which have been formerly described as specific chickpea symbionts. This is the first report on characterization of chickpea nodulating rhizobia covering soils of both northern and peninsular India. The collection of isolates, diverse in terms of species and symbiotic effectiveness holds a vast pool of genetic material which can be effectively used to yield superior inoculant strains.     

Multilocus sequence analysis reveals multiple symbiovars within Mesorhizobium species

The genus Mesorhizobium includes species nodulating several legumes, such as chickpea, which has a high agronomic importance. Chickpea rhizobia were originally described as either Mesorhizobium ciceri or M. mediterraneum. However, rhizobia able to nodulate chickpea have been shown to belong to several different species within the genus Mesorhizobium. The present study used a multilocus sequence analysis approach to infer a high resolution phylogeny of the genus Mesorhizobium and to confirm the existence of a new chickpea nodulating genospecies. The phylogenetic structure of the Mesorhizobium clade was evaluated by sequence analysis of the 16S rRNA gene, ITS region and the five core genes atpD, dnaJ, glnA, gyrB, and recA. Phylogenies obtained with the different genes are in overall good agreement and a well-supported, almost fully resolved, phylogenetic tree was obtained using the combined data. Our phylogenetic analyses of core genes sequences and their comparison with the symbiosis gene nodC, corroborate the existence of one new chickpea Mesorhizobium genospecies and one new symbiovar, M. opportunistum sv. ciceri. Furthermore, our results show that symbiovar ciceri spreads over six species of mesorhizobia. To our knowledge this study shows the most complete Mesorhizobium multilocus phylogeny to date and contributes to the understanding of how a symbiovar may be present in different species.     

Diversity of rhizobia nodulating wild shrubs of Sicily and some neighbouring islands

Legume shrubs have great potential for rehabilitation of semi-arid degraded soils in Mediterranean ecosystems as they establish mutualistic symbiosis with N-fixing rhizobia. Eighty-eight symbiotic rhizobia were isolated from seven wild legume shrubs native of Sicily (Southern Italy) and grouped in operational taxonomic units (OTU) by analysis of the ribosomal internal transcribed spacer (ITS) polymorphism. Partial sequencing of 16S rRNA gene of representative isolates of each OTU revealed that most Genisteae symbionts are related to Bradyrhizobium canariense, B. japonicum and B. elkanii. Teline monspessulana was the only Genistea nodulated by Mesorhizobium strains, and Anagyris foetida (Thermopsideae) was promiscuosly nodulated by Rhizobium, Mesorhizobium, Agrobacterium and Bradyrhizobium strains. Analysis of the nodulation gene nodA assigned most Mediterranean Genisteae bradyrhizobia to clade II but also to clades IV, I and III, which included, so far, sequences of (sub)tropical and Australian isolates. The high diversity and low host specificity observed in most wild legumes isolates suggest that preferential associations may establish in the field depending on differences in the benefits conferred to the host and on competition ability. Once identified, these beneficial symbiosis can be exploited for rehabilitation of arid, low productive and human-impacted soils of the Mediterranean countries.     

Diversity and geographical distribution of rhizobia associated with <Emphasis Type="Italic">Lespedeza</Emphasis> spp. in temperate and subtropical regions of China

Eighty-eight root-nodule isolates from Lespedeza spp. grown in temperate and subtropical regions of China were characterized by a polyphasic approach. Nine clusters were defined in numerical taxonomy and SDS-PAGE analysis of whole cell proteins. Based upon further characterizations of amplified 16S rDNA restriction analysis (ARDRA), PCR-based restriction fragment length polymorphism of ribosomal IGS, 16S rDNA sequence analysis and DNA-DNA hybridization, these isolates were identified as Bradyrhizobium japonicum, B. elkanii, B. yuanmingense, Mesorhizobium amorphae, M. huakuii, Sinorhizobium meliloti and three genomic species related to B. yuanmingense, Rhizobium gallicum and R. tropici. The Bradyrhizobium species and R. tropici-related rhizobia were mainly isolated from the subtropical region and the species of Mesorhizobium, S. meliloti and R. gallicum-related species were all isolated from the temperate region. Phylogenetic analyses of nifH and nodC indicated that the symbiotic genes of distinct rhizobial species associated with Lespedeza spp. might have different origins and there was no evidence for lateral gene transfer of symbiotic genes. The results obtained in the present study and in a previous report demonstrated that Lespedeza spp. are nodulated by rhizobia with diverse genomic backgrounds and these Lespedeza-nodulating rhizobia were not specific to the host species, but specific to their geographic origins. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. GenBank sequence accession numbers: The GenBank accession numbers were EF61095 through EF061114 and EF051240 for acquired 16S rDNA sequences; EF153395 through EF153402 for nifH sequences; and EF153403 through EF153410 for nodC sequences.     

Mesorhizobium ciceri LMS-1 expressing an exogenous 1-aminocyclopropane-1-carboxylate (ACC) deaminase increases its nodulation abilities and chickpea plant resistance to soil constraints

Aims: Our goal was to understand the symbiotic behaviour of a Mesorhizobium strain expressing an exogenous 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase, which was used as an inoculant of chickpea (Cicer arietinum) plants growing in soil. Methods and Results: Mesorhizobium ciceri LMS‐1 (pRKACC) was tested for its plant growth promotion abilities on two chickpea cultivars (ELMO and CHK3226) growing in nonsterilized soil that displayed biotic and abiotic constraints to plant growth. When compared to its wild‐type form, the M. ciceri LMS‐1 (pRKACC) strain showed an increased nodulation performance of c. 125 and 180% and increased nodule weight of c. 45 and 147% in chickpea cultivars ELMO and CHK3226, respectively. Mesorhizobium ciceri LMS‐1 (pRKACC) was also able to augment the total biomass of both chickpea plant cultivars by c. 45% and to reduce chickpea root rot disease susceptibility. Conclusions: The results obtained indicate that the production of ACC deaminase under free living conditions by Mesorhizobium strains increases the nodulation, plant growth abilities and biocontrol potential of these strains. Significance and Impact of the Study: This is the first study regarding the use of a transformed rhizobial strain expressing an exogenous ACC deaminase in different plant cultivars growing in soil. Hence, obtaining Mesorhizobium strains with high ACC deaminase activity is a matter of extreme importance for the development of inoculants for field applications.     

Potential role of rhizobia to enhance chickpea-growth and yield in low fertility-soils of Tunisia

Plant growth-promoting rhizobacteria are bacteria that improve plant growth and reduce plant pathogen damages. In this study, 100 nodule bacteria were isolated from chickpea, screened for their plant growth-promoting (PGP) traits and then characterised by PCR-RFLP of 16 S rDNA. Results showed that most of the slow-growing isolates fixed nitrogen but those exhibiting fast-growth did not. Fourteen isolates solubilized inorganic phosphorus, 16 strains produced siderophores, and 17 strains produced indole acetic acid. Co-culture experiments identified three strains having an inhibitory effect against Fusarium oxysporum, the primary pathogenic fungus for chickpea in Tunisia. Rhizobia with PGP traits were assigned to Mesorhizobium ciceri, Mesorhizobium mediterraneum, Sinorhizobium meliloti and Agrobacterium tumefaciens. We noted that PGP activities were differentially distributed between M. ciceri and M. mediterraneum. The region of Mateur in northern Tunisia, with clay–silty soil, was the origin of 53% of PGP isolates. Interestingly, we found that S. meliloti and A. tumefaciens strains did not behave as parasitic nodule-bacteria but as PGP rhizobacteria useful for chickpea nutrition and health. In fact, S. meliloti strains could solubilize phosphorus, produce siderophore and auxin. The A. tumefaciens strains could perform the previous PGP traits and inhibit pathogen growth also. Finally, one candidate strain of M. ciceri (LL10)—selected for its highest symbiotic nitrogen fixation and phosphorus solubilization—was used for field experiment. The LL10 inoculation increased grain yield more than three-fold. These finding showed the potential role of rhizobia to be used as biofertilizers and biopesticides, representing low-cost and environment-friendly inputs for sustainable agriculture.

     

The high diversity of <Emphasis Type="Italic">Lotus corniculatus</Emphasis> endosymbionts in soils of northwest Spain

The diversity of rhizobia that establish symbiosis with Lotus corniculatus has scarcely been studied. Several species of Mesorhizobium are endosymbionts of this legume, including Mesorhizobium loti, the type species of this genus. We analysed the genetic diversity of strains nodulating Lotus corniculatus in Northwest Spain and ten different RAPD patterns were identified among 22 isolates. The phylogenetic analysis of the 16S rRNA gene showed that the isolated strains belong to four divergent phylogenetic groups within the genus Mesorhizobium. These phylogenetic groups are widely distributed worldwide and the strains nodulate L. corniculatus in several countries of Europe, America and Asia. Three of the groups include the currently described Mesorhizobium species M. loti, M. erdmanii and M. jarvisii which are L. corniculatus endosymbionts. An analysis of the recA and atpD genes showed that our strains belong to several clusters, one of them very closely related to M. jarvisii and the remanining ones phylogenetically divergent from all currently described Mesorhizobium species. Some of these clusters include L. corniculatus nodulating strains isolated in Europe, America and Asia, although the recA and atpD genes have been sequenced in only a few L. corniculatus endosymbionts. The results of this study revealed great phylogenetic diversity of strains nodulating L. corniculatus, allowing us to predict that even more diversity will be discovered as further ecosystems are investigated.     

Diversity of rhizobia and interactions among the host legumes and rhizobial genotypes in an agricultural-forestry ecosystem

To investigate the diversity of rhizobia and interactions among the host legumes and rhizobial genotypes in the same habitat, a total of 97 rhizobial strains isolated from nine legume species grown in an agricultural-forestry ecosystem were identified into seven genomic species and 12 symbiotic genotypes within the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium based upon analyses of genomic DNA regions and symbiotic genes. The results evidenced that the symbiotic genotypes of rhizobia were consistent with their hosts of origin; revealed that vertical transfer was the main mechanism in rhizobia to maintain the symbiotic genes but lateral transfer of symbiotic genes might have happened between the closely related rhizobial species; suggested the existence of co-distribution and co-evolution among the legume hosts and compatible rhizobia. All of these data demonstrated that the biogeography of rhizobia was a result of interactions among the host legumes, bacterial genomic backgrounds and environments.     

Moderately acidophilic mesorhizobia isolated from chickpea

AIMS: Our aim was to evaluate the effect of acid and alkaline pH on chickpea rhizobia, and on chickpea-rhizobia symbiosis. METHODS AND RESULTS: Forty-seven rhizobia isolates obtained from 12 Portuguese soils were grown at pH 5, 7 and 9. Among these, 26 grew more at pH 5 than at 7, suggesting the existence of acidophiles. All isolates were identified as mesorhizobia by 16S rDNA partial sequence analysis. Molecular phylogeny of isolates based on partial 16S rDNA sequences suggests that pH tolerance might be species related. Further studies were conducted with six isolates, which were able to grow at acid pH. Isolates PT-35 and 64b grow optimally at pH 6-5-7, with a minimal pH range from 5 to 3, and may thus be considered as moderately acidophilic. Both isolates belong to a previously identified putative new Mesorhizobium species, based on 16S rDNA sequence. CONCLUSIONS: Two moderately acidophilic mesorhizobia isolated from chickpea were identified (PT-35 and 64b). A positive correlation was found between the symbiotic effectiveness at low pH and the acid tolerance of rhizobia isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on moderately acidophilic mesorhizobia, and is an important contribution for the development of highly effective inoculants for chickpea in acid soils.     

Genetic diversity and distribution of Bradyrhizobium and Azorhizobium strains associated with the herb legume Zornia glochidiata sampled from across Senegal

Herb legumes have great potential for rehabilitation of semi-arid degraded soils in Sahelian ecosystems as they establish mutualistic symbiosis with N₂-fixing rhizobia. A phylogenetic analysis was performed for 78 root nodule bacteria associated with the common Sahelian herb legume Zornia glochidiata Reichb ex DC in Senegal. Based on ITS (rDNA16S-23S) and recA sequences, these strains were shown to belong to the two genera Bradyrhizobium and Azorhizobium. Strains of this latter, although frequent, formed small and ineffective nodules and suggested a parasitism rather than a symbiotic association. A potential negative effect of Azorhizobium on Zornia growth was tested for when inoculated alone or in association with a Bradyrhizobium strain. Bradyrhizobium isolates were distributed in four groups. Groups A and B were two sister clades in a larger monophyletic group also including Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense, and Bradyrhizobium japonicum. Strains of cluster D fell in a sister clade of the photosynthetic Bradyrhizobium sp. group, including ORS278, whereas group C appeared to be divergent from all known Bradyrhizobium clusters. Amplified fragment length polymorphism (AFLP) clustering was congruent with ITS and recA phylogenies, but displayed much more variability. However, within the main Bradyrhizobium clades, no obvious relationship could be detected between clustering and geographical origin of the strains. Each sub-cluster included strains sampled from different locations. Conversely, Azorhizobium strains showed a tendency in the phylogeny to group together according to the site of sampling. The predominance of ineffective Azorhizobium strains in the nodules of Zornia roots, the large Bradyrhizobium genetic diversity and the geographical genetic diversity pattern are explored.     

Phylogeny of Symbiotic Genes and the Symbiotic Properties of Rhizobia Specific to Astragalus glycyphyllos L.

The phylogeny of symbiotic genes of Astragalus glycyphyllos L. (liquorice milkvetch) nodule isolates was studied by comparative sequence analysis of nodA, nodC, nodH and nifH loci. In all these genes phylograms, liquorice milkvetch rhizobia (closely related to bacteria of three species, i.e. Mesorhizobium amorphae, Mesorhizobium septentrionale and Mesorhizobium ciceri) formed one clearly separate cluster suggesting the horizontal transfer of symbiotic genes from a single ancestor to the bacteria being studied. The high sequence similarity of the symbiotic genes of A. glycyphyllos rhizobia (99–100% in the case of nodAC and nifH genes, and 98–99% in the case of nodH one) points to the relatively recent (in evolutionary scale) lateral transfer of these genes. In the nodACH and nifH phylograms, A. glycyphyllos nodule isolates were grouped together with the genus Mesorhizobium species in one monophyletic clade, close to M. ciceri, Mesorhizobium opportunistum and Mesorhizobium australicum symbiovar biserrulae bacteria, which correlates with the close relationship of these rhizobia host plants. Plant tests revealed the narrow host range of A. glycyphyllos rhizobia. They formed effective symbiotic interactions with their native host (A. glycyphyllos) and Amorpha fruticosa but not with 11 other fabacean species. The nodules induced on A. glycyphyllos roots were indeterminate with apical, persistent meristem, an age gradient of nodule tissues and cortical vascular bundles. To reflect the symbiosis-adaptive phenotype of rhizobia, specific for A. glycyphyllos, we propose for these bacteria the new symbiovar “glycyphyllae”, based on nodA and nodC genes sequences.