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.     

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.     

Diverse symbiovars nodulating cowpea (Vigna unguiculata L. Walp.) in highly adaptable agro-ecological zones in Mozambique

The presence of effective microsymbionts in the soil and their compatibility with the host plant are the key determinants to the N₂ fixation process. In Sub-Saharan Africa, nitrogen fixation in locally adapted cowpea and the distribution of their symbiovars are not well understood. The Aim of the study was to assess the distribution and symbiotic phylogenetic position of cowpea microsymbionts. Root nodules were sampled from various cowpea genotypes planted in Agro-Ecological Zone 7 and 8 (AEZ 7 and AEZ 8). Root-nodule bacteria were isolated and their molecular characterization was conducted. Physicochemical properties of soil were recorded. Enterobacterial Repetitive Intergenic Consensus (ERIC) distribution patterns in rhizobial genomes resulted in genetically diverse rhizobial population in Northern Mozambique. Principal component analysis showed that location-specific soil environment determined the presence of particular microsymbionts. Based on 16S rRNA and symbiotic gene analysis many diverse symbiovars were found in Mozambican soils. With few discrepancies, the results further confirmed the coevolution of the nifH, nodD, nodC and nodY/K genes, which was indicative of natural events such as vertical/horizontal gene transfer. The results suggested that ecological and phylogenetic studies of the microsymbionts are necessary to better reflect symbiovar identification and the ecological adaptation of the cowpea-nodulating rhizobial community.     

Genetic diversity and symbiotic evolution of rhizobia from root nodules of Coronilla varia

Ninety symbiotic rhizobial isolates from root nodules of Coronilla varia growing in the Shaanxi province of China were characterized. Combined with the results of RFLP patterns, six genotypes were defined among the rhizobial strains and they were divided into three genomic genera. These included Mesorhizobium sp., M. alhagi, M. amorphae, M. metallidurans/M. gobiense as the dominant group (86.7%), and Rhizobium yanglingense and Agrobacterium tumefaciens as the minor groups, according to analysis of the corresponding 16S rRNA, nodC and nifH genes. Five nodC types, which mainly grouped into the Mesorhizobium genus, were obtained from all the isolates examined, implying that nodC genes probably occurred from the native habitat through lateral transfer and long-term adaptation, finally evolving toward M. alhagi. Four different nifH types, displaying obvious differences compared to those of 16S rRNA and nodC, implied that possible lateral transfer of the symbiotic genes occurred between different genera. The association between soil components and the genetic diversity of the rhizobial population demonstrated that combined genotypes were positively correlated with the pH of soil samples.     

Microsymbionts of Phaseolus vulgaris in acid and alkaline soils of Mexico

In order to investigate bean-nodulating rhizobia in different types of soil, 41 nodule isolates from acid and alkaline soils in Mexico were characterized. Based upon the phylogenetic studies of 16S rRNA, atpD, glnII, recA, rpoB, gyrB, nifH and nodC genes, the isolates originating from acid soils were identified as the phaseoli symbiovar of the Rhizobium leguminosarum-like group and Rhizobium grahamii, whereas the isolates from alkaline soils were defined as Ensifer americanum sv. mediterranense and Rhizobium radiobacter. The isolates of “R. leguminosarum” and E. americanum harbored nodC and nifH genes, but the symbiotic genes were not detected in the four isolates of the other two species. It was the first time that “R. leguminosarum” and E. americanum have been reported as bean-nodulating bacteria in Mexico. The high similarity of symbiotic genes in the Rhizobium and Ensifer populations showed that these genes had the same origin and have diversified recently in different rhizobial species. Phenotypic characterization revealed that the “R. leguminosarum” population was more adapted to the acid and low salinity conditions, while the E. americanum population preferred alkaline conditions. The findings of this study have improved the knowledge of the diversity, geographic distribution and evolution of bean-nodulating rhizobia in Mexico.     

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.     

Adaptability to local conditions and phylogenetic differentiation of microsymbionts of TGx soybean genotypes in the semi-arid environments of Ghana and South Africa

The study of the nitrogen fixation and phylogenetic diversity of nodule microsymbionts of grain legumes in many parts of the globe is often carried out in order to identify legume-rhizobia combinations for agricultural sustainability. Several reports have therefore found that rhizobial species diversity is shaped by edapho-climatic conditions that characterize different geographic locations, suggesting that rhizobial communities often possess traits that aid their adaptation to their habitat. In this study, the soybean-nodulating rhizobia from semi-arid savannahs of Ghana and South Africa were evaluated. The authenticated rhizobial isolates were highly diverse based on their colony characteristics, as well as their BOX-PCR profiles and gene sequences. In the 16S rRNA phylogeny, the isolates were placed in the different clades Bradyrhizobium iriomotense and Bradyrhizobium jicamae together with two superclades Bradyrhizobium japonicum and Bradyrhizobium elkanii. The multilocus (atpD, glnII, gyrB, recA) phylogenetic analyses indicated the dominance of Bradyrhizobium diazoefficiens and putative new Bradyrhizobium species in the semi-arid Ghanaian region. The phylogenetic analyses based on the symbiotic genes (nifH and nodC) clustered the test isolates into different symbiovars (sv. glycinearum, sv. retame and sv. sojae). Principal component analysis (PCA) showed that soil factors played a significant role in favoring the occurrence of soybean-nodulating microsymbionts in the tested local conditions. The results suggested that isolates had marked local adaptation to the prevailing conditions in semi-arid regions but further studies are needed to confirm new Bradyrhizobium species.     

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.     

Legume growth-promoting rhizobia: An overview on the Mesorhizobium genus

The need for sustainable agricultural practices is revitalizing the interest in biological nitrogen fixation and rhizobia-legumes symbioses, particularly those involving economically important legume crops in terms of food and forage. The genus Mesorhizobium includes species with high geographical dispersion and able to nodulate a wide variety of legumes, including important crop species, like chickpea or biserrula. Some cases of legume-mesorhizobia inoculant introduction represent exceptional opportunities to study the rhizobia genomes evolution and the evolutionary relationships among species. Complete genome sequences revealed that mesorhizobia typically harbour chromosomal symbiosis islands. The phylogenies of symbiosis genes, such as nodC, are not congruent with the phylogenies based on core genes, reflecting rhizobial host range, rather than species affiliation. This agrees with studies showing that Mesorhizobium species are able to exchange symbiosis genes through lateral transfer of chromosomal symbiosis islands, thus acquiring the ability to nodulate new hosts. Phylogenetic analyses of the Mesorhizobium genus based on core and accessory genes reveal complex evolutionary relationships and a high genomic plasticity, rendering the Mesorhizobium genus as a good model to investigate rhizobia genome evolution and adaptation to different host plants. Further investigation of symbiosis genes as well as stress response genes will certainly contribute to understand mesorhizobia-legume symbiosis and to develop more effective mesorhizobia inoculants.     

Symbiotic efficiency and phylogeny of the rhizobia isolated from Leucaena leucocephala in arid–hot river valley area in Panxi, Sichuan, China

In search of effective nitrogen-fixing strains for inoculating Leucaena leucocephala, we assessed the symbiotic efficiency of 41 rhizobial isolates from root nodules of L. leucocephala growing in the arid–hot river valley area in Panxi, China. The genetic diversity of the isolates was studied by analyzing the housekeeping genes 16S rRNA and recA, and the symbiotic genes nifH and nodC. In the nodulation and symbiotic efficiency assay, only 11 of the 41 isolates promoted the growth of L. leucocephala while the majority of the isolates were ineffective in symbiotic nitrogen fixation. Furthermore, one fourth of the isolates had a growth slowing effect on the host. According to the 16S rRNA and recA gene analyses, most of the isolates were Ensifer spp. The remaining isolates were assigned to Rhizobium, Mesorhizobium and Bradyrhizobium. The sequence analyses indicated that the L. leucocephala rhizobia had undergone gene recombination. In contrast to the promiscuity observed as a wide species distribution of the isolates, the results implied that L. leucocephala is preferentially nodulated by strains that share common symbiosis genes. The symbiotic efficiency was not connected to chromosomal background of the symbionts and isolates carrying a similar nifH or nodC showed totally different nitrogen fixation efficiency.     

Genotypic diversity in native rhizobial population nodulating Vicia faba in arid and semi-arid regions of Haryana state (India)

A Rhizobium–legume interaction stands out from other plant–microbe interactions as one in which a true developmental mutualism occurs. To study the genotypic diversity in native population of rhizobia-nodulating Vicia faba plants, we retrieved 64 rhizobial isolates from root nodules of faba bean grown in pots holding soils collected from arid and semi-arid regions of the state of Haryana, India. The amplification of nodC in all the isolates authenticated these as rhizobia. The nitrogen-fixing potential of the isolates was tested by the amplification of the nifH gene. Only 50 isolates out of 64 showed nifH gene amplification. The characterization of the isolates by amplified 16S rDNA restriction analysis (ARDRA) categorized these into 36 16S rDNA genotypes using a combination of MspI and HaeIII restriction enzymes. Majority of the isolates resolved into separate genotypes, indicating a wide diversity among them, which seemed to arise from their geographical origin and soil characteristics. These findings may be immensely useful in agriculture towards developing rhizobial inoculants specific for faba beans under arid and semi-arid conditions.     

Core and symbiotic genes reveal nine Mesorhizobium genospecies and three symbiotic lineages among the rhizobia nodulating Cicer canariense in its natural habitat (La Palma,Canary Islands)

Cicer canariense is a threatened perennial wild chickpea endemic to the Canary Islands. In this study, rhizobia that nodulate this species in its natural habitats on La Palma (Canary Islands) were characterised. The genetic diversity and phylogeny were estimated by RAPD profiles, 16S-RFLP analysis and sequencing of the rrs, recA, glnII and nodC genes. 16S-RFLP grouped the isolates within the Mesorhizobium genus and distinguished nine different ribotypes. Four branches included minority ribotypes (3–5 isolates), whereas another five contained the predominant ribotypes that clustered with reference strains of M. tianshanense/M. gobiense/M. metallidurans, M. caraganae, M. opportunistum, M. ciceri and M. tamadayense. The sequences confirmed the RFLP groupings but resolved additional internal divergence within the M. caraganae group and outlined several potential novel species. The RAPD profiles showed a high diversity at the infraspecific level, except in the M. ciceri group. The nodC phylogeny resolved three symbiotic lineages. A small group of isolates had sequences identical to those of symbiovar ciceri and were only detected in M. ciceri isolates. Another group of sequences represented a novel symbiotic lineage that was associated with two particular chromosomal backgrounds. However, nodC sequences closely related to symbiovar loti predominated in most isolates, and they were detected in several chromosomal backgrounds corresponding to up to nine Mesorhizobium lineages. The results indicated that C. canariense is a promiscuous legume that can be nodulated by several rhizobial species and symbiotypes, which means it will be important to determine the combination of core and symbiotic genes that produce the most effective symbiosis.     

Diversity of endemic rhizobia on Christmas Island: Implications for agriculture following phosphate mining

Given that phosphate supplies may diminish and become uneconomic to mine after 2020, there is a compelling need to develop alternative industries to support the population on Christmas Island. Former mine sites could be turned into productive agricultural land, however, large-scale commercial agriculture has never been attempted, and, given the uniqueness of the island, the diversity of rhizobia prior to introducing legumes needed evaluation. Therefore, 84 rhizobia isolates were obtained from nine different hosts, both crop and introduced legumes, located at seven sites across the island. Based on 16S rRNA and recA gene sequence analysis, the isolates grouped into 13 clades clustering within the genus Bradyrhizobium, Ensifer, Cupriavidus and Rhizobium. According to the sequences of their symbiosis genes nodC and nifH, the isolates were classified into 12 and 11 clades, respectively, and clustered closest to tropical or crop legume isolates. Moreover, the symbiosis gene phylogeny and Multi Locus Sequence Analysis gene phylogeny suggested vertical transmission in the Alpha-rhizobia but horizontal transmission within the Beta-rhizobia. Furthermore, this study provides evidence of a large diversity of endemic rhizobia associated with both crop and introduced legumes, and highlights the necessity of inoculation for common bean, chickpea and soybean on the Island.     

Genetic diversity of soybean-nodulating rhizobia in Nepal in relation to climate and soil properties

Backgroud and aims

This study was conducted to reveal the genetic diversity of soybean-nodulating rhizobia in Nepal in relation to climate and soil properties.

Method

A total of 102 bradyrhizobial strains were isolated from the root nodules of soybeans cultivated in 12 locations in Nepal varying in climate and soil properties, and their genetic diversity was examined based on 16S rDNA, ITS regions of 16S–23S rDNA, nodC and nifH. In vitro growth properties of some representative strains were examined to elucidate their characteristic distribution in Nepal.

Results

Four species of the genus Bradyrhizobium were isolated, and B. japonicum dominated at temperate locations, while in subtropical locations, B. elkanii, B. yuanmingense, and B. liaoningense dominated at acidic, moderately acidic, and slightly alkaline soils, respectively. The relative nodule occupancies could not be fully explained by their in vitro growth properties. Similar nodC and nifH genes among the strains suggested co-evolution of these genes also in Nepal, probably through horizontal gene transfer.

Conclusions

The influence of climate and soil pH on diversity at the sub-species level was revealed. It is concluded that the highly diverse climate and soils in Nepal might be conducive for the existence of diverse soybean rhizobial strains.     

Retama species growing in different ecological–climatic areas of northeastern Algeria have a narrow range of rhizobia that form a novel phylogenetic clade within the Bradyrhizobium genus

Sixty-seven isolates were isolated from nodules collected on roots of Mediterranean shrubby legumes Retama raetam and Retama sphaerocarpa growing in seven ecological–climatic areas of northeastern Algeria. Genetic diversity of the Retama isolates was analyzed based on genotyping by restriction fragment length polymorphism of PCR-amplified fragments of the 16S rRNA gene, the intergenic spacer (IGS) region between the 16S and 23S rRNA genes (IGS), and the symbiotic genes nifH and nodC. Eleven haplotypes assigned to the Bradyrhizobium genus were identified. Significant biogeographical differentiation of the rhizobial populations was found, but one haplotype was predominant and conserved across the sites. All isolates were able to cross-nodulate the two Retama species. Accordingly, no significant genetic differentiation of the rhizobial populations was found in relation to the host species of origin. Sequence analysis of the 16S rRNA gene grouped the isolates with Bradyrhizobium elkanii, but sequence analyses of IGS, the housekeeping genes (dnaK, glnII, recA), nifH, and nodC yielded convergent results showing that the Retama nodule isolates from the northeast of Algeria formed a single evolutionary lineage, which was well differentiated from the currently named species or well-delineated unnamed genospecies of bradyrhizobia. Therefore, this study showed that the Retama species native to northeastern Algeria were associated with a specific clade of bradyrhizobia. The Retama isolates formed three sub-groups based on IGS and housekeeping gene phylogenies, which might form three sister species within a novel bradyrhizobial clade.     

Genetic Diversity and Evolution of Bradyrhizobium Populations Nodulating Erythrophleum fordii,an Evergreen Tree Indigenous to the Southern Subtropical Region of China

The nodulation of Erythrophleum fordii has been recorded recently, but its microsymbionts have never been studied. To investigate the diversity and biogeography of rhizobia associated with this leguminous evergreen tree, root nodules were collected from the southern subtropical region of China. A total of 166 bacterial isolates were obtained from the nodules and characterized. In a PCR-based restriction fragment length polymorphism (RFLP) analysis of ribosomal intergenic sequences, the isolates were classified into 22 types within the genus Bradyrhizobium. Sequence analysis of 16S rRNA, ribosomal intergenic spacer (IGS), and the housekeeping genes recA and glnII classified the isolates into four groups: the Bradyrhizobium elkanii and Bradyrhizobium pachyrhizi groups, comprising the dominant symbionts, Bradyrhizobium yuanmingense, and an unclassified group comprising the minor symbionts. The nodC and nifH phylogenetic trees defined five or six lineages among the isolates, which was largely consistent with the definition of genomic species. The phylogenetic results and evolutionary analysis demonstrated that mutation and vertical transmission of genes were the principal processes for the divergent evolution of Bradyrhizobium species associated with E. fordii, while lateral transfer and recombination of housekeeping and symbiotic genes were rare. The distribution of the dominant rhizobial populations was affected by soil pH and effective phosphorus. This is the first report to characterize E. fordii rhizobia.     

The large mimosoid genus Inga Mill. (tribe Ingeae,Caesalpinioideae) is nodulated by diverse Bradyrhizobium strains in its main centers of diversity in Brazil

Inga (Caesalpinioideae) is the type genus of the Ingeae tribe in the mimosoid clade. It comprises about 300 species, all trees or treelets, and has an exclusively neotropical distribution, with Brazil as its main center of diversity. In this study, we analyzed the diversity of 40 strains of rhizobia isolated from root nodules collected from ten species of Inga belonging to different types of vegetation in Brazil. Sequences of their housekeeping genes (dnaK, recA, rpoB, gyrB and glnII), 16S rRNA genes, internal transcribed spacer (ITS) regions, as well as their symbiosis-essential genes (nodC and nifH) were used to characterize them genetically. The ability of the rhizobia to form nodules on Inga spp., and on the promiscuous legume siratro (Macroptilium atropurpureum) was also evaluated. A multilocus sequence analysis (MLSA) combined with an analysis of the ITS region showed that the isolates were distributed into four main groups (A-D) within the large genus Bradyrhizobium. Analysis of the nodC and nifH genes showed that the isolates formed a separate branch from all described species of Bradyrhizobium, except for B. ingae. Most of the tested isolates formed nodules on siratro and all isolates tested nodulated Inga spp. Our results suggest a unique co-evolutionary history of Bradyrhizobium and Inga and demonstrate the existence of potential new species of microsymbionts nodulating this important and representative genus of leguminous tree from the Caesalpinioideae mimosoid clade.     

Chromosomal and Symbiotic Relationships of Rhizobia Nodulating Medicago truncatula and M. laciniata

Multilocus sequence typing (MLST) is a sequence-based method used to characterize bacterial genomes. This method was used to examine the genetic structure of Medicago-nodulating rhizobia at the Amra site, which is located in an arid region of Tunisia. Here the annual medics Medicago laciniata and M. truncatula are part of the natural flora. The goal of this study was to identify whether distinct chromosomal groups of rhizobia nodulate M. laciniata because of its restricted requirement for specific rhizobia. The MLST analysis involved determination of sequence variation in 10 chromosomal loci of 74 isolates each of M. laciniata and M. truncatula. M. truncatula was used as a control trap host, because unlike M. laciniata, it has relatively unrestricted rhizobial requirements. Allelic diversity among the plasmid nodC alleles in the isolates was also determined. The 148 isolates were placed into 26 chromosomal sequence types (STs), only 3 of which had been identified previously. The rhizobia of M. laciniata were shown to be part of the general Medicago-nodulating population in the soil because 99.95% of the isolates had chromosomal genotypes similar to those recovered from M. truncatula. However, the isolates recovered from M. laciniata were less diverse than those recovered from M. truncatula, and they also harbored an unusual nodC allele. This could perhaps be best explained by horizontal transfer of the different nodC alleles among members of the Medicago-nodulating rhizobial population at the field site. Evidence indicating a history of lateral transfer of rhizobial symbiotic genes across distinct chromosomal backgrounds is provided.     

Identification of the rhizobial symbiont of Astragalus glombiformis in Eastern Morocco as Mesorhizobium camelthorni

Astragalus gombiformis is a desert symbiotic nitrogen-fixing legume of great nutritional value as fodder for camels and goats. However, there are no data published on the rhizobial bacteria that nodulate this wild legume in northern Africa. Thirty-four rhizobial bacteria were isolated from root nodules of A. gombifomis grown in sandy soils of the South-Eastern of Morocco. Twenty-five isolates were able to renodulate their original host and possessed a nodC gene copy. The phenotypic and genotypic characterizations carried out illustrated the diversity of the isolates. Phenotypic analysis showed that isolates used a great number of carbohydrates as sole carbon source. However, although they were isolated from arid sandy soils, the isolates do not tolerate drought stress applied in vitro. The phenotypic diversity corresponded mainly to the diversity in the use of some carbohydrates. The genetic analysis as assessed by repetitive extragenic palindromic (REP)-polymerase chain reaction (PCR) showed that the isolates clustered into 3 groups at a similarity coefficient of 81 %. The nearly-complete 16S rRNA gene sequence from a representative strain of each PCR-group showed they were closely related to members of the genus Mesorhizobium of the family Phyllobactericeae within the Alphaproteobacteria. Sequencing of the housekeeping genes atpD, glnII and recA, and their concatenated phylogenetic analysis, showed they are closely related to Mesorhizobium camelthorni. Sequencing of the symbiotic nodC gene from each strain revealed they had 83.53 % identity with the nodC sequence of the type strain M. camelthorni CCNWXJ 40-4^T.     

Genetic markers for search of rhizobia based on symbiotic genes

The possible application of rhizobial symbiotic genes as markers for the search and primary identification of rhizobia from temperate-zone legumes was studied. It was shown that conservative sym genes nifH and nifD could be used as markers for rapid search of rhizobia among the analyzed isolates, while more variable genes nifK and nodC could be used for their primary identification. Efficiency of the proposed method was shown in analysis of bacterial isolates obtained from Onobrychis arenaria and Astragalus cicer root nodules.