Geographical and climatic distribution of lentil-nodulating rhizobia in Iran.

Lentil is one of the most important legumes cultivated in various provinces of Iran. However, there is limited information about the symbiotic rhizobia of lentils in this country. In this study, molecular identification of lentil-nodulating rhizobia was performed based on 16S-23S rRNA intergenic spacer (IGS) and recA, atpD, glnII, and nodC gene sequencing. Using PCR-RFLP analysis of 16S-23S rRNA IGS, a total of 116 rhizobia isolates were classified into 20 groups, leaving seven strains unclustered. Phylogenetic analysis of representative isolates revealed that the rhizobia strains belonged to Rhizobium leguminosarum and Rhizobium laguerreae, and the distribution of the species is partially related to geographical location. Rhizobium leguminosarum was the dominant species in North Khorasan and Zanjan, while R. laguerreae prevailed in Ardabil and East Azerbaijan. The distribution of the species was also influenced by agroecological climates; R. leguminosarum thrived in cold semiarid climates, whereas R. laguerreae adapted to humid continental climates. Both species exhibited equal dominance in the Mediterranean climate, characterized by warm, dry summers and mild, wet winters, in Lorestan and Kohgiluyeh-Boyer Ahmad provinces.

Major effect loci for plant size before onset of nitrogen fixation allow accurate prediction of yield in white clover.

KEY MESSAGE: Accurate genomic prediction of yield within and across generations was achieved by estimating the genetic merit of individual white clover genotypes based on extensive genetic replication using cloned material. White clover is an agriculturally important forage legume grown throughout temperate regions as a mixed clover-grass crop. It is typically cultivated with low nitrogen input, making yield dependent on nitrogen fixation by rhizobia in root nodules. Here, we investigate the effects of clover and rhizobium genetic variation by monitoring plant growth and quantifying dry matter yield of 704 combinations of 145 clover genotypes and 170 rhizobium inocula. We find no significant effect of rhizobium variation. In contrast, we can predict yield based on a few white clover markers strongly associated with plant size prior to nitrogen fixation, and the prediction accuracy for polycross offspring yield is remarkably high. Several of the markers are located near a homolog of Arabidopsis thaliana GIGANTUS 1, which regulates growth rate and biomass accumulation. Our work provides fundamental insight into the genetics of white clover yield and identifies specific candidate genes as breeding targets.

Phenotypic, stress tolerance, and plant growth promoting characteristics of rhizobial isolates of grass pea.

Grass pea (Lathyrus sativus L.) is widely cultivated for food and feed in some developing countries including Ethiopia. However, due to its overexaggerated neuro-lathyrism alkaloid causing paralysis of limbs, it failed to attract attention of the research community and is one of the most neglected orphan crops in the world. But, the crop is considered an insurance crop by resource-poor farmers due to its strong abiotic stress tolerance and ability to produce high yields when all other crops fail due to unfavorable environmental conditions. This study was aimed at screening rhizobial isolates of grass pea and evaluating their symbiotic nitrogen fixation efficiency and tolerance to abiotic stresses. Fifty rhizobial isolates collected from grass pea nodules were isolated, screened, and characterized based on standard microbiological methods. The rhizobial isolates showed diversity in nodulation, symbiotic nitrogen fixation, and nutrient utilization. The 16S rRNA gene sequencing of 14 rhizobial isolates showed that two of them were identified as Rhizobium leguminosarum and the remaining twelve as Rhizobium species. Based on their overall performance, strains AAUGR-9, AAUGR-11, and AAUGR-14 that performed top and identified as Rhizobium species were recommended for field trials. This study screened and identified effective and competitive rhizobial isolates enriched with high nitrogen-fixing and abiotic stress tolerant traits, which contributes much to the application of microbial inoculants as alternative to chemical fertilizers.

Search for Ancestral Features in Genomes of Rhizobium leguminosarum bv. viciae Strains Isolated from the Relict Legume Vavilovia formosa.

Vavilovia formosa is a relict leguminous plant growing in hard-to-reach habitats in the rocky highlands of the Caucasus and Middle East, and it is considered as the putative closest living relative of the last common ancestor (LCA) of the Fabeae tribe. Symbionts of Vavilovia belonging to Rhizobium leguminosarum bv. viciae compose a discrete group that differs from the other strains, especially in the nucleotide sequences of the symbiotically specialised (sym) genes. Comparison of the genomes of Vavilovia strains with the reference group composed of R. leguminosarum bv. viciae strains isolated from Pisum and Vicia demonstrated that the vavilovia strains have a set of genomic features, probably indicating the important stages of microevolution of the symbiotic system. Specifically, symbionts of Vavilovia (considered as an ancestral group) demonstrated a scattered arrangement of sym genes (>90 kb cluster on pSym), with the location of nodT gene outside of the other nod operons, the presence of nodX and fixW, and the absence of chromosomal fixNOPQ copies. In contrast, the reference (derived) group harboured sym genes as a compact cluster (<60 kb) on a single pSym, lacking nodX and fixW, with nodT between nodN and nodO, and possessing chromosomal fixNOPQ copies. The TOM strain, obtained from nodules of the primitive "Afghan" peas, occupied an intermediate position because it has the chromosomal fixNOPQ copy, while the other features, the most important of which is presence of nodX and fixW, were similar to the Vavilovia strains. We suggest that genome evolution from the ancestral to the derived R. leguminosarum bv. viciae groups follows the "gain-and-loss of sym genes" and the "compaction of sym cluster" strategies, which are common for the macro-evolutionary and micro-evolutionary processes. The revealed genomic features are in concordance with a relict status of the vavilovia strains, indicating that V. formosa coexists with ancestral microsymbionts, which are presumably close to the LCA of R. leguminosarum bv. viciae.

Rhizobia Isolated from the Relict Legume Vavilovia formosa Represent a Genetically Specific Group within Rhizobium leguminosarum biovar viciae.

Twenty-two rhizobia strains isolated from three distinct populations (North Ossetia, Dagestan, and Armenia) of a relict legume Vavilovia formosa were analysed to determine their position within Rhizobium leguminosarum biovar viciae (Rlv). These bacteria are described as symbionts of four plant genera Pisum, Vicia, Lathyrus, and Lens from the Fabeae tribe, of which Vavilovia is considered to be closest to its last common ancestor (LCA). In contrast to biovar viciae, bacteria from Rhizobium leguminosarum biovar trifolii (Rlt) inoculate plants from the Trifolieae tribe. Comparison of house-keeping (hkg: 16S rRNA, glnII, gltA, and dnaK) and symbiotic (sym: nodA, nodC, nodD, and nifH) genes of the symbionts of V. formosa with those of other Rlv and Rlt strains reveals a significant group separation, which was most pronounced for sym genes. A remarkable feature of the strains isolated from V. formosa was the presence of the nodX gene, which was commonly found in Rlv strains isolated from Afghanistan pea genotypes. Tube testing of different strains on nine plant species, including all genera from the Fabeae tribe, demonstrated that the strains from V. formosa nodulated the same cross inoculation group as the other Rlv strains. Comparison of nucleotide similarity in sym genes suggested that their diversification within sym-biotypes of Rlv was elicited by host plants. Contrariwise, that of hkg genes could be caused by either local adaptation to soil niches or by genetic drift. Long-term ecological isolation, genetic separation, and the ancestral position of V. formosa suggested that symbionts of V. formosa could be responsible for preserving ancestral genotypes of the Rlv biovar.

Analysis of metal tolerance in Rhizobium leguminosarum strains isolated from an ultramafic soil.

Natural habitats containing high amounts of heavy metals provide a valuable source of bacteria adapted to deal with metal toxicity. A functional analysis of the population of legume endosymbiotic bacteria in an ultramafic soil was undertaken by studying a collection of Rhizobium leguminosarum bv viciae (Rlv) isolates obtained using pea as trap plant. One of the isolates, Rlv UPM1137, was selected on the basis of its higher tolerance to nickel and cobalt and presence of inducible mechanisms for such tolerance. A random transposon mutagenesis of Rlv UPM1137 allowed the generation of 14 transposant derivatives with increased nickel sensitivity; five of these transposants were also more sensitive to cobalt. Sequencing of the insertion sites revealed that one of the transposants (D2250) was affected in a gene homologous to the cation diffusion facilitator gene dmeF first identified in the metal-resistant bacterium Cupriavidus metallidurans CH34. The symbiotic performance of D2250 and two other transposants bearing single transposon insertions was unaffected under high-metal conditions, suggesting that, in contrast to previous observations in other Rlv strain, metal tolerance in UPM1137 under symbiotic conditions might be supported by functional redundancy between several mechanisms.

Vicia faba L. in the Bejaia region of Algeria is nodulated by Rhizobium leguminosarum sv. viciae, Rhizobium laguerreae and two new genospecies.

Fifty-eight rhizobial strains were isolated from root nodules of Vicia faba cv. Equina and Vicia faba cv. Minor by the host-trapping method in soils collected from eleven sites in Bejaia, Eastern Algeria. Eleven genotypic groups were distinguished based on the combined PCR/RFLP of 16S rRNA, 16S-23S rRNA intergenic spacer and symbiotic (nodC and nodD-F) genes and further confirmed by multilocus sequence analysis (MLSA) of three housekeeping genes (recA, atpD and rpoB), the 16S rRNA gene and the nodulation genes nodC and nodD. Of the 11 genotypes, 5 were dominant and 2 were the most represented. Most of the strains shared high nodD gene sequence similarity with Rhizobium leguminosarum sv. viciae; their nodC sequences were similar to both Rhizobium leguminosarum and Rhizobium laguerreae. Sequence analyses of the 16S-23S rRNA intergenic spacer showed that all the new strains were phylogenetically related to those described from Vicia sativa and V. faba in several African, European, American and Asian countries, with which they form a group related to Rhizobium leguminosarum. Phylogenetic analysis based on MLSA of 16S rRNA, recA, atpD and rpoB genes allowed the affiliations of strain AM11R to Rhizobium leguminosarum sv. viciae and of strains EB1 and ES8 to Rhizobium laguerreae. In addition, two separate clades with <97% similarity may represent two novel genospecies within the genus Rhizobium.

Old meets new: most probable number validation of metagenomic and metatranscriptomic datasets in soil.

Metagenomics and metatranscriptomics provide insights into biological processes in complex substrates such as soil, but linking the presence and expression of genes with functions can be difficult. Here, we obtain traditional most probable number estimates (MPN) of Rhizobium abundance in soil as a form of sample validation. Our work shows that in the Highfield experiment at Rothamsted, which has three contrasting conditions (>50 years continual bare fallow, wheat and grassland), MPN based on host plant nodulation assays corroborate metagenomic and metatranscriptomic estimates for Rhizobium leguminosarum sv. trifolii abundance. This validation is important to legitimize soil metagenomics and metatranscriptomics for the study of complex relationships between gene function and phylogeny. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has demonstrated for the first time a functional assay validation of metagenomic and metatranscriptomic datasets by utilizing the clover and Rhizobium leguminosarum sv. trifolii mutualism. The results show that the Most Probable Number results corroborate the results of the 'omics approaches and gives confidence to the study of other biological systems where such a cross-check is not available.

Host-dependent symbiotic efficiency of Rhizobium leguminosarum bv. trifolii strains isolated from nodules of Trifolium rubens.

Trifolium rubens L., commonly known as the red feather clover, is capable of symbiotic interactions with rhizobia. Up to now, no specific symbionts of T. rubens and their symbiotic compatibility with Trifolium spp. have been described. We characterized the genomic diversity of T. rubens symbionts by analyses of plasmid profiles and BOX-PCR. The phylogeny of T. rubens isolates was inferred based on the nucleotide sequences of 16S rRNA and two core genes (atpD, recA). The nodC phylogeny allowed classification of rhizobia nodulating T. rubens as Rhizobium leguminosarum symbiovar trifolii (Rlt). The symbiotic efficiency of the Rlt isolates was determined on four clover species: T. rubens, T. pratense, T. repens and T. resupinatum. We determined that Rlt strains formed mostly inefficient symbiosis with their native host plant T. rubens and weakly effective (sub-optimal) symbiosis with T. repens and T. pratense. The same Rlt strains were fully compatible in the symbiosis with T. resupinatum. T. rubens did not exhibit strict selectivity in regard to the symbionts and rhizobia closely related to Rhizobium grahamii, Rhizobium galegae and Agrobacterium radiobacter, which did not nodulate Trifolium spp., were found amongst T. rubens nodule isolates.

Reclassification of the taxonomic status of SEMIA3007 isolated in Mexico B-11A Mex as Rhizobium leguminosarum bv. viceae by bioinformatic tools.

BACKGROUND: Evidence based on genomic sequences is extremely important to confirm the phylogenetic relationships within the Rhizobium group. SEMIA3007 was analyzed within the Mesorhizobium groups to define the underlying causes of taxonomic identification. We previously used biochemical tests and phenotypic taxonomic methods to identify bacteria, which can lead to erroneous classification. An improved understanding of bacterial strains such as the Mesorhizobium genus would increase our knowledge of classification and evolution of these species. RESULTS: In this study, we sequenced the complete genome of SEMIA3007 and compared it with five other Mesorhizobium and two Rhizobium genomes. The genomes of isolated SEMIA3007 showed several orthologs with M. huakuii, M. erdmanii and M. loti. We identified SEMIA3007 as a Mesorhizobium by comparing the 16S rRNA gene and the complete genome. CONCLUSION: Our ortholog, 16S rRNA gene and average nucleotide identity values (ANI) analysis all demonstrate SEMIA3007 is not Rhizobium leguminosarum bv. viceae. The results of the phylogenetic analysis clearly show SEMIA3007 is part of the Mesorhizobium group and suggest a reclassification is warranted.

Local genetic structure and worldwide phylogenetic position of symbiotic Rhizobium leguminosarum strains associated with a traditional cultivated crop, Vicia ervilia, from Northern Morocco.

A total of 212 symbiotic bacteria were isolated from nodules of Vicia ervilia, a traditional crop cultivated in Northern Morocco. The isolates were recovered from 10 different sites, trapped each time with the local cultivar grown in the same field. Four loci were sequenced in order to characterize the isolates, including two housekeeping genes (recA and glnII), one plasmidic symbiotic gene (nodC) and one locus from another plasmid (prL11). In several isolates, two different copies of glnII were detected and sequenced, suggesting a unique duplication event, which has never been reported previously. There was no correlation between the genetic differentiation among cultivars and among bacteria, showing that the evolution of the bacterial population was independent, at least partially, from the host plant. By placing the haplotypes in a wide-ranging phylogenetic reconstruction, it was shown that the diversity detected in Morocco was spread throughout the different clades detected worldwide. The differentiation between areas relied on frequency variations of haplotypes rather than a presence/absence pattern. This finding raises new questions concerning bacterial genetic resource preservation, and confirms the old tenet "everything is everywhere but the environment selects".

Rhizobium leguminosarum symbiovar trifolii, Ensifer numidicus and Mesorhizobium amorphae symbiovar ciceri (or Mesorhizobium loti) are new endosymbiotic bacteria of Lens culinaris Medik.

A total of 142 rhizobial bacteria were isolated from root nodules of Lens culinaris Medik endemic to Tunisia and they belonged to the species Rhizobium leguminosarum, and for the first time to Ensifer and Mesorhizobium, genera never previously described as microsymbionts of lentil. Phenotypically, our results indicate that L. culinaris Medik strains showed heterogenic responses to the different phenotypic features and they effectively nodulated their original host. Based on the concatenation of the 16S rRNA with relevant housekeeping genes (glnA, recA, dnaK), rhizobia that nodulate lentil belonged almost exclusively to the known R. leguminosarum sv. viciae. Interestingly, R. leguminosarum sv. trifolii, Ensifer numidicus (10 isolates) and Mesorhizobium amorphae (or M. loti) (9 isolates) isolates species, not considered, up to now, as a natural symbiont of lentil are reported. The E. numidicus and M. amorphae (or M. loti) strains induced fixing nodules on Medicago sativa and Cicer arietinum host plants, respectively. Symbiotic gene phylogenies showed that the E. numidicus, new symbiont of lentil, markedly diverged from strains of R. leguminosarum, the usual symbionts of lentil, and converged to the symbiovar meliloti so far described within E. meliloti Indeed, the nodC and nodA genes from the M. amorphae showed more than 99% similarity with respect to those from M. mediterraneum, the common chickpea nodulating species, and would be included in the new infrasubspecific division named M. amorphae symbiovar ciceri, or to M. loti, related to the strains able to effectively nodulate C. arietinum host plant. On the basis of these data, R. leguminosarum sv. trifolii (type strain LBg3 (T)), M. loti or M. amorphae sv. ciceri (type strain LB4 (T)) and E. numidicus (type strain LBi2 (T)) are proposed as new symbionts of L. culinaris Medik.

Genetic diversity of rhizobia nodulating native Vicia spp. in Sweden.

Despite the recognition that Rhizobium leguminosarum sv. viciae is the most common symbiont of Vicia species worldwide, there is no available information on rhizobia nodulating native Vicia species in Sweden. We have therefore studied the genetic diversity and phylogeny of root nodule bacteria isolated from V. cracca, V. hirsuta, V. sepium, V. tetrasperma and V. sylvatica growing in different locations in Sweden as well as an isolate each from V. cracca in Tromsø, Norway, and V. multicaulis in Siberia, Russia. Out of 25 isolates sampled from the six Vicia species in 12 different locations, there were 14 different genotypes based on the atpD, recA and nodA gene phylogenies. All isolates were classified into Rhizobium leguminosarum sv. viciae group based on the concatenated atpD and recA phylogeny and the nodA phylogeny.

Inoculation of new rhizobial isolates improve nutrient uptake and growth of bean (Phaseolus vulgaris) and arugula (Eruca sativa).

BACKGROUND: In the current agricultural model, the massive use of chemical fertilizer causes environmental and economic losses. Inoculation of plant-growth-promoting (PGP) nitrogen-fixing bacteria is an alternative to the use of nitrogen, phosphorus and potassium fertilizers. In this study, rhizobia strains isolated from common bean (Phaseolus vulgaris) root nodules were evaluated in an effort to identify an efficient nitrogen-fixing rhizobia strain able to improve bean germination and growth. RESULTS: Common bean plants were collected from seven sites in southern Brazil, and 210 native rhizobia isolates were obtained. Evaluation of PGP traits showed that most of the rhizobia isolates were non-siderophore producers and weak indolic compounds producers. Under laboratory conditions, rhizobia isolates E15 (Rhizobium leguminosarum) and L5 (Rhizobium radiobacter) increase germination percentage, length, and dry weight of common bean and arugula (Eruca sativa) seedlings. Under greenhouse conditions, common bean plants inoculated with the rhizobia isolates VC28 and L15 (both Rhizobium fabae) presented the highest nodule number and shoot dry matter, while VC28 also presented the highest values of shoot nitrogen and potassium. Isolate L17 presented highly effective N fixation, even with reduced nodulation. CONCLUSION: These new rhizobia isolates are attractive PGP alternatives to chemical fertilizers. © 2015 Society of Chemical Industry.

Cicer canariense, an endemic legume to the Canary Islands, is nodulated in mainland Spain by fast-growing strains from symbiovar trifolii phylogenetically related to Rhizobium leguminosarum.

Cicer canariense is a threatened endemic legume from the Canary Islands where it can be nodulated by mesorhizobial strains from the symbiovar ciceri, which is the common worldwide endosymbiont of Cicer arietinum linked to the genus Mesorhizobium. However, when C. canariense was cultivated in a soil from mainland Spain, where the symbiovar ciceri is present, only fast-growing rhizobial strains were unexpectedly isolated from its nodules. These strains were classified into the genus Rhizobium by analysis of the recA and atpD genes, and they were phylogenetically related to Rhizobium leguminosarum. The analysis of the nodC gene showed that the isolated strains belonged to the symbiovar trifolii that harbored a nodC allele (ß allele) different to that harbored by other strains from this symbiovar. Nodulation experiments carried out with the lacZ-labeled strain RCCHU01, representative of the ß nodC allele, showed that it induced curling of root hairs, infected them through infection threads, and formed typical indeterminate nodules where nitrogen fixation took place. This represents a case of exceptional performance between the symbiovar trifolii and a legume from the tribe Cicereae that opens up new possibilities and provides new insights into the study of rhizobia-legume symbiosis.

Genetic differentiation of Trifolium repens microsymbionts deriving from Zn-Pb waste-heap and control area in Poland.

The aim of this work was to determine the genetic structure of Rhizobium leguminosarum bv. trifolii population isolated from root nodules of Trifolium repens growing in heavy metal contaminated Boleslaw waste-heap area and compare it with that of an unpolluted control Bolestraszyce population. The 684-bp long dinitrogenase reductase (nifH) gene fragments were amplified in a PCR reaction and then sequenced. An analysis of nifH gene amplicons of 21 rhizobial strains from each of the studied populations revealed substantially reduced genotype (h) and nucleotide (π) diversities in the metallicolous Boleslaw population in comparison to the non-metallicolous Bolestraszyce one, and showed a significant genetic differentiation between these populations (F(ST) = 0.159, p = 0.018). Among the strains under investigation, six genotypes (A-F) with 95-99% nifH gene sequence identities were distinguished. Studied T. repens nodule isolates indicated the highest nifH gene sequence similarities (95-100%) with R. leguminosarum bv. trifolii reference strains and on nifH phylogram all these strains formed monophyletic, highly supported clade (100%). The decreased genotype and nucleotide diversities of the waste-heap R. leguminosarum bv. trifolii population, compared to that from the control area and substantial genetic differentiation between populations of nifH gene, is arguably the consequence of the random genetic drift (Tajima's D = 2.042, p = 0.99).

Rhizobium leguminosarum is the symbiont of lentils in the Middle East and Europe but not in Bangladesh.

Lentil is the oldest of the crops that have been domesticated in the Fertile Crescent and spread to other regions during the Bronze Age, making it an ideal model to study the evolution of rhizobia associated with crop legumes. Housekeeping and nodulation genes of lentil-nodulating rhizobia from the region where lentil originated (Turkey and Syria) and regions to which lentil was introduced later (Germany and Bangladesh) were analyzed to determine their genetic diversity, population structure, and taxonomic position. There are four different lineages of rhizobia associated with lentil nodulation, of which three are new and endemic to Bangladesh, while Mediterranean and Central European lentil symbionts belong to the Rhizobium leguminosarum lineage. The endemic lentil grex pilosae may have played a significant role in the origin of these new lineages in Bangladesh. The presence of R. leguminosarum with lentil at the center of origin and in countries where lentil was introduced later suggests that R. leguminosarum is the original symbiont of lentil. Lentil seeds may have played a significant role in the initial dispersal of this Rhizobium species within the Middle East and on to other countries. Nodulation gene sequences revealed a high similarity to those of symbiovar viciae.

Near-full length sequencing of 16S rDNA and RFLP indicates that Rhizobium etli is the dominant species nodulating Egyptian winter Berseem clover (Trifolium alexandrinum L.).

Egyptian winter Berseem clover (EWBC) is one of the main important forage legume crops in Egypt that is used for animal feeding in winter and it occupies about 2.5 million feddans (Feddan=4200m(2)) in winter agricultural rotation systems. Forty-eight rhizobial isolates that nodulated this legume host from different geographical regions within Egypt were isolated. RFLP analyses of 16S rDNA (1.5kb) and whole ribosomal DNA (5kb), the sequencing of 16S rDNA, and the sequencing of nodC, nifH and house keeping genes were used to identify these isolates. The RFLP analysis of 16S rDNA (1.5kb) among 15 representative strains with three enzymes generated two genotypes. The largest genotype was similar to Rhizobium etli CFN42T (93.33%) except for strain 902 that failed to re-nodulate EWBC. RFLP analysis of complete ribosomal DNA (5kb) produced five genotypes. The majority of tested strains shared the genotype with R. etli CFN42T (53.33%). Only one strain (1002) shared the genotype with Rhizobium leguminosarum sv. trifolii 3023. The other four strains were comprised of two unique genotypes. Phylogenetic analysis of 16S rDNA sequences revealed that seven representative strains could be divided into two genetic clusters sharing the ancestral clad with R. etli CFN42T. A phylogenetic tree based on nodC gene sequence confirmed that all the examined strains shared the genetic lineage with R. leguminosarum sv. trifolii WSM1325. The phylogenetic trees of house keeping genes are supported strongly the identification of majority of strains as a novel symbiovar of R. etli with new lineages.

Synergistic interaction of Rhizobium leguminosarum bv. viciae and arbuscular mycorrhizal fungi as a plant growth promoting biofertilizers for faba bean (Vicia faba L.) in alkaline soil.

Egyptian soils are generally characterized by slightly alkaline to alkaline pH values (7.5-8.7) which are mainly due to its dry environment. In arid and semi-arid regions, salts are less concentrated and sodium dominates in carbonate and bicarbonate forms, which enhance the formation of alkaline soils. Alkaline soils have fertility problems due to poor physical properties which adversely affect the growth and the yield of crops. Therefore, this study was devoted to investigating the synergistic interaction of Rhizobium and arbuscular mycorrhizal fungi for improving growth of faba bean grown in alkaline soil. A total of 20 rhizobial isolates and 4 species of arbuscular mycorrhizal fungi (AMF) were isolated. The rhizobial isolates were investigated for their ability to grow under alkaline stress. Out of 20 isolates 3 isolates were selected as tolerant isolates. These 3 rhizobial isolates were identified on the bases of the sequences of the gene encoding 16S rRNA and designated as Rhizobium sp. Egypt 16 (HM622137), Rhizobium sp. Egypt 27 (HM622138) and Rhizobium leguminosarum bv. viciae STDF-Egypt 19 (HM587713). The best alkaline tolerant was R. leguminosarum bv. viciae STDF-Egypt 19 (HM587713). The effect of R. leguminosarum bv. viciae STDF-Egypt 19 and mixture of AMF (Acaulospora laevis, Glomus geosporum, Glomus mosseae and Scutellospora armeniaca) both individually and in combination on nodulation, nitrogen fixation and growth of Vicia faba under alkalinity stress were assessed. A significant increase over control in number and mass of nodules, nitrogenase activity, leghaemoglobin content of nodule, mycorrhizal colonization, dry mass of root and shoot was recorded in dual inoculated plants than plants with individual inoculation. The enhancement of nitrogen fixation of faba bean could be attributed to AMF facilitating the mobilization of certain elements such as P, Fe, K and other minerals that involve in synthesis of nitrogenase and leghaemoglobin. Thus it is clear that the dual inoculation with Rhizobium and AMF biofertilizer is more effective for promoting growth of faba bean grown in alkaline soils than the individual treatment, reflecting the existence of synergistic relationships among the inoculants.

Rhizobium pisi sv. trifolii K3.22 harboring nod genes of the Rhizobium leguminosarum sv. trifolii cluster.

The taxonomic status of the Rhizobium sp. K3.22 clover nodule isolate was studied by multilocus sequence analysis (MLSA) of 16S rRNA and six housekeeping chromosomal genes, as well as by a subsequent phylogenic analysis. The results revealed full congruence with the Rhizobium pisi DSM 30132(T) core genes, thus supporting the same taxonomic position for both strains. However, the K3.22 plasmid symbiosis nod genes demonstrated high sequence similarity to Rhizobium leguminosarum sv. trifolii, whereas the R. pisi DSM 30132(T)nod genes were most similar to R. leguminosarum sv. viciae. The strains differed in the host range nodulation specificity, since strain K3.22 effectively nodulated red and white clover but not vetch, in contrast to R. pisi DSM 30132(T), which effectively nodulated vetch but was not able to nodulate clover. Both strains had the ability to form nodules on pea and bean but they differed in bean cultivar specificity. The R. pisi K3.22 and DSM 30132(T) strains might provide evidence for the transfer of R. leguminosarum sv. trifolii and sv. viciae symbiotic plasmids occurring in natural soil populations.