Characterization of rhizobial isolates of Phaseolus vulgaris by staircase electrophoresis of low-molecular-weight RNA

Low-molecular-weight (LMW) RNA molecules were analyzed to characterize rhizobial isolates that nodulate the common bean growing in Spain. Since LMW RNA profiles, determined by staircase electrophoresis, varied across the rhizobial species nodulating beans, we demonstrated that bean isolates recovered from Spanish soils presumptively could be characterized as Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum bv. viciae and bv. trifolii, and Sinorhizobium fredii.     

Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils

A collection of 160 isolates of rhizobia nodulating Phaseolus vulgaris in three geographical regions in Tunisia was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rDNA, nifH and nodC genes. Nine groups of rhizobia were delineated: Rhizobium gallicum biovar (bv.) gallicum, Rhizobium leguminosarum bv. phaseoli and bv. viciae, Rhizobium etli bv. phaseoli, Rhizobium giardinii bv. giardinii, and four groups related to species of the genus Sinorhizobium, Sinorhizobium meliloti, Sinorhizobium medicae and Sinorhizobium fredii. The most abundant rhizobial species were R. gallicum, R. etli, and R. leguminosarum encompassing 29–20% of the isolates each. Among the isolates assigned to R. leguminosarum, two-thirds were ineffective in nitrogen fixation with P. vulgaris and harbored a symbiotic gene typical of the biovar viciae. The S. fredii-like isolates did not nodulate soybean plants but formed numerous effective nodules on P. vulgaris. Comparison of nodC gene sequences showed that their symbiotic genotype was not related to that of S. fredii, but to that of the S. fredii-like reference strain GR-06, which was isolated from a bean plant grown in a Spanish soil. An additional genotype including 16% of isolates was found to be closely related to species of the genus Agrobacterium. However, when re-examined, these isolates did not nodulate their original host.     

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.     

Prevalence of the Rhizobium etli-Like Allele in Genes Coding for 16S rRNA among the Indigenous Rhizobial Populations Found Associated with Wild Beans from the Southern Andes in Argentina

A collection of rhizobial isolates from nodules of wild beans, Phaseolus vulgaris var. aborigineus, found growing in virgin lands in 17 geographically separate sites in northwest Argentina was characterized on the basis of host range, growth, hybridization to a nifH probe, analysis of genes coding for 16S rRNA (16S rDNA), DNA fingerprinting, and plasmid profiles. Nodules in field-collected wild bean plants were largely dominated by rhizobia carrying the 16S rDNA allele of Rhizobium etli. A similar prevalence of the R. etli allele was observed among rhizobia trapped from nearby soil. Intragroup diversity of wild bean isolates with either R. etli-like or Rhizobium leguminosarum bv. phaseoli-like alleles was generally found across northwest Argentina. The predominance of the R. etli allele suggests that in this center of origin of P. vulgaris the coevolution of Rhizobium spp. and primitive beans has resulted in this preferential symbiotic association.     

Rhizobium etli and Rhizobium gallicum Nodulate Common Bean (Phaseolus vulgaris) in a Traditionally Managed Milpa Plot in Mexico: Population Genetics and Biogeographic Implications

The stability of the genetic structure of rhizobial populations nodulating Phaseolus vulgaris cultivated in a traditionally managed milpa plot in Mexico was studied over three consecutive years. The set of molecular markers analyzed (including partial rrs, glnII, nifH, and nodB sequences), along with host range experiments, placed the isolates examined in Rhizobium etli bv. phaseoli and Rhizobium gallicum bv. gallicum. Cluster analysis of multilocus enzyme electrophoresis and plasmid profile data separated the two species and identified numerically dominant clones within each of them. Population genetic analyses showed that there was high genetic differentiation between the two species and that there was low intrapopulation differentiation of the species over the 3 years. The results of linkage disequilibrium analyses are consistent with an epidemic genetic structure for both species, with frequent genetic exchange taking place within conspecific populations but not between the R. etli and R. gallicum populations. A subsample of isolates was selected and used for 16S ribosomal DNA PCR-restriction fragment length polymorphism analysis, nifH copy number determination, and host range experiments. Plasmid profiles and nifH hybridization patterns also revealed the occurrence of lateral plasmid transfer among distinct multilocus genotypes within species but not between species. Both species were recovered from nodules of the same plants, indicating that mechanisms other than host, spatial, or temporal isolation may account for the genetic barrier between the species. The biogeographic implications of finding an R. gallicum bv. gallicum population nodulating common bean in America are discussed.     

Genetic diversity of rhizobia nodulating common bean (Phaseolus vulgaris L.) in the Central Black Sea Region of Turkey

We have analyzed 30 rhizobial isolates obtained from common bean (Phaseolus vulgaris L.) root nodules grown in the Middle Blacksea Region of Turkey, using ARDRA and nucleotide sequence data. ARDRA analysis with enzymes CfoI, HinfI, NdeII, MspI and PstI revealed three patterns. Based on sequence data from 16S rDNA, the patterns were identified as, Rhizobium leguminosarum bv. phaseoli (n = 16), R. etli bv. phaseoli (n = 8) and R. phaseoli (n = 6). On the other hand, nucleotide sequence phylogenies of housekeeping genes (recA, atpD and glnII) selected to confirm the 16S rDNA phylogeny revealed different evolutionary relationships. These results suggested the possibility of lateral transfers of these genes amongst different rhizobial species (including R. leguminosarum, R. etli and R. phaseoli) sharing the same ecological niche (nodulating P. vulgaris) which also indicates that there may be no true genetic barier among these species. Phylogenetic analysis based on DNA sequence data from the nodA and nifH genes showed that all rhizobial species obtained in this study were carrying nodA and nifH haplotypes which were the same or similar to those of CFN42 (R. etli type strain), suggesting a further support for the lateral transfer of CFN42 Sym plasmid, p42, amongst Turkish common bean nodulating rhizobial isolates.     

Phaseolus vulgaris seed-borne endophytic community with novel bacterial species such as Rhizobium endophyticum sp. nov.

The bacterial endophytic community present in different Phaseolus vulgaris (bean) cultivars was analyzed by 16S ribosomal RNA gene sequences of cultured isolates derived from surface disinfected roots and immature seeds. Isolated endophytes from tissue-macerates belonged to over 50 species in 24 different genera and some isolates from Acinetobacter, Bacillus, Enterococcus, Nocardioides, Paracoccus, Phyllobacterium, and Sphingomonas seem to correspond to new lineages. Phytate solubilizing bacteria were identified among Acinetobacter, Bacillus and Streptomyces bean isolates, phytate is the most abundant reserve of phosphorus in bean and in other seeds. Endophytic rhizobia were not capable of forming nodules. A novel rhizobial species Rhizobium endophyticum was recognized on the basis of DNA–DNA hybridization, sequence of 16S rRNA, recA, rpoB, atpD, dnaK genes, plasmid profiles, and phenotypic characteristics. R. endophyticum is capable of solubilizing phytate, the type strain is CCGE2052 (ATCC BAA-2116; HAMBI 3153) that became fully symbiotic by acquiring the R. tropici CFN299 symbiotic plasmid.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Isolation and Identification of Natural Endophytic Rhizobia from Rice (Oryza sativa L.) Through rDNA PCR-RFLP and Sequence Analysis

Three novel endophytic rhizobial strains (RRE3, RRE5, and RRE6) were isolated from naturally growing surface sterilized rice roots. These isolates had the ability to nodulate common bean (Phaseolus vulgaris). Polymerase chain reaction–restriction fragment length polymorphism and sequencing of 16S rDNA of these isolates revealed that RRE3 and RRE5 are phylogenetically very close to Burkholderia cepacia complex, whereas RRE6 has affinity with Rhizobium leguminosarum bv. phaseoli. Plant infection test using gusA reporter gene tagged construct of these isolates indicated that bacterial cells can go inside and colonize the rice root interiors. A significant increase in biomass and grain yield was also recorded in greenhouse-grown rice plants inoculated with these isolates.     

Rhizobia from Lanzarote,the Canary Islands,That Nodulate Phaseolus vulgaris Have Characteristics in Common with Sinorhizobium meliloti Isolates from Mainland Spain

The stable, low-molecular-weight (LMW) RNA fractions of several rhizobial isolates of Phaseolus vulgaris grown in the soil of Lanzarote, an island of the Canary Islands, were identical to a less-common pattern found within Sinorhizobium meliloti (assigned to group II) obtained from nodules of alfalfa and alfalfa-related legumes grown in northern Spain. The P. vulgaris isolates and the group II LMW RNA S. meliloti isolates also were distinguishable in that both had two conserved inserts of 20 and 46 bp in the 16S-23S internal transcribed spacer region that were not present in other strains of S. meliloti. The isolates from P. vulgaris nodulated bean but not Medicago sativa, while those recovered from Medicago, Melilotus, and Trigonella spp. nodulated both host legumes. The bean isolates also were distinguished from those of Medicago, Melilotus, and Trigonella spp. by nodC sequence analysis. The nodC sequences of the bean isolates were most similar to those reported for S. meliloti bv. mediterranense and Sinorhizobium fredii bv. mediterranense (GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"DQ333891","term_id":"84872507","term_text":"DQ333891"}}DQ333891 and {"type":"entrez-nucleotide","attrs":{"text":"AF217267","term_id":"13810627","term_text":"AF217267"}}AF217267, respectively). None of the evidence placed the bean isolates from Lanzarote in the genus Rhizobium, which perhaps is inconsistent with seed-borne transmission of Rhizobium etli from the Americas to the Canaries as an explanation for the presence of bean-nodulating rhizobia in soils of Lanzarote.A remarkable attribute of Phaseolus vulgaris (common bean) is its ability to nodulate with rhizobia from at least 20 different legume genera (summarized in reference 1). Of particular relevance is the report by Ishizawa (16), who described P. vulgaris nodulation ranging from doubtful to good by 14 strains recovered from Medicago sativa, Medicago denticulata, and Melilotus alba, while nodulation of the latter three legumes by four bean strains was negative.At the time of the host range experiments, such as those described by Ishizawa (16), rhizobial nomenclature depended on the legume host of origin; the taxonomy of the strains was based on cross-inoculation groups. Consequently, no information was available about the genetic relationships among the rhizobial strains that originated from the different host legume genera and formed nodules on P. vulgaris. Eventually, rhizobial nomenclature based on the cross-inoculation groups was abandoned because of the many unexplainable and incongruous nodulation data (44). The cross-inoculation groups consisted of different rhizobial species within the single genus Rhizobium. Eventually, rhizobial taxonomy was expanded to several different genera based on estimates of their phylogeny (38). Phylogenies of bean-nodulating rhizobia were estimated from variations in the 16S rRNA gene sequence (39), even though subsequently it became clear that this method is significantly limited by histories of genetic exchange and recombination (6, 40). Most reported phylogenies of rhizobia nodulating P. vulgaris have placed them in the genus Rhizobium (3, 39), but several surveys with isolates from North Africa and Spain have demonstrated that rhizobia in the genus Sinorhizobium also nodulate this legume species (12, 23, 24, 25, 41), supporting the nodulation data originally published by Ishizawa (16). The number of isolates described as originating from nodules of P. vulgaris in the genus Sinorhizobium is small, and for the most part, from the published evidence, it has been suggested that they are affiliated with Sinorhizobium fredii. However, nodules of P. vulgaris growing in a single Tunisian soil where beans are cultivated yielded four isolates that, according to the data, appeared to support an affiliation with Sinorhizobium meliloti rather than S. fredii (25). Whether these four cultures were of the same rhizobial genotype constituting a single example of S. meliloti isolated from P. vulgaris is unknown.P. vulgaris was introduced into Europe as a crop plant as early as the 16th century (31) but never became a very important part of agriculture in Lanzarote, one of the Canary Islands that lie in the Atlantic Ocean to the west of the North African coast. Since there is no record of any nodulation studies with P. vulgaris cultivated on Lanzarote Island, the first objective of this study was to examine bean plants that had grown in Lanzarote soil for nodulation. Considering that the diversity of rhizobia able to nodulate bean plants is extremely wide, the second objective was to characterize the isolates originating from the nodules of plants grown in Lanzarote soil.(Part of this work was presented as a poster at the First International Meeting on Microbial Phosphate Solubilization, Salamanca, Spain, July 2002.)     

The Diversity of Phaseolus-Nodulating Rhizobial Populations Is Altered by Liming of Acid Soils Planted with Phaseolus vulgaris L. in Brazil

PCR-mediated restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA internally transcribed spacer (ITS) region and the 16S rRNA gene indicated that the rhizobial populations isolated from common bean (Phaseolus vulgaris L.) nodules in the unlimed soil from a series of five lime rates applied 6 years previously to plots of an acidic oxisol had less diversity than those from plots with higher rates of liming. Isolates affiliated with Rhizobium tropici IIB and Rhizobium leguminosarum bv. phaseoli were predominant independent of lime application. An index of richness based on the number of ITS groups increased from 2.2 to 5.7 along the soil liming gradient, and the richness index based on “species” types determined by RFLP analysis of the 16S rRNA gene varied from 0.5 to 1.4. The Shannon index of diversity, based on the number of ITS groups, increased from 1.8 in unlimed soil to 2.8 in limed soil, and, based on RFLP analysis of the 16S rRNA gene, ranged from 0.9 to 1.4. In the limed soil, the subpopulation of R. tropici IIB pattern types contained the largest number of ITS groups. In contrast, there were more R. leguminosarum bv. phaseoli types in the unlimed soil with the lowest pH than in soils with the highest pH. The number of ITS (“strain”) groups within R. leguminosarum bv. phaseoli did not change with increased abundance of rhizobia in the soil, while with R. tropici IIB, the number of strain groups increased significantly. Some cultural and biochemical characteristics of Phaseolus-nodulating isolates were significantly related to changes in soil properties caused by liming, largely due to changes in the predominance of the rhizobial species groups.     

Genetic diversity and symbiotic effectiveness of Phaseolus vulgaris-nodulating rhizobia in Kenya

Phaseolus vulgaris (common bean) was introduced to Kenya several centuries ago but the rhizobia that nodulate it in the country remain poorly characterised. To address this gap in knowledge, 178 isolates recovered from the root nodules of P. vulgaris cultivated in Kenya were genotyped stepwise by the analysis of genomic DNA fingerprints, PCR-RFLP and 16S rRNA, atpD, recA and nodC gene sequences. Results indicated that P. vulgaris in Kenya is nodulated by at least six Rhizobium genospecies, with most of the isolates belonging to Rhizobium phaseoli and a possibly novel Rhizobium species. Infrequently, isolates belonged to Rhizobium paranaense, Rhizobium leucaenae, Rhizobium sophoriradicis and Rhizobium aegyptiacum. Despite considerable core-gene heterogeneity among the isolates, only four nodC gene alleles were observed indicating conservation within this gene. Testing of the capacity of the isolates to fix nitrogen (N₂) in symbiosis with P. vulgaris revealed wide variations in effectiveness, with ten isolates comparable to Rhizobium tropici CIAT 899, a commercial inoculant strain for P. vulgaris. In addition to unveiling effective native rhizobial strains with potential as inoculants in Kenya, this study demonstrated that Kenyan soils harbour diverse P. vulgaris-nodulating rhizobia, some of which formed phylogenetic clusters distinct from known lineages. The native rhizobia differed by site, suggesting that field inoculation of P. vulgaris may need to be locally optimised.     

Genetic Diversity of a Natural Population of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis> Nodulating Plants of <Emphasis Type="Italic">Vicia faba</Emphasis> in the Vesuvian Area

A total of 98 rhizobial strains, isolated during the winter of the years 2003 (35 isolates), 2004 (33 isolates), and 2005 (30 isolates) were analyzed to determine the genetic diversity of the natural population nodulating Vicia faba plants and to identify dominant genotypes. All isolates were identified as Rhizobium leguminosarum bv. viciae by biovar-specific polymerase chain reaction amplification of the nodC gene. Intraspecific DNA polymorphism was evaluated through the restriction endonucleases analysis combined with pulsed-field gel electrophoresis. Four genotypes characterized 53% of the isolates, showing a high occurrence; moreover, they were recovered over the 3 years, thus showing a lasting persistence in the soil, which could mean a high degree of saprophytic competitiveness. The richness, diversity, and dominance indexes of genotypes were calculated to monitor the evolution of the rhizobial population during the 3 years. The genetic diversity of the analyzed strains decreased along the 3 years. In fact, the biodiversity index H′ decreased from 2.6 in the first and second year to 1.9 in the third year; probably, as a result of bean monocropping, specific genotypes of Rh. leguminosarum bv. viciae were naturally selected.     

Isolation and Identification of Natural Endophytic Rhizobia from Rice (Oryza sativa L.) Through rDNA PCR-RFLP and Sequence Analysis

Three novel endophytic rhizobial strains (RRE3, RRE5, and RRE6) were isolated from naturally growing surface-sterilized rice roots. These isolates had the ability to nodulate common bean (Phaseolus vulgaris). Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) and sequencing of 16S rDNA of these isolates revealed that RRE3 and RRE5 are phylogenetically very close to Burkholderia cepacia complex, whereas RRE6 has affinity with Rhizobium leguminosarum bv. phaseoli. Plant infection test using gusA reporter gene–tagged construct of these isolates indicated that bacterial cells can go inside and colonize the rice root interiors. A significant increase in biomass and grain yield was also recorded in greenhouse-grown rice plants inoculated with these isolates.     

Genetic diversity of Vicia faba L. and Pisum sativum L. nodulating rhizobia in the central Black Sea region of Turkey

In this study, we obtained a total of 60 rhizobial isolates from root nodules of Vicia faba L. (n = 30) and Pisum sativum L. (n = 30) grown in the Central Black Sea region of Turkey. The 16S rDNA PCR-RFLP analysis with enzymes CfoI, HinfI, NdeII and MspI revealed a single pattern. Moreover, nucleotide sequence phylogenies based on both the 16S rDNA and recA suggested that these isolates belonged to Rhizobium leguminosarum. Phylogenetic analysis showed that some of our V. faba L.-originated isolates were closely related, indicating molecular evidence for the selection of some special R. leguminosarum bv. viciae isolates by V. faba L., as suggested in previous studies. Network analysis based on recA sequences revealed a common evolutionary history for Turkish, European, North and South American, and Jordanian R. leguminosarum bv. viciae isolates. We isolated four haplotypes using nodA and nifH nucleotide sequence data, i.e. four types of sym plasmids. Two of these types were common to rhizobial isolates from both V. faba L. and P. sativum L., indicating that nodulation factors may not be the mechanism for selection of the special R. leguminosarum bv. viciae populations by V. faba L.     

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.     

Rhizobium sophorae is the dominant rhizobial symbiont of Vicia faba L. In North China

Faba bean (Vicia faba L.) is a major introduced grain-legume crop cultivated in China. In this study, rhizobia that nodulated faba bean grown in soils from three sites in North China (Hebei Province) were isolated and characterized. Firstly, isolates were categorized into genotypes by ribosomal IGS PCR-RFLP analysis, then representatives of the different IGS genotypes were further identified by phylogenetic analyses of 16S rRNA, housekeeping (atpD, recA) and nodulation (nodC) gene sequences. Rhizobial distribution based on the IGS genotype was related to the different soil physicochemical features by redundancy analysis. IGS typing and phylogenetic analyses of 16S rRNA and concatenated housekeeping gene sequences affiliated the 103 rhizobial strains isolated into four Rhizobium species/genospecies. A total of 69 strains of 3 IGS types were assigned to R. sophorae, 20 isolates of 5 IGS types to R. changzhiense and 9 isolates of 3 IGS types to R. indicum. The representative strain of the five remaining isolates (1 IGS type) was clearly separated from all Rhizobium type strains and was most closely related to defined genospecies according to the recently described R. leguminosarum species complex. Rhizobium sophorae strains (67% of total isolates) were common in all sites and shared an identical nodC sequence typical of faba bean symbionts belonging to symbiovar viciae. In this first study of rhizobia nodulating faba bean in Hebei Province, China, R. sophorae was found to be the dominant symbiont in contrast to other countries.     

Influence of calcium and rhizobial infections (Rhizobium leguminosarum) on the dynamics of nitric oxide (NO) content in roots of etiolated pea (Pisum sativum L.) seedlings

The effect of exogenous calcium (Ca²⁺) and rhizobial infections (Rhizobium leguminosarum bv viceae) on the dynamics of the level of nitric oxide (NO) was studied in cross cuts of roots of two-day-old etiolated pea seedlings (Pisum sativum L.) using a DAF-2DA fluorescent probe. Fluctuations of the NO level, indicating the presence of a rhythm in the generation of NO in roots, were observed during the incubation of seedlings in water, a CaCl₂ solution, and with rhizobial infections. Exogenous factors (Ca²⁺ and two rhizobial stamms) change the time dynamics of the NO level in comparison with the control (water).     

Genotypic and phenotypic characterization of rhizobia that nodulate snap bean (Phaseolus vulgaris L.) in Egyptian soils

Snap bean fields in 12 of the 25 governorates of Egypt were surveyed to determine the distribution and taxonomy of snap bean-nodulating rhizobia. Nodulation rates in the field were very low, indicating that Egyptian soils do not have sufficient numbers of snap bean-compatible Rhizobium spp. A total of 87 rhizobial isolates were assayed on the most commonly grown cultivars in order to identify the most effective strains. The five most effective isolates (R11, R13, R28, R49 and R52) were fast-growing and utilized a wide range of carbon and nitrogen sources. A phylogenetic assignment of these strains by analysis of the 16S ribosomal RNA gene suggested that all fell within the Rhizobium etli–Rhizobium leguminosarum group. Strains R11, R49 and R52 all clustered with other identified R. etli strains, while strains R13 and R28 were more distinct. The distinctness of R13 and R28 was supported by physiological characteristics, such as their ability to utilize citrate, erythritol, dulcitol and lactate. Strains R13 and R28 also yielded the highest plant nitrogen content of all isolates.The highly effective strains isolated in this study, in particular strains R13 and R28, are promising candidates for improving crop yields. The data also suggested that these two strains represented a novel sub-group within the R. etli–R. leguminosarum group. As snap bean is a crop of great economic value to Egypt, the identification of highly effective rhizobial strains adapted to Egyptian soils, such as strains R13 and R28, is of great interest.     

Molecular Diversity of Rhizobia Occurring on Native Shrubby Legumes in Southeastern Australia

The structure of rhizobial communities nodulating native shrubby legumes in open eucalypt forest of southeastern Australia was investigated by a molecular approach. Twenty-one genomic species were characterized by small-subunit ribosomal DNA PCR-restriction fragment length polymorphism and phylogenetic analyses, among 745 rhizobial strains isolated from nodules sampled on 32 different legume host species at 12 sites. Among these rhizobial genomic species, 16 belonged to the Bradyrhizobium subgroup, 2 to the Rhizobium leguminosarum subgroup, and 3 to the Mesorhizobium subgroup. Only one genomic species corresponded to a known species (Rhizobium tropici). The distribution of the various genomic species was highly unbalanced among the 745 isolates, legume hosts, and sites. Bradyrhizobium species were by far the most abundant, and Rhizobium tropici dominated among the Rhizobium and Mesorhizobium isolates in the generally acid soils where nodules were collected. Although a statistically significant association occurred between the eight most common genomic species and the 32 hosts, there was sufficient overlap in distributions that no clear specificity between rhizobial genomic species and legume taxa was observed. However, for three legume species, some preference for particular genomic species was suggested. Similarly, no geographical partitioning was found.