攀枝花市银合欢根瘤菌遗传多样性

【目的】研究分离自四川攀枝花的银合欢根瘤菌的遗传多样性。【方法】采用联合16S rDNA RFLP和IGS RFLP的综合聚类分析(16S-IGS RFLP)、AFLP及多位点持家基因(16S rDNA,atpD,recA)序列的联合分析对供试银合欢根瘤菌进行研究。【结果】31株未知菌具有15种16S-IGS遗传图谱类型、27种AFLP类型。16S-IGS RFLP结果表明,没有未知菌与Bradyrhizobium的参比菌株聚在一起。在71.4%的相似水平上,31个未知菌按属的水平分成3个分支:S、M和R,分别分布在Sinorhizobium属(28株)、Mesorhizobium属(2株)和Rhizobium属(1株)。S分支的28个菌在84%的相似水平上,16S-IGS RFLP聚类图中构成3个群:群S1、群S2、群S3;在AFLP聚类图中构成9个AFLP群:S1–S9。多位点基因序列表明,代表菌株SCAU215、SCAU231分别与M.Plurifarium、R.huautlense亲缘关系最近。而分布于Sinorhizobium属SCAU222和SCAU228、SCAU213、SCAU216可能代表Sinorhizobium的3个新类群。【结论】攀枝花市银合欢根瘤菌遗传多样性丰富,分布于Sinorhizobium、Mesorhizobium和Rhizobium三个属,且优势类群为Sinorhizobium。     

Genetic diversity of rhizobia from Leucaena leucocephala nodules in Mexican soils

Leucaena species are leguminous plants native to Mexico. Using two L. leucocephala cultivars grown in different soils, we obtained 150 isolates from the nodules. Twelve rDNA types were identified which clustered into groups corresponding to Mesorhizobium, Rhizobium , and Sinorhizobium by restriction fragment length polymorphism (RFLP) of amplified 16S rRNA genes. Types 2, 4, 5, 6, 10, 11, and 12 were distinct from all the defined species. Others had patterns indistinguishable from some recognized species. Most of the isolates corresponded to Sinorhizobium . Forty-one electrophoretic types (ETs) were identified among the isolates based on the different combinations of electrophoretic patterns of 13 metabolic enzymes. ETs were clustered into groups in general agreement with the rDNA types. Diverse plasmid patterns were obtained among the isolates, but common plasmids were observed among most isolates within rDNA types 5, 10, and 11. The symbiotic plasmids were identified among most of the isolates, except for the Mesorhizobium isolates. The affinities of host cultivars for different rhizobial groups and the impact of soil cultivation on the soil populations of rhizobia were analysed from the estimation of isolation frequencies and diversity. The results showed differences in rhizobial populations in cultivated and uncultivated soils and also differences in rhizobia trapped by L. leucocephala cv. Cunningham or Peruvian.     

Symbiotic competence and genetic diversity of Rhizobium strains used as inoculants for alfalfa and berseem clover

To provide the necessary information for strain improvement and the development of competitive strains, characterization of some agronomically important inoculant Rhizobium strains specific for either alfalfa or berseem clover is an ongoing project in this laboratory. Plasmid DNA content and identification of symbiosis-controlling plasmids were previously determined. In this study, to determine their symbiotic competence and their apparent genetic diversity, plant tests and RFLP analysis of total DNAs were conducted. Symbiotic effectiveness varied significantly among the Rh. meliloti strains but not among the berseem clover symbionts. Only one strain, Rh. meliloti ARC 104, was symbiotically ineffective. RFLP analysis using a ribosomal RNA operon probe, with three different enzyme digests, produced four related groups which correlated with plasmid profiles. The use of a nifHD gene probe produced similar results which clustered the berseem clover microsymbionts but more clearly separated two distinct groups of Rh. meliloti. A cosmid clone, from a Rh. leguminosarum biovar trifolii T24 gene library, distinguished each strain. This new information will be useful in identifying and producing improved recombinant strains by plasmid transfer.     

Phylogenetic relationships and host range of Rhizobium spp. that nodulate Phaseolus vulgaris L.

We determined the nucleotide sequences of 16S rRNA gene segments from five Rhizobium strains that have been isolated from tropical legume species. All share the capacity to nodulate Phaseolus vulgaris L., the common bean. Phylogenetic analysis confirmed that these strains are of two different chromosomal lineages. We defined the host ranges of two strains of Rhizobium etli and three strains of R. tropici, comparing them with those of the two most divergently related new strains. Twenty-two of the 43 tested legume species were nodulated by three or more of these strains. All seven strains have broad host ranges that include woody species such as Albizia lebbeck, Gliricidia maculata, and Leucaena leucocephala.     

Nodulation of acacia species by fast- and slow-growing tropical strains of Rhizobium

Thirteen Acacia species were classified into three groups according to effective nodulation response patterns with fast- and slow-growing tropical strains of Rhizobium. The first group nodulated effectively with slow-growing, cowpea-type Rhizobium strains; the second, with fast-growing Rhizobium strains; and the third, with both fast- and slow-growing Rhizobium strains. The Rhizobium requirements of the Acacia species of the second group were similar to those of Leucaena leucocephala.     

Comparison of geographically distant populations of Rhizobium isolated from root nodules of Phaseolus vulgaris

Seventy-two rhizobial strains were isolated from the root nodules of french beans ( Phaseolus vulgaris ). They were sampled from two geographically distant field populations and 18 additional different sites in France. They were characterized by a) plasmid profiles, (b) RFLP analysis of total cellular DNA using various chromosomal and symbiotic gene probes (including nif H from Rhizobium etli bv. phaseoli ) and c) their ability to nodulate a potential alternative host, L. leucocephala. Over half of the isolates were ascribed to Rhizobium leguminosarum bv. phaseoli on the basis of the hybridization analysis, the possession of multiple copies of nif H and their inability to nodulate L. leucocephala. The remaining isolates belonged to 2 groups which were shown to be genomically distinct from R. leguminosarum bv. phaseoli, R. etli bv. phaseoli and R. tropici. Most members of these two groups shared with R. tropici the ability to nodulate L. leucocephala and, for isolates of only one of these groups, the presence of one copy of nif H. Members of each of the 3 taxa were widely distributed in France and circumstantial evidence of pSym transfer between them was shown. R. leguminosarum bv. phaseoli and one of the two novel groups co-occurred within the two geographically distant populations. Individual genotypes were conserved between them. The finding of a third taxon at various other locations indicated additional diversity among rhizobia nodulating beans.     

Multiple copies of nodD in Rhizobium tropici CIAT899 and BR816.

Rhizobium tropici strains are able to nodulate a wide range of host plants: Phaseolus vulgaris, Leucaena spp., and Macroptilium atropurpureum. We studied the nodD regulatory gene for nodulation of two R. tropici strains: CIAT899, the reference R. tropici type IIb strain, and BR816, a heat-tolerant strain isolated from Leucaena leucocephala. A survey revealed several nodD-hybridizing DNA regions in both strains: five distinct regions in CIAT899 and four distinct regions in BR816. Induction experiments of a nodABC-uidA fusion in combination with different nodD-hybridizing fragments in the presence of root exudates of the different hosts indicate that one particular nodD copy contributes to nodulation gene induction far more than any other nodD copy present. The nucleotide sequences of both nodD genes are reported here and show significant homology to those of the nodD genes of other rhizobia and a Bradyrhizobium strain. A dendrogram based on the protein sequences of 15 different NodD proteins shows that the R. tropici NodD proteins are linked most closely to each other and then to the NodD of Rhizobium phaseoli 8002.     

Heterologous exopolysaccharide production in Rhizobium sp. strain NGR234 and consequences for nodule development.

Rhizobium sp. strain NGR234 produces large amounts of acidic exopolysaccharide. Mutants that fail to synthesize this exopolysaccharide are also unable to nodulate the host plant Leucaena leucocephala. A hybrid strain of Rhizobium sp. strain NGR234 containing exo genes from Rhizobium meliloti was constructed. The background genetics and nod genes of Rhizobium sp. strain NGR234 are retained, but the cluster of genes involved in exopolysaccharide biosynthesis was deleted. These exo genes were replaced with genes required for the synthesis of succinoglycan exopolysaccharide from R. meliloti. As a result of the genetic manipulation, the ability of these hybrids to synthesize exopolysaccharide was restored, but the structure was that of succinoglycan and not that of Rhizobium sp. strain NGR234. The replacement genes were contained on a cosmid which encoded the entire known R. meliloti exo gene cluster, with the exception of exoB. Cosmids containing smaller portions of this exo gene cluster did not restore exopolysaccharide production. The presence of succinoglycan was indicated by staining with the fluorescent dye Calcofluor, proton nuclear magnetic resonance spectroscopy, and monosaccharide analysis. Although an NGR234 exoY mutant containing the R. meliloti exo genes produced multimers of the succinoglycan repeat unit, as does the wild-type R. meliloti, the deletion mutant of Rhizobium sp. strain NGR234 containing the R. meliloti exo genes produced only the monomer. The deletion mutant therefore appeared to lack a function that affects the multiplicity of succinoglycan produced in the Rhizobium sp. strain NGR234 background. Although these hybrid strains produced succinoglycan, they were still able to induce the development of an organized nodule structure on L. leucocephala. The resulting nodules did not fix nitrogen, but they did contain infection threads and bacteroids within plant cells. This clearly demonstrated that a heterologous acidic exopolysaccharide structure was sufficient to enable nodule development to proceed beyond the developmental barrier imposed on mutants of Rhizobium sp. strain NGR234 that are unable to synthesize any acidic exopolysaccharide.     

Competitiveness of Rhizobium sp. [Leucaena leucocephala (Lam.) Dewit] strains and their genetically marked derivatives

D.M. SWELIM, L.D. KUYKENDALL, F.M. HASHEM, S.M. ABDEL-WAHAB AND N.I. HEGAZI. 1996. The competitiveness of wild-type strains of Rhizobium sp. ( Leucaena ) and their genetically marked double mutants was examined in mixed infection experiments in the greenhouse. Antibiotic resistance markers were selected for use in strain identification, but these genetic markers apparently lowered both competitiveness and effectiveness, except in the case of strain DS 144/2 where the genetically marked derivative was evidently superior to the wild-type parent strain in effectiveness. Four wild-type strains and their genetically marked derivatives were carefully evaluated using double reciprocal pairs, the results of which nevertheless allowed the formulation of some conclusions. Strains DS 65 and DS 78 were more competitive than strain DS 144/2; only strain DS 78 was more competitive than DS 158; and strains DS 158 and DS 65 were equally competitive. There was no correlation between nodule number and competitiveness. Shoot dry weight and nitrogen mass, as well as nitrogenase activity, decreased with some strain mixtures indicating that relatively ineffective symbioses had formed, as compared with single-strain inoculations using symbiotically competent strains.     

Phenotypic and genotypic analysis of rhizobia isolated from pasture legumes native of Sardinia and Asinara Island

Thirty-five rhizobial strains were isolated from nodules of Lotus edulis, L. ornithopodioides, L. cytisoides, Hedysarum coronarium, Ornithopus compressus and Scorpiurus muricatus growing in Sardinia and Asinara Island. Basic characteristics applied to identification of rhizobia such as symbiotic properties, antibiotic- and salt-resistance, temperate-sensitivities, utilization of different sources of carbon and nitrogen were studied. The results from the 74 metabolic tests were used for cluster analysis of the new rhizobial isolates and 28 reference strains, belonging to previously classified and unclassified fast-, intermediate- and slow-growing rhizobia. All strains examined were divided into two large groups at a linkage distance of 0.58. None of the reference strains clustered with the new rhizobial isolates, which formed five subgroups almost respective of their plant origin. RFLP analysis of PCR-amplified 16S-23S rDNA IGS showed that the levels of similarity between rhizobial isolates from Ornithopus, Hedysarum and Scorpiurus, and the type strains of Rhizobium leguminosarum, Mesorhizobium loti, M. ciceri, M. mediterraneum, Sinorhizobium meliloti and Bradyrhizobium japonicum were not more than 30%. Thus, it can be assumed that these groups of new rhizobial isolates are not closely related to the validly described rhizobial species.     

Classification of the uptake hydrogenase-positive (Hup+) bean rhizobia as Rhizobium tropici.

Phenotypic and genetic characterization indicated that Hup+ bean rhizobial strains are type IIA and type IIB Rhizobium tropici. The Hup+ strain USDA 2840, which did not cluster with either of the two types of R. tropici in a restriction fragment length polymorphism analysis, had electrophoretic patterns of PCR products generated with primers for repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences similar to those of three reference strains of R. tropici type IIA. The Hup+ strain USDA 2738, which clustered with the reference strain of R. tropici IIB in a restriction fragment length polymorphism analysis, had electrophoretic patterns of PCR products generated with primers for repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences more closely resembling those of the reference strains of R. tropici type IIA than those of type IIB. DNA amplification with the Y1 and Y2 primers to generate a portion of the 16S rDNA operon was useful to distinguish R. tropici type IIA strains from other bean rhizobial strains. The phylogenetic position of the type IIA strain of R. tropici USDA 2840, determined from the partial 16S rDNA sequence, indicated a more distant relationship with the type IIB strain of R. tropici CIAT899 than with the as yet unnamed rhizobial species of Leucaena leucocephala, TAL 1145. Therefore, we suggest that it may be appropriate either to separate R. tropici types IIA and IIB into two different species or to identify TAL 1145 to the species level as a third type of R. tropici.     

Variation of clonal, mesquite-associated rhizobial and bradyrhizobial populations from surface and deep soils by symbiotic gene region restriction fragment length polymorphism and plasmid profile analysis

Genetic characteristics of 14 Rhizobium and 9 Bradyrhizobium mesquite (Prosopis glandulosa)-nodulating strains isolated from surface (0- to 0.5-m) and deep (4- to 6-m) rooting zones were determined in order to examine the hypothesis that surface- and deep-soil symbiont populations were related but had become genetically distinct during adaptation to contrasting soil conditions. To examine genetic diversity, Southern blots of PstI-digested genomic DNA were sequentially hybridized with the nodDABC region of Rhizobium meliloti, the Klebsiella pneumoniae nifHDK region encoding nitrogenase structural genes, and the chromosome-localized ndvB region of R. meliloti. Plasmid profile and host plant nodulation assays were also made. Isolates from mesquite nodulated beans and cowpeas but not alfalfa, clover, or soybeans. Mesquite was nodulated by diverse species of symbionts (R. meliloti, Rhizobium leguminosarum bv. phaseoli, and Parasponia bradyrhizobia). There were no differences within the groups of mesquite-associated rhizobia or bradyrhizobia in cross-inoculation response. The ndvB hybridization results showed the greatest genetic diversity among rhizobial strains. The pattern of ndvB-hybridizing fragments suggested that surface and deep strains were clonally related, but groups of related strains from each soil depth could be distinguished. Less variation was found with nifHDK and nodDABC probes. Large plasmids (>1,500 kb) were observed in all rhizobia and some bradyrhizobia. Profiles of plasmids of less than 1,000 kb were related to the soil depth and the genus of the symbiont. We suggest that interacting selection pressures for symbiotic competence and free-living survival, coupled with soil conditions that restrict genetic exchange between surface and deep-soil populations, led to the observed patterns of genetic diversity.     

Vesicular arbuscular mycorrhizal fungi improves establishment of micropropagated Leucaena leucocephala plantlets

Investigations were carried out to achieve cent per cent transplantation success of micropropagated Leucaena leucocephala (a fast growing multipurpose leguminous tree species) plantlets using two vesicular arbuscular mycorrhizal fungi, Glomus fasciculatum and Glomus macrocarpum. Plantlets were obtained by rooting the shoots [obtained through; hypocotyl callus in presence of 10^-5M BAP + 10^-6M NAA; and axillary bud sprouting from cotyledonary and other nodes in presence of 10^-5M BAP, on Gamborg's B5 medium], on half strength B5 medium supplemented with 5×10^-6M IBA. Subsequent to the nodulation of their roots with Rhizobium (strain PRGL 001)in soilrite, these plantlets were tranferred to sterilized garden soil by laying inoculum of either Glomus fasciculatum or Glomus macrocarpum around their roots. Only 20% of the plantlets survived in soils lacking VAM fungus. In contrast, cent per cent of the plantlets of Leucaena leucocephala established very well and showed good growth in VAM inoculated soil. Roots of the later plantlets showed presence of both external and internal hyphae with well formed arbuscules and vesicles confirming the establishment of good mycorrhizal association. These studies convincingly demonstrate that the mycorrhizal association help in successful establishment of tissue culture raised plantlets of Leucaena leucocephala in the field conditions by alleviating the transplantation shock. This revised version was published online in June 2006 with corrections to the Cover Date.     

Distribution and diversity of rhizobia nodulating agroforestry legumes in soils from three continents in the tropics

The natural rhizobial populations of Calliandra calothyrsus, Gliricidia sepium, Leucaena leucocephala and Sesbania sesban were assessed in soils from nine sites across tropical areas of three continents. The rhizobial population size varied from undetectable numbers to 1.8 x 104 cells/g of soil depending on the trap host and the soil. Calliandra calothyrsus was the most promiscuous legume, nodulating in eight soils, while S. sesban nodulated in only one of the soils. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analyses of the 16S rRNA gene and the internally transcribed spacer (ITS) region between the 16S and 23S rRNA genes were used to assess the diversity and relative abundance of rhizobia trapped from seven of the soils by C. calothyrsus, G. sepium and L. leucocephala. Representatives of the 16S rRNA RFLP groups were also subjected to sequence analysis of the first 950 base pairs of the 16S rRNA gene. Eighty ITS groups were obtained, with none of the ITS types being sampled in more than one soil. RFLP analysis of the 16S rRNA yielded 23 'species' groups distributed among the Rhizobium, Mesorhizobium, Sinorhizobium and Agrobacterium branches of the rhizobial phylogenetic tree. The phylogeny of the isolates was independent of the site or host of isolation, with different rhizobial groups associated with each host across the soils from widely separated geographical regions. Although rhizobial populations in soils sampled from the centre of diversity of the host legumes were the most genetically diverse, soil acidity was highly correlated with the diversity of ITS types. Our results support the hypothesis that the success of these tree legumes in soils throughout the tropics is the result of their relative promiscuity (permissiveness) allowing nodulation with diverse indigenous rhizobial types.     

Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium loti strain R7A VirB/D4 type IV secretion system

The symbiosis island of Mesorhizobium loti strain R7A contains genes with strong similarity to the structural vir genes (virB1-11; virD4) of Agrobacterium tumefaciens that encode the type IV secretion system (T4SS) required for T-DNA transfer to plants. In contrast, M. loti strain MAFF303099 lacks these genes but contains genes not present in strain R7A that encode a type III secretion system (T3SS). Here we show by hybridization analysis that most M. loti strains contain the VirB/D4 T4SS and not the T3SS. Strikingly, strain R7A vir gene mutants formed large nodules containing bacteroids on Leucaena leucocephala in contrast to the wild-type strain that formed only uninfected tumour-like structures. A rhcJ T3SS mutant of strain MAFF303099 also nodulated L. leucocephala, unlike the wild type. On Lotus corniculatus, the vir mutants were delayed in nodulation and were less competitive compared with the wild type. Two strain R7A genes, msi059 and msi061, were identified through their mutant phenotypes as possibly encoding translocated effector proteins. Both Msi059 and Msi061 were translocated through the A. tumefaciens VirB/D4 system into Saccharomyces cerevisiae and Arabidopsis thaliana, as shown using the Cre recombinase Reporter Assay for Translocation (CRAfT). Taken together, these results suggest that the VirB/D4 T4SS of M. loti R7A plays an analogous symbiotic role to that of T3SS found in other rhizobia. The heterologous translocation of rhizobial proteins by the Agrobacterium VirB/D4 T4SS is the first demonstration that rhizobial effector proteins are translocated into plant cells and confirms functional conservation between the M. loti and A. tumefaciens T4SS.     

Diversity within Serogroups of Rhizobium leguminosarum biovar viceae in the Palouse Region of Eastern Washington as Indicated by Plasmid Profiles, Intrinsic Antibiotic Resistance, and Topography

Serology, plasmid profiles, and intrinsic antibiotic resistance (IAR) were determined for 192 isolates of Rhizobium leguminosarum biovar viceae from nodules of peas (Pisum sativum L.) grown on the south slope and bottomland topographic positions in eastern Washington State. A total of 3 serogroups and 18 plasmid profile groups were identified. Nearly all isolates within each plasmid profile group were specific for one of the three serogroups. Cluster analysis of IAR data showed that individual clusters were dominated by one serogroup and by one or two plasmid profile groups. Plasmid profile analysis and IAR analysis grouped 72% of the isolates similarly. Most plasmid profile groups and several IAR clusters favored either the south slope or the bottomland topographic position. These findings show that certain intraserogroup strains possess a greater competitiveness for nodulation and/or possess a greater ability to survive in adjacent soil environments.     

Nitrogen fixation ability of exopolysaccharide synthesis mutants of Rhizobium sp. strain NGR234 and Rhizobium trifolii is restored by the addition of homologous exopolysaccharides.

Several transposon Tn5-induced mutants of the broad-host-range Rhizobium sp. strain NGR234 produce little or no detectable acidic exopolysaccharide (EPS) and are unable to induce nitrogen-fixing nodules on Leucaena leucocephala var. Peru or siratro plants. The ability of these Exo- mutants to induce functioning nodules on Leucaena plants was restored by coinoculation with a Sym plasmid-cured (Nod- Exo+) derivative of parent strain NGR234, purified EPS from the parent strain, or the oligosaccharide from the EPS. Coinoculation with EPS or related oligosaccharide also resulted in formation of nitrogen-fixing nodules on siratro plants. In addition, an Exo- mutant (ANU437) of Rhizobium trifolii ANU794 was able to form nitrogen-fixing nodules on white clover in the presence of added EPS or related oligosaccharide from R. trifolii ANU843. These results demonstrate that the absence of Rhizobium EPSs can result in failure of effective symbiosis with both temperate and subtropical legumes.     

Diversity of Plasmid Profiles and Conservation of Symbiotic Nitrogen Fixation Genes in Newly Isolated Rhizobium Strains Nodulating Sulla (Hedysarum coronarium L.)

Forty-five Rhizobium strains nodulating sulla (Hedysarum coronarium L.), isolated from plants grown in different sites in Menorca Island and southern Spain, were examined for plasmid content and the location and organization of nif (nitrogen fixation) and nod (nodulation) sequences. A great diversity in both number and size of the plasmids was observed in this native population of strains, which could be distributed among 19 different groups according to their plasmid profiles. No correlation was found between plasmid profile and geographical origin of the strains. In each strain a single plasmid ranging from 187 to 349 megadaltons hybridized to Rhizobium meliloti nifHD and nodD DNA, and in three strains the spontaneous loss of this plasmid resulted in the loss of the nodulation capacity. In addition to the symbiotic plasmid, 18 different cryptic plasmids were identified. A characteristic cryptic plasmid of >1,000 megadaltons was present in all strains. Total DNA hybridization experiments, with nifHD and portions of nodC and nodD genes (coding for common nodulation functions) from R. meliloti as probes, demonstrated that both the sequence and organization of nif and common nod genes were highly conserved within rhizobia nodulating sulla. Evidence for reiteration of nodD sequences and for linkage of nodC to at least one copy of nodD was obtained for all the strains examined. From these results we conclude that Rhizobium strains nodulating sulla are a homogeneous group of symbiotic bacteria that are closely related to the classical fast-growing group of rhizobia.     

Evidence of an American origin for symbiosis-related genes in Rhizobium lusitanum

Randomly amplified polymorphic DNA (RAPD) analysis was used to investigate the diversity of 179 bean isolates recovered from six field sites in the Arcos de Valdevez region of northwestern Portugal. The isolates were divided into 6 groups based on the fingerprint patterns that were obtained. Representatives for each group were selected for sequence analysis of 4 chromosomal DNA regions. Five of the groups were placed within Rhizobium lusitanum, and the other group was placed within R. tropici type IIA. Therefore, the collection of Portuguese bean isolates was shown to include the two species R. lusitanum and R. tropici. In plant tests, the strains P1-7, P1-1, P1-2, and P1-16 of R. lusitanum nodulated and formed nitrogen-fixing symbioses both with Phaseolus vulgaris and Leucaena leucocephala. A methyltransferase-encoding nodS gene identical with the R. tropici locus that confers wide host range was detected in the strain P1-7 as well as 24 others identified as R. lusitanum. A methyltransferase-encoding nodS gene also was detected in the remaining isolates of R. lusitanum, but in this case the locus was that identified with the narrow-host-range R. etli. Representatives of isolates with the nodS of R. etli formed effective nitrogen-fixing symbioses with P. vulgaris and did not nodulate L. leucocephala. From sequence data of nodS, the R. lusitanum genes for symbiosis were placed within those of either R. tropici or R. etli. These results would support the suggestion that R. lusitanum was the recipient of the genes for symbiosis with beans from both R. tropici and R. etli.