Diversity among Bradyrhizobium isolates nodulating yardlong bean and sunnhemp in Guam

AIMS: To isolate and characterize bradyrhizobia that nodulate yardlong bean and sunnhemp in Guam. METHODS AND RESULTS: Bradyrhizobia populations that nodulate yardlong bean and sunnhemp in Guam were examined for genetic diversity and their relatedness to Bradyrhizobium japonicum and B. elkanii reference strains. Genomic DNA of 58 isolates of Bradyrhizobium spp. was hybridized with B. japonicum nodY and B. elkanii nodK genes. Based on the hybridization patterns, the isolates were classified into three nodY-nodK hybridizing groups. Group I comprised the majority of the isolates and hybridized with nodY whereas group II isolates hybridized with nodK. The group III isolates, that did not hybridize with either nodY or nodK, formed nitrogen-fixing nodules on cowpea but did not nodulate soybean. DNA sequence analysis of a 280-bp fragment of the variable region of the 16S rRNA gene of a few group III isolates showed that these isolates were more similar to Bradyrhizobium spp. than to B. japonicum or B. elkanii. CONCLUSIONS: The majority of the isolates nodulating yardlong bean and sunnhemp in Guam are similar to B. japonicum, although some isolates are similar to Bradyrhizobium spp. that nodulate a miscellaneous group of legumes including cowpea. SIGNIFICANCE AND IMPACT OF THE STUDY: Since both yardlong bean and sunnhemp are nodulated by a range of bradyrhizobia, selection of superior strains may be based on nodulation effectiveness on both legumes.     

Phylogenetic relationship of Lotus uliginosus symbionts with bradyrhizobia nodulating genistoid legumes

Lotus species are legumes with potential for pastures in soils with low-fertility and environmental constraints. The aim of this work was to characterize bacteria that establish efficient nitrogen-fixing symbiosis with the forage species Lotus uliginosus. A total of 39 isolates were obtained from nodules of L. uliginosus naturally growing in two different locations of Portugal. Molecular identification of the isolates plus the commercial inoculant strain NZP2039 was performed by REP-PCR, 16S rRNA RFLP, and 16S rRNA, glnII and recA sequence analyses. Limited genetic diversity was found among the L. uliginosus symbionts, which showed a close phylogenetic relationship with the species Bradyrhizobium japonicum. The symbiotic nifH, nodA and nodC gene sequences were closely related with the corresponding genes of various Bradyrhizobium strains isolated from Lupinus and other genistoid legumes and therefore were phylogenetically separated from other Lotus spp. rhizobia. The L. uliginosus bradyrhizobia were able to nodulate and fix nitrogen in association with L. uliginosus, could nodulate Lotus corniculatus with generally poor nitrogen-fixing efficiency, formed nonfixing nodules in Lotus tenuis and Lupinus luteus roots and were unable to nodulate Glycine soja or Glycine max. Thus, L. uliginosus rhizobia seem closely related to B. japonicum biovar genistearum strains.     

Competition among<Emphasis Type="Italic">Bradyrhizobium</Emphasis> strains for nodulation of green gram (<Emphasis Type="Italic">Vigna radiata</Emphasis>): Use of dark-nodule strain

The competitiveness of dual-strain inoculum of Bradyrhizobium strains S24 and GR4 was demonstrated for nodulation of green gram (Vigna radiata). Strain S24 formed pink nodules, GR4 produced visually distinguishable dark-brown nodules. When a mixture of these Bradyrhizobium strains was applied as inoculum, nodules of both pink and dark-brown types were formed on the same root. The strain GR4, which was less competitive than strain S24, was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine to obtain pigment-diverse mutants and six selected mutants were screened for symbiotic parameters. One mutant produced pink nodules and appreciably increased plant dry mass. The competitive ability of this mutant lacking brown pigment was compared with that of strain S24 by using antibiotic resistance markers; it showed increased nodulation competitiveness than its parent strain GR4. The dark-brown nodule-phenotype could be useful in evaluating nodulation competitiveness of "cowpea miscellany" bradyrhizobia in soil where dark-brown nodule-forming strains are not indigenous.     

Strain-specific antisera to identify Thai Bradyrhizobium japonicum strains in preserved soybean nodules

Strain-specific antisera were produced against six Bradyrhizobium japonicum strains using two immunization procedures. These specific antisera were used for detection of bradyrhizobia in preserved soybean nodules. Antisera specific for two of these strains were either conjugated with a fluorescent dye or used with a fluorescent secondary antibody for identification of bradyrhizobia in soybean nodules that were preserved in four different storage conditions. Results show that soybean nodules dried in the oven, stored under room temperature, or at –20 °C are as suitable as fresh nodules for strain identification using fluorescent antisera.     

A Selective Medium for the Isolation and Quantification of Bradyrhizobium japonicum and Bradyrhizobium elkanii Strains from Soils and Inoculants

The ecological examination of members of the family Rhizobiaceae has been hampered by the lack of a selective medium for isolation of root nodule bacteria from soil. A novel non-antibiotic-containing medium has been developed which allows selective isolation of Bradyrhizobium japonicum and B. elkanii strains from soil and inoculants. The medium, BJSM, is based on the resistance of B.japonicum and B. elkanii strains to more than 40 μg of the metals ions Zn²⁺ and Co²⁺ per ml. BJSM does not allow growth of Rhizobium sp. strains. We used BJSM to isolate bacteria from a Hubbard soil and from several commercially prepared soybean inoculants. Ninety-eight percent of the isolates obtained from Hubbard soil nodulated Glycine max cv. Kasota, and between 55 and 95% of the isolates from the commercial inoculants had the ability to nodulate soybeans. Numbers of bradyrhizobia obtained by using BJSM, strain-specific fluorescent antibodies, and the most-probable-number plant infection assay indicated that the three techniques were comparable in quantifying B. japonicum strains in soils and inoculants, although most-probable-number counts were generally 0.5 order of magnitude greater than those obtained by using BJSM. Results of our studies indicate that BJSM is useful for direct isolation and quantification of B. japonicum and B. elkanii from natural soils and inoculants. This medium may prove to be an important tool for autecological and enumeration studies of diverse populations of bradyrhizobia and as a quality control method for soybean inoculants.     

Electrophoretic patterns of lipopolysaccharides and antigenic analysis of soybean bradyrhizobia

Several serological methods have been used for the characterization and identification of soybean bradyrhizobia. However, some problem were non-reactivity of certain strains and cross-reactivity among others. Since lipopolysaccharide (LPS) can often be used in strain identification, the objective was to investigate the antigenic properties and polyacrylamide gel electrophoretic pattern of 12 Brazilian strains of Bradyrhizobium japonicum that nodulate soybean and to compare them to standard strains. The close correlation between the LPS patterns obtained by SDS-PAGE and the serological analysis permitted us to assign the strains to nine groups different or the same as the standard strains.     

一株能在大豆上结瘤的苜蓿中华根瘤菌

苜蓿中华根瘤菌(Sinorhizobium meliloti)XJ96077分离自新疆的苜蓿根瘤中,其原宿主为紫花苜蓿(Medicago sativa)。交叉结瘤试验发现,它既可在苜蓿上又能在大豆上结瘤固氮。DNA(G C)mol％分析表明,XJ96077的DNA(G C)mol％为61．9％,与已报道的根瘤菌属的DNA(G C)mol％范围(59％-64％)相符。DNA同源性分析表明,XJ96077与苜蓿中华根瘤菌USDA1002^T和042BM的同源性分别达到93％和80％,说明XJ96077归属于苜蓿中华根瘤菌。应用绿色荧光蛋白基因标记XJ96077,得到重组菌株XJ96077(G)。将其接种普通紫花苜蓿,通过激光共聚焦荧光显微镜可以检测到标记基因的表达。接种北引1号大豆上,同样可以清楚地观察到标记基因在根瘤中的表达,从而确证了XJ96077能同时在苜蓿和大豆上结瘤。通过不同品种大豆的结瘤试验,发现XJ96077对大豆品种的结瘤能力不同。     

Subgroups of the Cowpea Miscellany: Symbiotic Specificity within Bradyrhizobium spp. for Vigna unguiculata, Phaseolus lunatus, Arachis hypogaea, and Macroptilium atropurpureum

Rhizobia classified as Bradyrhizobium spp. comprise a highly heterogeneous group of bacteria that exhibit differential symbiotic characteristics on hosts in the cowpea miscellany cross-inoculation group. To delineate the degree of specificity exhibited by four legumes in the cowpea miscellany, we tested the symbiotic characteristics of indigenous cowpea bradyrhizobia on cowpea (Vigna unguiculata), siratro (Macroptilium atropurpureum), lima bean (Phaseolus lunatus), and peanut (Arachis hypogaea). The most-probable-number counts of indigenous bradyrhizobia at three sites on Maui, Hawaii, were substantially different on the four hosts: highest on siratro, intermediate on cowpea, and significantly lower on both lima bean and peanut. Bradyrhizobia from single cowpea nodules from the most-probable-number assays were inoculated onto the four hosts. Effectiveness patterns of these rhizobia on cowpea followed a normal distribution but were strikingly different on the other legumes. The effectiveness profiles on siratro and cowpea were similar but not identical. The indigenous cowpea-derived bradyrhizobia were of only moderate effectiveness on siratro and were in all cases lower than the inoculant-quality reference strain. Between 5 and 51% of the bradyrhizobia, depending on site, failed to nodulate peanut, whereas 0 to 32% failed to nodulate lima bean. No significant correlation was observed between the relative effectiveness of the bradyrhizobia on cowpea and their corresponding effectiveness on either lima bean or peanut. At all sites, bradyrhizobia that were ineffective on cowpea but that effectively nodulated lima bean, peanut, or both were found. Eighteen percent or fewer of the bradyrhizobia were as effective on lima bean as the reference inoculant strain; 44% or fewer were as effective on peanut as the reference strain. Only 18% of all cowpea-derived bradyrhizobia tested were able to form N(2)-fixing nodules on both lima bean and peanut. These results indicate the need to measure indigenous bradyrhizobial population characteristics directly with the crop of interest to obtain an accurate assessment of the need to inoculate.     

Variation in the nod gene RFLPs, nucleotide sequences of 16S rRNA genes, Nod factors, and nodulation abilities of Bradyrhizobium strains isolated from Thai Vigna plants

The analysis of nod genes and 16S rRNA gene regions, Nod factors, and nodulation abilities of Brady rhizobium strains isolated from tropical Thai Vigna species is reported. A total of 55 Bradyrhizobium strains isolated from two cultivated and six wild Vigna species growing in central and northern Thailand were evaluated. Thai Vigna spp. Bradyrhizobium strains showed higher levels of nod gene RFLP diversity compared with Thai soybean Brady rhizobium strains or temperate strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii. Analysis of the 16S rRNA gene region using selected strains also suggests a high genetic diversity of the Thai Vigna-Bradyrhizobium association. Based on thin-layer chromatography analysis, Nod factors produced by tropical Thai Vigna spp. Brady rhizobium strains are more diverse than temperate Japanese and US strains of B. japonicum and B. elkanii. Thai Vigna spp. Bradyrhizobium strains showed variation in nodulation ability and affinity, estimated by the number of normal nodules versus green nodules in an inoculation study. There are some Bradyrhizobium-host combinations that could not form any nodules, suggesting that some genetic differentiation has evolved in their host range. However, most of the Thai Vigna spp. Bradyrhizobium strains formed nodules on the cultigens soybean (Glycine max), mungbean (Vigna radiata), azuki bean (Vigna angularis), and cowpea (Vigna unguiculata). This is the first study on Bradyrhizobium strains associated with a range of cultivated and wild Vigna and reveals that these Bradyrhizobium strains are diverse and may provide novel sources of useful variation for the improvement of symbiotic systems.     

一株能在苜蓿上结瘤的费氏中华根瘤菌

费氏中华根瘤菌 (Sinorhizobiumfredii) 0 4 2BS分离自新疆的苜蓿根瘤 ,通过交叉结瘤试验 ,发现它既可在苜蓿上又可在大豆上结瘤固氮。 1 6SrDNAPCR RFLP分析表明 ,0 4 2BS与费氏中华根瘤菌模式菌株USDA2 0 5的 4种限制性酶切图谱完全一致。其G +Cmol%为 60 0 ,与费氏中华根瘤菌USDA2 0 5和USDA1 91的DNA同源性分别为 84 9%和89 6% ,表明 0 4 2BS属于费氏中华根瘤菌。应用绿色荧光蛋白基因标记 0 4 2BS ,得到重组菌株 0 4 2BSG。将其接种保定苜蓿和北引 1号大豆 ,并重新分离出根瘤菌 ,利用激光共聚焦荧光显微镜检测到标记基因的表达 ,从而确证了 0 4 2BS能在苜蓿和大豆上结瘤。而且 ,0 4 2BS对不同苜蓿品种的结瘤能力不同     

Genetic diversity of indigenous Bradyrhizobium nodulating promiscuous soybean [Glycine max (L) Merr.] varieties in Kenya: Impact of phosphorus and lime fertilization in two contrasting sites

While soybean is an exotic crop introduced in Kenya early last century, promiscuous (TGx) varieties which nodulate with indigenous rhizobia have only recently been introduced. Since farmers in Kenya generally cannot afford or access fertilizer or inoculants, the identification of effective indigenous Bradyrhizobium strains which nodulate promiscuous soybean could be useful in the development of inoculant strains. Genetic diversity and phylogeny of indigenous Bradyrhizobium strains nodulating seven introduced promiscuous soybean varieties grown in two different sites in Kenya was assayed using the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of the 16S-23S rDNA intergenic spacer region and 16S rRNA gene sequencing. PCR-RFLP analysis directly applied on 289 nodules using Msp I distinguished 18 intergenic spacer groups (IGS) I–XVIII. Predominant IGS groups were I, III, II, IV and VI which constituted 43.9%, 24.6%, 8.3% 7.6% and 6.9% respectively of all the analyzed nodules from the two sites while IGS group VII, IX, X, XI, XII, XIV, XVI, XVII, XVIII each constituted 1% or less. The IGS groups were specific to sites and treatments but not varieties. Phylogenetic analysis of the 16S rRNA gene sequences showed that all indigenous strains belong to the genus Bradyrhizobium. Bradyrhizobium elkanii, Bradyrhizobium spp and Bradyrhizobium japonicum related strains were the most predominant and accounted for 37.9%, 34.5%, and 20.7% respectively while B. yuanmigense related accounted for 6.9% of all strains identified in the two combined sites. The diversity identified in Bradyrhizobium populations in the two sites represent a valuable genetic resource that has potential utility for the selection of more competitive and effective strains to improve biological nitrogen fixation and thus increase soybean yields at low cost.     

香菇印gpd-Le和ras-Le启动子的功能分析

利用从香菇菌丝体中克隆的启动子片段gpd-Le(613bp)和ras-Le(715bp)分别连接于报告基因gfp(绿色荧光蛋白基因)的上游,构建了启动子功能活性检测表达质粒pLg-gfp和pLr-gfp。采用PEG介导法把表达质粒pLg-gfp和pLr-gfp分别与辅助质粒pCc1001(含有trp1基因)共转化进色氨酸营养缺陷型的灰盖鬼伞粉孢子的原生质体中。经过选择培养基筛选、假定转化子的分子鉴定以及GFP荧光检测。结果表明:香菇gpd-Le启动子在灰盖鬼伞的菌丝中具有较强驱动外源gfp基因表达的活性,在荧光显微镜和共聚焦显微镜下观察到gfp基因表达的绿色荧光。而香菇ras-Le启动子没有检测到有驱动外源gfp基因表达的活性。     

灰树花gpd-Gf启动子的功能分析

利用从灰树花菌丝体中克隆的gpd-Gf（615bp）启动子片段串联于报告基因gfp上游,构建启动子功能活性检测表达质粒pGg-gfp。采用PEG介导法把表达质粒pGg-gfp与辅助质粒pCc1001（含有trp1基因）共转化进色氨酸营养缺陷型的灰盖鬼伞粉孢子的原生质体中。经过选择培养基筛选、假定转化子的分子鉴定以及GFP荧光检测, 结果表明：灰树花gpd-Gf启动子在灰盖鬼伞菌丝中具有较强驱动gfp基因表达的活性,在荧光显微镜和共聚焦显微镜下可以观察到转化子菌丝发出的强烈荧光。     

Obtaining of fluorescent-labeled nodule bacteria strains of wild legumes for their detection in vive and in vitro

A series of expression vectors containing genes of fluorescent proteins TurboGFP and TurboRFP under the phage T5 constitutive promoter regulation, intended for lifetime marking of nodule bacteria is created: a series of vectors based on a broad-host-range replicon BBRI, for marking strains with an expression of reporter gene from a transformed plasmid and a series of vectors based on a plasmid pRL765gfp for marking strains by introduction genes of fluorescent proteins in a bacterial chromosome. It was shown that transformation is the most preferable method of constructions transfer in nodule bacteria cells, as in the presence of mob locus in the vectors necessary for conjugation, exists the possibility of occasional plasmid mobilization and its transition from marked strain cells in other soil bacteria. With application of the created vector constructions we obtained fluorescent tagged strains of Rhizobium sp., Mesorhizobium sp., Ensifer (Sinorhizobium) sp., Bradyrhizobium sp., Phyllobacterium sp., Agrobacterium sp. Also their suitability for experiments in vivo and in vitro is shown.     

In most Bradyrhizobium groups sequence comparison of 16S-23S rDNA internal transcribed spacer regions corroborates DNA-DNA hybridizations

In an extension of a previous small-scale test to assess the use of 16S-23S rDNA internal transcribed spacer (ITS) sequences for rapid grouping of bradyrhizobia, we have sequenced the ITS region of 32 isolates of Bradyrhizobium that had previously been studied using AFLP and DNA-DNA hybridizations. We also included representatives of Afipia and Rhodopseudomonas. Our results indicate that ITS sequences are very diverse among bradyrhizobia. Nevertheless, for most of the bradyrhizobia, the grouping of ITS sequences was in line with AFLP results and DNA-DNA hybridization data. Strains that have at least 95.5% ITS sequence similarity belong to the same genospecies, i.e. they have more than 60% DNA-DNA hybridization values. The ITS sequences can therefore provide a relatively fast way to guide strain identification and aid selection of the reference groups that should be included in DNA-DNA hybridization experiments for precise genotypic identification. The Bradyrhizobium strains isolated from Aeschynomene species showed a much larger diversity in ITS sequences than other bradyrhizobia, possibly as a result of lateral exchange. The above ITS sequence similarity criterion for genospecies therefore does not apply to them, but they can easily be distinguished from other Bradyrhizobium genospecies because they have a distinct tRNA(ala) gene.     

Rhizobitoxine production and symbiotic compatibility of Bradyrhizobium from Asian and North American lineages of amphicarpaea.

Reciprocal inoculations with Bradyrhizobium sp. isolates from the North American legume Amphicarpaea bracteata (L.) Fern. (Phaseoleae-Glycininae) and from a Japanese population of its close relative Amphicarpaea edgeworthii (Benth.) var. japonica were performed to analyze relative symbiotic compatibility. Amphicarpaea edgeworthii plants formed few or no nodules with any North American bradyrhizobial strains isolated from A. bracteata, but all A. bracteata lineages formed effective nitrogen-fixing nodules with Japanese Bradyrhizobium isolates from A. edgeworthii. However, one group of A. bracteata plants (lineage Ia) when inoculated with Japanese bradyrhizobia developed a striking leaf chlorosis similar to that known to be caused by rhizobitoxine. The beta-cystathionase inhibition assay demonstrated that significant amounts of rhizobitoxine were present in nodules formed by these Japanese bradyrhizobia. No North American bradyrhizobial isolate from A. bracteata induced chlorosis on any plants, and the beta-cystathionase assay failed to detect rhizobitoxine in nodules formed by these isolates. The role of rhizobitoxine in A. edgeworthii nodulation development was tested by inoculating plants with a Bradyrhizobium elkanii rhizobitoxine-producing strain, USDA 61, and two mutant derivatives, RX17E and RX18E, which are unable to synthesize rhizobitoxine. Amphicarpaea edgeworthii inoculated with wild-type USDA 61 developed >150 nodules per plant, while plants inoculated with RX17E and RX18E developed fewer than 10 nodules per plant. Thus, efficient nodule development in A. edgeworthii appears to be highly dependent on rhizobitoxine production by Bradyrhizobium strains.     

Expression by Soil Bacteria of Nodulation Genes from Rhizobium leguminosarum biovar trifolii

Gram-negative, rod-shaped bacteria from the soil of white clover-ryegrass pastures were screened for their ability to nodulate white clover (Trifolium repens) cultivar Grasslands Huia and for DNA homology with genomic DNA from Rhizobium leguminosarum biovar trifolii ICMP2668 (NZP582). Of these strains, 3.2% were able to hybridize with strain ICMP2668 and nodulate white clover and approximately 19% hybridized but were unable to nodulate. Strains which nodulated but did not hybridize with strain ICMP2668 were not detected. DNA from R. leguminosarum biovar trifolii (strain PN165) cured of its symbiotic (Sym) plasmid and a specific nod probe were used to show that the relationship observed was usually due to chromosomal homology. Plasmid pPN1, a cointegrate of the broad-host-range plasmid R68.45 and a symbiotic plasmid pRtr514a, was transferred by conjugation to representative strains of nonnodulating, gram-negative, rod-shaped soil bacteria. Transconjugants which formed nodules were obtained from 6 of 18 (33%) strains whose DNA hybridized with that of PN165 and 1 of 9 (11%) strains containing DNA which did not hybridize with that of PN165. The presence and location of R68.45 and nod genes was confirmed in transconjugants from three of the strains which formed nodules. Similarly, a pLAFR1 cosmid containing nod genes from a derivative of R. leguminosarum biovar trifolii NZP514 formed nodules when transferred to soil bacteria.     

Nodulation of Cyclopia spp. (Leguminosae, Papilionoideae) by Burkholderia tuberum

BACKGROUND AND AIMS: Species of the genus Burkholderia, from the Betaproteobacteria, have been isolated from legume nodules, but so far they have only been shown to form symbioses with species of Mimosa, sub-family Mimosoideae. This work investigates whether Burkholderia tuberum strains STM678 (isolated from Aspalathus carnosa) and DUS833 (from Aspalathus callosa) can nodulate species of the South African endemic papilionoid genera Cyclopia (tribe Podalyrieae) and Aspalathus (Crotalarieae) as well as the promiscuous legume Macroptilium atropurpureum (Phaseoleae). METHODS: Bacterial strains and the phylogeny of their symbiosis-related (nod) genes were examined via 16S rRNA gene sequencing. Seedlings were grown in liquid culture and inoculated with one of the two strains of B. tuberum or with Sinorhizobium strain NGR 234 (from Lablab purpureus), Mesorhizobium strain DUS835 (from Aspalathus linearis) or Methylobacterium nodulans (from Crotalaria podocarpa). Some nodules, inoculated with green fluorescence protein (GFP)-tagged strains, were examined by light and electron microscopy coupled with immunogold labelling with a Burkholderia-specific antibody. The presence of active nitrogenase was checked by immunolabelling of nitrogenase and by the acetylene reduction assay. B. tuberum STM678 was also tested on a wide range of legumes from all three sub-families. KEY RESULTS: Nodules were not formed on any of the Aspalathus spp. Only B. tuberum nodulated Cyclopia falcata, C. galioides, C. genistoides, C. intermedia and C. pubescens. It also effectively nodulated M. atropurpureum but no other species tested. GFP-expressing inoculant strains were located inside infected cells of C. genistoides, and bacteroids in both Cyclopia spp. and M. atropurpureum were immunogold-labelled with antibodies against Burkholderia and nitrogenase. Nitrogenase activity was also shown using the acetylene reduction assay. This is the first demonstration that a beta-rhizobial strain can effectively nodulate papilioinoid legumes. CONCLUSIONS: Papilionoid legumes from widely different tribes can be nodulated by beta-rhizobia, forming both indeterminate (Cyclopia) and determinate (Macroptilium) nodules.     

Genetic diversity of symbiotic Bradyrhizobium elkanii populations recovered from inoculated and non-inoculated Acacia mangium field trials in Brazil

Acacia mangium is a legume tree native to Australasia. Since the eighties, it has been introduced into many tropical countries, especially in a context of industrial plantations. Many field trials have been set up to test the effects of controlled inoculation with selected symbiotic bacteria versus natural colonization with indigenous strains. In the introduction areas, A. mangium trees spontaneously nodulate with local and often ineffective bacteria. When inoculated, the persistence of inoculants and possible genetic recombination with local strains remain to be explored. The aim of this study was to describe the genetic diversity of bacteria spontaneously nodulating A. mangium in Brazil and to evaluate the persistence of selected strains used as inoculants. Three different sites, several hundred kilometers apart, were studied, with inoculated and non-inoculated plots in two of them. Seventy-nine strains were isolated from nodules and sequenced on three housekeeping genes (glnII, dnaK and recA) and one symbiotic gene (nodA). All but one of the strains belonged to the Bradyrhizobium elkanii species. A single case of housekeeping gene transfer was detected among the 79 strains, suggesting an extremely low rate of recombination within B. elkanii, whereas the nodulation gene nodA was found to be frequently transferred. The fate of the inoculant strains varied depending on the site, with a complete disappearance in one case, and persistence in another. We compared our results with the sister species Bradyrhizobium japonicum, both in terms of population genetics and inoculant strain destiny.