Competition For Nodule Occupancy Research Articles

Comparative genomic analysis of Bradyrhizobium strains with natural variability in the efficiency of nitrogen fixation, competitiveness, and adaptation to stressful edaphoclimatic conditions.

Bradyrhizobium is known for fixing atmospheric nitrogen in symbiosis with agronomically important crops. This study focused on two groups of strains, each containing eight natural variants of the parental strains, Bradyrhizobium japonicum SEMIA 586 (=CNPSo 17) or Bradyrhizobium diazoefficiens SEMIA 566 (=CNPSo 10). CNPSo 17 and CNPSo 10 were used as commercial inoculants for soybean crops in Brazil at the beginning of the crop expansion in the southern region in the 1960s-1970s. Variants derived from these parental strains were obtained in the late 1980s through a strain selection program aimed at identifying elite strains adapted to a new cropping frontier in the central-western Cerrado region, with a higher capacity of biological nitrogen fixation (BNF) and competitiveness. Here, we aimed to detect genetic variations possibly related to BNF, competitiveness for nodule occupancy, and adaptation to the stressful conditions of the Brazilian Cerrado soils. High-quality genome assemblies were produced for all strains. The core genome phylogeny revealed that strains of each group are closely related, as confirmed by high average nucleotide identity values. However, variants accumulated divergences resulting from horizontal gene transfer, genomic rearrangements, and nucleotide polymorphisms. The B. japonicum group presented a larger pangenome and a higher number of nucleotide polymorphisms than the B. diazoefficiens group, possibly due to its longer adaptation time to the Cerrado soil. Interestingly, five strains of the B. japonicum group carry two plasmids. The genetic variability found in both groups is discussed considering the observed differences in their BNF capacity, competitiveness for nodule occupancy, and environmental adaptation.IMPORTANCEToday, Brazil is a global leader in the study and use of biological nitrogen fixation with soybean crops. As Brazilian soils are naturally void of soybean-compatible bradyrhizobia, strain selection programs were established, starting with foreign isolates. Selection searched for adaptation to the local edaphoclimatic conditions, higher efficiency of nitrogen fixation, and strong competitiveness for nodule occupancy. We analyzed the genomes of two parental strains of Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens and eight variant strains derived from each parental strain. We detected two plasmids in five strains and several genetic differences that might be related to adaptation to the stressful conditions of the soils of the Brazilian Cerrado biome. We also detected genetic variations in specific regions that may impact symbiotic nitrogen fixation. Our analysis contributes to new insights into the evolution of Bradyrhizobium, and some of the identified differences may be applied as genetic markers to assist strain selection programs.

Widespread occurrence of Sinorhizobium meliloti strains with a type IV secretion system

Improving symbiotic nitrogen fixation would reduce reliance on synthetic fertilizers, but establishment of effective bacterial strains is hampered due to competition by indigenous, less effective rhizobia. This study investigated the origins, diversity, and competitiveness of Sinorhizobium meliloti (syn. Ensifer meliloti) strains isolated from root nodules of alfalfa (lucerne; Medicago sativa L.) grown in soils that had not been in alfalfa cultivation for over 30 years. Sets of PCR primers were developed to identify Sinorhizobium spp. and to identify strains with virD 4 , the defining gene for a type IV secretion system (T4SS), which has been implicated in increasing strain competitiveness for nodule colonization of M. truncatula, an annual species closely related to alfalfa. A collection of S. meliloti isolates was made from nodules of field-grown plants and from seeds used for establishing field plots that had been inoculated with rhizobia by the manufacturer prior to packaging. Diversity among strains was examined by repetitive element palindromic PCR (rep-PCR) DNA fingerprinting using the BOXA1R primer. Strains originating from the seed inoculant could not be detected in nodules, even in the first year of alfalfa establishment, which were occupied exclusively by indigenous rhizobia originating from soil. Field strains were very diverse within and among field sites, and genetically distinct from inoculant strains. Approximately 33% of field strains were positive for virD 4 , a component of the T4SSb gene cluster, which putatively mobilizes effector proteins across the bacterial cell envelop into the host cell. Strains with virD 4 , the presence of which suggests that they have a functional T4SS, had more rapid nodulation kinetics than did those lacking virD 4 . These results provide additional support for the role of the S. meliloti T4SS in competitiveness for nodule occupancy.

Diversity patterns of Rhizobiaceae communities inhabiting soils, root surfaces and nodules reveal a strong selection of rhizobial partners by legumes.

Current knowledge about rhizobial diversity patterns in non-nodule habitats is scarce, limiting our understanding of basic aspects of rhizobial ecology like competitiveness for nodule occupancy and host effects on community structure. We used a combination of cultivation-dependent and independent approaches to analyse alpha and beta diversity patterns of Rhizobiaceae communities from a conserved seasonally dry tropical forest site in central Mexico and two nearby agricultural fields. Lineage-specific recA amplicon libraries were generated from soil DNA and their sequences compared with those from root surface and nodule isolates recovered in trapping experiments from two native Acacia species and two Phaseolus vulgaris cultivars. Rarefaction analyses revealed that Rhizobiaceae diversity in soils is larger than on root surfaces, and smallest in nodules. A 'rare biosphere'-like distribution of species was found in the three habitats. Multivariate statistical analyses demonstrated that the plant genus exerted a stronger influence than the land-usage regime on the diversity of rhizobia associated with hosts. Rhizobium etli was the dominant Rhizobiaceae found in the soil libraries. It dominated nodulation of Acacia spp. and predominately harboured symbiovar mimosae-like nodC genes. A novel Rhizobium lineage (Rsp1) dominated bean nodulation. Specialist and generalist genotypes for host nodulation were detected in both species.

Exopolysaccharide production in response to medium acidification is correlated with an increase in competition for nodule occupancy.

Sinorhizobium meliloti strains unable to utilize galactose as a sole carbon source, due to mutations in the De-Ley Doudoroff pathway (dgoK), were previously shown to be more competitive for nodule occupancy. In this work, we show that strains carrying this mutation have galactose-dependent exopolysaccharide (EPS) phenotypes that were manifested as aberrant Calcofluor staining as well as decreased mucoidy when in an expR(+) genetic background. The aberrant Calcofluor staining was correlated with changes in the pH of the growth medium. Strains carrying dgoK mutations were subsequently demonstrated to show earlier acidification of their growth medium that was correlated with an increase expression of genes associated with succinoglycan biosynthesis as well as increased accumulation of high and low molecular weight EPS in the medium. In addition, it was shown that the acidification of the medium was dependent on the inability of S. meliloti strains to initiate the catabolism of galactose. To more fully understand why strains carrying the dgoK allele were more competitive for nodule occupancy, early nodulation phenotypes were investigated. It was found that strains carrying the dgoK allele had a faster rate of nodulation. In addition, nodule competition experiments using genetic backgrounds unable to synthesize either succinoglycan or EPSII were consistent with the hypothesis that the increased competition phenotype was dependent upon the synthesis of succinoglycan. Fluorescent microscopy experiments on infected root-hair cells, using the acidotropic dye Lysotracker Red DND-99, provide evidence that the colonized curled root hair is an acidic compartment.

Competition for nodule occupancy between introduced and native strains of Rhizobium leguminosarum biovar trifolii

The aim of this work was to evaluate the competitive ability between Rhizobium leguminosarum bv trifolii strain U204 used as commercial inoculants in Uruguay for Trifolium repens L. and Trifolium pratense L. and two native strains isolated from inoculated pastures of T. pratense. T126 is an efficient nitrogen fixer and a melanin producer strain; T70 is inefficient and a melanin non-producer strain; and U204 is very efficient in both hosts but is a melanin non-producer strain. Competitiveness between the strains was determined in experiments in pots and in growth pouches under controlled conditions. In the last experiment, we evaluated pH of plant nutrient solution and inoculum ratios. Plant dry weight was determined, and the identification of nodule bacteria was done using melanin production and DNA fingerprinting (GTG5-PCR). The U204 symbiotic efficiency was not affected by the co-inoculation with the others two native strains. The T70 strain was a poor competitor when was co-inoculated with one of the effective strains in both experiments. Our results confirmed a “selective nodulation” because an effective symbiosis occurred preferentially over an ineffective one in Trifolium species. The native effective strain competed with U204 for nodule formation in both clovers species, but the nodule occupancy depended on the inoculum ratio. The pH of nutritive solution did not affect competition ability of the studied strains. It may be possible to isolate efficient, competitive, and genetically different native rhizobial strains to be used as inoculant strains for clover pastures in Uruguay. Both (GTG)5-PCR and melanin production were useful methods to identify nodulating bacteria in competition studies.

The diversity of rhizobia nodulating chickpea ( Cicer arietinum) under water deficiency as a source of more efficient inoculants

Chickpea is an important pulse crop grown mainly in the arid and semi-arid regions. The effect of water deficiency on nodulation, biomass production, and competition for nodule occupancy was evaluated in three different soils with two chickpea cultivars, Amdoun I and Chetoui. Two watering regimes were considered; a control that was irrigated three times per week and a water-deficient treatment that was irrigated only one time a week. Results showed that water deficiency significantly decreased the nodule number and the shoot dry weight for both cultivars. Root-nodule bacteria were isolated and characterized by PCR-RFLP of 16S rDNA and nifD-K intergenic spacer. The results show that water deficiency affects the diversity of nodulating rhizobia. The nodulation by Mesorhizobium mediterraneum was reduced while inefficient nodulation by Ensifer meliloti was favoured. In both treatments, chickpea was preferentially nodulated by nifD-K type N6. Analysis for NaCl tolerance showed that most of rhizobia nodulating chickpea under water deficiency are NaCl tolerant. Inoculation with the selected salt-tolerant strain of M. mediterraneum LILM10 increased significantly nodule number and grain yield in the field.

Lack of trehalose catabolism in Sinorhizobium species increases their nodulation competitiveness on certain host genotypes.

The role of host and bacterial genotypes in determining the competitiveness of trehalose utilization mutants of Sinorhizobium meliloti and Sinorhizobium medicae was investigated here. Trehalose utilization mutants of S. meliloti and S. medicae were obtained by mutagenesis of their trehalose utilization gene thuB. The mutant strains and the wild type were coinoculated on three cultivars of alfalfa (Medicago sativa) and two cultivars of Medicago truncatula and assessed for competitiveness in root colonization, and nodule occupancy. The thuB mutants formed more nodules than their parent strains on two of the three alfalfa lines tested and on one of the two M. truncatula lines tested. They were not more competitive on the other alfalfa and M. truncatula lines. Their competitiveness for nodule occupancy did not correlate positively with their ability to colonize these roots but correlated with the extent of thuB induction in the infection threads. Induction of thuB was shown to be dependent on the concentration of trehalose in the environment. These results suggest a direct role for host trehalose metabolism in early plant-symbiont interactions and show that the ability to manage host-induced stresses during infection, rather than the ability to colonize the root, is critical for competitive nodulation.

Competition for Nodule Occupancy Between Introduced Strains of Rhizobium leguminosarum Biovar trifolii and its Influence on Plant Production

Interactions among white clover ( Trifolium repens L.) and selected Rhizobium leguminosarum biovar trifolii strains, and related clover/grass production, were studied in a field experiment during three growing seasons in Iceland. Weather conditions in this country are harsh and rhizobia are rare or absent in the soils. White clover and red fescue ( Festuca rubra L. ‘Leik’) were established in the field in May 1994, using a white clover breeding line (HoKv 9238) which originated from a breeding programme aimed at developing commercial cultivars for cold climates. Rhizobia were initially absent from the field site and clover was inoculated with one of three R. leguminosarum bv. trifolii strains, or a mixture of all three strains. White clover and grass yields were measured during three growing seasons after the establishment year. Nodule occupancy by the three Rhizobium strains was studied by ERIC-PCR DNA fingerprinting. In the mixed inoculum treatment, R. leguminosarum bv. trifolii strain 20-15 occupied 80–90% of the nodules tested, while strain 32-28 was present in up to 3% of the nodules tested. The dominance of strain 20-15 persisted and tended to increase throughout the 4 year experimental period. The introduced rhizobia persisted in high numbers in the soil 1 year after clover was removed and a non-host crop had been grown. ERIC-PCR was an efficient and reliable method by which to identify the Rhizobium strains from cells as well as directly from nodule suspensions. The selected breeding line of white clover persisted well under Icelandic conditions but produced low yield throughout the entire experimental period. Mixed strain inoculation had a positive effect, resulting in both higher white clover yields and higher total plant yields than on plots treated with single strain inoculum.

Competition for Nodule Occupancy Between Introduced Strains of Rhizobium leguminosarum Biovar trifolii and its Influence on Plant Production

Competition for Nodule Occupancy Between Introduced Strains of Rhizobium leguminosarum Biovar trifolii and its Influence on Plant Production

Competition for nodule occupancy on Phaseolus vulgaris by Rhizobium etli and Rhizobium tropici strains can be efficiently monitored in an ultisol during the early stages of growth using a constitutive GUS gene fusion

The GUS gene fusion (pKW107) was transferred into 4 Rhizobium phaseoli strains (KIM5s, CIAT 895, CIAT 7202, CIAT 151) and 1 Rhizobium tropici strain (CIAT 899) in order to determine the nodulation competitiveness of these strains on common bean ( Phaseolus vulgaris) in soil. DNA was fingerprinted with the different mutants to verify correct integration of the glucuronidase gene in the strains' genomes. Co-inoculation trials in growth pouches proved that the GUS + mutant strains were unaltered in their nodulation capabilities. Plant studies in a non-sterile ultisol examined the value of GUS-marked Rhizobium strains for monitoring nodule occupancy under tropical conditions. At 14 and 21 days after planting (DAP), blue nodule color allowed consistently clear and reliable differentiation between nodules formed by GUS + rhizobia and indigenous soil rhizobia. At 30 DAP, however, expression of the GUS trait was inconsistent. Older nodules formed by the GUS + rhizobia were only a faint green after incubation in detection buffer, probably because of the restriction of gusA gene expression in the construct to free-living rhizobia. Nodules formed by GUS-harboring inocula strains were differentiated from ineffective green nodules formed by native rhizobia in 30 DAP nodules in microplates by crushing nodules before adding detection buffer. Data obtained by use of the GUS gene fusion were confirmed by data derived using the ELISA technique and antibiotic resistance markers. The GUS gene fusion was found to be a powerful tool to examine competitiveness of inocula strains in a tropical soil.

Effect of pH on competition for nodule occupancy by type I and type II strains ofRhizobium leguminosarum bv.phaseoli

The effect of soil pH on the competitive abilities of twoRhizobium leuminosarum bv.phaseoli type I and one type II strains was examined in a nonsterile soil system.Phaseolus vulgaris seedlings, grown in unlimed (pH 5.2) or limed (pH 7.6) soil, were inoculated with a single-strain inoculum containing 1 × 106 cells mL−1 of one of the three test strains or with a mixed inoculum (1:1, type I vs. type II) containing the type II strain CIAT 899 plus one type I strain (TAL 182 or CIAT 895). At harvest, nodule occupants were determined. In a separate experiment, a mixed suspension (1:1, type I vs. type II) of CIAT 899 paired with either TAL 182 or CIAT 895 was used to inoculateP. vulgaris seedlings grown in sterile, limed or unlimed soil. The numbers of each strain in the rhizosphere were monitored for 10 days following inoculation. The majority of nodules (> 60%) formed on plants grown in acidic soil were occupied by CIAT 899, the type II strain. This pattern of nodule occupancy changed in limed soil. When CIAT 899 was paired with TAL 182, the type I strain formed 78% of the nodules. The number of nodules formed by CIAT 899 and CIAT 895 (56% and 44%, respectively) were not significantly different. The observed patterns of nodule occupancy were not related to the relative numbers or specific growth rates of competing strains in the host rhizosphere prior to nodulation. The results indicate that soil pH can influence which symbiotype ofR. leguminosarum bv.phaseoli will competitively nodulateP. vulgaris.

Environmental effects on competition for nodule occupancy between introduced and indigenous rhizobia and among introduced strains

Understanding the impact of environmental variables on interstrain competition is important to ensure the successful use of rhizobial inoculant. In eight inoculation trials conducted at five diverse sites on Maui, Hawaii, equal numbers of three serologically distinct strains of effective, homologous rhizobia in a peat-based inoculant were applied to seeds of soybean, bush bean, cowpea, lima bean, peanut, leucaena, clover, and tinga pea. We studied the influence of environmental variables on interstrain competition between applied and indigenous rhizobia and among the three strains comprising the inoculum. Although temperature and soil fertility were correlated with nodule occupancy by inoculant strains in a few cases, the most significant environmental variable controlling their competitive success was the size of the indigenous rhizobial population. Nodule occupancy was best described (r2 = 0.51, p < 0.001) by the equation y = 97.88 − 15.03(log10(x + 1)), where y is percent nodule occupancy by inoculant rhizobia and x is the most probable number of indigenous rhizobia per gram soil. For each legume, one of the three inoculant strains was a poor competitor across sites. Competition between the other two strains varied between sites, but was infrequently related to environmental variables. Results indicated that competitive strains could be selected that perform well across a range of environments. Key words: competition, rhizobial ecology, inoculation response, competitiveness index.

A nolC-lacZ gene fusion in Rhizobium fredii facilitates direct assessment of competition for nodulation of soybean

A constitutively expressed chromosomal gene, nolC, regulates the ability of Rhizobium fredii strain USDA257 to nodulate certain agronomically improved soybean cultivars. Other advanced cultivars, as well as unimproved varieties such as 'Peking', form nitrogen-fixing nodules with parental strain USDA257 and with nolC mutants. We have used 257DH512, a derivative of USDA257 that contains a nolC-lacZ fusion, to directly measure strain competition for nodule occupancy. Seedlings of the soybean cultivar 'Peking' were inoculated with mixtures containing various ratios of 257DH512 and R. fredii USDA208; nodules were harvested 14 days later and stained histochemically with X-gal, a chromogenic substrate for the β-galactosidase product of lacZ. We were able to stain and evaluate batches of nearly 500 nodules in just 4 h, without the use of expensive equipment. Under the conditions of these experiments, USDA208 was considerably more competitive than 257DH512, even when it had been present in the inoculum at a numerical disadvantage. We suggest that gene fusions can be used to greatly simplify the assessment of nodulation competitiveness in rhizobia. Key words: competition, nolC-lacZ fusion, soybean – Rhizobium fredii, nodule occupancy.

Response of Soybean to Inoculation with Efficient and Inefficient Bradyrhizobium japonicum Variants

Soybean [Glycine max (L.) Merr.] plants are grown in soils containing large indigenous bradyrhizobial populations. Individual strains within these populations differ in symbiotic efficiency and competitiveness for nodule occupancy. The purpose of this study was to determine the effect of mixed inocula of an efficient and an inefficient variant of Bradyrhizobim japonicum on symbiotic performance in soybean. Lee soybean was initially inoculated with either an efficient or an inefficient USDA II0 colony morphology variant. The opposite variant was then applied after 0, 2, 4, 8, or 13 d. Delayed inoculation with the efficient variant following the inefficient variant resulted in progressively decreased symbiotic performance. In a subsequent competition experiment, soybean cultivars Lee and Ransom were inoculated with efficient and inefficient variants at ratios of 1:1, 1:10 and 10:1. More nodules were formed by the efficient variant than were expected by chance. Significantly reduced dry weight and whole plant N contents were noted when the inefficient variant was present in more than 50% of the nodules. Split‐root experiments were conducted and either type of variant was capable of inhibiting the other after a 7‐d delay in inoculation. In contrast, when both sides of the split‐root were simultaneously inoculated, nodule numbers were similar; however, nodules formed by the efficient variant were larger. This increase in size indicated a preferential partitioning of photosynthate to the nodules formed by the efficient variant. These results together indicate that N2 fixation is enhanced with increased nodule occupancy by superior variants due to more effective strain‐cultivar interactions.

Impairment of transposon-induced mutants of Rhizobium leguminosarum

Tn 5 mutagenesis was investigated as a technique to genetically mark Rhizobium for soil microcosm studies. Sixteen mutants created by Tn 5 insertion or suicide vector integration were analyzed to determine how the mutations affected several phenotypic traits. These included growth rate in culture, symbiotic effectiveness, and competitiveness for nodule occupancy. Seven of 10 Tn 5-containing strains and 5 of 6 vector-integrate strains were impaired in one or more phenotypic traits in comparison to their parent. These results illustrate the need to carefully characterize genetically marked organisms instead of assuming the marked organism is simply an antibiotic-resistant strain equal to the parent.

Influence of elevation and applied nitrogen on rhizosphere colonization and competition for nodule occupancy by different rhizobial strains on field-grown soybean and common bean

In the absence of indigenous rhizobial populations, the pattern of competition between inoculum strains for nodule occupancy is found to be a stable characteristic, independent of rhizosphere population size, nitrogen application, or elevation. Soybean (Glycine max (L.) Merrill) and common bean (Phaseolus vulgaris L.), inoculated with peat-based rhizobia, were grown with three nitrogen levels at 320- and 150-m sites along an elevational transect on the island of Maui, Hawaii. Rhizosphere soil of 8-day-old plants was examined by immunofluorescence for populations of three strains each of Bradyrhizobium japonicum and Rhizobium leguminosarum bv. phaseoli, which made up the inocula applied to the respective host legumes at planting. Nodules were examined for occupancy by specific strains at two sampling times. Site differences and nitrogen treatment had no significant effect on rhizosphere colonization or nodule occupancy by the three strains. The three inoculum strains colonized their respective host rhizosphere in approximately equal numbers. In soybean, strain TAL 102(USDA 110) occupied most of the nodules, while strain TAL 379 (USDA 1366) was the least competitive in nodule formation in all treatments. In common bean, TAL 182 outcompeted the other two strains in nodule formation, while TAL 1797 (CIAT 899) occupied the least number of nodules. High elevation decreased nodulation by soybean more than that by common bean. Nitrogen application reduced nodule number and mass of both legumes more at the low-elevation than the high-elevation site; the reductions in nodule mass were more pronounced in common bean. The pattern of nodule occupancy remained stable regardless of the differences in nodule number and mass between elevations, N levels, and sampling times. Key words: rhizobial ecology, rhizosphere colonization, interstrain competition, immunofluorescence, legumes.

Root-temperature effects on competition for nodule occupancy between two Rhizobium meliloti strains

Axenically grown alfalfa (Medicago sativa L. var. Peace) was simultaneously inoculated with Canadian commercial Rhizobium meliloti strains NRG-185 and BALSAC. The plants were grown for 7 weeks in sealed units at five different root temperatures (8°, 13°, 17°, 21°, and 25°C) and at a relatively constant air temperature (24°–30°C). Nodule occupancy by each strain was determined by enzyme-linked immunosorbent assay (ELISA). Nitrogenase activity, nodule fresh weight, and plant dry weight were also measured. The lowest root-temperature regime (8°C) resulted in substantially lower nodule numbers and weights, and plant dry weights, than the higher temperature regimes. Development of nitrogenase activity was completely inhibited at 8°C. The immunoassay of nodule-strain occupancy showed markedly different strain-nodulation responses to the various root-temperature regimes. At 8°C, 63% of nodules were occupied by both strains. Dual strain occupancy decreased from 63% to 2% with increasing root-growth temperature, while the proportion of nodules containing only strain NRG-185 increased from 9% to 75%. Nodules containing only strain BALSAC remained relatively constant at 25% from 8° to 21°C, decreasing slightly at 25°C. The results suggest that root-environment temperatures during the period of nodule formation may have major differential effects on the success of competing rhizobial strains. If this is so, then selection of Rhizobium strains with enhanced low-temperature nodulation capabilities should be possible.

Response of soybean [ Glycine max (L.) merrill.] to Bradyrhizobium japonicum inoculation in three soils in ghana

Three antigenically distinguishable Bradyrhizobium japonicum streptomycin-resistant mutant strains (SRJ-S, S14-S and SX-S) were evaluated in three soil series in Ghana (Tikobo, pH 4.3; Amo, pH 5.4; Akuse, pH 6.1), for nodulating ability and competitiveness for nodule occupancy on soybean var. Williams. Few nodules developed on plants grown in the uninoculated soils. The most probable number counts on sand-grown soybean indicated 23, 280 and 330 native B. japonicum cells g −in the Tikobo, Akuse and Amo soils, respectively. Inoculation with single or mixed strain combinations of SRJ-S. S14-S and SX-S, at 10 5 cells g −1 soil greatly improved nodulation (>10-fold), N accretion (50–470%) and shoot dry weight (40–90%). When independently inoculated into soil, these strains formed between 75–100% of the soybean nodules, with no significant nodulation differences between any of the single strain inoculation treatments in each soil. However, strain identification from nodules formed by mixed strain inoculation treatments (in population ratios of 1:1 or 1:1:1) revealed marked variations in competitiveness between the three strains for nodule occupancy. Nodules formed by the most competitive strain, SRJ-S (obtained from Kenya), were also the most effective in N 3 fixation (as measured by the total N in plants). Of the locally isolated strains, S14-S was more competitive than SX-S. The nodulation, N content and dry matter yield results showed that, in general, mixed strain inoculation resulted in similar effects as those by the best individual in a particular strain-combination.