Competitiveness of indigenous populations of Bradyrhizobium japonicum serocluster 123 as determined using a root-tip marking procedure in growth pouches

Soil Bradyrhizobium populations limit nodule occupancy of soybean by symbiotically-superior inoculant strains throughout much of the American midwest. In this study, the competitiveness of indigenous populations of B. japonicum serocluster 123 from Waukegan and Webster soils was evaluated in growth pouches using a root-tip marking procedure. The native rhizobia were from soils incubated 0–8 h in soybean root exudate (SRE) or plant nutrient solution (PNS) prior to inoculation. Populations of serocluster 123 strains in soil and nodule occupancy by these strains were assessed using fluorescent antibodies prepared against B. japonicum USDA 123. There were no significant differences in populations that came from SRE or PNS incubated soils: both populations increased in number over the incubation period. Nodule occupancy by both populations in growth pouches was similar to that previously encountered in field studies with these two soils. With the Waukegan soil, the serocluster 123 population dominated nodulation forming 69 and 62% of taproot nodules above and below the root tip mark, respectively. However, for the more alkaline Webster soil, serocluster 123 strains were much less competitive, producing only 9 and 13%, respectively, of the nodules formed above and below the root tip mark. In growth pouches, soil populations of bradyrhizobia from the Webster soil produced significantly more nodules than those from the Waukegan soil, but both strains and a pure culture of USDA 110 had a similar distribution of nodules.     

Competitive Ability and Efficiency in Nodule Formation of Strains of Bradyrhizobium japonicum

In the American Midwest, superior N₂-fixing inoculant strains of Bradyrhizobium japonicum consistently fail to produce the majority of nodules on the roots of field-grown soybean. Poor nodulation by inoculant strains is partly due to their inability to stay abreast of the expanding soybean root system in numbers sufficient for them to be competitive with indigenous bradyrhizobia. However, certain strains are noncompetitive even when numerical dominance is not a factor. In this study, we tested the hypothesis that the nodule occupancy achieved by strains is related to their nodule-forming efficiency. The nodulation characteristics and competitiveness of nine strains of B. japonicum were compared at both 20 and 30°C. The root tip marking technique was used, with the nodule-forming efficiency of each strain estimated from the average position of the uppermost nodule and the number of nodules formed above the root tip mark. The competitiveness of the nine strains relative to B. japonicum USDA 110 was determined by using immunofluorescence to identify nodule occupants. The strains differed significantly in competitiveness with USDA 110 and in nodulation characteristics, strains that were poor competitors usually proving to be inferior in both the average position of the uppermost root nodule and the number of nodules formed above the root tip mark. Thus, competitiveness was correlated with both the average position of the uppermost nodule (r = 0.5; P = 0.036) and the number of nodules formed above the root tip mark (r = 0.64; P = 0.005), while the position of the uppermost nodule was also correlated to the percentage of plants nodulated above the root tip mark (r = 0.81; P < 0.001) and the percentage of plants nodulated on the taproot (r = 0.67; P = 0.002).     

Nodulation and Competition for Nodulation of Selected Soybean Genotypes among Bradyrhizobium japonicum Serogroup 123 Isolates

Twenty recently obtained field isolates of Bradyrhizobium japonicum serogroup 123 were tested for their nodule mass production on the standard commercial soybean (Glycine max (L.) Merr. cv. Williams) and on two soybean plant introduction (PI) genotypes previously determined to restrict nodulation by strain USDA 123. Four of the field isolates showed similar restricted nodulation on the two genotypes, while all 20 isolates produced a normal amount of nodules on G. max cv. Williams. Serological analyses with adsorbed fluorescent antibodies showed that members of the 123 serotype ranked low in nodulation of the two PIs, in contrast to members of serotypes 127 and 129. Competition studies on the PIs indicated that isolates which were restricted were not competitive for nodule occupancy against strain USDA 110. However, unrestricted isolates of serogroup 123 were very competitive against USDA 110. On G. max cv. Williams, all serogroup 123 isolates tested were very competitive against USDA 110.     

Competition of Rhizobium japonicum Strains in Early Stages of Soybean Nodulation

The effects of preexposure of soybean (Glycine max L. Merrill) roots to Rhizobium japonicum strains and subsequent establishment of other strains in the nodules were investigated by using combinations of effective strains (USDA 110 and USDA 138) and effective-ineffective strains (USDA 110 and SM-5). Strain USDA 110 was a better competitor than either USDA 138 or SM-5 on cultivars Lee and Peking. However, when either of the two less-competitive strains was inoculated into 2-day-old seedlings before USDA 110 was, their nodule occupancy increased significantly on both cultivars. With USDA 138 as the primary inoculum and USDA 110 delayed for 6, 48, and 168 h, the incidence of USDA 138 nodules increased on cultivar Peking from 6% (at zero time) to 28, 70, and 82% and on cultivar Lee from 17% (at zero time) to 32, 88, and 95% for the three time delays, respectively. Preexposure of 2-week-old roots of cultivar Lee to USDA 138 had essentially the same effect: the incidence of USDA 138 nodules increased from 23% at zero time to 89 and 97% when USDA 110 was delayed for 24 and 72 h, respectively. When the ineffective strain SM-5 was used as the primary inoculum, followed by USDA 110 72 h later, the percentage of nodules containing SM-5 increased from 7 to 76%. These results indicate that the early events in the nodulation process of soybeans are perhaps the most critical for competition among R. japonicum strains.     

Genistein preincubation of Bradyrhizobium japonicum cells improves strain competitiveness under greenhouse,but not field conditions

In the soybean-B. japonicum symbiosis, genistein has been identified as one of the major compounds in soybean seed and root exudates responsible for inducing expression of the B. japonicum nodYABC operon. In this study, we have tested the possibility that genistein treatment prior to inoculation can increase the competitiveness of the treated B. japonicum strain under both greenhouse and field conditions. Two mutants of the two B. japonicum strains each with a different antibiotic resistant marker were selected. They were tested with one or the other treated with genistein. The results showed genistein treated mutants had higher levels of nodule occupancy than the untreated mutant or parent strain under greenhouse conditions. Mutants from 532C had higher nodule occupancies than mutants from USDA110, especially at 15 °C. In the more complex field environment, genistein treated mutants formed fewer nodules than the untreated mutants. The contradictory results of strain competitiveness for greenhouse and field experiments are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

N2 fixation by soybean-Bradyrhizobium combinations under acidity,low P and high Al stresses

Five strains of Bradyrhizobium japonicum (USDA 6, 110, 122, 138, and 143) were screened in cell culture for tolerance to acidity (pH 4.2, 4.4, and 4.6) and Al (0, 3, 4, 5, and 6 mg L^–1) under low P conditions. Each strain was later grown in association with seven soybean [Glycine max. (L) Merr.] cultivars which were also screened for tolerance to the same stresses in nutrient culture to determine which soybean-Bradyrhizobium combinations would establish the most effective symbiotic N₂ fixing relationships. Results indicated that strains USDA 110 and 6 were more tolerant than USDA 122, 138 and 143 with USDA 110 being the most tolerant. Acidity appeared to be the more severe stress; but even when strains showed tolerance to the stresses, cell numbers were significantly reduced. This suggests that colonization of soils and soybean roots can be adversely affected under similar conditions in the field which may result in reduced nodulation. The strains found to be more tolerant to the stresses were more effective N₂ fixers in symbiosis with all soybean cultivars, with USDA 110 being definitely superior. The association between the more tolerant strains and cultivars had the largest nitrogenase activity. Further studies on the inclusion of tolerant Bradyrhizobium strains in inoculum used on tolerant soybean cultivars in the field are warranted.     

Regulation of nodule formation in soybean-Bradyrhizobium symbiosis is controlled by shoot or/and root signals

Two strains of Bradyrhizobium japonicum wereevaluated with five commercial cultivars of soybean(Clark, Crauford, Davis, Centaur, and Nessen) and onehypernodulating mutant NOD1-3. The hypernodulatingNOD1-3 produced 30–50 times more nodules thancommercial cultivars either inoculated with B.japonicum strain USDA 123 or RCR 3409. The currentexperiments were extended to determine if therestricted nodulation of commercial cultivars could be overcome by grafting them to a hypernodulated shoot (NOD1-3). Grafting of NOD1-3 shoots to Clark and Davis roots induced hypernodulation on roots of Clark and Davis but did not enhance nodulation when grafted onto the roots of Crauford, Centaur, and Nessen. The shoots of Clark, Davis, Centaur and Nessen significantlyinhibited nodule formation on the root of NOD1-3,while Crauford shoots did not alter nodule formationon the roots of NOD1-3 as compared with self-grafts ofNOD1-3. It appears that the shoot of NOD1-3 has theability to alter autoregulatory control of nodulationof Clark and Davis cultivars, but did not withCrauford, Centaur and Nessen. The results suggestedthat the regulation of nodulation in soybean cultivarsClark and Davis is controlled by the shoot factors,while the Crauford was root controlled.Reciprocal-grafts between NOD1-3 and Centaur or Nessenindicate that both shoot and root factors involved inregulation of nodulation and the regulation ofnodulation did not depend on bradyrhizobial strains. Isoflavonoid analyses from extracts of grafted plantsshowed that NOD1-3 shoots had markedly higher rootisoflavonoid concentrations in roots of both Clark andNOD1-3. The shoot control of hypernodulation may becausally related to differential root isoflavonoidlevels, which are also controlled by the shoot. Thecurrent work was extended to investigate the effect ofapplication of an isoflavonoid (daidzein) on nodulationand nitrogen fixation of soybean cultivars Clark andCentaur as well as in vitro growth of Bradyrhizobium japonicum. Application of theisoflavonoid (daidzein) significantly enhanced thenodulation and nitrogenase activity of Clark but notof Centaur indicating that this character is notrelated to isoflavonoids. Therefore, autoregulationin Clark and Centaur plants may be separate events inlegume-rhizobia symbiosis and regulated by differentkinds of signals. Addition of daidzein to yeastmannitol broth medium promoted the growth of B.japonicum strain USDA 123 and RCR 3409. It seemsthat this compound is able to help the nodulation ofsoybean cv Clark by a Bradyrhizobium strain. Understanding the signaling pathways between rhizobiaand their host plants may allow modifications of thisinteraction to improve symbiotic performance.     

Host Plant Effects on Nodulation and Competitiveness of the Bradyrhizobium japonicum Serotype Strains Constituting Serocluster 123

Strains in Bradyrhizobium japonicum serocluster 123 are the major indigenous competitors for nodulation in a large portion of the soybean production area of the United States. Serocluster 123 is defined by the serotype strains USDA 123, USDA 127, and USDA 129. The objective of the work reported here was to evaluate the ability of two soybean genotypes, PI 377578 and PI 417566, to restrict the nodulation and reduce the competitiveness of serotype strains USDA 123, USDA 127, and USDA 129 in favor of the highly effective strain CB1809 and to determine how these soybean genotypes alter the competitive relationships among the three serotype strains in the serocluster. The soybean genotypes PI 377578 and PI 417566 along with the commonly grown cultivar Williams were planted in soil essentially free of soybean rhizobia and inoculated with single-strain treatments of USDA 123, USDA 127, USDA 129, or CB1809 and six dual-strain competition treatments of USDA 123, USDA 127, or USDA 129 versus CB1809, USDA 123 versus USDA 127, USDA 123 versus USDA 129, and USDA 127 versus USDA 129. PI 377578 severely reduced the nodulation and competitiveness of USDA 123 and USDA 127, while PI 417566 similarly affected the nodulation and competitiveness of USDA 129. Thus, the two soybean genotypes can reduce the nodulation and competitiveness of each of the three serocluster 123 serotype strains. Our results indicate that host control of restricted nodulation and reduced competitiveness is quite specific and effectively discriminates between B. japonicum strains which are serologically related.     

Exclusion of inefficient Bradyrhizobium japonicum serogroups by soybean genotypes

Soybean [Glycine max (L.) Merr.] forms a symbiosis with serogroups of Bradyrhizobium japonicum that differ in their dinitrogen fixing abilities. The objectives of this study were to identify soybean genotypes that would restrict nodulation by relatively inefficient serogroups indigenous to a large portion of the southeastern USA, and then characterize the nodulation responses of selected genotypes with specific bradyrhizobial strains under controlled conditions. From field screening trials followed by controlled single and competitive inoculations of serogroups USDA 31, 76 and 110, twelve soybean genotypes out of 382 tested were identified with varying levels of exclusion abilities. Soybean nodule occupancies and nodulation characteristics were influenced by plant genotype, environment (i.e. field or greenhouse), bradyrhizobial serogroup, and location of nodules (i.e. tap or lateral root). The cultivar Centennial sustains high seed yields even though it nodulates to a high degree with the inefficient serogroup USDA 31. In contrast, data from the released cultivars Braxton, Centennial and Coker 368 indicate that they may have been selected to exclude the inefficient serogroup USDA 76 from their tap root nodules, possibly contributing to high seed yield.     

Response of introduced Bradyrhizobium strains infectinga promiscuous soybean cultivar

Two field experiments were established to assess the competitiveness of foreign bradyrhizobia in infecting the promiscuous soybean cultivar TGX 536-02D. Seeds were inoculated with antibiotic mutants of the bradyrhizobia strains before planting after land preparation. Soybean plants were harvested at pre-determined days after planting for estimating nodule number, nodule dry weight, nodule occupancy, shoot dry weight and seed yield. Results show that nodule number and dry weight significantly increased and showed great variability at 84 days after planting (DAP), probably due to differences in the ability of inoculant bradyrhizobia to form nodules with the soybean cultivar TGX 536-02D. Increased shoot dry weight, %N, total N and seed yield were a result of increased nodulation by the effective and competitive inoculant Bradyrhizobium strains. Strain USDA 110 occupied the highest percentage of nodule sites because it was more competitive than the other Bradyrhizobium strains. These results show that there was high potential for increasing growth and seed yield of the promiscuous soybean cultivar TGX 536-02D by inoculation with foreign Bradyrhizobium strains.     

Influence ofGlycine max nodulation on the persistence in soil of a genetically markedBradyrhizobium japonicum strain

Since competition with indigenous strains limits nodule occupancy by bacteria applied to seeds, the ecology of Bradyrhizobium inoculum strains used for soybean is of concern. A genetically marked strain,B. japonicum I-110 ARS, was directly enumerated from soil on selective medium. A clear long-term positive influence of even limitedGlycine max nodulation was shown by comparisons of population densities obtained with or without plant removal prior to nodule senescence in the first year and with an incompatible as well as a compatible soybean variety after 5 years.     

Survival and Competitiveness of Bradyrhizobium japonicum Strains 20 Years after Introduction into Field Locations in Poland

It was previously demonstrated that there are no indigenous strains of Bradyrhizobium japonicum forming nitrogen-fixing root nodule symbioses with soybean plants in arable field soils in Poland. However, bacteria currently classified within this species are present (together with Bradyrhizobium canariense) as indigenous populations of strains specific for nodulation of legumes in the Genisteae tribe. These rhizobia, infecting legumes such as lupins, are well established in Polish soils. The studies described here were based on soybean nodulation field experiments, established at the Poznań University of Life Sciences Experiment Station in Gorzyń, Poland, and initiated in the spring of 1994. Long-term research was then conducted in order to study the relation between B. japonicum USDA 110 and USDA 123, introduced together into the same location, where no soybean rhizobia were earlier detected, and nodulation and competitive success were followed over time. Here we report the extra-long-term saprophytic survival of B. japonicum strains nodulating soybeans that were introduced as inoculants 20 years earlier and where soybeans were not grown for the next 17 years. The strains remained viable and symbiotically competent, and molecular and immunochemical methods showed that the strains were undistinguishable from the original inoculum strains USDA 110 and USDA 123. We also show that the strains had balanced numbers and their mobility in soil was low. To our knowledge, this is the first report showing the extra-long-term persistence of soybean-nodulating strains introduced into Polish soils and the first analyzing the long-term competitive relations of USDA 110 and USDA 123 after the two strains, neither of which was native, were introduced into the environment almost 2 decades ago.     

Enhanced nodulation of soybean by Bradyrhizobium in the presence ofPseudomonas fluorescens

Experiments were undertaken to determine the effect ofPseudomonas fluorescens on nodulation of soybean by two strains ofBradyrhizobium japonicum, USDA I-110 and 61A76.Pseudomonas fluorescens can enhance the nodulation ability ofB. japonicum. Preincubation ofB. japonicum withP. fluorescens before inoculation further increased the level of nodulation.     

Bradyrhizobium japonicum-Environment Interactions: Nodulation and Interstrain Competition in Soils along an Elevational Transect

The effects of temperature and soil type on interstrain competition of Bradyrhizobium japonicum and on nodulation and nitrogen accumulation in five soybean varieties belonging to four maturity groups were investigated at three sites devoid of soybean rhizobia along an elevational transect in Hawaii. Competition patterns of the three B. japonicum strains were unaffected by soil type or soil temperature. Strain USDA 110 was the best competitor, occupying on the average 81 and 64% of the nodules in the field and greenhouse experiments, respectively. Strain USDA 138 was the least successful in the field (4%), although it formed 34% of the nodules in the greenhouse. Nodule occupancy by B. japonicum strains was found to be related to soybean maturity group. Strain USDA 110 formed 61, 71, 88, 88, and 98% of the nodules in the field on Clay (00), Clark (IV), D68-0099 (VI), N77-4262 (VI), and Hardee (VIII), respectively. Strain USDA 136b formed few nodules on Hardee, an Rj2 soybean variety incompatible with that strain, in both experiments. Nodule number and weight at the 1,050-m site were reduced to 41 and 27%, respectively, of those at the 320-m site because of the decrease in temperature. Nodule number increased with increasing maturity group number at each site; however, there was not a corresponding increase in nodule weight. Nitrogen accumulation decreased from 246 mg of N per plant at the lowest elevation site to 26 mg of N per plant at the highest elevation. While soil type and temperature had no effect on strain competition, temperature had a profound influence on nodule parameters and plant growth.     

Competition among Rhizobium leguminosarum bv. phaseoli strains for nodulation of common bean.

Six effective Rhizobium leguminosarum bv. phaseoli strains were examined for nodulation competitiveness on common bean (Phaseolus vulgaris L.), using all possible two-strain combinations of inoculum. Nodule occupancy was determined with strain-specific fluorescent antibodies. The strains were divided into three groups according to their overall competitive abilities on pole bean cv. Kentucky Wonder and bush bean cv. Bountiful. Strains TAL 182 and TAL 1472 were highly competitive (greater than 70% nodule occupancy); strains KIM-5, Viking 1, and CIAT 899 were moderately competitive (approximately 50% nodule occupancy); and strain CIAT 632 was poorly competitive (less than 5% nodule occupancy). The competitiveness of the six strains was similar on the two host cultivars. The proportion of competing strains in the inoculum influenced the nodule occupancy of the highly competitive and moderately competitive strains, but not that of the poorly competitive strain. Two outstanding strains (TAL 182 and TAL 1472) were identified as ideal model strains for molecular and genetic studies on nodulation competitiveness.     

Short communication: Screening of native and foreign Bradyrhizobium japonicum strains for high N2 fixation in soybean

Four local rhizobia isolates selected after two screening experiments and five USDA Bradyrhizobium japonicum strains were estimated for N₂ fixation in soybean using the ¹⁵N isotope dilution technique. Strain USDA 110 was superior to the local isolates in nodulation and N₂ fixation when inoculated onto soybean cv TGX 1497-ID in a Nigerian soil and could therefore be used as an inoculant for enhanced N₂ fixation in soybean in Nigeria.     

Characterization ofR. fredii QB1130, a strain effective on commercial soybean cultivars

Summary Two rhizobial strains (QB1130 and C3A) from northeast China were identified asRhizobium fredii on the basis of growth rate, media acidification and growth on a wide range of carbon substrates. The strains were shown to be distinct from USDA 191 on the basis of plasmid number and size. Bothnif and commonnod genes were located on the 295 kb plasmid of strains QB1130 and USDA 191, while onlynif genes were identified on this plasmid in C3A. When used to inoculate four commercial soybean (Glycine max) cultivars, one of the strains (C3A) was found to be ineffective, while the other (QB1130) was at least as effective as USDA 191, a strain ofR. fredii reported to be widely effective on North American cultivars of soybean. Further, QB1130 was capable of more effective nodulation of cowpea or the uncultivated soybean line, Peking, than either USDA 191 or the slow-growingBradyrhizobium japonicum USDA 16. Strain QB1130 should be useful for studies directed at improving symbiotic performance in soybean, or for studies of the comparative physiology and genetics of FG and SG strains on a single host.     

Soybean response to nodulation by bradyrhizobia differing in rhizobitoxine phenotype

Rhizobitoxine-producing (RT⁺) strains of Bradyrhizobium japonicum, differing in their abilities to induce foliar chlorosis with ‘Forrest’ soybean (Glycine max [L.] Merr.), were evaluated for effects on short term shoot productivity, nodulation, N₂ fixation, and nodule protein production under greenhouse conditions. Soybeans were singly inoculated with washed suspensions of (Group II) USDA strains 31, 46, 76, 94, 110, 123 or 130. Strains USDA 110 and USDA 123 (Group I/Ia) were included as RT-controls. The plants were cultured in the absence of combined N in horticultural-grade vermiculite for 49 days. Beginning 21 days after planting, plants were evaluated weekly for chlorophyll, leaf protein and biomass accumulation, nodular contents of leghemoglobin, soluble protein and RT, and total shoot N content. Rhizobitoxine was detected in nodules of all RT⁺ strains with the exception of USDA 31. However, only USDA 76 and USDA 94 produced both quantifiable concentrations of RT and symptoms of RT-induced chlorosis. Coincident with moderate to severe chlorosis were reductions in chlorophyll concentrations, shoot and nodule dry weight, leaf protein and total N₂ fixation. During extended periods of severe chlorosis, reductions in Lb and soluble nodular protein were observed. Based on carbon accumulation, all non-chlorotic treatments were statistically more productive than the chlorotic treatments. Similarly, non-chlorotic Group II treatments tended to fix less carbon relative to the RT-Group I/Ia controls, although these differences were not statistically significant. The results of this study suggest that, in the absence of discernable foliar chlorosis, the effect of RT⁺ (Group II) nodulation on short term soybean productivity is minimal. Published as Miscellaneous Paper No. 1439 of the Delaware Agricultural Experiment Station. Published as Miscellaneous Paper No. 1439 of the Delaware Agricultural Experiment Station.     

Symbiotic Effectiveness and Host-Strain Interactions of Rhizobium fredii USDA 191 on Different Soybean Cultivars

Nodulation, acetylene reduction activity, dry matter accumulation, and total nitrogen accumulation by nodulated plants growing in a nitrogen-free culture system were used to compare the symbiotic effectiveness of the fast-growing Rhizobium fredii USDA 191 with that of the slow-growing Bradyrhizobium japonicum USDA 110 in symbiosis with five soybean (Glycine max (L.) Merr.) cultivars. Measurement of the amount of nitrogen accumulated during a 20-day period of vegetative growth (28 to 48 days after transplanting) showed that USDA 110 fixed 3.7, 39.1, 4.6, and 57.3 times more N₂ than did USDA 191 with cultivars Pickett 71, Harosoy 63, Lee, and Ransom as host plants, respectively. With the unimproved Peking cultivar as the host plant, USDA 191 fixed 3.3 times more N₂ than did the USDA 110 during the 20-day period. The superior N₂ fixation capability of USDA 110 with the four North American cultivars as hosts resulted primarily from higher nitrogenase activity per unit nodule mass (specific acetylene reduction activity) and higher nodule mass per plant. The higher N₂-fixation capability of USDA 191 with the Peking cultivar as host resulted primarily from higher nodule mass per plant, which was associated with higher nodule numbers. There was significant variation in the N₂-fixation capabilities of the four North American cultivar-USDA 191 symbioses. Pickett 71 and Lee cultivars fixed significantly more N₂ in symbiosis with USDA 191 than did the Harosoy 63 and Ransom cultivars. This quantitative variation in N₂-fixation capability suggests that the total incompatibility (effectiveness of nodulation and efficiency of N₂ fixation) of host soybean plants and R. fredii strains is regulated by more than one host plant gene. These results indicate that it would not be prudent to introduce R. fredii strains into North American agricultural systems until more efficient N₂-fixing symbioses between North American cultivars and these fast-growing strains can be developed. When inoculum containing equal numbers of USDA 191 and of strain USDA 110 was applied to the unimproved Peking cultivar in Perlite pot culture, 85% of the 160 nodules tested were occupied by USDA 191. With Lee and Ransom cultivars, 99 and 85% of 140 and 96 nodules tested, respectively, were occupied by USDA 110.     

Rapid Colored-Nodule Assay for Assessing Root Exudate-Enhanced Competitiveness of Bradyrhizobium japonicum

The effects of root exudate (RE) treatment on nodule occupancy by Bradyrhizobium japonicum were investigated by a rapid colored-nodule assay, which is based on the observation that B. japonicum L-110 and its antibiotically marked derivatives form dark-red nodules on certain soybean (Glycine max) cultivars, whereas other strains form beige nodules. The efficacy of the assay was confirmed by direct immunofluorescence and by antibiotic platings of nodule bacteria. Both logarithmic- and stationary-phase cultures of the reference strain, L-110Nal, were used in paired-competition studies with RE-treated or untreated cells of seven challenge strains. On the basis of field and greenhouse competition studies, these strains were placed into three competitiveness groups: high (AN-11, AN-16aStrRif, and AN-6), intermediate (AN-3 and 122SR), and low (I-110ARS and AN-18). Seedlings of G. max cv. Centennial were inoculated with two ratios of challenge to reference strain, 1:1 and 1:9, and nodule occupancy was determined after the V4 to V5 stage of ontogeny. Two of the strains showed increased occupancy in response to RE treatment at the 1:1 inoculation ratio. Logarithmic- and stationary-phase cultures of AN-6 showed increased occupancy, from 22 to 38% (P < 0.10) and from 23 59 39% (P < 0.05), respectively. While the maximum increase for stationary-phase cultures of AN-16aStrRif was from 34 to 47% (P < 0.05), logarithmic-phase cultures failed to respond to RE treatment. The results of these studies indicate that RE treatment increases the nodule occupancy of some, but not all, B. japonicum strains and that the colored-nodule assay could be rapidly and reliably used to determine the competitive ability of B. japonicum.