Identification of Bradyrhizobium japonicum Nodule Isolates from Wisconsin Soybean Farms

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a more discriminating method than serotyping for identifying strains of Bradyrhizobium japonicum. Analysis of 543 nodule isolates from southeastern Wisconsin soybean farms revealed that none of the isolates were formed by any of the inoculant strains supplied by either of two inoculant companies. Twenty-nine indigenous strains and six inoculant strains were identified. Strain 61A76, the most competitive indigenous strain, formed 21% of the nodules. Indigenous strains 3030, 3058, 0336, and 3052 formed 15, 11, 9, and 9% of the nodules, respectively. These predominant strains were not associated with a particular soybean cultivar, soil type, or farm location.     

Bradyrhizobium japonicum Inoculant Mobility, Nodule Occupancy, and Acetylene Reduction in the Soybean Root System

In the American Midwest, superior inoculant rhizobia applied to soybeans usually occupy only 5 to 20% of nodules, and response to inoculation is the exception rather than the rule. Attempts to overcome this problem have met with limited success. We evaluated the ability of Bradyrhizobium japonicum, supplied as a seed coat inoculant, to stay abreast of the infectible region of the developing soybean root system. The rhizoplane population of the inoculant strain declined with distance from site of placement, the decrease being more pronounced on lateral than on taproots. This decline was paralleled by a decrease in inoculant-strain nodule occupancy. Inoculant bradyrhizobia contributed little to nodulation of lateral roots, which at pod-fill accounted for more than 50% of nodule number and mass, and were major contributors to acetylene reduction activity. From these data, it appears that inoculant bradyrhizobia are competitive with indigenous soil strains at the point of placement in the soil but have limited mobility and so are incapable of sustaining high populations throughout the developing root system. The result is low nodule occupancy by the inoculant strain in the tapand lateral roots. Future studies should address aspects of inoculant placement and establishment.     

Competition among Strains of Rhizobium leguminosarum biovar trifolii and Use of a Diallel Analysis in Assessing Competition

Competition between indigenous Rhizobium leguminosarum biovar trifolii strains and inoculant strains or between mixtures of inoculant strains was assessed in field and growth-room studies. Strain effectiveness under competition was compared with strain performance in the absence of competition. Field inoculation trials were conducted at Elora, Ontario, Canada, with soil containing indigenous R. leguminosarum biovar trifolii. The indirect fluorescent-antibody technique was used for the identification of nodule occupants. Treatments consisted of 10 pure strains, a commercial peat inoculant containing a mixture of strains, and an uninoculated control. Inoculant strains occupied 17.5 to 85% of nodules and resulted in increased dry weight and nitrogen content, as compared with the uninoculated control. None of the strains was capable of completely overcoming resident rhizobia, which occupied, on average, 50% of the total nodules tested. In growth-room studies single commercial strains were mixed in all possible two-way combinations and assessed in a diallel mating design. Significant differences in plant dry weight of red clover were observed among strain combinations. Specific combining ability effects were significant at the 10% level, suggesting that the effectiveness of strain mixtures depended on the specific strain combinations. Strains possessing superior effectiveness and competitive abilities were identified by field and growth-room studies. No relationship was detected between strain effectiveness and competitive ability or between strain recovery and host cultivar. The concentration of indigenous populations was not considered to be a limiting factor in the recovery of introduced strains at this site.     

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).     

Effectiveness of Rhizobium Strains Used in Inoculants after Their Introduction into Soil

Rhizobium strains used in inoculants for Trifolium spp., Medicago spp., Glycine max, and Lotus pedunculatus were isolated from nodules of these legumes grown in soils into which the rhizobia had been introduced 4 to 8 years before. Isolations were made from a total of 420 nodules. Nodule occupancy by the inoculant strains varied from 17.7% for a soybean strain to 100% in the case of L. pedunculatus whose specific rhizobia did not occur in the soils studied. In general, inoculant strains isolated from nodules did not differ in effectiveness from cultures of the same strains concurrently maintained in lyophilized form. The average effectiveness of all of the isolates (identified and unidentified) from a legume was 7.1 to 73.3% higher than that of the unidentified isolates alone, demonstrating the prolonged effect that a single-seed inoculation has on the rhizobial population in a soil which had not been planted with legumes before. Relatively weak recovery of a Rhizobium japonicum strain introduced into soil 4 years after soybean seed inoculated with a different strain had been planted in the same soil confirmed the advantage of a resident population over an introduced inoculant strain.     

Influence of Glycine spp. on Competitiveness of Bradyrhizobium japonicum and Rhizobium fredii

The displacement of indigenous Bradyrhizobium japonicum in soybean nodules with more effective strains offers the possibility of enhanced N₂ fixation in soybean (Glycine max (L.) Merr.). Our objective was to determine whether the wild soybean (G. soja Sieb. & Zucc.) genotype PI 468397 would cause reduced competitiveness of important indigenous B. japonicum strains USDA 31, 76, and 123 and thereby permit nodulation by Rhizobium fredii, the fast-growing microsymbiont of soybean. In an initial experiment, PI 468397 nodulated and fixed moderate amounts of N₂ with USDA 31 and 76 but, despite the formation of nodules, fixed essentially no N₂ with USDA 123. In contrast, PI 468397 formed a highly effective symbiosis with R. fredii strain USDA 193. In two subsequent experiments, Williams soybean and PI 468397 were grown in a pasteurized soil mixture or in soybean rhizobium-free soil and inoculated with both USDA 123 and USDA 193. In each experiment, more than 90% of the nodules of Williams contained USDA 123, while only a maximum of 2% were occupied with USDA 193. In contrast, in the two experiments, 16 and 11%, respectively, of the nodules produced on PI 468397 were occupied by USDA 123, while in both experiments 87% contained USDA 193. Thus, in relation to the cultivar Williams, which is commonly grown and used as a parent in soybean breeding programs in the United States, PI 468397 substantially reduced the competitive ability of B. japonicum strain USDA 123 in relation to R. fredii strain USDA 193.     

Single-Strain versus Multistrain Inoculation: Effect of Soil Mineral N Availability on Rhizobial Strain Effectiveness and Competition for Nodulation on Chick-Pea, Soybean, and Dry Bean

The nitrogen-fixing effectiveness of multistrain inoculants was found to be determined by both the effectiveness of the component strains and the percentage of the nodules occupied by them. Multistrain formulations were always either as good as the most effective single-strain inoculant or intermediate between the most and the least effective. The percentage of nodules occupied and the amount of nitrogen fixed by the component strains of a multistrain inoculant showed highly significant linear correlation. The availability of soil N had a significant influence on the nitrogen fixation potential of each strain. The mineral N status of the soil was clearly a significant factor in affecting the competition pattern of Rhizobium loti (chick-pea) and Bradyrhizobium japonicum strains. Differences between the effectiveness of strains were masked under conditions of soil N availability. However, when soil N was immobilized with sugarcane bagasse, the differences became significant. In the chick-pea system, R. loti TAL 1148 (Nit 27A8) was the most effective but not the most competitive of the three strains used. In the soybean and dry bean systems, B. japonicum TAL 102 (USDA 110) and R. leguminosarum bv. phaseoli TAL 182, respectively, were consistently the most effective and, more often than not, the most competitive of the strains used for each species.     

Assessment of faba bean (Vicia faba) response to inoculation with Rhizobium leguminosarum in clay loam Nile Delta soil

A field experiment was conducted to assess the response to inoculation with rhizobia in a clay loam soil of the Nile Delta using faba bean (Vicia faba) for two successive winter seasons (1985/6 and 1986/7). Three selected strains of Rhizobium leguminosarum, TAL 634, NRC 65 and TAL 1400, were used singly or in combination as peat-based inocula in 1985/6 winter season. Strain TAL 1400 was replaced by strain F9 in the 1986/7 winter season. A significant seed yield response was obtained only with strain TAL 1400, in the 1985/6 season. In the 1986/7 season, no significant yield response was observed with any of the strains. The serotyping of nodules collected in the 1985/6 season showed that strain TAL 1400 was more competitive than either the indigenous rhizobia or the two inoculant strains. However, the majority of nodules formed in the 1986/7 season were formed from strains other than the inoculant ones.     

Nodulation of Glycine max by Six Bradyrhizobium japonicum Strains with Different Competitive Abilities

The root nodule locations of six Bradyrhizobium japonicum strains were examined to determine if there were any differences which might explain their varying competitiveness for nodule occupancy on Glycine max. When five strains were added to soybeans in plastic growth pouches in equal proportions with a reference strain (U.S. Department of Agriculture, strain 110), North Carolina strain 1028 and strain 110 were the most competitive for nodule occupancy, followed by U.S. Department of Agriculture strains 122, 76, and 31 and Brazil strain 587. Among all strains, nodule double occupancy was 17% at a high inoculum level (10⁷ CFU pouch⁻¹) and 2% at a low inoculum level (10⁴ CFU pouch⁻¹). The less competitive strains increased their nodule representation by an increase in the doubly occupied nodules at the high inoculum level. Among all strains, the number of taproot and lateral root nodules was inversely related at both the high and low inoculum levels (r = −0.62 and −0.69, respectively; P = 0.0001). This inverse relationship appeared to be a result of the plant host control of bacterial infection. Among each of the six strains, greater than 95% of the taproot nodules formed at the high inoculum density were located on 25% of the taproot length, the nodules centering on the position of the root tip at the time of inoculation. No differences among the six strains were observed in nodule initiation rates as measured by taproot nodule position. Taproot nodules were formed in the symbiosis before lateral root nodules. One of the poorly competitive strains (strain 76) occupied three times as many taproot nodules as lateral root nodules when competing with strain 110 (nodules were harvested from 4-week-old plants). Among these six wild-type strains of B. japonicum, competitive ability evidently is not related to nodule initiation rates.     

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.     

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.     

Competition Among Rhizobium spp. for Nodulation of Leucaena leucocephala in Two Tropical Soils

The successful nodulation of legumes by a Rhizobium strain is determined by the competitive ability of that strain against the mixture of other native and inoculant rhizobia. Competition among six Leucaena rhizobial strains in single and multistrain inoculants were studied. Field inoculation trials were conducted in an oxisol and a mollisol soil, both of which contained indigenous Leucaena-nodulating rhizobia. Strain-specific fluorescent antibodies were used for the identification of the strains in Leucaena nodules. Mixtures of three recommended inoculum strains for Leucaena spp. (TAL82, TAL582, and TAL1145) were used in peat-based inocula either alone or with one of the three other strains isolated from the sites, B213, B214, and B215. Each of these latter three strains was also used as single-strain inocula to study their competition with the native rhizobia in the two soil systems. In the oxisol soil, strains B213 and B215, when used as single-strain inocula, outcompeted the native rhizobia and formed 92 and 62% of the nodules, respectively. Strain B214 was the least competitive in oxisol soil, where it formed 30% of the nodules, and the best in mollisol soil, where it formed 70% of the nodules. The most successful competitor for nodulation in multistrain inocula was strain TAL1145, which outcompeted native and other inoculum Leucaena rhizobia in both soils. None of the strains in single or multistrain inoculants was capable of completely overcoming the resident rhizobia, which formed 4 to 70% of the total nodules in oxisol soil and 12 to 72% in mollisol soil. No strong relationship was detected between the size of the rhizosphere population of a strain and its successful occupation of nodules.     

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.     

Influence of Environmental Factors on Interstrain Competition in Rhizobium japonicum

The effect of several biotic and abiotic factors on the pattern of competition between two strains of Rhizobium japonicum was examined. In two Minnesota soils, Waseca and Waukegan, strain USDA 123 occupied 69% (Waseca) and 24% (Waukegan) of the root nodules on Glycine max L. Merrill cv. Chippewa. USDA 110 occupied 2% of the root nodules in the Waseca soil and 12% of the nodules in the Waukegan soil. Under a variety of other growth conditions—vermiculite, vermiculite amended with Waseca soil, and two Hawaiian soils devoid of naturalized Rhizobium japonicum strains—USDA 110 was more competitive than USDA 123. The addition of nitrate to or the presence of antibiotic-producing actinomycetes in the rhizosphere of soybeans did not affect the pattern of competition between the two strains. However, preexposure of young seedings to USDA 110 or USDA 123 before transplantation into soil altered the pattern of competition between the two strains significantly. In the Waseca soil, preexposure of cv. Chippewa to USDA 110 for 72 h increased the percentage of nodules occupied by USDA 110 from 2 to 55%. Similarly, in the Hawaiian soil Waimea, nodule occupancy by USDA 123 increased from 7 to 33% after a 72-h preexposure.     

Elevated CO2 induces differences in nodulation of soybean depending on bradyrhizobial strain and method of inoculation

High CO₂ has been shown to increase plant growth and to affect symbiotic activity in many legumes species, including soybean (Glycine max [L.] Merr.). In order to assess the interaction between elevated CO₂ and rhizobial symbionts on soybean growth and nodulation, we combined the effects of CO₂ with those of different bradyrhizobial strains and methods of inoculation. Soybean seeds were sown in agricultural soil in pots and inoculated with three strains of Bradyrhizobium japonicum (5Sc2 and 12NS14 indigenous to Quebec soils, and 532c, a reference strain), the inoculum being either applied directly to the seed or incorporated into the soil. Plants were grown in growth chambers (22/17ºC) for 6 weeks, under either near ambient (400 μmol mol^?1) or elevated (800 μmol mol^?1) concentrations of CO_2. Elevated CO₂ increased mass (63%) and number (50%) of soybean nodules, particularly medium and large, allowed a deeper nodule development, and increased shoot dry weight (+30%), shoot C uptake (+33%) and shoot N uptake (+78%), compared to ambient CO₂. The two indigenous strains induced more medium and large nodules under elevated CO₂ than the reference strain and showed the greatest increases in shoot dry weight. Soil inoculation induced higher number of small nodules than seed inoculation, specifically for the two indigenous strains, but did not affect plant growth parameters. We conclude that soybean yield enhancements due to elevated CO₂ are associated with the production of large and medium-size nodules and a deep nodulation, that the two indigenous strains better respond to elevated CO₂ than the reference strain, and that the method of inoculation has little influence on this response.     

Effect of Microbial Inoculants on the Indigenous Actinobacterial Endophyte Population in the Roots of Wheat as Determined by Terminal Restriction Fragment Length Polymorphism

The effect of single actinobacterial endophyte seed inoculants and a mixed microbial soil inoculant on the indigenous endophytic actinobacterial population in wheat roots was investigated by using the molecular technique terminal restriction fragment length polymorphism (T-RFLP). Wheat was cultivated either from seeds coated with the spores of single pure actinobacterial endophytes of Microbispora sp. strain EN2, Streptomyces sp. strain EN27, and Nocardioides albus EN46 or from untreated seeds sown in soil with and without a commercial mixed microbial soil inoculant. The endophytic actinobacterial population within the roots of 6-week-old wheat plants was assessed by T-RFLP. Colonization of the wheat roots by the inoculated actinobacterial endophytes was detected by T-RFLP, as were 28 to 42 indigenous actinobacterial genera present in the inoculated and uninoculated plants. The presence of the commercial mixed inoculant in the soil reduced the endophytic actinobacterial diversity from 40 genera to 21 genera and reduced the detectable root colonization by approximately half. The results indicate that the addition of a nonadapted microbial inoculum to the soil disrupted the natural actinobacterial endophyte population, reducing diversity and colonization levels. This was in contrast to the addition of a single actinobacterial endophyte to the wheat plant, where the increase in colonization level could be confirmed even though the indigenous endophyte population was not adversely affected.     

Rhizobial position as a main determinant in the problem of competition for nodulation in soybean

Selected Bradyrhizobium japonicum strains inoculated on soybean seeds often fail to occupy a significant proportion of nodules when a competitor rhizobial population is established in the soil. This competition problem could result from a genetic/ physiological advantage of the adapted soil population over the introduced inoculant or from a positional advantage, as the soil population already occupies the soil profile where the roots will penetrate, whereas the inoculant remains concentrated around the seeds. Here, we have assessed the contribution of these factors with a laboratory model in which a rhizobial population is established in sterile vermiculite. We observed that the wild-type strain B. japonicum LP 3004 was able to grow in pots with N-free plant nutrient solution-watered vermiculite for six or seven generations with a duplication rate of at least 0.7 day(-1). In addition, the rhizobial population persisted for 3 months with 10(6)-10(7) colony-forming units ml(-1) of the vermiculite-retained solution. N-starved, young rhizobial cultures are more efficient in performing several steps along their early association with soybean roots. However, N starvation during growth of rhizobia used for seed inoculation did not enhance their competitiveness against a 1 month vermiculite-established rhizobial population, which occupied more than 72% of the nodules. When a similarly established rhizobial population was recovered from the vermiculite and homogeneously suspended in plant nutrient solution, these cells were significantly less competitive (29% of nodules occupied) than rhizobia obtained from a fresh, logarithmic culture in a N-poor minimal medium, thus indicating that cell position rather than intrinsic competitiveness was the determinant for nodule occupation.     

Evaluation of strain competitiveness in Rhizobium leguminosarum bv phaseoli using a nod + fix− natural mutant

Competition from native soil rhizobia is likely to be an important factor limiting Phaseolus vulgaris L. inoculant response in Latin America. We used UMR 1116, a nod ⁺ fix^– natural mutant of Rhizobium leguminosarum bv phaseoli strain CC511, as a reference strain to study competition for nodulation sites in this species. When P. vulgaris cv Carioca was planted in soils containing different proportions of UMR 1116 and the effective and competitive strain UMR 1899, UMR 1116 occupied more than 50% of the nodules at all inoculant ratios tested, though increasing the proportion of UMR 1899 in the inoculant did enhance the number and percentage of effective nodules and plant dry weight. Sixty two strains of bean rhizobia were tested in competition with UMR 1116. An inoculant ratio of 1:1 was used, with all strains applied to the soil rather than to seeds. Strains varied in the number and percentage of effective nodules produced in competition with UMR 1116, and in plant dry weight, and there was a strong correlation between variation in each of these traits and plant N accumulation. Seven of the strains (UMR 1073, 1084, 1102, 1125, 1165, 1378 and 1384) were identified as both superior in competitive ability and active in N₂ fixation. Site of placement of the inoculant and ambient temperature influenced strain response.Journal paper 16736, Agricultural Experiment Station, University of Minnesota, St. Paul, MN 55108, USA     

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.