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.     

光敏生物素标记总DNA探针对大豆根瘤菌的检测

以光敏生物素标记慢生型大豆根瘤菌(Bradyrhizobium japonicum)USDA110总DNA作为探针,与快生型大豆根瘤菌杂交时,没有杂交斑点形成,而与慢生型大豆根瘤菌中的部分菌株能形成杂交斑点,表明该探针具有种和部分菌株特异性,用该探针与压碎的根瘤汁液进行DNA杂交,检测USDA110在不灭菌的盆栽土壤中的竞争结瘤能力,发现USDA110在大豆不同生育期的占瘤率为70%～90%。     

Soybean cultivars affect nodulation competition of Bradyrhizobium japonicum strains

The influence of five Thai soybean cultivars on nodulation competitiveness of four Bradyrhizobium japonicum strains was investigated. Cultures of B. japonicum strains THA5, THA6, USDA110 and SEMIA5019 were mixed with each other prior to inoculating germinated soybean seeds growing in Leonard jars with nitrogen-free nutrient solution. At harvest, nodule occupancy by each strain was determined by a fluorescent antibody technique. The term ‘general competitive ability’ was introduced to describe the average competitive nodule occupancy of a strain in paired co-inoculation with a number of strains on soybean. The nodule occupancies by an individual strain were directly correlated with the proportions of that strain in the inoculum mixtures. USDA110 showed higher nodulation competitiveness than the other strains on three of the five cultivars. The Thai strain THA6 appeared to be more competitive than USDA110 on cultivar SJ5. Thus, nodulation competitiveness of the B. japonicum strains was affected by the cultivars of soybean used. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

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.     

Phylogenetic analyses of Bradyrhizobium strains nodulating soybean (Glycine max) in Thailand with reference to the USDA strains of Bradyrhizobium.

To elucidate the phylogenetic relationships between Thai soybean bradyrhizobia and USDA strains of Bradyrhizobium, restriction fragment length polymorphism (RFLP) analysis using the nifDK gene probe and sequencing of the partial 16S rRNA gene were performed. In our previous work, Thai isolates of Bradyrhizobium sp. (Glycine max) were separated clearly from Bradyrhizobium japonicum and Bradyrhizobium elkanii based on the RFLP analysis using the nodDYABC gene probe. RFLP analysis using the nifDK gene probe divided 14 Thai isolates and eight USDA strains of B. japonicum into different groups, respectively, but categorized into the same cluster. All of seven strains within these Thai isolates had the same sequence of the partial 16S rRNA gene, and it was an intermediate sequence between those of B. japonicum USDA 110 and B. elkanii USDA 76T. Furthermore, three USDA strains of B. japonicum, USDA of (B. japonicum ATCC 10324T), USDA 115 and USDA 129, had the same partial 16S rRNA gene sequence that seven Thai isolates had. These results suggest that Thai isolates of Bradyrhizobium sp. (Glycine max) are genetically distinct from USDA strains of B. japonicum and B. elkanii, but also indicate a close relationship between Thai isolates and USDA strains of B. japonicum.     

Diversity among Field Populations of Bradyrhizobium japonicum in Poland

Genetic structure in field populations of Bradyrhizobium japonicum isolated in Poland was determined by using several complementary techniques. Of the 10 field sites examined, only 4 contained populations of indigenous B. japonicum strains. The Polish bradyrhizobia were divided into at least two major groups on the basis of protein profiles on polyacrylamide gels, serological reaction with polyclonal antisera, repetitive extragenic palindromic PCR fingerprints of genomic DNA, and Southern hybridization analyses with nif and nod gene probes. Serological analyses indicated that 87.5% of the Polish B. japonicum isolates tested were in serogroups 123 and 129, while seven (12.5%) of the isolates tested belonged to their own unique serogroup. These seven strains also could be grouped together on the basis of repetitive extragenic palindromic PCR fingerprints, protein profiles, and Southern hybridization analyses. Cluster analyses indicated that the seven serologically undefined isolates were genetically dissimilar from the majority of the Polish B. japonicum strains. Moreover, immuno-cross-adsorption studies indicated that although the Polish B. japonicum strains reacted with polyclonal antisera prepared against strain USDA123, the majority failed to react with serogroup 123- and 129-specific antisera, suggesting that Polish bradyrhizobia comprise a unique group of root nodule bacteria which have only a few antigens in common with strains USDA123 and USDA129. Nodulation studies indicated that members of the serologically distinct group were very competitive for nodulation of Glycine max cv. Nawiko. None of the Polish serogroup 123 or 129 isolates were restricted for nodulation by USDA123- and USDA129-restricting soybean plant introduction genotypes. Taken together, our results indicate that while genetically diverse B. japonicum strains were isolated from some Polish soils, the majority of field sites contained no soybean-nodulating bacteria. In addition, despite the lack of long-term soybean production in Poland, field populations of unique B. japonicum strains are present in some Polish soils and these strains are very competitive for nodulation of currently used Polish soybean varieties.     

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.     

Effect of soil bradyrhizobia on the success of soybean inoculant strain CB 1809

Four decades of soybean [Glycine max (L.) Merr.] cultivation in South Africa has resulted in the establishment of populations of bradyrhizobia against which the recently introduced inoculant strain CB 1809 must compete. Serological and DNA fingerprinting methods were used to study the diversity of nodule isolates from soils at Bergville, Koedoeskop and Morgenzon. Dominant serogroups included Bradyrhizobium elkanii serotype 76 at Bergville (67%), Bradyrhizobium japonicum serotype 123 at Morgenzon (81%) and B. japonicum serotype 135 at Koedoeskop (100%). Their origin is unknown as they do not correspond in serotype to strains used in previous inoculants. A small percentage of isolates from Bergville (13%) and Morgenzon (16%) were serologically homologous to strain WB 1 (serotype 31/76), applied for two decades before CB 1809 (serotype 122). Nitrogen-fixing effectiveness of CB 1809 was superior to 60% of the isolates tested from Bergville and Morgenzon, but similar to 73% of the Koedoeskop isolates. Seed and liquid-in-furrow application methods increased CB 1809 nodule occupancy at least three-fold above background levels at Bergville (pH 5.16) and Morgenzon (pH 6.33). Inoculation did not, however, increase CB 1809 nodule occupancy at Koedoeskop (pH 7.76), possibly because alkaline soil conditions favoured the serotype 135 population predominant at this site.     

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.     

N2-fixation ability of Brazilian soybean cultivars with Sinorhizobium fredii and Sinorhizobium xinjiangensis

The main N₂-fixing symbiotic associations with soybean (Glycine max (L.) Merrill) plants are realized with bacteria belonging to the species Bradyrhizobium japonicum and B. elkanii. However, in 1982, fast-growing rhizobia were isolated from soybean root nodules collected in The People's Republic of China and these bacteria are today classified as Sinorhizobium fredii and S. xinjiangensis. The fast growing strains formed an effective symbiosis with primitive soybean cultivars such as Peking, but not with most North American cultivars, which are the progenitors of almost all Brazilian cultivars. The main purpose of this study was to evaluate the ability of 80 soybean cultivars from the Brazilian germplasm bank to produce effective nodules when inoculated with S. fredii or S. xinjiangensis strains. Sixty-six percent of the Brazilian genotypes formed effective nodules with both Sinorhizobium species. However, when 20 Fix⁺ genotypes were inoculated with a mixture of B. elkanii and S. fredii, at a ratio of 1:1, most or all nodules were occupied by B. elkanii. Consequently, there was no relationship between the growth rate in vitro and the ability to compete for nodule occupancy. Fast-growing strains have also been isolated from soybean nodules in Brazil, but the ecological importance of these symbiotic associations is still to be determined.     

Expression of nir, nor and nos denitrification genes from Bradyrhizobium japonicum in soybean root nodules

Expression of Bradyrhizobium japonicum wild-type strain USDA110 nirK , norC and nosZ denitrification genes in soybean root nodules was studied by in situ histochemical detection of β -galactosidase activity. Similarly, P_nirK- lacZ , P_norC- lacZ , and P_nosZ- lacZ fusions were also expressed in bacteroids isolated from root nodules. Levels of β -galactosidase activity were similar in both bacteroids and nodule sections from plants that were solely N₂-dependent or grown in the presence of 4 m M KNO₃. These findings suggest that oxygen, and not nitrate, is the main factor controlling expression of denitrification genes in soybean nodules. In plants not amended with nitrate, B. japonicum mutant strains GRK308, GRC131, and GRZ25, that were altered in the structural nirK , norC and nosZ genes, respectively, showed a wild-type phenotype with regard to nodule number and nodule dry weight as well as plant dry weight and nitrogen content. In the presence of 4 m M KNO₃, plants inoculated with either GRK308 or GRC131 showed less nodules, and lower plant dry weight and nitrogen content, relative to those of strains USDA110 and GRZ25. Taken together, the present results revealed that although not essential for nitrogen fixation, mutation of either the structural nirK or norC genes encoding respiratory nitrite reductase and nitric oxide reductase, respectively, confers B. japonicum reduced ability for nodulation in soybean plants grown with nitrate. Furthermore, because nodules formed by each the parental and mutant strains exhibited nitrogenase activity, it is possible that denitrification enzymes play a role in nodule formation rather than in nodule function.     

Development and application of a multilocus sequence analysis method for the identification of genotypes within genus Bradyrhizobium and for establishing nodule occupancy of soybean (Glycine max L. Merr)

A multilocus sequence typing (MLST) method based on allelic variation of seven chromosomal loci was developed for characterizing genotypes (GT) within the genus Bradyrhizobium. With the method, 29 distinct multilocus GT were identified among 190 culture collection soybean strains. The occupancy of 347 nodules taken from uninoculated field-grown soybean plants also was determined. The bacteroid GT were either the same as or were closely related to GT identified among strains in the culture collection. Double-nodule occupancy estimates of 2.9% were much lower than values published based on serology. Of the 347 nodules examined, 337 and 10 were occupied by Bradyrhizobium japonicum and B. elkanii, respectively. The collection strains within the species B. japonicum and B. elkaniialso were compared with Bradyrhizobium cultures from other legumes. In many cases, the observed GT varied more according to their geographic origin than by their trap hosts of isolation. In other cases, there were no apparent relationships with either the legume or geographic source. The MLST method that was developed should be a useful tool in determining the influence of geographic location, temperature, season, soil type, and host plant cultivar on the distribution of GT of Bradyrhizobium spp.     

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.     

Evaluation of commercial soybean inoculants from Argentina

Eighteen samples of soybean inoculants representative of the major manufacturing companies in Argentina were purchased from the market and evaluated using plate counts, most probable number (MPN) of Bradyrhizobium japonicum on plants and time of nodule appearance. One or two B. japonicum isolates per product were isolated and typed by analysis of their DNA patterns. The Log10 numbers of B. japonicum obtained were in the range of 0 to 6/soybean seed, with only two products above 1 × 106 bacteria/seed. Of 18 products, 17 were contaminated, and of these 14 contained more contaminants than B. japonicum. The time of nodule appearance varied between 8 and 16 days, indicating a great difference in microbial activity between products. The strains were found to be similar to USDA 138 (five isolates), E45-INTA Argentina (two isolates), USDA 142 (four isolates) and E4-INTA (one isolate). Thus, even if most of the typed strains are considered as good N2-fixing strains, the average quality of the analysed samples was low, and could not support efficient inoculation of soybean.     

Efficiency of different formulations of Bradyrhizobium japonicum and effect of co-inoculation of Bacillus subtilis with two different strains of Bradyrhizobium japonicum

A key constraint in successfully obtaining an effective inoculant is overcoming difficulties in formulating a viable and user-friendly final product and maintaining the microbial cells in a competent state. Co-cultures of rhizobia and PGPR (Plant Growth Promoting Rhizobacteria) are a logical next subject for formulation researchers as they can influence the efficacy of rhizobia. A greenhouse experiment was set to assess the formulation effect of one strain i.e. Bradyrhizobium japonicum, 532c (granules, liquid and broth) and also to determine the efficiency of co-inoculation of Bacillus with two commercial strains of B. japonicum (532c and RCR 3407) on 2 soybean (Glycine max L.) varieties. PCR-RFLP analysis was used to determine the nodule occupancy in each treatment. Most of the inoculants showed increased nodulation and biomass yields (by approximately 2-5 and 4-10 g plant(-1) respectively) as compared to the uninoculated controls. TGx1740-2F showed no significant differences in nodule fresh weights for the formulation effect while the co-inoculants increased the nodule fresh weights by up to 4 g plant(-1). The liquid and granule-based inoculants induced higher biomass yields (4-8 g plant(-1)) suggesting a possible impact of formulation on the effectiveness of the inoculants. The co-inoculants also gave higher yields but showing no significant differences to the rhizobial inoculants. Nodule occupancy was 100 % for the rhizobial inoculants as well as the co-inoculants emphasizing the infectivity and high competitiveness of 532c and RCR 3407 strains despite the high population of indigenous rhizobia.     

我国南北大豆产区慢生大豆根瘤菌的遗传多样性和系统发育研究

利用16S rRNA基因RFLP、16S rRNA基因序列分析以及16S-23S rRNA IGS PCR RFLP技术对分离自我国南北大豆产区的慢生大豆根瘤菌进行了群体遗传多样性和系统发育研究。16S rRNA基因PCR RFLP分析以及16S rRNA基因序列分析结果表明:所有供试慢生大豆根瘤菌可分为B.japonicum和B.elkanii两个类群,其中属于B.japonicum的为优势种群,占供试菌株的91%,属于B.elkanii的仅占9%,多样性水平较低。16S-23S rRNA IGS PCRRFLP研究结果表明:属于B.japonicum的慢生根瘤菌具有较丰富的遗传多样性,在69%的相似性水平上可分为群Ⅰ和群Ⅱ两大类群。群I的菌株以分离自黑龙江和河北等北部区域的菌株为代表,群Ⅱ的菌株以分离自广西和江苏等南部地域的菌株为代表,反映出明显的地域特征。两群菌株在系统发育上均与USDA6、USDA110和USDA122等B.japonicum的模式或代表菌株有差异。     

Competition between introduced <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> strains and indigenous bradyrhizobia in Minnesota organic farming systems

Organic farmers recognize the importance of rhizobial associations with legume plants to help meet N fertility and plant productivity needs. A field experiment was done at three organic fields in Minnesota to assess the effect of indigenous Bradyrhizobium japonicum ORGS3 and ORGS5 and reference USDA 110 strains on the growth and yield performance of soybean. Soybean genotypes MN1505SP and Lambert inoculated with B. japonicum ORGS3 had significantly greater (P < 0.01) nodule numbers (42.1 ± 2.5), herbage N-contents (4.02 ± 0.01%), dry biomass (12.60 ± 1.45 g), and plant populations (117,890 ± 288.13 plant/acre) compared with the un-inoculated control. Grain yields were not affected by inoculation. Most nodules formed on non-inoculated Lambert (70%) and MN1505SP (53%) were occupied by strain ORGS5. The inoculant strains USDA110 and ORGS5 increased nodule occupancy by 10% on MN1505SP and Lambert. In contrast, strain ORGS3, and the combination of strains ORGS5 plus ORGS3, increased nodules occupancy on Lambert by 23 and 20%, respectively, compared with the control. The majority of nodules on Lambert (59%) and MN1505SP (52%) in the Farmington and Lamberton fields, respectively, were occupied by ORGS5. In contrast, 41 and 45% of nodules formed on Lambert and MN1505SP at Rosemount, respectively, were occupied by strain ORGS3. The lowest percentage of nodules formed on Lambert (4%) and MN1505SP (5%), in the Farmington field, were occupied by USDA110. These results showed that Bradyrhizobium strains ORGS3 and ORGS5 can be used to enhance N fixation and productivity of organically-grown soybeans grown in Minnesota fields.     

The Rhizobial hemA Gene Is Required for Symbiosis in Species with Deficient [delta]-Aminolevulinic Acid Uptake Activity

Most rhizobial hemA mutants induce root nodules on their respective legume hosts that lack nitrogen fixation activity and leghemoglobin expression. However, a Bradyrhizobium japonicum hemA mutant elicits effective nodules on soybean, and we proposed previously that synthesis and uptake of the heme precursor [delta]-aminolevulinic acid (ALA) by the plant and bacterial symbiont, respectively, allow mutant rescue (I. Sangwan, M.R. O'Brian [1991] Science 251: 1220-1222). In the present work, the B. japonicum hemA mutant MLG1 elicited normal nodules on three hosts, including cowpea, a plant that is not effectively nodulated by a hemA mutant of Rhizobium sp. These data indicate that B. japonicum rather than soybean possesses the unique trait that allows normal nodule development by a hemA mutant. Cowpea expressed glutamate-dependent ALA formation activity in nodules induced by B. japonicum strains I110 or MLG1 and by Rhizobium sp. ANU240. Exogenous ALA was taken up by B. japonicum bacteroids isolated from soybean or cowpea nodules, and the kinetics of uptake were biphasic. By comparison, Rhizobium sp. ANU240 had very low ALA uptake activity. In addition, ALA uptake was observed in cultured cells of B. japonicum but not in cultured cells of three other rhizobial species tested. We suggest that the differential success of legume-rhizobial hemA symbioses is due to an ALA uptake activity in B. japonicum that is deficient in other rhizobia, thereby further validating the ALA rescue hypothesis.     

Persistence of introduced Bradyrhizobium japonicum strains in forming nodules in subsequent years after inoculation in Wisconsin soils

Nodulation of soybeans by indigenous and inoculum strains of Bradyrhizobium japonicum was studied in field experiments in Wisconsin from 1983 to 86. Aqueous suspensions of bacteria were applied to seeds at the time of planting at levels of 7?×?10(7)-10(10) bacteria per 2.5-cm row. The predominant indigenous serogroup was 123 in these soils. Six different inoculum strains were used (two from serocluster 123, two from serogroup 110, and one each from serogroups 122 and C1). Nodule occupants were identified using spontaneous antibiotic-resistant mutations in the inoculum strains, phage typing, and serotyping. In the 1983 experiment, the majority of nodules were formed by the inoculum strains in almost all cases (up to 100% in some cases), in two different soils containing 3.5?×?10(5) indigenous B. japonicum per gram. After 2 years without inoculation at the same two site, the inoculum strains did not form many nodules on uninoculated soybeans (less than 10% in most cases; less than 30% in all cases). In inoculation experiments carried out in 1985 and 1986, four inoculum strains were used (3 members of 123 serocluster and USDA 110str); inocula containing 10(8) bacteria per 2.5-cm row formed less than42%ofthe nodules in soils containing 1?×?10(4)-4?×?10(4)B. japonicum per gram. The major conclusions are (i) the success of inoculation in Midwestern U.S. soils is highly variable, even with members of the (highly competitive) 123 serocluster, and (ii) successful inoculation in 1 year in a Wisconsin soil does not ensure that the inoculated strain will persist in forming nodules in that field in subsequent years without further inoculation. Key words: Bradyrhizobium japonicum, strain persistence, field trials.