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.     

The Soybean Rj4 Allele Restricts Nodulation by Bradyrhizobium japonicum Serogroup 123 Strains

Of nine Bradyrhizobium japonicum serogroup 123 strains examined, 44% were found to be restricted for nodulation by cultivar Hill. Nodulation studies with soybean isoline BARC-2 confirmed that the soybean Rj4 allele restricts nodulation by the same serogroup 123 isolates. Immunological analyses indicated that B. japonicum strains in serogroups 123 and 31 share at least one surface somatic antigen.     

Lysogeny in Bradyrhizobium japonicum and Its Effect on Soybean Nodulation

Rhizobiophage V, isolated from soil in the vicinity of soybean roots, was strongly lytic on Bradyrhizobium japonicum 123B (USDA 123) but only mildly lytic on strain L4-4, a chemically induced small-colony mutant of 123. Numerous bacteriophage-resistant variants were isolated from L4-4 infected with phage V; two were studied in detail and shown to be lysogenic. The two, L4-4 (V5) and L4-4 (V12), are the first reported examples of temperate-phage infection in B. japonicum. Phage V and its derivative phages V5 and V12 were closely related on the basis of common sensitivity to 0.01 M sodium citrate and phage V antiserum, phage immunity tests, and apparently identical morphology when examined by electron microscopy. However, the three phages differed in host range and in virulence. Lysogens L4-4 (V5) and L4-4 (V12) were immune to infection by phages V and V5 but not to infection by V12. Southern hybridization analysis confirmed the incorporation of phage V into the genomes of strains L4-4(V5) and L4-4(V12) and also demonstrated the incorporation of phage V into the genome of a phage V-resistant derivative of USDA 123 designated 123 (V2). None of the three lysogens, L4-4(V5), L4-4(V12), or 123B(V2), was able to nodulate soybean plants. However, Southern hybridization profile data indicated that phage V had not incorporated into any of the known B. japonicum nodulation genes.     

Early Infection and Competition for Nodulation of Soybean by Bradyrhizobium japonicum 123 and 138

Interactions of soybean with Bradyrhizobium japonicum 123 (serogroup 123) and 138 (serogroup c1) were used to examine the relationship between early infection rates, competition for nodulation, and patterns of nodule occupancy. Both strains formed more infections in autoclaved soil (sterile soil) than in untreated soil (unsterile soil). Inoculation did not increase numbers of infection threads in unsterile soil-grown plants, where infection of proximal portions of primary roots was complete by 5 days after planting. Both strains infected and nodulated at similar rates in sterile soil. Nodules were always clustered on the upper root system, regardless of inoculation and soil treatment. Sixty-seven percent of the nodules of uninoculated plants grown in unsterile soil were occupied by rhizobia belonging to serogroups other than 123 or c1. Inoculation with strain 123 or 138 increased occupancy by that strain at the expense of residency by other rhizobia. Eighty-three percent of all nodules on plants dually inoculated with both strains in sterile soil contained strain 138. The corresponding value for plants inoculated in unsterile soil was 31%. Neither inoculum strain dominated occupancy of first-formed nodules in unsterile soil. It appears that north central Missouri soil may not have populations of highly competitive serogroup 123 and that early infection and nodulation rates do not contribute to the competitive success of strain 138.     

Host-Controlled Restriction of Nodulation by Bradyrhizobium japonicum Strains in Serogroup 110

We previously reported the identification of a soybean plant introduction (PI) genotype, PI 417566, which restricts nodulation by Bradyrhizobium japonicum MN1-1c (USDA 430), strains in serogroup 129, and USDA 110 (P. B. Cregan, H. H. Keyser, and M. J. Sadowsky, Appl. Environ. Microbiol. 55:2532-2536, 1989, and Crop Sci. 29:307-312, 1989). In this study, we further characterized nodulation restriction by PI 417566. Twenty-four serogroup 110 isolates were tested for restricted nodulation on PI 417566. Of the 24 strains examined, 62.5% were restricted in nodulation by the PI genotype. The remainder of the serogroup 110 strains tested (37.5%), however, formed significant numbers of nodules on PI 417566, suggesting that host-controlled restriction of nodulation by members of serogroup 110 is strain dependent. Analysis of allelic variation at seven enzyme-encoding loci by multilocus enzyme electrophoresis indicated that the serogroup 110 isolates can be divided into two major groups. The majority of serogroup 110 isolates which nodulated PI 417566 belonged to the same multilocus enzyme electrophoresis group. B. japonicum USDA 110 and USDA 123 were used as coinoculants in competition-for-nodulation studies using PI 417566. Over 98% of the nodules formed on PI 417566 contained USDA 123, whereas less than 2% contained USDA 110. We also report the isolation of a Tn5 mutant of USDA 110 which has overcome nodulation restriction conditioned by PI 417566. This mutant, D4.2-5, contained a single Tn5 insertion and nodulated PI 417566 to an extent equal to that seen with the unrestricted strain USDA 123. The host range of D4.2-5 on soybean plants and other legumes was unchanged relative to that of USDA 110, except that the mutant nodulated Glycine max cv. Hill more efficiently. While strain USDA 110 has the ability to block nodulation by D4.2-5 on PI 417566, the nodulation-blocking phenomenon was not seen unless strain USDA 110 was inoculated at a 100-fold greater concentration than the mutant strain.     

Genotypic Diversity among Strains of Bradyrhizobium japonicum Belonging to Serogroup 110

Thirty-three strains of Bradyrhizobium japonicum within serogroup 110 were examined for genotypic diversity by using DNA-DNA hybridization analyses. The analysis of the DNA from 15 hydrogen-uptake-negative strains with the bradyrhizobial uptake hydrogenase probe pHU52 showed variation in degree of homology and restriction fragment length polymorphism of EcoRI-restricted DNA. Clustering analysis of the 33 strains on the basis of DNA-DNA hybridization analysis with four restriction enzymes and with the bradyrhizobial nodulation locus, pRJUT10, as probe indicated the existence of four groups of strains, which were less than 70% similar. Restriction digestion of genomic DNA with BamHI and DNA-DNA hybridization with pRJUT10 permitted classification of each of the strains according to a specific fingerprint pattern.     

Phenotypic Diversity among Strains of Bradyrhizobium japonicum Belonging to Serogroup 110

Thirty-four strains of Bradyrhizobium japonicum within serogroup 110 were examined for phenotypic diversity. The strains differed in their abilities to nodulate and fix dinitrogen with Glycine max (L.) Merr. cv. Williams. Thirteen strains expressed uptake hydrogenase activity when induced as free-living cultures in the presence of 2% hydrogen and oxygen. Six bacteriophage susceptibility reactions were observed. Each of the strains produced either a large, mucoid or a small, dry colony morphology, but colony type was not related to effectiveness for nitrogen fixation.     

Bradyrhizobium japonicum Serocluster 123 and Diversity among Member Isolates

Diversity was examined within a group of 79 isolates of Bradyrhizobium japonicum reactive to fluorescent antibodies (FAs) prepared against B. japonicum USDA 123. Analyses were by means of cross-adsorbed FAs, bacteriophage typing, and endonuclease restriction digest patterns. Serogroups 127 and 129 shared antigenic determinants with serogroup 123 but not with each other. Bacteriophage and DNA digest patterns reflected more common features between serogroups 123 and 127 than between 123 and 129. Serogroups 129 and 122 showed FA cross-reactivity. The term serocluster was proposed to reflect interrelationships observed among the serogroups.     

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.     

DNA Hybridization Probe for Use in Determining Restricted Nodulation among Bradyrhizobium japonicum Serocluster 123 Field Isolates

Several soybean plant introduction (PI) genotypes have recently been described which restrict nodulation of Bradyrhizobium japonicum serocluster 123 in an apparently serogroup-specific manner. While PI 371607 restricts nodulation of strains in serogroup 123 and some in serogroup 127, those in serogroup 129 are not restricted. When DNA regions within and around the B. japonicum I-110 common nodulation genes were used as probes to genomic DNA from the serogroup strains USDA 123, USDA 127, and USDA 129, several of the probes differentially hybridized to the nodulation-restricted and -unrestricted strains. One of the gene regions, cloned in plasmid pMJS12, was subsequently shown to hybridize to 4.6-kilobase EcoRI fragments from DNAs from nodulation-restricted strains and to larger fragments in nodulation-unrestricted strains. To determine if the different hybridization patterns could be used to predict nodulation restriction, we hybridized pMJS12 to EcoRI-digested genomic DNAs from uncharacterized serocluster 123 field isolates. Of the 36 strains examined, 15 were found to have single, major, 4.6-kilobase hybridizing EcoRI fragments. When tested for nodulation, 80% (12 of 15) of the strains were correctly predicted to be restricted for nodulation of the PI genotypes. In addition, hybridization patterns obtained with pMJS12 and nodulation phenotypes on PI 371607 indicated that there are at least three types of serogroup 127 strains. Our results suggest that the pMJS12 gene probe may be useful in selecting compatible host-strain combinations and in determining the suitability of field sites for the placement of soybean genotypes containing restrictive nodulation alleles.     

Improved Method of Typing Bradyrhizobium japonicum in Soybean Nodules

An improved method for antibiotic resistance recovery of Bradyrhizobium japonicum from soybean (Glycine max (L.) Merr.) nodules that is simple, time saving, and economical was developed. This technique involves the use of two 96-well microtiter plates as a multinodule sterilization chamber and a template and a third plate as a 16-point replicator constructed with steel nails affixed to the plate with epoxy cement. With this system a team of four technicians could type 3,000 nodules per day. This method was useful in assessing strain establishment and interstrain competition when one or more uniquely labeled strains of B. japonicum were inoculated onto either growth-room- or field-grown soybeans. Contamination was low and reproducibility across replicates approached the theoretical upper limit. Simplicity in design and use made this recovery method especially adaptable for field studies in which large numbers of nodules were required to provide a representative statistical sample offering good precision.     

Cloning and Mapping of a Novel Nodulation Region from Bradyrhizobium japonicum by Genetic Complementation of a Deletion Mutant

The phenotypes of a set of Bradyrhizobium japonicum 110 mutants with large deletions in the region of symbiotic gene cluster I were tested. The majority of the mutants showed a delayed nodulation on soybean and, by mixed-infection experiments, were found to be strongly reduced in their competitiveness. Phenotypic comparison of mutants with different deletion endpoints allowed a preliminary localization of two genomic regions, called nod-1 and nod-2, which were required for normal nodulation on soybean. Loss of nod-1 was found to result in a Nod⁻ phenotype on cowpea, mung bean, and siratro. A recombinant cosmid was identified which fully restored nodulation ability of a mutant lacking nod-1. Using Tn5-containing derivatives and subclones of this cosmid for complementation, we delimited the nod-1 region to a DNA segment of 3.1 to 3.5 kilobase pairs.     

Improved Soybean Root Association of N-Starved Bradyrhizobium japonicum

In this study, we addressed the effects of N limitation in Bradyrhizobium japonicum for its association with soybean roots. The wild-type strain LP 3001 grew for six generations with a growth rate of 1.2 day(-1) in a minimal medium with 28 mM mannitol as the carbon source and with the N source [(NH(4))(2)SO(4)] limited to only 20 microM. Under these conditions, the glutamine synthetase (GS) activity was five to six times higher than in similar cultures grown with 1 or 0.1 mM (NH(4))(2)SO(4). The NtrBC-inducible GSII form of this enzyme accounted for 60% of the specific activity in N-starved rhizobia, being negligible in the other two cultures. The exopolysaccharide (EPS) and capsular polysaccharide (CPS) contents relative to cell protein were significantly higher in the N-starved cultures, but on the other hand, the poly-3-hydroxybutyrate level did not rise in comparison with N-sufficient cultures. In agreement with the accumulation of CPS in N-starved cultures, soybean lectin (SBL) binding as well as stimulation of rhizobial adsorption to soybean roots by SBL pretreatment were higher. The last effect was evident only in cultures that had not entered stationary phase. We also studied nodC gene induction in relation to N starvation. In the chromosomal nodC::lacZ fusion Bj110-573, nodC gene expression was induced by genistein 2.7-fold more in N-starved young cultures than in nonstarved ones. In stationary-phase cultures, nodC gene expression was similarly induced in N-limited cultures, but induction was negligible in cultures limited by another nutrient. Nodulation profiles obtained with strain LP 3001 grown under N starvation indicated that these cultures nodulated faster. In addition, as culture age increased, the nodulation efficiency decreased for two reasons: fewer nodules were formed, and nodulation was delayed. However, their relative importance was different according to the nutrient condition: in older cultures the overall decrease in the number of nodules was the main effect in N-starved cultures, whereas a delay in nodulation was more responsible for a loss in efficiency of N-sufficient cultures. Competition for nodulation was studied with young cultures of two wild-type strains differing only in their antibiotic resistance, the N-starved cultures being the most competitive.     

Influence of Bradyrhizobium japonicum Location and Movement on Nodulation and Nitrogen Fixation in Soybeans

The influence of seed and soil inoculation on bradyrhizobial migration, nodulation, and N₂ fixation was examined by using two Bradyrhizobium japonicum strains of contrasting effectiveness in N₂ fixation. Seed-inoculated strains formed fewer nodules on soybeans (mostly restricted to the tap and crown roots within 0 to 5 cm from the stem base) than did bradyrhizobia distributed throughout the soil or inoculated at specific depths. Nodulation was greater below the depths at which bradyrhizobial cells were located rather than above, even though watering was done from below to minimize passive bradyrhizobial migration with percolating water. The most profuse nodulation occurred within approximately 5 cm below the point of placement and was generally negligible below 10 cm. These and other results suggest that bradyrhizobial migration from the initial point of placement was very limited. Nevertheless, the more competitive strain, effective strain THA 7, migrated into soil to a greater extent than the ineffective strain THA 1 did. Nitrogen fixation resulting from the dual-strain inoculations differed depending on the method of inoculation. For example, the amount of N₂ fixed when both strains were slurried together onto the seed was about half that obtained from mixing the effective strain into the soil with the ineffective strain on the seed. The results indicate the importance of rhizobial distribution or movement into soil for nodulation, nodule distribution, strain competitiveness, and N₂ fixation in soil-grown legumes.     

Genetic Variation in Two Cultures of Bradyrhizobium japonicum 110 Differing in Their Ability to Impart Drought Tolerance to Soybean

The polymerase chain reaction with arbitrary primers (RAPD) discriminated between two separately maintained cultures of Bradyrhizobium japonicum USDA 110 differing in symbiotic performance under drought conditions. Since strain 110 is used in inoculum production, the use of RAPD to monitor inoculum cultures could help to preserve their genetic composition and prevent the loss of important symbiotic properties. The use of RAPD could also be extended to other B. japonicum strains currently used in inoculum production. Received: 19 May 1997 / Accepted: 27 June 1997     

Bradyrhizobium japonicum Survival in and Soybean Inoculation with Fluid Gels

The utilization of gels, which are used for fluid drilling of seeds, as carriers of Bradyrhizobium japonicum for soybean (Glycine max (L.) Merr.) inoculation was studied. Gels of various chemical composition (magnesium silicate, potassium acrylate-acrylamide, grafted starch, and hydroxyethyl cellulose) were used, although the hydroxyethyl cellulose gels were more extensively investigated. Gel inocula were prepared by mixing gel powder with liquid cultures of B. japonicum (2% [wt/vol]). The population of B. japonicum USDA 110 did not change in each gel type during 8 days of incubation at 28°C. These fluid gels were prepared with late-exponential-growth-phase cells that were washed and suspended in physiological saline. Mid-exponential-growth-phase B. japonicum USDA 110, 123, and 138 grew in cellulose gels prepared with yeast extract-mannitol broth as well as or better than in yeast extract-mannitol broth alone for the first 10 days at 28°C. Populations in these cellulose gels after 35 days were as large as when the gels had originally been prepared, and survival occurred for at least 70 days. Soybeans grown in sand in the greenhouse had greater nodule numbers, nodule weights, and top weights with gel inoculants compared with a peat inoculant. In soil containing 10³ indigenous B. japonicum per g of soil, inoculation resulted in increased soybean nodule numbers, nodule weights, and top weights, but only nodule numbers were greater with gel than with peat inoculation. The gel-treated seeds carried 10² to 10³ more bacteria per seed (10⁷ to 10⁸) than did the peat-treated seeds.     

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.     

Effects of Bradyrhizobium japonicum and Soybean (Glycine max (L.) Merr.) Phosphorus Nutrition on Nodulation and Dinitrogen Fixation

Cells of Bradyrhizobium japonicum were grown in media containing either 1.0 mM or 0.5 μM phosphorus. In growth pouch experiments, infection of the primary root of soybean (Glycine max (L.) Merr.) by B. japonicum USDA 31, 110, and 142 was significantly delayed when P-limited cells were applied to the root. In a greenhouse experiment, B. japonicum USDA 31, 110, 122, and 142 grown with sufficient and limiting P were used to inoculate soybeans which were grown with either 5 μM or 1 mM P nutrient solution. P-limited cells of USDA 31 and 110 formed significantly fewer nodules than did P-sufficient cells, but P-limited cells of USDA 122 and 142 formed more nodules than P-sufficient cells. The increase in nodule number by P-limited cells of USDA 142 resulted in significant increases in both nodule mass and shoot total N. In plants grown with 1 mM P, inoculation with P-limited cells of USDA 110 resulted in lower total and specific nitrogenase activities than did inoculation with P-sufficient cells. Nodule numbers, shoot dry weights, and total N and P were all higher in plants grown with 1 mM P, and plants inoculated with USDA 31 grew poorly relative to plants receiving strains USDA 110, 122, and 142. Although the effects of soybean P nutrition were more obvious than those of B. japonicum P nutrition, we feel that it is important to develop an awareness of the behavior of the bacterial symbiont under conditions of nutrient limitation similar to those found in many soils.     

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.