Analysis of the Symbiotic Performance of Bradyrhizobium japonicum USDA 110 and Its Derivative I-110 and Discovery of a New Mannitol-Utilizing, Nitrogen-Fixing USDA 110 Derivative

Previously, Bradyrhizobium japonicum USDA 110 was shown to contain colony morphology variants which differed in nitrogen-fixing ability. Mannitol-utilizing derivatives L1-110 and L2-110 have been shown to be devoid of symbiotic nitrogen fixation ability, and non-mannitol-utilizing derivatives I-110 and S-110 have been shown to be efficient at nitrogen fixation. The objectives of this study were to determine the effect of media carbon sources on the symbiotic N₂-fixing ability of strain USDA 110 and to compare the effectiveness of strain USDA 110 and derivative I-110. Based on acetylene reduction activity and the nitrogen content of 41-day-old soybean plants, neither derivative I-110 nor cultures of USDA 110 grown in media favoring non-mannitol-using derivatives had symbiotic nitrogen fixation that was statistically superior to that of cultures of USDA 110 grown in media favoring mannitol-using derivatives. In another experiment 200 individual nodules formed by strain USDA 110 grown in yeast extract gluconate were screened for colony morphology of occupying variant(s) and acetylene reduction activity. Nodules occupied by mannitol-using derivatives (large colony type on 0.1% yeast extract-0.05% K₂HPO₄-0.08% MgSO₄ · 7H₂O-0.02% NaCl-0.001% FeCl₃ · 6H₂O [pH 6.7] with 1% mannitol [YEM] plates) had a mean acetylene reduction activity equal to that of nodules occupied by non-mannitol-using derivatives (small colony type on YEM plates). A total of 20 large colonial derivatives and 10 small colonial derivatives (I-110-like) were isolated and purified by repeated culture in YEM and YEG (same as YEM except 1% gluconate instead of 1% mannitol) media, respectively, followed by dilution in solutions containing 0.05% Tween 40. After 25 days of growth, soybean plants inoculated with the large colony isolates had mean whole-plant acetylene reduction activity, whole-plant dry weight, and whole-plant nitrogen contents equal to or better than those of plants inoculated with either the small colony isolates (I-110-like) or the I-110 (non-mannitol-using) derivative. Hence, the existence of a mannitol-utilizing derivative that fixes nitrogen in a culture of strain USDA 110 obtained from the U.S. Department of Agriculture, Beltsville, Md., was established. This new USDA 110 derivative was designated as MN-110 because it was a mannitol-utilizing nitrogen-fixing USDA 110 derivative. This derivative was morphologically indistinguishable from the non-nitrogen-fixing derivative L2-110 found in cultures obtained earlier from the U.S. Department of Agriculture, Beltsville. DNA-DNA homology and restriction enzyme analyses indicated that MN-110 is genetically related to other USDA 110 derivatives that have been characterized previously.     

Detection of genetic variation in Bradyrhizobium japonicum USDA 110 variants using DNA fingerprints generated with GC rich arbitrary PCR primers

Bradyrhizobium japonicum USDA 110 has been shown to contain several genetically similar naturally occurring colony morphology variants. These variants differ in symbiotic nitrogen fixation ability and in the utilization of various carbon substrates. They have been shown to share extensive DNA homology and appear to be derived from a common ancestor. Despite these similarities certain B. japonicum USDA 110 variants have been shown to be devoid of symbiotic nitrogen fixation. One of these variants (L2-110), however, was recently shown to possess significant levels of explanta nitrogen fixation and to synthesize functional dinitrogenase enzyme within bacteroids. In an effort to identify genetic markers which could explain differences in symbiotic nitrogen fixation between B. japonicum variants, DNA fingerprints were generated by PCR using arbitrary primers. Two of these primers with GC rich sequences were able to differentiate between B. japonicum USDA 110 variants I-110, L2-110, and MN-110. Unique markers have now been identified which could be examined further to determine if they explain the differences in symbiotic nitrogen fixation between USDA 110 variants.     

Characterization of a Mannitol-Utilizing, Nitrogen-Fixing Bradyrhizobium japonicum USDA 110 Derivative

We have isolated a colonial derivative of Bradyrhizobium japonicum USDA 110 (designated MN-110) that is both mannitol utilizing and N(2) fixing. Derivative MN-110 showed growth on mannitol and glucose similar to that of non-N(2)-fixing, mannitol-utilizing L2-110. Derivative MN-110 showed high constitutive and induced d-mannitol dehydrogenase activity (similar to L2-110) relative to N(2)-fixing, non-mannitol-utilizing I-110. Hybridization to EcoRI and HindIII total DNA digests with cloned USDA 110 nif DK and nif H genes revealed similar patterns for non-N(2)-fixing mannitol-utilizing derivative L1-110 and derivative MN-110. Symbiotic tests with soybean cultivars Ransom and Lee indicate MN-110 to be a superior N(2)-fixing derivative compared with derivative I-110 and the parent strain USDA 110. However, these differences were not revealed when comparing 28-day-old soybean-B. japonicum associations but were apparent in 49-day-old associations. It was apparent from this work that mannitol utilization was not necessarily correlated to symbiotic effectiveness in B. japonicum and that gene rearrangements were not responsible for differences in N(2) fixation between L1-110 or L2-110 and MN-110.     

nif Gene expression in a Nif+, Fix−Bradyrhizobium japanicum variant

Abstract Bradyhizobium japonicum USDA 110 has been shown to contain several genetically similar, naturally occurring colony morphology variants. One of these variants, L2-110, although devoid of symbiotic nitrogen fixation, retains significant levels of explanta nitrogen fixation ability relative to other symbiotically competent USDA 110 variants (MN-110 and I-110). Interestingly, Northern blot analyses revealed that L2-110 nodules, despite their lack of symbiotic nitrogen fixation, contained 65% the level of mRNA for dinitrogenase ( nif DK) and 64% the level of dinitrogenase reductase ( nif H) mRNA relative to MN-110 nodules. Western blot analyses of tissue from the same nodules detected 32% the level of dinitrogenase and 31% the level of dinitrogenase reductase in L2-110 relative to MN-110. L2-110 appears to be a new class of mutant based on the complete absence of symbiotic nitrogen fixation (Fix⁻ and the presence of significant exanta nitrogen fixation (Nif⁺).     

Symbiotic Potential, Competitiveness, and Serological Properties of Bradyrhizobium japonicum Indigenous to Korean Soils

The symbiotic potential of Bradyrhizobium japonicum isolates indigenous to seven Korean soils was evaluated by inoculating soybeans with 10- and 1,000-fold-diluted soil suspensions (whole-soil inocula). At both levels, significant differences in the symbiotic potential of the indigenous B. japonicum isolates were demonstrated. The relationship between rhizobial numbers in the whole-soil inocula (x) and nitrogen fixation parameters (y) was best predicted by a straight line (y = a + bx) when the numbers in the inocula were 100 to 10,000 ml^-1, while the power curve (y = ax^b) predicted the variation when the numbers were 1 to 100 ml^-1. Thirty isolates from three soils showed wide differences in effectiveness (measured as milligrams of shoot N per plant), and several were of equal or greater effectiveness than reference strain B. japonicum USDA 110 on soybean cultivars Clark and Jangbaekkong. On both of the soybean cultivars grown in a Hawaiian mollisol, the Korean B. japonicum isolate YCK 213 and USDA 110 were of equal effectiveness; USDA 110 was the superior strain in colonization (nodule occupancy). Korean isolates YCK 117 and YCK 141 were superior colonizers compared with USDA 110. However, B. japonicum USDA 123 was the superior colonizer compared with isolates YCK 213, YCK 141, and YCK 117. In an immunoblot analysis of 97 indigenous Korean isolates of B. japonicum, 41% fell into the USDA 110 and USDA 123 serogroups. Serogroups USDA 110 and USDA 123 were represented in six of the seven soils examined. In one Korean soil, 100% of the B. japonicum isolates reacted only with antisera of YCK 117, an isolate from the same soil.     

Generation of Bradyrhizobium japonicum Mutants with Increased N2O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

We obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N₂O) reductase (N₂OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQ gene (pKQ2) and performing enrichment culture under selection pressure for N₂O respiration. Mutation of dnaQ, which encodes the epsilon subunit of DNA polymerase III, gives a strong mutator phenotype in Escherichia coli. pKQ2 introduction into B. japonicum USDA110 increased the frequency of occurrence of colonies spontaneously resistant to kanamycin. A series of repeated cultivations of USDA110 with and without pKQ2 was conducted in anaerobic conditions under 5% (vol/vol) or 20% (vol/vol) N₂O atmosphere. At the 10th cultivation cycle, cell populations of USDA110(pKQ2) showed higher N₂OR activity than the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed 7 to 12 times the N₂OR activity of the wild-type USDA110. Although two mutants had a weak or null fix phenotype for symbiotic nitrogen fixation, the remaining two (5M09 and 5M14) had the same symbiotic nitrogen fixation ability and heterotrophic growth in culture as wild-type USDA110.     

Duplication of DNA regions carrying repetitive sequence RSα in Bradyrhizobium japonicum 110

L. D. Kuykendall, M. E. Barnett and J. N. Mathis. 1997. RSα is a repeated DNA sequence found within the nitrogen-fixation gene cluster of Bradyrhizobium japonicum , a symbiotic nitrogen-fixing bacterium that nodulates soybean. Bradyrhizobium japonicum strain 110 spc 4 contains 12 repeats, each located on a separate Xho I DNA restriction fragment between 1.2 and 14 kb in length. Although Fix⁺ and Fix⁻ derivatives of B. japonicum USDA 110 were first reported more than two decades ago, genotypic differentiation, on the basis of RSα hybridization pattern, was reported only recently. Bradyrhizobium japonicum strain USDA 110 had only single copies of the RSα-hybridizing bands, but a particular Fix⁻ derivative, MSDJGl, carried doublets of two distinct Xho I fragments that carry RSα3 and RSα4. In this study, RSα hybridization patterns were analysed further in both Fix⁺ and Fix⁻ derivatives of strain 110 to test for duplication of these particular genomic regions. It was concluded that the duplication, or not, of genetic regions carrying RSα3 and RSα4 in strain USDA 110 derivatives is unrelated to symbiotic nitrogen-fixation ability. Like Fix⁻ MSDJGl, Fix⁺ strain 110 derivatives I-110 and MN-110 had duplications of the Xho I DNA restriction fragments carrying RSα3 and RSα4, but Fix⁻ strain 110 derivative L2–110 lacked these duplications. Thus, it is now clear that Fix⁻ derivatives MSDJG1 and L2–110 arose via distinct genetic mechanisms. Interestingly, Fix⁺ derivatives of strain 110 from the laboratories of Elkan and Hennecke differed in RSα hybridization profile.     

Conservation of a Symbiotic DNA Region in Soybean Root Nodule Bacteria

Bradyrhizobium japonicum USDA 3I1b110 contains a DNA region in which symbiotic genes and many repeated sequences are closely linked. Hybridization analysis revealed that this region was highly conserved in some B. japonicum strains (USDA 24, USDA 122, USDA 123, ATCC 10324, 61A24) but not in others (USDA 76, 61A76, 61A101). The genomic presence of multiple copies of one of the repeated sequences (RSα) appeared to be specifically characteristic for soybean root nodule bacteria, including the fast-growing Rhizobium fredii, which carries most of these RSα copies on the symbiotic plasmid.     

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.     

Comparative genomics of Bradyrhizobium japonicum CPAC 15 and Bradyrhizobium diazoefficiens CPAC 7: elite model strains for understanding symbiotic performance with soybean

Background

The soybean-Bradyrhizobium symbiosis can be highly efficient in fixing nitrogen, but few genomic sequences of elite inoculant strains are available. Here we contribute with information on the genomes of two commercial strains that are broadly applied to soybean crops in the tropics. B. japonicum CPAC 15 (=SEMIA 5079) is outstanding in its saprophytic capacity and competitiveness, whereas B. diazoefficiens CPAC 7 (=SEMIA 5080) is known for its high efficiency in fixing nitrogen. Both are well adapted to tropical soils. The genomes of CPAC 15 and CPAC 7 were compared to each other and also to those of B. japonicum USDA 6^T and B. diazoefficiens USDA 110^T.

Results

Differences in genome size were found between species, with B. japonicum having larger genomes than B. diazoefficiens. Although most of the four genomes were syntenic, genome rearrangements within and between species were observed, including events in the symbiosis island. In addition to the symbiotic region, several genomic islands were identified. Altogether, these features must confer high genomic plasticity that might explain adaptation and differences in symbiotic performance. It was not possible to attribute known functions to half of the predicted genes. About 10% of the genomes was composed of exclusive genes of each strain, but up to 98% of them were of unknown function or coded for mobile genetic elements. In CPAC 15, more genes were associated with secondary metabolites, nutrient transport, iron-acquisition and IAA metabolism, potentially correlated with higher saprophytic capacity and competitiveness than seen with CPAC 7. In CPAC 7, more genes were related to the metabolism of amino acids and hydrogen uptake, potentially correlated with higher efficiency of nitrogen fixation than seen with CPAC 15.

Conclusions

Several differences and similarities detected between the two elite soybean-inoculant strains and between the two species of Bradyrhizobium provide new insights into adaptation to tropical soils, efficiency of N₂ fixation, nodulation and competitiveness.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-420) contains supplementary material, which is available to authorized users.     

Coordination between Bradyrhizobium and Pseudomonas alleviates salt stress in soybean through altering root system architecture

It is a well accepted strategy to improve plant salt tolerance through inoculation with beneficial microorganisms. However, its underlying mechanisms still remain unclear. In the present study, hydroponic experiments were conducted to evaluate the effects of Bradyrhizobium japonicum USDA 110 with salt-tolerant Pseudomonas putida TSAU1 on growth, protein content, nitrogen, and phosphorus uptake as well as root system architecture of soybean (Glycine max L.) under salt stress. The results indicated that the combined inoculation with USDA 110 and TSAU1 significantly improved plant growth, nitrogen and phosphorus contents, and contents of soluble leaf proteins under salt stress compared to the inoculation with the symbiont alone or compared to un-inoculated ones. The root architectural traits, like root length, surface area, project area, and root volume; as well as nodulation traits were also significantly increased by co-inoculation with USDA 110 and TSAU1. The plant-growth promoting rhizobacteria (PGPR) P. putida strain TSAU1 could improve the symbiotic interaction between the salt-stressed soybean and B. japonicum USDA 110. In conclusion, inoculation with B. japonicum and salt-tolerant P. putida synergistically improved soybean salt tolerance through altering root system architecture facilitating nitrogen and phosphorus acquisition, and nodule formation.     

Enhanced competitiveness of a Bradyrhizobium japonicum mutant strain improved for nodulation and nitrogen fixation

Bradyrhizobium japonicum strain TA-11NOD⁺, with altered indole biosynthesis, exhibited enhanced nodulation and nitrogen fixation on soybean in previous greenhouse studies. In this study, field experiments were conducted at Upper Marlboro, Maryland, in the summers of 1988 and 1993. In 1988, the site used was essentially free of soybean-nodulating bacteria and seed yield in plots inoculated with either I-110ARS or TA-11NOD⁺ was significantly higher by 12 or 20%, respectively, than that of the uninoculated controls. The 1993 site had an indigenous soil population (about 10⁴ cells g^-1) of symbiotically ineffective soybean-nodulating bacteria. Nevertheless, six-week-old Morgan soybean plants inoculated with strain TA-11NOD⁺ had 44% more nodules and exhibited 50% more nitrogen fixation by acetylene reduction when compared with plants that received the parental strain I-110ARS. Nodule occupancy, as determined using genetic markers for rifampicin and streptomycin resistance, was significantly higher for strain TA-11NOD⁺ than for strain I-110ARS. Overall, for the two years and the two soybean genotypes, the yield obtained with TA-11NOD⁺ was 6% higher than that obtained with I-110ARS. Competition experiments were conducted in the greenhouse and strain TA-11NOD⁺ was significantly more competitive than strain I-110ARS in competition with strains USDA 6 or USDA 438.     

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.     

Enhanced Nodulation and Nitrogen Fixation by a Revertant of a Nodulation-Defective Bradyrhizobium japonicum Tryptophan Auxotroph

In greenhouse studies, the symbiotic properties of a prototrophic revertant (TA11 NOD⁺) of a nodulation defective tryptophan auxotroph of Bradyrhizobium japonicum were compared with those of the normally nodulating wild-type strain, B. japonicum I-110 ARS. Strain I-110 ARS was the parent of auxotrophic mutant TA11. Plants inoculated with TA11 NOD⁺ contained significantly more nitrogen per plant than did plants inoculated with wild-type bacteria (275.9 ± 35 versus 184 ± 18 mg). Also, plants that received the revertant were larger, averaging 8.4 ± 0.9 g (dry weight) versus 6.4 ± 0.6 g for those that received the wild-type bacterial strain. Additionally, plants that received the NOD⁺ strain had 56% more nodules and 41% more nodule mass than did control plants. With both inocula, average nodule size and amount of nitrogen fixed per gram of nodule were about the same. These data indicated that the improvement in nitrogen fixation observed with the TA11 NOD⁺ resulted from an increase in the overall nodule number. The physiological basis for this increase in nodulation is not known, but enhanced tryptophan catabolism does not appear to be involved.     

Regulation of nodulation in the soybean-Rhizobium symbiosis : strain and cultivar variability

Double inoculation (15 h apart) of the soybean cultivar Williams with Bradyrhizobium japonicum I-110ARS reveals a rapid regulatory plant response that inhibits nodulation of distal portions of the primary root (M Pierce, WD Bauer 1984 Plant Physiol 73: 286-290). Only living, homologous rhizobia elicit the response. We conducted similar double inoculation experiments to test the hypothesis that this is a universal phenomenon in soybean symbioses. We investigated interactions of the cultivar McCall with the slow-growing strain Bradyrhizobium sp. 3185 (=3G4b16) and strains of the fast-growing soybean symbiont, Rhizobium fredii (USDA191 [Nod⁺ on McCall] and USDA257 [Nod⁻ on McCall]). Nodulation was not detectably inhibited when USDA257 was included in various combinations with an inoculum of USDA191. Strain USDA257 cohabited nodules with strain USDA191 when plants were inoculated sequentially with both strains, but USDA257 did not nodulate McCall when a sterile culture filtrate of USDA191 was added to USDA257 inoculum. There was only a slight inhibition of nodulation of distal portions of the primary root in double inoculation experiments with McCall and strain 3185. Because these results were unexpected, we repeated the experiments with Williams and strain I-110ARS. The response was similar to that observed in the McCall × 3185 interaction. Regulation of nodulation on the primary root thus appears to be variable and depend on strain X cultivar interactions.     

Symbiotic efficiency and phylogeny of the rhizobia isolated from Leucaena leucocephala in arid–hot river valley area in Panxi, Sichuan, China

In search of effective nitrogen-fixing strains for inoculating Leucaena leucocephala, we assessed the symbiotic efficiency of 41 rhizobial isolates from root nodules of L. leucocephala growing in the arid–hot river valley area in Panxi, China. The genetic diversity of the isolates was studied by analyzing the housekeeping genes 16S rRNA and recA, and the symbiotic genes nifH and nodC. In the nodulation and symbiotic efficiency assay, only 11 of the 41 isolates promoted the growth of L. leucocephala while the majority of the isolates were ineffective in symbiotic nitrogen fixation. Furthermore, one fourth of the isolates had a growth slowing effect on the host. According to the 16S rRNA and recA gene analyses, most of the isolates were Ensifer spp. The remaining isolates were assigned to Rhizobium, Mesorhizobium and Bradyrhizobium. The sequence analyses indicated that the L. leucocephala rhizobia had undergone gene recombination. In contrast to the promiscuity observed as a wide species distribution of the isolates, the results implied that L. leucocephala is preferentially nodulated by strains that share common symbiosis genes. The symbiotic efficiency was not connected to chromosomal background of the symbionts and isolates carrying a similar nifH or nodC showed totally different nitrogen fixation efficiency.     

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.     

Strain-Specific Inhibition of nod Gene Induction in Bradyrhizobium japonicum by Flavonoid Compounds

A broad-host-range plasmid, pEA2-21, containing a Bradyrhizobium japonicum nodABC'-'lacZ translational fusion was used to identify strain-specific inhibitors of the genes required for soybean nodulation, the common nod genes. The responses of type strains of B. japonicum serogroups USDA 110, USDA 123, USDA 127, USDA 129, USDA 122, and USDA 138 to nod gene inhibitors were compared. Few compounds inhibited nod gene expression in B. japonicum USDA 110. In contrast, nod gene expression in strains belonging to several other serogroups was inhibited by most of the flavonoids tested. However, the application of two of these strain-specific compounds, chrysin and naringenin, had little effect on the pattern of competition between indigenous and inoculum strains of B. japonicum in greenhouse and field trials. Preliminary studies with radiolabeled chrysin and naringenin suggest that the different responses to nod gene inhibitors may be partly due to the degree to which plant flavonoids can be metabolized by each strain.     

Altered exopolysaccharides of Bradyrhizobium japonicum mutants correlate with impaired soybean lectin binding, but not with effective nodule formation

 The exact mechanism(s) of infection and symbiotic development between rhizobia and legumes is not yet known, but changes in rhizobial exopolysaccharides (EPSs) affect both infection and nodule development of the legume host. Early events in the symbiotic process between Bradyrhizobium japonicum and soybean (Glycinemax [L.] Merr.) were studied using two mutants, defective in soybean lectin (SBL) binding, which had been generated from B. japonicum 2143 (USDA 3I-1b-143 derivative) by Tn5 mutagenesis. In addition to their SBL-binding deficiency, these mutants produced less EPS than the parental strain. The composition of EPS varied with the genotype and with the carbon source used for growth. When grown on arabinose, gluconate, or mannitol, the wild-type parental strain, B. japonicum 2143, produced EPS typical of DNA homology group I Bradyrhizobium, designated EPS I. When grown on malate, strain 2143 produced a different EPS composed only of galactose and its acetylated derivative and designated EPS II. Mutant 1252 produced EPS II when grown on arabinose or malate, but when grown on gluconate or mannitol, mutant 1252 produced a different EPS comprised of glucose, galactose, xylose and glucuronic acid (1:5:1:1) and designated EPS III. Mutant 1251, grown on any of these carbon sources, produced EPS III. The EPS of strain 2143 and mutant 1252 contained SBL-binding polysaccharide. The amount of the SBL-binding polysaccharide produced by mutant 1252 varied with the carbon source used for growth. The capsular polysaccharide (CPS) produced by strain 2143 during growth on arabinose, gluconate or mannitol, showed a high level of SBL binding, whereas CPS produced during growth of strain 2143 on malate showed a low level of SBL binding. However, the change in EPS composition and SBL binding of strain 2143 grown on malate did not affect the wild-type nodulation and nitrogen fixation phenotype of 2143. Mutant 1251, which produced EPS III, nodulated 2 d later than parental strain 2143, but formed effective, nitrogen-fixing tap root nodules. Mutant 1252, which produced either EPS II or III, however nodulated 5–6 d later and formed few and ineffective tap root nodules. Restoration of EPS I production in mutant 1252 correlated with restored SBL binding, but not with wild-type nodulation and nitrogen fixation. Received: 6 October 1999 / Accepted: 18 November 1999     

Relationship of the Presence and Copy Number of Plasmids to Exopolysaccharide Production and Symbiotic Effectiveness in Rhizobium fredii USDA 206

Rhizobium fredii USDA 206 harbors four large plasmids, one of which carries nodulation and nitrogen fixation genes. Previously isolated groups of plasmid-cured derivatives of strain USDA 206 were compared with each other to determine possible plasmid functions. Mutant strain 206CANS was isolated as a nonmucoid (Muc⁻) derivative of strain 206CA, a mutant that was cured of two plasmids. The Muc⁻ phenotype of 206CANS was only expressed when the strain was grown on certain media, particularly those with polyols as carbon sources. Plasmid pRj206b of strain 206CANS was previously shown to have a higher copy number than the same plasmid in strains USDA 206 and 206CA. When this plasmid was transferred to Muc⁺ strains, it conferred a nonmucoid phenotype on recipient strains. The symbiotic effectiveness of the wild-type and cured strains was compared. Overall, few differences were shown, but strains 206CA and 206CANS were found to have higher nitrogenase activities than the other strains. Thus, there appeared to be a possible relationship among exopolysaccharide synthesis, plasmid copy number, and symbiotic effectiveness.