Strain selection for improvement of Bradyrhizobium japonicum competitiveness for nodulation of soybean

A Bradyrhizobium japonicum USDA 110-derived strain able to produce wider halos in soft-agar medium than its parental strain was obtained by recurrent selection. It was more chemotactic than the wild type towards mannitol and three amino acids. When cultured in minimal medium with mannitol as a single carbon-source, it had one thick subpolar flagellum as the wild type, plus several other flagella that were thinner and sinusoidal. Root adsorption and infectivity in liquid media were 50-100% higher for the selected strain, but root colonization in water-unsaturated vermiculite was similar to the wild type. A field experiment was then carried out in a soil with a naturalized population of 1.8 x 10(5) soybean-nodulating rhizobia g of soil(-1). Bradyrhizobium japonicum strains were inoculated either on the soybean seeds or in the sowing furrows. Nodule occupation was doubled when the strains were inoculated in the sowing furrows with respect to seed inoculation (significant with P<0.05). On comparing strains, nodule occupation with seed inoculation was 6% or 10% for the wild type or selected strains, respectively, without a statistically significant difference, while when inoculated in the sowing furrows, nodule occupation increased to 12% and 22%, respectively (differences significant with P<0.05).     

Effects of N-starvation and C-source on Bradyrhizobium japonicum exopolysaccharide production and composition, and bacterial infectivity to soybean roots

The exopolysaccharide (EPS) is an extracellular molecule that in Bradyrhizobium japonicum affects bacterial efficiency to nodulate soybean. Culture conditions such as N availability, type of C-source, or culture age can modify the amount and composition of EPS. To better understand the relationship among these conditions for EPS production, we analyzed their influence on EPS in B. japonicum USDA 110 and its derived mutant ΔP22. This mutant has a deletion including the 3′ region of exoP, exoT, and the 5′ region of exoB, and produces a shorter EPS devoid of galactose. The studies were carried out in minimal media with the N-source at starving or sufficient levels, and mannitol or malate as the only C-source. Under N-starvation there was a net EPS accumulation, the levels being similar in the wild type and the mutant with malate as the C-source. By contrast, the amount of EPS diminished in N-sufficient conditions, being poyhydroxybutyrate accumulated with culture age. Hexoses composition was the same in both N-situations, either with mannitol or malate as the only C-source, in contrast to previous observations made with different strains. This result suggests that the change in EPS composition in response to the environment is not general in B. japonicum. The wild type EPS composition was 1 glucose:0.5 galactose:0.5 galacturonic acid:0.17 mannose. In ΔP22 the EPS had no galactose but had galacturonic acid, thus indicating that it was not produced from oxidation of UDP-galactose. Infectivity was lower in ΔP22 than in USDA 110. When the mutant infectivity was compared between N-starved or N-sufficient cultures, the N-starved were not less infective, despite the fact that the amounts of altered EPS produced by this mutant under N-starvation were higher than in N-sufficiency. Since this altered EPS does not bind soybean lectin, the interaction of EPS with this protein was not involved in increasing ΔP22 infectivity under N-starvation.     

Agglutination ofRhizobium japonicum 3I1b110 by soybean lectin

Summary Conditions leading to agglutination ofRhizobium japonicum 3I1b110 with soybean seed lectin were examined. Ability of cells to be agglutinated was transient and was optimal for cultures grown for 4–5 days on yeast extract mannitol plates. Similar lectin-binding results were obtained with cells from the same cultures using fluorescence microscopy with fluorescein isothiocyanate-labelled lectin. These results revise the previous model for soybean lectin-R. japonicum interactions, since it was based on the inability of soybean lectin to agglutinate these bacteria.     

Chemical control of interstrain competition for soybean nodulation by Bradyrhizobium japonicum

Previous research has shown that a significant limitation to the agricultural use of improved rhizobial inoculant strains is competition from the indigenous soil population. In this work, we sought to test whether chemical inhibitors of flavonoid-induced nod gene expression in Bradyrhizobium japonicum could be identified and utilized to affect interstrain competition for nodulation of soybeans. Approximately 1,000 structural and functional analogs of the known, natural inducers of nod gene expression were tested on six strains of B. japonicum containing a nodY-lacZ fusion. We successfully identified effective inhibitors of nodY expression. The addition of the inhibitor 7-hydroxy-5-methylflavone significantly inhibited nodulation by a sensitive strain and could be used to effectively manipulate the competition between strains for soybean nodulation. However, this work also uncovered significant limitations for the practical use of this methodology. For example, despite the almost universal induction response to the identified natural inducers, there was a wide variability among strains in their response to any specific inhibitor. Given this unexpected variability, the cost of registration of an agronomic chemical, and the potential for the development of resistant field populations, it is unlikely that chemical inhibitors can be successfully applied to a field situation.     

The effect of growth medium on lectin binding byRhizobium japonicum

Summary A comparative study of soybean lectin binding byRhizobium japonicum 61A76 grown in yeast extract mannitol medium supplemented with soil extract showed that it produced cells with greater lectin binding capacity, larger cell size and a higher percentage of coccoid forms than those produced in yeast extract mannitol medium alone.     

Chemical control of interstrain competition for soybean nodulation by Bradyrhizobium japonicum.

Previous research has shown that a significant limitation to the agricultural use of improved rhizobial inoculant strains is competition from the indigenous soil population. In this work, we sought to test whether chemical inhibitors of flavonoid-induced nod gene expression in Bradyrhizobium japonicum could be identified and utilized to affect interstrain competition for nodulation of soybeans. Approximately 1,000 structural and functional analogs of the known, natural inducers of nod gene expression were tested on six strains of B. japonicum containing a nodY-lacZ fusion. We successfully identified effective inhibitors of nodY expression. The addition of the inhibitor 7-hydroxy-5-methylflavone significantly inhibited nodulation by a sensitive strain and could be used to effectively manipulate the competition between strains for soybean nodulation. However, this work also uncovered significant limitations for the practical use of this methodology. For example, despite the almost universal induction response to the identified natural inducers, there was a wide variability among strains in their response to any specific inhibitor. Given this unexpected variability, the cost of registration of an agronomic chemical, and the potential for the development of resistant field populations, it is unlikely that chemical inhibitors can be successfully applied to a field situation.     

Effect of lectin on nodulation by wild-type Bradyrhizobium japonicum and a nodulation-defective mutant

The nodulation characteristics of wild-type Bradyrhizobium japonicum USDA 110 and mutant strain HS111 were examined. Mutant strain HS111 exhibits a delayed-nodulation phenotype, a result of its inability to initiate successful nodulation promptly following inoculation of the soybean root. Previously, we showed that the defect in initiation of infection leading to subsequent nodulation which is found in HS111 can be phenotypically reversed by pretreatment with soybean root exudate or soybean seed lectin. This effect is not seen after pretreatment with root exudates and lectins obtained from other plant species. Treatment of strain HS111 with as little as 10 soybean seed lectin molecules per bacterium (3.3 X 10 (-12) M) resulted in enhancement of nodule formation. Pretreatment of wild-type B. japonicum USDA 110 with soybean root exudate or seed lectin increased nodule numbers twofold on 6-week-old plants. Wild-type strain USDA 110 cells inoculated at 10(4) cells per seedling exhibited a delay in initiation of infection leading to subsequent nodulation. Wild-type cells pretreated in soybean root exudates or seed lectin did not exhibit a delay in nodulation at this cell concentration. Mutant strain HS111 pretreated in seed lectin for 0 or 1 h, followed by washing with the hapten D-galactose to remove the lectin, exhibited a delay in initiation of nodulation. Phenotypic reversal of the delayed-nodulation phenotype exhibited by strain HS111 was seen if incubation was continued for an additional 71 h in plant nutrient solution following 1 h of lectin pretreatment. Reversal of the delayed-nodulation phenotype of HS111 through lectin pretreatment was prevented by chloramphenicol or rifampin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and use of actinomycetes for enhanced nodulation of soybean co-inoculated with Bradyrhizobium japonicum

The utilization of actinomycetes as potential soybean (Glycine max (L.)) co-inoculants was evaluated. Soil samples from Carbondale and Belleville, Ill., were used to inoculate pre-germinated soybean plants to determine antibiotic sensitivity in the native Bradyrhizobium japonicum population. Sensitivity was in the order kanamycin > tetracycline > oxytetracycline > rifampicin > neomycin. Antagonism by five actinomycete cultures toward seven test strains of B. japonicum was also assessed. The ranking average inhibition (across all seven B. japonicum strains) by these actino mycetes was Streptomyces kanamyceticus = Streptomyces coeruleoprunus > Streptomyces rimosus > Streptomyces sp. > Amy colatopsis mediterranei. Ten antibiotic combinations were used to isolate antibiotic-resistant mutants of B. japonicum I-110 and 3I1B-110 via successive cycles of mutation. Eighty-one antibiotic-resistant strains were isolated and tested for symbiotic competency; nine of which were selected for further characterization in a greenhouse pot study. Few differences in nodule number were caused by these treatments. Nodule occupancy varied from 0% to 18.3% when antibiotic-resistant strains of B. japonicum were used as the sole inoculants. However, when three mutant strains of B. japonicum were co-inoculated with S. kanamyceticus, significant increases in nodule occupancy (up to 55%) occurred. Increases in shoot nitrogen composition (27.1%-40.9%) were also caused by co-inoculation with S. kanamyceticus.     

Increased nodulation of soybean by a strain of Bradyrhizobium japonicum with altered tryptophan metabolism

A strain of Bradyrhizobium japonicum was isolated that accumulated anthranilic acid, indole, 3-indoleacetic acid, 3-indolelactic acid and 3-indolepyruvic acid in culture. Such accumulations are indicative of altered tryptophan metabolism. Soybean plants inoculated with these bacteria formed more nodules (349 vs 159 per plant) and had more nodule mass (3.9 vs 2.2 g wet wt per plant) than plants inoculated with the wild-type strain.     

Effect of lectin on nodulation by wild-type Bradyrhizobium japonicum and a nodulation-defective mutant.

The nodulation characteristics of wild-type Bradyrhizobium japonicum USDA 110 and mutant strain HS111 were examined. Mutant strain HS111 exhibits a delayed-nodulation phenotype, a result of its inability to initiate successful nodulation promptly following inoculation of the soybean root. Previously, we showed that the defect in initiation of infection leading to subsequent nodulation which is found in HS111 can be phenotypically reversed by pretreatment with soybean root exudate or soybean seed lectin. This effect is not seen after pretreatment with root exudates and lectins obtained from other plant species. Treatment of strain HS111 with as little as 10 soybean seed lectin molecules per bacterium (3.3 X 10 (-12) M) resulted in enhancement of nodule formation. Pretreatment of wild-type B. japonicum USDA 110 with soybean root exudate or seed lectin increased nodule numbers twofold on 6-week-old plants. Wild-type strain USDA 110 cells inoculated at 10(4) cells per seedling exhibited a delay in initiation of infection leading to subsequent nodulation. Wild-type cells pretreated in soybean root exudates or seed lectin did not exhibit a delay in nodulation at this cell concentration. Mutant strain HS111 pretreated in seed lectin for 0 or 1 h, followed by washing with the hapten D-galactose to remove the lectin, exhibited a delay in initiation of nodulation. Phenotypic reversal of the delayed-nodulation phenotype exhibited by strain HS111 was seen if incubation was continued for an additional 71 h in plant nutrient solution following 1 h of lectin pretreatment. Reversal of the delayed-nodulation phenotype of HS111 through lectin pretreatment was prevented by chloramphenicol or rifampin.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Characterization of the Bradyrhizobium japonicum galE gene: its impact on lipopolysaccharide profile and nodulation of soybean

The galE gene from Bradyrhizobium japonicum 61A101C, a soybean endosymbiont, was cloned and characterized. Its deduced amino-acid sequence showed a high similarity with that of other rhizobia. Functional identification of the galE gene was achieved by complementation of a galE mutant strain, PL2, with a series of pKM subclones. Disruption of the B. japonicum galE gene affects the lipopolysaccharide profile compared with that of the wild type, suggesting that galE is responsible for alteration of lipopolysaccharide structure. Examination of nodule formation by the wild-type and galE mutant revealed that the former displayed normal nodule development on soybean roots, whereas the latter showed no nodule formation at all time points examined except for 20 days after inoculation when <10% of soybean formed pseudo-nodules.     

Increase of Bradyrhizobium japonicum numbers in soils and enhanced nodulation of soybean (Glycine max (L) merr.) using granular inoculants amended with nutrients

Abstract: Mineral microgranules, amended with nutrients and inoculated with either peat or liquid Bradyrhizobium japonicum inoculants, increased the growth and recovery of the bacterium during laboratory incubation in unsterilized soil. Increases in the range of 1 log unit per g or ml inoculant used were observed in different soil types. B. japonicum showed better survival with nutrient-amended granules than in unamended ones, in soil undergoing desiccation. In a growth chamber experiment, the number of nodules per plant were significantly increased by nutrient-amendment of the granules, but only under suboptimal conditions for nodulation. Nutrient-amended granules significantly enhanced early nodulation of soybean and increased N content of the grain at harvest in four field trials. All these effects were obtained using an average of 10 kg granules amended with 1.14 kg glycerol and 0.16 kg sodium glutamate per hectare. The possible use of nutrient-amended granules to improve efficacy and reliability of microbial inoculation is discussed.     

Effect of organic N source on bacterial growth, lipo-chitooligosaccharide production, and early soybean nodulation by Bradyrhizobium japonicum

Production of Bradyrhizobium japonicum inoculants is problematic because high inoculation rates are necessary but expensive, while production of rhizobial Nod factors (lipo-chitooligosaccharides (LCOs)), key signal molecules in the establishment of legume-rhizobia symbioses, may be inhibited at high culture cell densities. We conducted experiments to determine the effects of growth medium N source on B. japonicum growth, LCO production, and early nodulation of soybean. We found that 1.57 mmol ammonium nitrate x L(-1) resulted in less rhizobial growth and rhizobial capacity to produce LCOs (on a per cell basis) than did 0.4 g yeast extract x L(-1), which contained the same amount of N as the ammonium nitrate. Increasing yeast extract to 0.8 g x L(-1) increased rhizobial growth and LCO production on a volume basis (per litre of culture) and did not affect cell capacity to produce LCOs; however, at 1.4 g yeast extract x L(-1) per cell, production was reduced. A mixture of 0.8 g yeast extract x L(-1) and 1.6 g casein hydrolysate x L(-1) resulted in the greatest bacterial growth and LCO production on a volume basis but reduced LCO production per cell. Changes in organic N level and source increased production of some of the measured LCOs more than others. LCO production was positively correlated with cell density when expressed on a volume basis; however, it was negatively correlated on a per cell basis. We conclude that although quorum sensing affected Nod factor production, increased levels of organic N, and specific compositions of organic N, increased LCO production on a volume basis. Greenhouse inoculation experiments showed that the medium did not modify nodule number and N fixation in soybean, suggesting that it could have utility in inoculant production.     

Suppression of nodulation in soybeans by superoptimal inoculation with Bradyrhizobium japonicum

Symbiotic nodulation of the primary roots of soybeans ( Glycine max L. Merrill cv. Pride 216) is regulated by the plant, and is suppressed in response to a high inoculum dose of Bradyrhizobium japonicum USDA strain I–110 (ARS)⁺ applied at one time to the root. If an optimal dose is followed 10 h later by a superoptimal dose, nodules from the first inoculum near the base of the primary root are suppressed in a dose-dependent way similar to that observed after single inoculations. The nodules which appear are probably derived from infections initiated by the bacteria in both inocula.     

Associative effect ofAzospirilium brasilense withRhizobium japonicum on nodulation and yield of soybean (Glycine max)

Summary Azospirillum was associated with nodules of soybean. In general, seed inoculation with a broth culture ofAzospirillum brasilense alone significantly increased nodulation and grain yield of soybean grown in pots in unsterilized soil with different levels of urea ranging from 0 to 80 kg N/ha. This trend was significantly reproducible in a second experiment when a carrier based inoculant of the bacterium was used for seed inoculation.Inoculation withRhizobium japonicum andA. brasilense in combination generally increased grain yield in both the experiments, although the data were not significant.     

Concentration-dependent influence of quercetin on nodulation process and the main characteristics of soybean inoculated with Bradyrhizobium japonicum

Soybean plants cv. Corsoy were grown in greenhouse conditions on sterilized quartz sand. They were inoculated with Bradyrhizobium japonicum, strain 542. The plants were treated with different concentrations of quercetin (ranging from 10 nM to 1M) at regular intervals during the experiment. The experiment was terminated at flower development. The following parameters, important for symbiosis efficiency were determined: shoot, root and nodule weights, nodule number, total leghemoglobin in the nodules,total nitrogen and soluble protein concentrations in shoots and roots, as well as chlorophyll concentration in the leaves.The results obtained partly confirmed the earlier findings that quercetin inhibits nodulation since increasing quercetin concentration decreased the number of nodules. However, at very low concentrations, quercetin stimulated the number of nodules. Quercetin also exerted a stimulating influence on other characteristics of the plant and nodules which did not correlate with nodule number and quantity of N fixed. These are: nodule weight, leghemoglobin concentration, total soluble protein content in shoots and roots as well as shoot and root weight.     

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