Rhizobitoxine Production by Bradyrhizobium elkanii Enhances Nodulation and Competitiveness on Macroptilium atropurpureum

Application of 1-aminoocyclopropane-1-carboxylic acid, an ethylene precursor, decreased nodulation of Macroptilium atropurpureum by Bradyrhizobium elkanii. B. elkanii produces rhizobitoxine, an ethylene synthesis inhibitor. Elimination of rhizobitoxine production in B. elkanii increased ethylene evolution and decreased nodulation and competitiveness on M. atropurpureum. These results suggest that rhizobitoxine enhances nodulation and competitiveness of B. elkanii on M. atropurpureum.     

DNA Sequence and Mutational Analysis of Rhizobitoxine Biosynthesis Genes in Bradyrhizobium elkanii

We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii. The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of rtxA. A large-deletion mutant of B. elkanii, USDA94Δrtx::Ω1, which lacks rtxA, ORF1 (rtxC), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxA and these ORFs, complemented rhizobitoxine production in USDA94Δrtx::Ω1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the rtxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that rtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.     

Effects of ethylene precursor and inhibitors for ethylene biosynthesis and perception on nodulation in Lotus japonicus and Macroptilium atropurpureum

Inhibitors of ethylene synthesis or its physiological function enhanced nodulation in Lotus japonicus and Macroptilium atropurpureum. In contrast, the application of 1-aminocyclopropane-1-carboxylic acid, a precursor of ethylene biosynthesis, reduced the nodule number in these legumes. These results suggest that an ethylene-mediated signaling pathway is involved in the nodulation process even in the determinate nodulators.     

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).     

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.     

Nitrogen-fixing bacteria communities occurring in soils under different uses in the Western Amazon Region as indicated by nodulation of siratro (Macroptilium atropurpureum)

Understanding native communities is a crucial step for the management of biological nitrogen fixation, since they may be either a source of efficient strains or a limiting factor when efficient strains need to be introduced. This work aimed to evaluate the density, diversity and efficiency of Leguminosae nodulating bacterial (LNB) communities and their component strains in soils under various land use systems (LUSs): pristine forest, agriculture, pasture, agroforestry, young secondary forest, and old secondary forest,. The LNB communities were trapped from these soils by using the promiscuous host siratro under controlled conditions. We also studied their relationships with physical and chemical attributes of the soil. Agroforestry and agriculture soil samples induced the highest number of nodules in siratro, while forest soil samples induced the lowest number of nodules. No relationship was found between LNB and Leguminosae species diversity in the LUSs. The soil chemical variables that were most related to differences in nodule number and shoot dry matter weight of plants inoculated with soil suspensions of the LUSs were, respectively: Ca²⁺, Mg²⁺, base saturation, exchangeable bases and Cu²⁺; and pH, cation exchange capacity, B, Cu²⁺ and clay. Although, LNB communities from all LUSs were efficient under controlled and similar conditions, they were found to be composed of strains with variable efficiency: inefficient, efficient, highly efficient and superior efficiency. Efficient strains occurred at the highest frequency in all LUSs. The isolated strains presented similar and new sequences that were phylogenetically related to well known LNB genera in α-and β-Proteobacteria. Unusual genera in these branches, as well as in other branches, which are probably endophytic bacteria, were also isolated from nodules. These data support siratro as a useful trap species to study the LNB biodiversity of diverse ecosystems in tropical soils. The fact that the highest diversity and nodulation were seen in managed systems such as agriculture and agroforestry suggests a high resilience of LNB communities to changes in land use after deforestation in a region where large forest areas are still preserved and can be a source of propagules.     

Influence of Water Stress on Flowering and Seed Production of Macroptilium atropurpureum cv. Siratro

Macroptilium atropurpureum cv. Siratro was grown in large soilbeds with a constant water table below, developing a dawn leafwatêr potential of --0.25 MPa. Water stresses equivalentto -0.7 or -1.0 MPa were developed over 14 d, causing reducedstem and bud elongation, leaf expansion, and bud differentiationand survival. Apex size, the proportion of buds which were floralor vegetative, the early phases of floral initiation, and seedformation on advanced inflorescences were little affected duringthe water deficit period. Upon rewatering previously stressed plants showed increasesrelative to control plants in the rates of shoot appearance,leaf expansion and new node appearance. The ratio of buds becomingfloral was independent of watering treatment, and the enhancedrate of floral bud production in the previously-stressed treatmentswas due to higher rates of total bud differentiation which persistedfor up to six weeks after rewatering. Survival of floral budswas reduced by previous stress, but number of flowers per inflorescence,pod setting, seed number per pod and 100-seed weight were independentof treatment. Seed production was controlled by inflorescencedensity. Rate of seed production was independent of treatmentduring water deficit and four weeks subsequently, and was thenenhanced by 46 and 54 per cent relative to the control in the–0.7 and –1.0 MPa treatments respectively. Macroptilium atropurpureum, Siratro, floral initiation, flowering, seed production, water stress, bud development     

Rhizobitoxine production and symbiotic compatibility of Bradyrhizobium from Asian and North American lineages of amphicarpaea.

Reciprocal inoculations with Bradyrhizobium sp. isolates from the North American legume Amphicarpaea bracteata (L.) Fern. (Phaseoleae-Glycininae) and from a Japanese population of its close relative Amphicarpaea edgeworthii (Benth.) var. japonica were performed to analyze relative symbiotic compatibility. Amphicarpaea edgeworthii plants formed few or no nodules with any North American bradyrhizobial strains isolated from A. bracteata, but all A. bracteata lineages formed effective nitrogen-fixing nodules with Japanese Bradyrhizobium isolates from A. edgeworthii. However, one group of A. bracteata plants (lineage Ia) when inoculated with Japanese bradyrhizobia developed a striking leaf chlorosis similar to that known to be caused by rhizobitoxine. The beta-cystathionase inhibition assay demonstrated that significant amounts of rhizobitoxine were present in nodules formed by these Japanese bradyrhizobia. No North American bradyrhizobial isolate from A. bracteata induced chlorosis on any plants, and the beta-cystathionase assay failed to detect rhizobitoxine in nodules formed by these isolates. The role of rhizobitoxine in A. edgeworthii nodulation development was tested by inoculating plants with a Bradyrhizobium elkanii rhizobitoxine-producing strain, USDA 61, and two mutant derivatives, RX17E and RX18E, which are unable to synthesize rhizobitoxine. Amphicarpaea edgeworthii inoculated with wild-type USDA 61 developed >150 nodules per plant, while plants inoculated with RX17E and RX18E developed fewer than 10 nodules per plant. Thus, efficient nodule development in A. edgeworthii appears to be highly dependent on rhizobitoxine production by Bradyrhizobium strains.     

Partitioning and redistribution of sulphur during S-stress in Macroptilium atropurpureum cv. Siratro

During the first 7 d of sulphate-deprivation stored SO₄^2- wasredistributed and assimilated into organic forms in the tropicallegume Macroptilium atropurpu-reum cv. Siratro. However, whilstthe sulphate content of all tissues declined after removingthe external SO₄^2- supply this was slowest in mature leaves.By contrast, the total S content of mature leaves declined markedlyin the absence of external sulphate whilst that of both youngleaves and roots increased. Furthermore, when radiolabelledSO₄^2- was applied to abraded surfaces of mature leaves, mostof the translocated label was recovered in the root following2 d SO₄^2- deprivation. By contrast, radiolabelled SO₄^2-appliedto young leaves was mostly retained in these tissues and nottranslocated. Within 3 d of removing the SO₄^2- supply there was a large increasein extractable APS-sulphotransferase activity in roots accompaniedby a decline in nitrate reductase activity, but these effectswere not seen in leaves. Five days after the removal of SO₄^2-there was a large increase in the content of asparagine in roots. The results are discussed in relation to the co-ordination ofNO₃^- and SO₄^2- uptake and assimilation and the partitioningof sulphur during S-stress. Key words: Sulphate supply, stomatal conductance, ATP-sulphurylase, APS-sulphotransferase, nitrate reductase     

Stimulation of adhesiveness, infectivity, and competitiveness for nodulation of Bradyrhizobium japonicum by its pretreatment with soybean seed lectin

Soybean seed lectin stimulates adsorption of Bradyrhizobium japonicum to its host roots. Pretreatment of the rhizobia with soybean seed lectin for at least 6-12 h previous to their interaction with the plants was required to detect the stimulatory effect. This activity could be observed with as few as 1000 soybean seed lectin molecules per bacterium, and required specific carbohydrate binding. Infectivity and competitiveness for nodulation were also stimulated by preincubation of the rhizobia either with soybean seed meal extract or soybean seed lectin, the extract being more effective in enhancing competitiveness.     

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.     

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)     

Isolation and characterization of low-indole-3-acetic acid-producing mutants from Bradyrhizobium elkanii

We isolated 11 low-indole-3-acetic acid (IAA)-producing mutants of Bradyrhizobium elkanii by Tn5 mutagenesis. The amount of IAA produced by each mutant was 2.2-13.6% of that of the wild-type. It was found by resting cell reactions that the biosynthetic step to convert indole-3-pyruvic acid to indole-3-acetaldehyde was blocked in all the mutants.     

A Selective Medium for the Isolation and Quantification of Bradyrhizobium japonicum and Bradyrhizobium elkanii Strains from Soils and Inoculants

The ecological examination of members of the family Rhizobiaceae has been hampered by the lack of a selective medium for isolation of root nodule bacteria from soil. A novel non-antibiotic-containing medium has been developed which allows selective isolation of Bradyrhizobium japonicum and B. elkanii strains from soil and inoculants. The medium, BJSM, is based on the resistance of B.japonicum and B. elkanii strains to more than 40 μg of the metals ions Zn²⁺ and Co²⁺ per ml. BJSM does not allow growth of Rhizobium sp. strains. We used BJSM to isolate bacteria from a Hubbard soil and from several commercially prepared soybean inoculants. Ninety-eight percent of the isolates obtained from Hubbard soil nodulated Glycine max cv. Kasota, and between 55 and 95% of the isolates from the commercial inoculants had the ability to nodulate soybeans. Numbers of bradyrhizobia obtained by using BJSM, strain-specific fluorescent antibodies, and the most-probable-number plant infection assay indicated that the three techniques were comparable in quantifying B. japonicum strains in soils and inoculants, although most-probable-number counts were generally 0.5 order of magnitude greater than those obtained by using BJSM. Results of our studies indicate that BJSM is useful for direct isolation and quantification of B. japonicum and B. elkanii from natural soils and inoculants. This medium may prove to be an important tool for autecological and enumeration studies of diverse populations of bradyrhizobia and as a quality control method for soybean inoculants.     

The Involvement of Indole-3-Acetic Acid Produced by Bradyrhizobium elkanii in Nodule Formation

IAA-deficient mutants T10, T27 and TN3 were isolated from B.elkanii USDA 31 by spontaneous and/or N-methyl-N'-nitro-N-nitrosoguanidine(NTG) mutageneses. Inoculation with these mutants significantlyreduced the nodule number on soybean roots when compared tothat of the parent strain. Furthermore, exogenous IAA applicationrestored the nodule number of soybeans inoculated with TN3 tothe original level. (Received July 18, 1994; Accepted September 13, 1994)