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.     

Interactions ofRhizobium japonicum strains in soybean nodules and their contribution to nitrogen fixation

Summary Rhizobium japonicum strain 8-0 Str^R applied as inoculum to Clark 63 soybeans formed small ineffective nodules which had very low nitrogenase activity compared to nodules formed by two effective strains, 110 Tet^R and 138 Kan^R. Mean numbers of cells per milligram of nodule tissue for plants up to 34 days old were 7.7×10⁶ for 8-0 Str^R, 4.1×10⁸ for 110 Tet^R and 7.6×10⁸ for 138 Kan^R. Cell counts per unit mass of nodule were independent of plant age for strains 110 Tet^R and 138 Kan^R, however, for strain 8-0 Str^R, 25 and 34 days old plants had fewer viable cells per nodule mass than 18 day old plants. When a mixture of two effective strains was used, the nodules of individual plants were predominantly caused by either 110 Tet^R or 138 Kan^R. In one experiment the predominance was random, but in another, strain 110 Tet^R clearly dominated. Strain 138 Kan^R was absent in some nodules on 18 day old plants, and in others, less than 10² cells per nodule were found. When strains 8-0 Str^R and 138 Kan^R were used as mixed inoculum, most of the nodules had strain 8-0 Str^R but strain 138 Kan^R was detected in many nodules and was generally evident in the largest nodules. Nitrogenase activity by many individual nodules was low except for nodules which had cells of 138 Kan^R. Nitrogenase activity by whole root systems of these plants was relatively high and similar to plants that had only nodules of strain 138 Kan^R. Similar relationships were observed for a mixed inoculum of 8-0 Str^R and 110 Tet^R. In general, mixed inoculations resulted in nodules with a particular strain being dominant for each individual plant. Double infections within individual nodules were not uncommon and such nodules often had disproportionate numbers of cells of two competingR. japonicum strains.Contribution from the Laboratory of Soil Microbiology, Department of Agronomy, Missouri Agricultural Experiment Station. Missouri Journal Series Number 7967.     

Denitrification of nitrate to nitrogen gas by washed cells ofRhizobium japonicum and by bacteroids fromGlycine max

Nitrate, nitrite and nitrous oxide were denitrified to N₂ gas by washed cells ofRhizobium japonicum CC706 as well as by bacteroids prepared from root nodules ofGlycine max (L.) Merr. (CV. Clark 63). Radiolabelled N₂ was produced from either K¹⁵NO₃ or Na¹⁵NO₂ by washed cells ofRh. japonicum CC705 grown with either nitrate only (5 mM) or nitrate (5 mM) plus glutamate (10 mM). Nitrogen gas was also produced from N₂O. Similar results were obtained with bacteroids ofG. max. The stoichiometry for the utilization of¹⁵NO ₃ ^- or¹⁵NO ₂ ^- and the produciton of¹⁵N₂ was 2:1 and for N₂O utilization and N₂ production it was 1:1. Some of the¹⁵N₂ gas produced by denitrification of¹⁵NO ₃ ^- in bacteroids was recycled via nitrogenase into cell nitrogen.     

Strain-specific antisera to identify Thai Bradyrhizobium japonicum strains in preserved soybean nodules

Strain-specific antisera were produced against six Bradyrhizobium japonicum strains using two immunization procedures. These specific antisera were used for detection of bradyrhizobia in preserved soybean nodules. Antisera specific for two of these strains were either conjugated with a fluorescent dye or used with a fluorescent secondary antibody for identification of bradyrhizobia in soybean nodules that were preserved in four different storage conditions. Results show that soybean nodules dried in the oven, stored under room temperature, or at –20 °C are as suitable as fresh nodules for strain identification using fluorescent antisera.     

Survival and multiplication ofRhizobium japonicum strains in silt loam

Summary Antibiotic resistant mutants 8-0 Str^R, 110 Tet^R and 138 Kan^R derived from wild typeRhizobium japonicum strains were inoculated into silt loam soil to cell concentrations greater than 2×10⁸/g of soil. Population changes were monitored using antibiotic media and strain identification was done using immunodiffusion assay on microcores of soil. Immunodiffusion bands formed by the mutant strains with homologous antisera essentially duplicated bands formed by the parent strain. Strains 110 Tet^R and 8-0 Str^R had cross reacting antigens whereas antigens of strain 138 Kan^R reacted only with the homologous antiserum. Populations ofR. japonicum strains introduced into sterile soil increased over a period of four weeks under both single and mixed culture inoculations. All populations decreased by the end of six weeks and thereafter remained constant. When theseR. japonicum strains were introduced into non-sterile soil, the population did not increase over the initial population added. Population decreased gradually for two weeks and then maintained thereafter. It was possible to recover very low populations of antibiotic resistantR. japonicum strains from both sterile and unsterile soils using media containing specific antibiotics. Detection ofR. japonicum strains by immunodiffusion was accomplished only when the population was 10⁹ cells/g of soil. The method using antibiotic resistant mutants permitted an evaluation of the interactions of variousR. japonicum strains in soil with respect to their survival and multiplication.     

Structural requirements for the binding of oligosaccharides to immobilized lectin ofErythrina variegata (Linn) var.Orientalis

The structural requirements for the interaction of asparagine-linked oligosaccharide moieties of glycoproteins withErythrina variegata agglutinin (EVA) were investigated by means of affinity chromatography on an EVA-Sepharose column. Some of the branched poly-N-acetyllactosamine-type oligosaccharides obtained from human erythrocyte band 3 glycoprotein were found to show high affinity to EVA-Sepharose, whereas complex-type oligosaccharides were shown to have low affinity. Hybrid type, oligomannose-type and unbranched poly-N-acetyllactosamine-type oligosaccharides bound very little or not at all to EVA-Sepharose. To further study the carbohydrate-binding specificity of this lectin, we investigated the interaction of immobilized EVA and oligosaccharide fragments obtained through partial hydrolysis from branched poly-N-acetyllactosamine-type oligosaccharides. Branched poly-N-acetyllactosamine-type oligosaccharides were subjected to limited hydrolysis with 0.1% trifluoroacetic acid at 100°C for 40 min and then separated on an amino-bonded silica column. One of pentasaccharides thus prepared strongly bound to the EVA-Sepharose column. Structural analysis of this pentasaccharide showed that the Gal1-4GlcNAc1-3(Gal1-4GlcNAc1-6)Gal sugar sequence, which is an l-antigen determinant, was essential for the high affinity binding of the oligosaccharides to the EVA-Sepharose column.Abbreviations EVA Erythrina variegata agglutinin - WGA wheat germ agglutinin - STA potato lectin - LEA tomato lectin - DSA Datura stramonium agglutinin - PBS 0.01 M sodium phosphate buffer, pH 7.3, containing 0.15 M NaCl - Galol galactitol     

Nodulation of soybean byRhizobium japonicum mutants with altered capsule synthesis

Spontaneous mutants with altered capsule synthesis were isolated from a marked strain of the symbiont,Rhizobium japonicum. Differential centrifugation was used to enrich serially for mutants incapable of forming capsules. The desired mutants were detected by altered colony morphology and altered ability to bind host plant lectin. Three mutants failed to form detectable capsules at any growth phase when cultured in vitro or in association with the host (soybean,Glycine max (L.) Merr.) roots. These mutants were all capable of nodulating and attaching to soybean roots, indicating that the presence of a capsule physically surrounding the bacterium is not required for attachment or for infection and nodulation. Nodulation by several of the mutants was linearly proportional to the amount of acidic exopolysaccharide that they released into the culture medium during the exponential growth phase, indicating that such polysaccharide synthesis is important and perhaps required for nodulation. Two of the mutants appeared to synthesize normal lectin-binding capsules when cultured in association with host roots, but not when cultured in vitro. Nodulation by these mutants appeared to depend on how rapidly after inoculation they synthesized capsular polysaccharide.Abbreviations CPS capsular polysaccharide - EPS exopolysaccharide - FITC fluorescein isothiocyanate Contribution No. 719 of the C.F. Kettering Research Laboratory     

Relationship between raman spectroscopic lines and growth ofRhizobium japonicum

The Raman spectroscopic lines of liquid cultures ofRhizobium japonicum have been compared with electron microscopic examinations and growth measurements of these cells. The results showed that the significant Raman lines are related to the reproduction activities of the procaryotic cells.     

Soil temperature effects on competitiveness and growth ofRhizobium japonicum and on Rhizobium-induced chlorosis of soybeans

Summary The effects of temperature on growth in broth and soil and on competition for nodule formation betweenRhizobium japonicum serotypes USDA 76 and 94 compared to 6 and 110 were studied. Increasing root temperatures of Lee soybean from 20 to 35°C increased the competitiveness of 76 and 94 relative to 6 and 110 for all inoculum ratios such that at 30 and 35°C symptoms ofRhizobium-induced chlorosis appeared. Tolerance to elevated temperatures was exhibited by 76 and 110, but not 94 and 6 in broth and soil which suggested that increased competitiveness of 76 and 94 at high soil temperatures was not dependent upon growth at elevated temperatures. Nodulation and vegetative growth of Lee soybeans were at a minimum at 20°C and optimum at 30°C. Differences in competitiveness of 6 to previous studies indicated the need to standardize temperatures of assays. Differences in growth responses of 76 and 94 to temperature from a previous study suggested a confounding effect on different carbon sources in growth media. Scientific Article No. A-3721 Contribution No. 6697 of the Maryland Agric Exp Sta, Dept of Agronomy, College Park, MD 20742 and the USDA, ARS, Beltsville, MD 20705. Part of a thesis submitted by the senior author in partial fulfillment of the requirements for the M.S. Degree.     

Carbohydrate exposure of human promyelocytic HL60 cells and histiocytic U937 cells during phagocytic differentiation assessed with fluoresceinated lectins

The display of carbohydrate structures was measured in promyelocytic HL60 cells and in histiocytic U937 cells induced to differentiate to phagocytic cellsin vitro during three to seven days of cultivation in the presence of dimethylsulfoxide (DMSO). It was assessed by micro-or spectrofluorometric quantification of the binding of fluorescent lectins. Changes in the cell size and the association and uptake of IgG-or complementopsonized yeast cells (Saccharomyces cerevisiae) were used as signs of phagocyte differentiation.The binding of wheat germ agglutinin (WGA), concanavalin A (Con A),Ricinus communis agglutinin-I (RCA-I) andUlex europaeus agglutinin-I (UEA-I) varied due to the presence of DMSO during cultivation, and without DMSO also on the number of days in culture and the type of cell.Abbreviations DMSO dimethylsulfoxide - PMA phorbol 12-myristate 13-acetate - KRG Krebs-Ringer phosphate buffer with glucose - WGA wheat germ agglutinin - Con A concanavalin A - RCA-I Ricinus communis agglutinin-I - UEA-I Ulex europaeus agglutinin-I     

Adsorption ofRhizobium meliloti to alfalfa roots: Dependence on divalent cations and pH

Adsorption ofRhizobium meliloti L5-30 in low numbers to alfalfa (Medicago sativa L.) roots was dependent on the presence of divalent cations, and required neutral pH. Adsorption was proportional to Ca and/or Mg concentrations up to 1.5 mM. Ca was not substituted by Sr, Ba or Mn. Adsorption was abolished and viability decreased at pH6. When lowering pH, higher Ca concentrations were required to attain similar adsorption levels, indicating a marked interactive effect between Ca and H ions. Pretreatment of the roots with Ca and low pH did not affect subsequent adsorption of the bacteria. However, Ca pretreatment ofR. meliloti sustained further adsorption at low Ca levels and low pH substantially affected their ability to adsorb. Low pH appears to affect the stability of binding causing desorption of the previously bound bacteria. The presence of saturating concentrations of heterologousR. leguminosarum bv.trifolii A118, did not prevent the expression of divalent cations and pH requirements, as well as their interaction. Our results suggest that rhizobial binding to the root surface already shows the Ca and pH dependence of alfalfa nodulation, which was generally associated to some event prior to rhizobial penetration of root hairs.     

Establishment of Bradyrhizobium japonicum for soybean by inoculation of a preceding wheat crop

On fields with no history of soybean (Glycine max (L.) Merr.) production, inoculation alone is often inadequate to provide for adequate nodulation the first time this crop is grown. The objective of this study was to determine if inoculation of spring wheat (Triticum aestivum L.) seed with Bradyrhizobium japonicum would lead to an increase of B. japonicum numbers in the soil, and improve nodulation of a subsequent soybean crop. In the greenhouse, wheat seed inoculation increased B. japonicum numbers from undetectable numbers to >9000 g^–1 of soil, whereas the numbers of introduced B. japonicum declined in unseeded pots. In the field, inoculation of wheat seed increased B. japonicum numbers in the soil from undetectable levels to >4000 g^–1 the following year. When soybean seed was inoculated, but grown in soil devoid of B. japonicum, nodules formed only near the point of seed placement. The heaviest nodulation, and widest distribution of nodules in the topsoil were found whenB. japonicum was established the year before by wheat seed inoculation, plus soybean seed inoculation. Wheat seed inoculation the year before growing soybean, combined with proper soybean seed inoculation, should provide for abundant nodulation the first time soybean is grown on a field.     

Preincubation of B. japonicum cells with genistein reduces the inhibitory effects of mineral nitrogen on soybean nodulation and nitrogen fixation under field conditions

Genistein is the major root produced isoflavonoid inducer of nod genes in the symbiosis between B. japonicum and soybean plants. Reduction in the isoflavonoid content of the host plants has recently been suggested as a possible explanation for the inhibition of mineral nitrogen (N) on the establishment of the symbiosis. In order to determine whether genistein addition could overcome this inhibition, we incubated B. japonicum cells (strain 532C) with genistein. Mineral N (in the form of NH₄NO₃) was applied at 0, 20 and 100 kg ha^-1. The experiments were conducted on both a sandy-loam soil and a clay-loam soil. Preincubation of B. japonicum cells with genistein increased soybean nodule number and nodule weight, especially in the low-N-containing sandy-loam soil and the low N fertilizer treatment. Plant growth and yield were less affected by genistein preincubation treatments than nitrogen assimilation. Total plant nitrogen content was increased by the two genistein preincubation treatments at the early flowering stage. At maturity, shoot and total plant nitrogen contents were increased by the 40 μM genistein preincubation treatment at the sandy-loam soil site. Total nitrogen contents were increased by the 20 μM genistein preincubation treatment only at the 0 and 20 kg ha^-1 nitrate levels in clay-loam soil. Forty μM genistein preincubation treatment increased soybean yield on the sandy-loam soil. There was no difference among treatments for 100-seed weight. The results suggest that preincubation of B. japonicum cells with genistein could improve soybean nodulation and nitrogen fixation, and at least partially overcome the inhibition of mineral nitrogen on soybean nodulation and nitrogen fixation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Detection of lectins in nodulated peanut and soybean plants

The direct double-antibody enzymelinked immunosorbent assay system was used in the detection and measurement of seed lectins from peanut (Arachis hypogaea L.) and soybean (Glycine max L.) plants (PSL and SBL, respectively) that had been inoculated with their respective rhizobia. Concentrations of PSL dropped to undetectable levels in peanut roots at 9 d and stems and leaves at 27 d after planting; SBL could no longer be detected in soybean roots at 9 d and in stems and leaves at 12 d. A lectin antigenically similar to PSL was first detected in root nodules of peanuts at 21 d reaching a maximum of 8 g/g at 29 d then decreasing to 2.5 g/g at 60 d. There was no evidence of a corresponding lectin in soybean nodules.Sugar haemagglutination inhibition tests with neuraminidase-treated human blood cells established that PSL and the peanut nodule lectin were both galactose/lactose-specific. Further tests with rabbit blood cells demonstrated a second mannosespecific lectin in peanut nodule extracts that was not detected in root extracts of four-week-old inoculated plants or six-week-old uninoculated plants, although six-week-old root extracts from inoculated plants showed weak lectin activity. The root extracts from both nodulated and uninoculated plants contained another peanut lectin that agglutinated rabbit but not human blood cells. Haemagglutination by this lectin was, however, not inhibited by simple sugars but a glycoprotein, asialothyroglobulin, was effective in this respect.Abbreviations DAS double antibody sandwich - ELISA enzyme-linked immunosorbent assay - PBS phosphate-buffered saline - PSL peanut seed lectin - SBL soybean lectin     

Tolerance ofRhizobium leguminosarum bv.phaseoli to acidity and drought

Ten strains ofRhizobium leguminosarum bv.phaseoli isolated from soils of Morocco were more tolerant than three culture collection strains to acid conditions in culture media or in sterile soil. The survival rate of a tolerant strain in a sandy acid soil was greater than a sensitive strain at different humidity levels. These properties should give locally selected strains an advantage in nodulatingPhaseolus vulgaris roots in soils similar to those used here.     

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.     

Relationships between cell membrane stability, exudate content and infectivity of Bradyrhizobium japonicum strain 639 to boron starved soybean plants

The influence of boron starvation on the root exudates content in soybean seedlings (Glycine max. L. Merr.) and the effect of exudates pretreatment on the pre-infection processes in symbiotic system Br. japonicum strain 636 and soybean were investigated. Root cell membrane stability of boron starved soybean plants (-B) decreased compared to the control. The concentrations of all analyzed metabolites (reducing sugars, free amino acids, organic acids, soluble phenols and total flavonoids) from root exudates of -B plants were lower than the control concentrations. Analysis of polyphenols after HPLC chromatography of root exudates showed significant difference of peak numbers between chromatograms of exudates obtained from boron starved and from control plants. Bacterial culture treatment with root exudates from -B plants showed decreased growth, chemotaxis and attachment ability toward the host root compared to the control exudate treatments. These changes were accompanied by decreased nodulation and acetylene reduction activity of boron starved soybean plants.     

Identification and characterization of plasmids in hydrogen uptake positive and hydrogen uptake negative strains ofRhizobium japonicum

Modifications were made of published procedures to allow routine isolation of plasmids fromRhizobium japonicum. The plasmid profiles of a series of H₂ uptake positive and H₂ uptake negative strains were compared. None of the strains ofR. japonicum with high H₂ uptake activities exhibited discernible plasmids, while most of the strains, with little or no H₂ uptake activity, showed plasmids with molecular weights ranging from approximately 49–290 x10⁶. An examination of H₂ uptake negative mutants derived from an H₂ uptake positive parent revealed two discernible plasmid bands in nonrevertible mutants but no detectable plasmids in revertible mutants or in the parent strain from which mutants were derived.     

Lack of specific binding of labeledRhizobium japonicum lipopolysaccharides to wild soybean (Glycine soja) roots

Summary Lipopolysaccharides (LPS) were extracted from two strains ofRhizobium japonicum (61A76NS and 3I1b110-I). The extracted LPS was purified by gel filtration column chromatography and the amount of 2-keto-3-deoxyoctonate (KDO) was determined. Column purified LPS from both strains were conjugated to rhodamine isothiocyanate on celite to examine binding of this purified, labeled surface component to aseptically grownGlycine soja (wild soybean) seedlings as a basis for symbiotic specificity using fluorescent microscopy. Rhodamine conjugated LPS from both strains ofRhizobium japonicum did not exhibit specific binding to wild soybean seedling roots.Paper no. 8130 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27650, USA.     

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.