Strains Of Rhizobium Japonicum Research Articles

Enhancement of specific nitrogenase activity in Azospirillum brasilense and Klebsiella pneumoniae, inhibition in Rhizobium japonicum under air by phenol.

Specific nitrogenase activity in Azospirillum brasilense ATCC 29145 in surface cultures under air is enhanced from about 50 nmol C2H4 X mg protein -1 X h-1 to 400 nmol C2H4 by the addition of 1 mM phenol. 0.5 and 2 mM phenol added increase the rate 5-fold and 4-fold. This enhancement effect is observed only between 2 and 3 days after inoculation, with only a small reduction of the growth of the cells by the phenol added. In surface cultures under 1% O2, nitrogenase activity is slightly reduced by the addition of 1-0.01 mM phenol. Utilization of succinate is enhanced during the period of maximum enhancement of nitrogenase activity by 60% by addition of 1 mM phenol. The cells did not produce 14CO2 from [U-14C] phenol, neither in surface cultures nor in liquid cultures and less than 0.1% of the phenol was incorporated into the cells. A smaller but significant enhancement of nitrogenase activity by about 100% in surface cultures under air was found with Klebsiella pneumoniae K 11 after addition of 1 mM phenol. However, in Rhizobium japonicum 61-A-101 all phenol concentrations above 0.01 mM reduced nitrogenase activity. With 1 mM phenol added activity was reduced to less than 10% with no effect on the growth in the same cultivation system. With this Rhizobium japonicum strain significant quantities of phenol (25 mumol in 24 h by 2 X 10(12) cells) were metabolized to 14CO2, with phenol as sole carbon source. With Azospirillum brasilense in liquid culture under 1% and 2% O2 in the gas phase, no enhancement of nitrogenase activity by phenol was noticed.

Quantifying dinitrogen (N2) fixation in soybeans by 15N isotope dilution: the question of the nonfixing control plant

The stable isotope 15N was used to trace the contribution of soil, fertilizer, and atmospheric N to the plant N of soybeans (Glycine max L. Merrill). 15N isotope dilution is the best technique to quantify N2 fixation (percent nitrogen derived from the atmosphere, % Ndfa) but requires reference to an appropriate nonfixing control plant so that the contribution of soil and (or) fertilizer N to the N yield of the fixing plant may be determined. The nodulating soybean line in nonfixing mode, i.e., uninoculated or inoculated with an ineffective strain of Rhizobium japonicum, was shown to be the best nonfixing control plant. The nonnodulating isoline, even when near isogenic, overestimated the % Ndfa and was judged to be an improper control. Of three nonlegumes tested only Hordeum vulgare was useful as a nonfixing control plant. In two lysimeter studies in a growth room the % Ndfa of Glycine max cv. Harosoy was 59% while that of cv. X005 was 38%. The average % Ndfa of cv. Chippewa soybeans under field conditions was 17%. The precision of the isotopic dilution technique, as measured by the coefficient of variation, was as good as 1.3% in one lysimeter study but averaged 6.2% in growth room lysimeters or in the field.

Hydrogen Uptake (HUP) Activity of Rhizobium japonicum from Major U.S. Soybean Production Areas

The purpose of these experiments was to determine the distribution of the hydrogen uptake (Hup) system among Rhizobium japonicum isolated from major soybean production areas of the USA. Soybean nodules were collected from approximately 100 different locations during the 1979 and 1980 growing seasons. The major conclusion is that a majority (> 75 %) of the Rhizobium japonicum strains isolated from major soybean producing areas are missing H2 uptake activity. The role of the H2 uptake system in energy efficiency of nitrogen fixation is discussed.

Glutamate as a differential nitrogen source for the characterization of acetylene‐reducing Rhizobium strains

A majority (36 of 44) of Rhizobium japonicum strains tested reduced acetylene asymbiotically when grown on an agar medium containing 0.1% (w/v) L‐glutamate as a sole nitrogen (N) source. Glutamate as N source led to pinpoint colonies and uniform glutamine synthetase activity of three selected, slow‐growing acetylene‐reducing strains (R. japonicum L‐259 and 110 and a cowpea‐type Rhizobium 32H1). The three test strains were characterized further by antibiotic resistance, colony type, cellular morphology, and differential growth on different N sources. The evidence suggests that, in an agar medium, glutamate creates a growth condition leading to acetylene reduction activity, pinpoint colonies and pleomorphism.

Clays and the Survival of Rhizobium in Soil During Desiccation

AbstractA study was conducted to assess the effect of drying on populations of two strains of Rhizobium japonicum and two cowpea rhizobia. The numbers of these bacteria fell markedly in soils undergoing drying. The extent of the decline varied with the strain and the soil. The decline in the population of a cowpea rhizobium occurred during the time of rapid water loss from four soils. The fall in numbers of three of the strains during desiccation was greater if the organisms were on glass beads than in soil. Illite did not increase survival on the beads, and kaolinite reduced the number of survivors of three strains dried on fine sand. Montmorillonite at the same concentrations protected against viability loss of two rhizobia undergoing desiccation on fine sand, but had no such effect on the more desiccation‐sensitive strain. The percent survival of three strains in a soil and in sand‐montmorillonite mixtures was directly related to the amount of water remaining after drying and was also affected by relative humidity. A possible relation between tolerance to desiccation and cell water content is suggested.

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.

Desiccation tolerance of four strains of Rhizobium japonicum

The survival of different strains of Rhizobium japonicum was evaluated in three soils with two matric- and three osmotic-induced moisture potentials. Both drying and added NaCl significantly decreased populations. Strains CC 709 and USDA 110 were less affected by the matric- or osmotic-induced desiccation than strains CB 1809 and USDA 123. The survival of CC 709 and USDA 110 at 2 weeks with 0.7% added NaCI was 33 and 46% of initial counts in soils undergoing drying, and 70 and 69% in soils maintained at the 30kPa (0.3 bar) potential. Comparable survivals of CB 1809 and USDA 123 were 15 and 18% and 56 and 59%, respectively. The soil with the greatest clay and organic C contents maintained the highest populations during desiccation. Turbidity measurements indicated similar rates of growth of the four strains at a water activity ( A w ) of 0.999 in yeast mannitol broth (YMB). When the YMB was adjusted with glycerol to lower water activities, strains CC 709 and USDA 110 consistently showed greater growth than did strains CB 1809 and USDA 123. No growth of CB 1809 and USDA 123 was observed at an ( A w ) of 0.975. Water adsorption isotherms of freeze-dried cells showed that the more desiccation-susceptible strains (CC 709 and USDA 110) retained greater amounts of water at a given relative vapor pressure than did the two more tolerant strains.

Effectivity of Cross-Infective Rhizobia in Heterologous Legume Hosts in vivo

Summary Modulation and nitrogen-fixing ability of cross-infective strains of Rhizobium japonicum and Rhiz . sp. ‘cow pea group’ were tested in different hosts of two groups. It was observed that in distantly related hosts the cross nodulating bacteria were often non-effective in nitrogen fixation. Therefore, Rhizobium strain, genetically manipulated for wide host infectivity, cannot be of much practical advantage in use as legume inoculant for hosts of more than one cross inoculation group, unless the nitrogen-fixing ability of the bacterium is also manipulated.

Response of Soybean Plants to High Root Temperature as Affected by Plant Cultivar and Rhizobium Strain1

AbstractThis study examined the effect of high root temperatures on nodulation, dinitrogen fixation, and growth of five soybean [Glycine max (L.) Merr.] cultivars associated with either of two Rhizobium japonicum strains of contrasting temperature tolerances.In each of three greenhouse experiments, soybeans growing in pots containing vermiculite were submitted to root temperatures of 28, 33, and 37 C or 28, 33, and 35 C by submerging the pots in thermostatically controlled water baths. In the first experiment, cvs. ‘Hutton’ and ‘Ransom’ were inoculated with either strain USDA 110 or 587, while in the second experiment the same strains were used to inoculate cvs. ‘Gasoy‐17’ and ‘Bragg.’ In a third experiment cvs. Bragg, Gasoy‐17, ‘Lee,’ and Ransom were grown in association with strain 587. Plants were harvested 34 days after germination.The superiority in high temperature tolerance of strain 587 over USDA 110, found in earlier work with cv. Lee, was confirmed for cvs. Gasoy‐17, Hutton, and Ransom. Results with cultivar Bragg were inconsistent for the two experiments in which that genotype was included.The results indicate that soybean genotype influences the response of the symbiotic system to high root temperatures. Gasoy‐17 and Lee appeared to be more tolerant to high root temperature than the other cultivars. That characteristic was enhanced when Gasoy‐17 was associated with the tolerant strain 587. Selection of soybean cultivars with tolerance to high root temperature in symbiotic association with R. japonicum appears to be a feasible alternative to overcome high soil temperature stress and needs to be done in conjunction with strain selection.

Soil microorganisms antagonistic towards Rhizobium japonicum

Success in introducing Rhizobium japonicum strains into soil is related to their interaction with native microorganisms including some that are antagonistic. Actinomycetes, bacteria, fungi and rhizobiophages antagonistic towards strains of R. japonicum were counted directly using soil samples from field plots under different crop and soil management systems. The antagonistic actinomycete population varied from 1.3 × 10 3 to 4.5 × 10 5 g −1 dry soil and ranged up to 90% of total actinomycetes. Soybean rhizosphere soil samples included antagonistic actinomycetes ranging up to 70% of total actinomycetes. The antagonistic bacterial population was less than 10% of total bacteria and the proportion did not vary significantly with crop or soil management practices. Antagonistic fungi were observed for many of the soils examined but they could not be counted. There were few rhizobiophages and they were found most frequently in soybean rhizospheres. Occasional bacterial and actinomycete colonies that stimulated growth of R. japonicum were randomly observed among the soil samples tested.

Survival rates of Rhizobium japonicum populations introduced into different soils

A 5-yr experiment was carried out to follow the survival rate of Rhizobium japonicum strains introduced into different French soils, using the plant enumeration technique. Rhizobia have a good survival rate in these soils (⩾10 4 bacteria g −1 of dry soil after 5 yr) even without any soybean crop during the experiment. Certain hypotheses resulting from this experiment were then examined in the laboratory in a controlled environment using the quantitative fluorescent antibody membrane filter technique to count rhizobia. In laboratory conditions whatever the initial population introduced, its number reached an equilibrium value according to a mathematical logistic model. We noticed a significant morphological bacteria change during the incubation.

Antibiotic-resistant mutants identified from nodules of uninoculated soybeans grown in a strongly acidic soil

Two hypotheses that antibiotic-resistant nodule isolates from uninoculated soybeans grown in a strongly acidic soil were naturally occurring rhizobia which had acquired resistance to spectinomycin and streptomycin or were contaminants from adjacent, inoculated treatments, were tested in laboratory experiments. Soybean nodule isolates (166) as well as 48 cowpea and 89 Rhizobium japonicum strains were used in tests of resistance to six concentrations (0–500 μg ml −1) of kanamycin, spectinomycin and streptomycin, tolerance of stresses of pH 4.6, with or without 50 μ m Al, and serological cross-reactivity. More strains from the strongly acidic soil were resistant to the antibiotics than from slightly acidic soils, but resistance to antibiotics did not necessarily entail resistance to pH 4.6 or to 50 μ m Al. Twenty-three nodule isolates which were resistant to spectinomycin or streptomycin cross-reacted with antisera of the inoculum strains, indicating that they were contaminants. None of 59 antibiotic-sensitive nodule isolates from uninoculated plants and none of 31 from inoculated plants cross-reacted with the antisera. All 53 antibiotic-resistant isolates from nodules of plants in inoculated plots cross-reacted with test antisera, indicating stability of the antibiotic markers. Cowpea rhizobia were generally more resistant to the antibiotics and more tolerant of pH 4.6 and 50 μ m Al than were R. japonicum. Among strains of R. japonicum, slow growers were more resistant to antibiotics than moderately fast growers.

Effect of High Root Temperature and Rhizobium Strain on Nodulation, Nitrogen Fixation, and Growth of Soybeans

AbstractThe effects of high root temperatures on nodulation, nitrogen (N) fixation, and dry matter production of ‘Lee’ soybean [Glycine max (L.) Merr.] plants inoculated with 13 different strains of Rhizobium japonicum were studied in the greenhouse. Plants were grown in vermiculite, and their root systems were submerged in thermostatically controlled water baths. A treatment without inoculation in which 12 mM of KNO3 was supplied with the nutrient solution was included.Increasing root temperatures from 28 to 40°C had a detrimental effect on the number of nodules, the specific nitrogenase activity, the N content, and the dry weight of tops and roots of inoculated plants. The magnitude of this effect was highly dependent on the rhizobial strain.For some strains, higher nodule weights and higher total nitrogenase (C2H2 reduction) activity were found at 33 than at 28°C, whereas for other strains, these variables declined with increasing temperatures. It appeared that, with increasing temperatures, the processes determining nodule initiation became susceptible at lower temperatures than those associated with nodule growth.When plants formed nodules under nonlimiting temperatures and then were transferred to stressing root temperatures, appearance of additional nodules, N fixation, and plant growth were restricted by high root temperature. These results indicate that the detrimental effect of increasing root temperatures on inoculated plants is due in part to the reduction in number of nodules formed and also to limitations imposed on other processes of the soybean‐Rhizobium association.The growth of plants supplied with combined N was also adversely affected by continuous high root temperatures. However, while a temperature of 34°C restricted the growth of plants depending on N fixation, it was stimulatory for the growth of plants supplied with combined N. Therefore, the effect of temperatures near 34°C on the growth of nodulated plants without combined N was due mainly to the effect on the symbiotic system and not on other physiological processes of the plant.The response of different Rhizobium strains in symbiosis with soybean plants to increasing temperature was related to their response to temperature in pure culture and modified the adverse effect of temperature on plant development. Therefore, pure culture screening of R. japonicum may be a useful tool to aid in the selection of strains better suited for soil environments where high temperature is a limiting factor for the symbiosis.

Tests for a Diffusable Compound Endowing rj1‐incompatible Strains of Rhizobium japonicum with the Ability to Nodulate the rj1rj1 Soybean Genotype1

The nodulation restrictive genotype of soybeans [Glycine max (L.) Merr.] rj1 rj1 is nodulated by some strains of Rhizobium japonicum. Inoculation with mixed cultures of rj1‐compatible and rj1‐incompatible strains has shown that the presence of an rj1‐compatible strain facilitates nodulation of rj1rj1 plants by an rj1‐incompatible strain. This study was undertaken to determine whether rj1‐compatible strains of R. japonicum produce a diffusable product capable of endowing rj1‐incompatible strains with the ability to nodulate the rj1rj1 genotype. An rj1‐incompatible strain remained unchanged in ability to nodulate rj1rj1 plants when: (a) grown in culture filtrates of rj1‐compatible strains, (b) cultured in U‐tubes across a 0.08 mμ pore size polycarbonate membrane from rj1‐compatible strains, or (c) cultured in dialysis tubing suspended in broth cultures of an rj1‐compatible strain. It was concluded that the rj1‐compatible strains tested apparently do not produce a diffusable compound in vitro capable of endowing rj1‐incompatible strains with the ability to nodulate the rj1rj1 genotype. The rj1‐compatible Rhizobium appear to exert their effect, in close proximity to the nodulation site.

Phosphorus Nutrition of Rhizobium japonicum: Strain Differences in Phosphate Storage and Utilization

AbstractPhosphate accumulation from defined liquid medium was assessed in six strains of Rhizobium japonicum grown at high P (2 × 10−3M orthophosphate in solution). The total cell P concentration ranged from 1.6 to 2.4% dry mass depending on strain, and correlated with subsequent growth after transfer to medium without added P. Stored cell P could support up to four or five generations. Steps to ensure high‐P storage in inoculant cultures might have practical utility.Growth ceased when total cell P dropped to 0.3%. Electron microscopy indicated a tendency of deficient cells to become elongated, distorted, and packed with lightly staining granules (perhaps poly β hydroxybutyrate).High‐P cells of all the strains contained from 5 to 7 electrondense granules. Their total volume varied between 0.1 and 0.5% of the cell, depending on strain; and related linearly to percent of P in the cell. The granules occurred only when cell P was already high (> 1.5%) and disappeared within 1 day after transfer to medium without P. They may be polyphosphate storage granules. They provided P sufficient at most for 1 to 2 generations.Cells grown at an intermediate external solution‐P concentration buffered at 6 × 10−6M, resembling that in fertile soil, had few granules and moderate stored P (1.3 to 1.7%). Invasion of P‐depleted rhizospheres by rhizobia from soil probably cannot depend on previously stored P: it must also require efficient uptake from very dilute‐P concentrations.External gum was abundantly produced at low‐ and moderate‐P supply, but not at the high P concentrations routinely supplied in laboratory media.

Effect of Cadmium on Symbiotic Soybean Plants

AbstractThe potential for environmental contamination by cadmium (Cd) has increased significantly in recent years. Since Cd may be hazardous to living systems, a study was conducted to investigate: (i) the existence of Rhizobium japonicum strains tolerant to Cd, (ii) the Cd effect on the symbiosis between host soybean plants (Glycine max (L.) Merr.) and R. japonicum strains with different Cd tolerances, and (iii) interactions and distribution of Cd and other elements in the host when biomass is decreased by Cd.The existence of R. japonicum strains tolerant to Cd was assessed by comparing growth curves of 10 different strains growing in a basal medium with gluconate, yeast extract, L‐arabinose, and Cd (0, 3.5, 7, 14, and 28 µg/ml). Strain 3Ilb110 (110) exhibited a tolerance to Cd after an initial lag in growth, while strain 3Ilb123 (123) was susceptible.Strains 110 and 123 were used to inoculate plants growing in nutrient solution with Perlite as a support medium to evaluate the plant‐rhizobial interactions in the presence of 0, 2.2, 6.7, 10.1, and 20.2 µg Cd/ml, respectively. Plants were harvested and tops, roots, and nodules were analyzed 23 days after starting Cd application.Cadmium decreased dry matter production of tops, roots, and nodules. Plants inoculated with strain 123 accumulated significantly more dry matter in nodules than those with strain 110. However, in the presence of 0 and 2.2 µg Cd/ml, plants inoculated with strain 110 accumulated significantly more N than plants inoculated with strain 123.Nutrient imbalances were observed in the presence of Cd. It is suggested that the interactions of Cd with some nutrients may contribute to Cd toxicity in soybean plants. In this study the most pronounced Cd effect was on Fe and Mn nutrition, rather than Zn as had been previously reported.

Respiration and efficiency of N2fixation by nodules formed with a H2-uptake positive strain ofRhizobium japonicum

Abstract It is well known that nodules of certain legumes fix H2 and CO2 as well as N2 (1–5). A H2 uptake positive strain of Rhizobium japonicum was reported to be linked with ATP formation through oxidative phosphorylation (6).

Fungal Effects on Rhizobium japonicum‐Soybean Symbiosis1

AbstractSoil fungi are known to produce mycotoxins which could inhibit growth and activity of other microorganisms in the soil. This study was undertaken to examine antagonistic effects of mycotoxins on growth of Rhizobium japonicum and on nodulation and growth of soybeans [Glycine max (L.)].Seventeen common fungi were isolated from the surface horizon of an Udollic ochraqualf and examined in laboratory and growth chamber studies to determine effects of these fungi on Rhizobium japonicum strains USDA 110 and 138. Several fungi were found to be antagonistic towards rhizobia due to mycotoxins which were exuded from the mycelia into the surrounding environment. During preliminary screening experiments Trichoderma viride, Rhizopus nigricans, and Mucor vesiculosis were observed to be consistently antagonistic to R. japonicum. Subsequently, these three fungi were inoculated simultaneously with rhizobia into perlite which was seeded to soybeans. The effect of each fungus on nodulation and growth of the soybeans during culture for 9 weeks was determined. The nodulation of soybeans by strains 110 and 138 was reduced by Trichoderma viride. Nodules per plant for strain 138 were reduced from a control value of 54 to 34. Total nodule weight and N2(C2H2) reduction also decreased, however, no changes were observed in plant shoot and root weight or in total N. Mucor vesiculosis increased nodulation and plant growth, while Rhizopus nigricans had no effect.In non‐sterile soil, Trichoderma viride was the only fungal isolate capable of reducing the R. japonicum population. The population was reduced to one tenth that of the control soil. Trichoderma viride also decreased nodule number which was accompanied by a decrease in N2(C2H2) reduction per soybean plant from a control value of 1.07 to 0.65 µmoles/hour. The antagonistic effects of Trichoderma viride on rhizobia1 activity were paralled by decreases in plant root and shoot weights. These plant parameters were similarly decreased, although not as severely, by Mucor vesiculosus and Rhizopus nigricans

Regulation of the expression of leghaemoglobin genes in effective and ineffective root nodules of soybean

The expression of leghaemoglobin genes in effective and ineffective (unable to fix nitrogen) root nodules of soybean developed by Rhizobium japonicum strains 61A76, 61A24 and SM5 was measured by using a cDNA probe or a cloned leghaemoglobin sequence and in vitro translation of Lb-mRNA. Hybridization of the poly(A)-containing nodule polysomal RNA from 3-week-old nodules with a kinetically purified Lb-cDNA or with plasmid (pLbl) containing a leghaemoglobin sequence showed that Lb-mRNA is present in ineffective nodules formed by strains SM5 and 61A24 at reduced levels. Of the two major classes of electrophoretically distinguishable leghaemoglobins in soybean, LbS was not synthesized in 3-week-old strain 61A24-induced nodules while both sorts of leghaemoglobin were synthesized and accumulated in ineffective nodules formed by strain SM5 of Rhizobium. Ineffective nodules formed by strain 61A24 are green inside and do not appear to accumulate leghaemoglobin as measured by the haemochromogen assay, although low levels of apoleghaemoglobin were detected using leghaemoglobin antibodies. SM5-induced nodules were found to have about half as much as leghaemoglobin of that of effective (61A76-induced) nodules. This study demonstrates that while the appearance of leghaemoglobin is independent of nitrogenase activity in bacteroids, its synthesis is influenced to different degrees both by a mutation (SM5) and incompatibility (61A24) of Rhizobium. The primary regulation appears to be at the level of transcription or processing of mRNA since ineffective nodules contain Lb-mRNA approximately in proportion to the amount of apoleghaemoglobin present in these nodules.

Plasmid transfer within and between serologically distinct strains of Rhizobium japonicum, using antibiotic resistance mutants and auxotrophs.

Methionine-requiring and pantothenic acid-requiring auxotrophs of Rhizobium japanicum USDA 31, as well as highly antibiotic-resistant mutants of R. japonicum strains USDA 31, USDA 110, USDA 138, and Webster 48, were isolated. These mutants were used to transfer the P-1 group plasmids R68.45 and RP4 within and between strains USDA 31, USDA 110, and Webster 48. Attempts to demonstrate transfer of either plasmid to strain USDA 138 were unsuccessful.