Strains Of Rhizobium Japonicum Research Articles

Unidentified compound specifically accumulated in soybean nodules formed with H2-uptake negativerhizobium japonicumstrains

A large quantity of an unidentified compound was detected by a fluorogenic method in the soybean nodules formed with H2-uptake negative Rhizobium japonicum strains. The unidentified compound (compound X) reacted with o-phthalaldehyde in the presence of 2-mercaptoethanol giving rise to a fluorescent compound, and was not hydrolyzed in strong acid and strong base. Therefore, the compound X was considered to be one of amines and amino acids. The content of the compound X in the nodules formed with H2-uptake positive strains was remarkably lower than that in the nodules formed with H2-uptake negative strains. It was suggested that the compound X may be involved in the process of hydrogen metabolism in soybean nodules. The compound X was not detected in leaves, stems, petioles, and roots of soybean plants, but detected in H2-uptake negative strains of free-living R. japonicum. In nodules, a great portion of the compound X was distributed into cytosol fraction.

Persistence and Recovery of Rhizobium japonicum Inoculum in a Field Soil1

AbstractInoculation of soybean (Glycine max L.) rarely increases seed yields in fields where this crop has been previously grown. The inability of the inoculum strain to persist in soil could be a factor in this poor response. The persistence of the inoculant strain Rhizobium japonicum USDA 110 was followed for 56 weeks in a field soil (Typic Haplaquoll) using a fluorescent antibody technique. Statistically significant increases in the population of R. japonicum strain USDA 110 in soil were observed for the first 7 weeks after inoculation. When USDA 110 was applied at 2 × 106 cells g−1 soil the population of this organism in soil reached 1 × 107 cells g−1 soil 1 week after inoculation; a value significantly higher than observed in other inoculation and fertilization treatments. The population observed for the uninoculated control varied nonsignificantly from 6 × 104 to 2.6 × 105 cells g−1 during both growing seasons. No significant differences due to inoculation were observed for seed yield, or total aboveground N in either year. Even though the soil population was increased significantly, inoculation of strain USDA 110 at high rates produced no significant alteration in strain distribution in the nodules of soybeans in either year. Rhizobium japonicum strain USDA 110 was observed mostly in doubly infected nodules with strain USDA 123 which may have minimized the effect of strain USDA 110 on N2 fixation and seed yield.

Isolation and expression of Rhizobium japonicum cloned DNA encoding an early soybean nodulation function.

A first visible step in the nodulation of legumes by Rhizobium spp. is the deformation and curling of root hairs. We have identified and cloned DNA sequences encoding this function from two strains of Rhizobium japonicum (USDA 122 and USDA 110) with a weakly homologous probe from Rhizobium meliloti. Root hair curling encoded by the cloned DNA fragments was examined on soybeans (Glycine soja ) after conjugative transfer of these sequences in broad-host-range vectors to various bacterial genera. Pseudomonas putida gave unambiguous expression of the root hair curling genes. This enabled us to identify the 8.7-kilobase EcoRI fragments encoding root hair curling from each strain. The phenotypes encoded by the plasmids pBS1 (derived from strain USDA 122) and pBS2 (derived from strain USDA 110) are distinct and represent a phenotype characteristic of their parent R. japonicum strains. Subclones of pBS1 and pBS2 were generated in single and multicopy vectors, and their expression was analyzed in P. putida. We established that a 4.2-kilobase internal Sa/I fragment of pBS1 and a 3.5-kilobase SstI -EcoRI fragment of pBS2 are sufficient to confer root hair curling on soybeans.

Suppression of Nodule Development of One Side of a Split-Root System of Soybeans Caused by Prior Inoculation of the Other Side

In a split-root system of soybeans (Glycine max L. Merr), inoculation of one half-side suppressed subsequent development of nodules on the opposite side. At zero time, the first side of the split-root system of soybeans received Rhizobium japonicum strain USDA 138 as the primary inoculum. At selected time intervals, the second side was inoculated with the secondary inoculum, a mixture of R. japonicum strain USDA 138 and strain USDA 110. In a short-day season, nodulation by the secondary inoculum was inhibited 100% when inoculation was delayed 10 days. Nodulation on the second side was significantly suppressed when the secondary inoculum was delayed for only 96 hours. In a long-day season, nodule suppression on the second side was highly significant, but not always 100%. Nodule suppression on the second side was not related to the appearance of nodules or nitrogenase activity on the side of split-roots which were inoculated at zero time. When the experiments were done under different light intensities, nodule suppression was significantly more pronounced in the shaded treatments.

Multistrain vs. Single Strain Rhizobium japonicum Inoculants for Early Maturing (00 and 000) Soybean Cultivars: N2Fixation Quantified by 15N Isotope Dilution1

AbstractEarly maturing soybean cultivars (groups 00 and 000) adapted to western Canada are now available but since the soils contain no indigenous Rhizobium japonicum, it is important to determine if strains selected for efficient N2fixation with American soybean cultivars are also efficient with Canadian cultivars and if single strain are superior to multistrain inoculants. Accordingly, a 2‐year evaluation was made of the effect of up to eight single strains and two commercial multistrain combinations of R. japonicumgranular inoculants on maturity, seed yield, oil and protein content, and N2fixation (as determined for the first time by 15N isotope dilution) for three promising soybean lines. Rhizobium japonicumstrains 61A148 (USDA 142) and 61A118 (USDA 138) were consistently superior in N2fixation with all soybean cultivars, resulting in seed yields and seed protein higher than those of the other strains. Strain 61A101 was consistently an inefficient N2fixer. These differences in efficiency between strains were not related to documented ability to take up H2evolved by nitrogenase because, although strain 61A148 is considered Hup+, strain 61A118 is Hup–. Several strains resulted in percent plant N derived from the atmosphere exceeding 50% with N2fixed as high as 151 kg N ha−1for strain 61A148 on King Grain line X005. However, over all strains, Maple Amber was superior for supporting N2fixation, averaging 91 kg while X005 had 83 kg and Maple Presto 60 kg N fixed ha−1. Line strain combinations resulting in lower levels of N2fixation assimilated more soil N such that total N yields averaged over lines and strains were generally similar. These strains selected for U. S. soybean cultivars are also acceptable for Canadian soybeans. Interstrain competition occurred with one multistrain inoculant, to the detriment of plant yield. A commercial mix containing four strains (61A101, 61A118, 61A124, 61A148) had low N2fixation similar to that of strain 61A101. This suggested that strain 61A101 was highly competitive and may have formed a majority of the nodules thereby excluding the highly efficient strains 61A118 and 61A148. However, a new commercial mix containing three strains which were clones of 61A101, 61A118, and 61A124 did not exhibit any detrimental interstrain competition. Thus, the new multistrain soybean inoculant can be recommended for Canadian soybean cultivars.

Exopolysaccharides and lipopolysaccharides from a fast-growing strain of Rhizobium japonicum (USDA191)

Analysis of the exopolysaccharides from Rhizobium japonicum USDA191 showed substantial amounts of mannose (22.5%) and uronic acids (13.4%). These sugars are normally absent in the fast growers like Rhizobium meliloti. In addition USDA191 contained pyruvate (5.1%), which is normally absent in slow-growing strains of Rhizobium japonicum. The heterogeneity in the exopolysaccharide composition of strain 191 indicates a closer similarity with the slow-growing Rhizobium japonicum. SDS-urea polyacrylamide gel analysis of the lipopolysaccharides also reflects this heterogeneity.

Exopolysaccharides and lipopolysaccharides from a fast-growing strain ofRhizobium japonicum(USDA191)

Analysis of the exopolysaccharides from Rhizobium japonicum USDA191 showed substantial amounts of mannose (22.5%) and uronic acids (13.4%). These sugars are normally absent in the fast growers like Rhizobium meliloti. In addition USDA191 contained pyruvate (5.1%), which is normally absent in slow-growing strains of Rhizobium japonicum. The heterogeneity in the exopolysaccharide composition of strain 191 indicates a closer similarity with the slow-growing Rhizobium japonicum. SDS-urea polyacrylamide gel analysis of the lipopolysaccharides also reflects this heterogeneity.

Role of Lectins in the Specific Recognition of Rhizobium by Lotononis bainesii

Fluorescein isothiocyanate (FITC)-labeled lectin purified from the root of Lotononis bainesii Baker was bound by cells of five out of seven L. bainesii-nodulating strains of Rhizobium under culture conditions. With the exception of a strain of Rhizobium leguminosarum, strains of noninfective rhizobia failed to bind the root lectin under these conditions. The two nonlectin binding L. bainesii-specific strains did not bind root lectin on the L. bainesii rhizoplane although this was observed with three other L. bainesii-nodulating strains. A single Rhizobium japonicum strain bound root lectin on the L. bainesii rhizoplane. There was no evidence of an interaction between the L. bainesii seed lectin and the Rhizobium strains tested.Root lectin-specific FITC-labeled antibodies were bound to the tips of developing root hairs and lateral growth points of more mature root hairs of L. bainesii seedlings. The damaged edges of severed root hairs always bound FITC-labeled root lectin antibody. Seed lectin-specific FITC-labeled antibodies were not bound to the roots of L. bainesii. The preemergent root hair region of L. bainesii was most susceptible to infection by rhizobia but nodules also emerged in the developing and mature root hair regions. Lectin exposed at growth points on L. bainesii root hairs may provide a favorable site for host plant recognition of infective strains of Rhizobium.

Spheroplasts of Rhizobium japonicum

Speroplasts of Rhizobium japonicum strains 61A76, USDA 31, and 110 were prepared by culturing cells in the presence of glycine, followed by treatment with lysozyme. The cells were examined by scanning electron microscopy before, during, and after becoming spheroplasts and found to be morphologically similar to the bacteroid forms found in soybean root nodules. Some similarities of spheroplast and bacteroid formation are discussed.

Enhancing growth and nitrogen uptake by soybeans using pesticides

Benomyl applied to the seeds of soybean (Glycine max L. Merrill) inoculated with a benomyl resistant strain ofRhizobium japonicum increased the relative abundance of nodules formed by the inoculum strain and the numbers of the added rhizobium on the roots, the total N content, the percentage N, the yield at one plant density and, in one of four soils, the pod weight of soybeans grown in the greenhouse. Oxamyl applied to the seeds, foliage or both of soybeans inoculated with an oxamyl resistant strain ofR. japonicum increased the yield, N content, percentage N, and weight of nodules, pods and grain and enhanced the relative frequency of nodules formed by the inoculum strain. It is suggested that pesticides or other antimicrobial agents and rhizobia resistant to these inhibitors may provide a new means for increasing nitrogen fixation by soybeans.

Soybean root response to infection by Rhizobium japonicum: mannoconjugate turnover in effective and ineffective nodules

Crude organelle preparations from uninfected Glycine max root tissue, or from root tissue infected with the nod +/nif + (effective) or the nod +/nif − (ineffective) strains of Rhizobium japonicum all assembled mannose from GDP-mannose into trichloroacetic acid-precipitable forms. Cellular fractionation studies showed that the enzyme responsible for the initial event in the mannoconjugate assembly process was located exclusively in the endoplasmic reticulum (ER) ofthe host cell. Exogenous 14C-labelled mannose, when supplied to intact tissue for short periods, tended to accumulate at this location. After longer labelling periods the product was found to be present in both heavier and lighter membrane fractions, which corresponded with the subcellular sites of the enzyme α-mannosidase. The mannose-containing material assembled by the ER in vivo acted as a substrate for the α-mannosidase-containing fractions. During nodule development α-mannosidase activity showed two maxima, the early concomitant with, and the latter preceded by, an increase in ER marker enzyme activity. In these changes tissue infected with ineffective strains of R. japonicum showed parallel, but less pronounced biochemical trends. These data suggest that in nod +/nif + symbioses mannoconjugate assembly is stimulated and that this extra product is transferred through the cell where structures formed only in nod +/nif + symbioses may be responsible for its disassembly or modification.

Effect of herbicides on survival of rhizobia and nodulation of peas, groundnuts and lucerne

Thirteen herbicides were tested for toxicity against strains of Rhizobium used in South African legume inoculants for lucerne, clover, soybeans, groundnuts and lupins, respectively. Each of alachlor, bromoxynil, proprop, metolachlor, naptalam + dinoseb, and trifluralin inhibited at least two of the strains after a contact period of ca 10 s. No strain survived 42 h in contact with any of these herbicides. Atrazine and terbutryn were relatively non-toxic. The slow-growing strains of Rhizobium japonicum, Rhizobium lupini and Rhizobium sp. (groundnuts) were less affected by at least two of the herbicides tested than strains of the fast-growing R. meliloti and R. trifolii. Toxicity of a herbicide to rhizobia in vitro did not necessarily correlate with its effect on nodulation and some are considered suitable for field application. S. Afr. J. Plant Soil 1984, 1: 135–138

Soybean root response to infection by Rhizobium japonicum: saccharidases in root and nodule tissue

Two saccharidases, from soybean root nodules infected with Rhizobium japonicum strains effective or ineffective in N 2-fixation, have been investigated. One, α-galactosidase, was plant-derived and the other, α-glucosidase, was found largely in the bacterial fraction. Values of K m of these enzymes towards their paranitrophenyl (PNP) substrates and pH optima were determined and compared with those found for the previously partially characterized α-mannosidase from this tissue. The affinity of each enzyme for its PNP substrate was decreased by the addition of the appropriate monosaccharide, but these inhibitors were not interchangeable, i.e. galactose affected only α-galactosidase, not α-glucosidase or α-mannosidase and so forth. The levels of enzyme activity varied as nodules developed following root infection. Generally α-galactosidase activity in infected tissue showed trends similar to those reported previously for α-mannosidase. The activity of α-glucosidase also fluctuated but in a different pattern which in nodules infected with wild-type R. japonicum appeared to coincide with nitrogenase activity. These variations were not found in free-living strains of R. japonicum, either by derepressing nitrogenase or by varying the carbon sources.

Glutamine synthetase, glutamate synthase and glutamate dehydrogenase in Rhizobium japonicum strains grown in cultures and in bacteroids from root nodules of Glycine max

The growth yields of three strains of Rhizobium japonicum (CB 1809, CC 723, CC 705) in culture solutions containing L-glutamate were about twice those grown with ammonium. The activities of glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.4) were dependent on the nitrogen source in the medium and also varied with growth. Both NADPH-and NADH-dependent glutamate synthase (GOGAT; EC 1.4.1.13) and NADPH-dependent GDH were found in strains grown with either glutamate or ammonium but NADH-linked GDH was only detected in glutamate-grown cells. Glutamine synthetase was adenylylated in cells grown with NH 4 (+) (90%) and to lesser extent in those grown with L-glutamate (50%). In root nodules produced by the three strains in Glycine max (L.) Merr., the bulk of GS was located in the nodule cytosol (60-85%). The enzyme was adenylylated in bacteroids (43-75%) and in the nodule tissues (52-68%). The enzyme in cell-free extracts of Rh. japonicum (CC 705) grown in culture solutions containing glutamate and in bacteroids (CC 705) was deadenylylated by snake-venom phosphodiesterase. L-methionine-DL-sulfoximine restricted the incoporation of (15)N-labelled (NH4)2SO4 into cells of strains CB 1809 and CC 705, as well as in bacteroids of strain CC 705. It is noteworthy that appreciable activities for GDH were found in the free-living rhizobia grown on glutamate. Thus the presence of an enzyme does not necessarily imply that a particular pathway is operative in assimilating ammonium into cell nitrogen. Based on (15)N studies, the GS-GOGAT pathway of rhizobia (strains CB 1809 and CC 705) is important when grown in culture solutions as well as in bacteroids from root nodules of G. max.

Mobilization of 15N from Soybean Leaves as Influenced by Rhizobial Strains1

Nitrogen nutrition of soybeans [Glycine max (L.) Merr.] during pod development is important in determining seed yield. Soybeans utilize leaf N to meet the N needs of developing fruit which may reduce yield potential. A field experiment was undertaken using 15N to test the hypothesis that high N2‐fixing rhizobia would delay mobilization of N from leaves to developing pods. Three strains of Rhizobium japonicum, USDA strains 110, 31, and an ineffective mutant of strain 8‐0, varying in N2‐fixation effectiveness were used as inoculum to determine their influence on the utilization and translocation of N during pod fill. Foliarly applied 15N urea was sprayed onto leaves five times prior to pod formation to label vegetative N. Total application of N was approximately 3 kg/ha. During the first 17 days of pod development approximately 25% of the incorporated 15N was mobilized to the pods regardless of the rhizobial strain. During the final stages of pod development, mobilization of labelled N continued at nearly a linear rate for all rhizobial treatments. for USDA strain 110, there was an insignificant net loss in leaf and petiole N during the first 17 days of pod development which indicated there was an exchange between N2 recently fixed and N previously incorporated into leaves. The rate of N2‐fixation by both effective strains of rhizobia increased during pod development. Even though strain 110 fixed more total N than strain 31 the final seed yield for both treatments was similar due to differential partitioning of N. Future research is needed to elucidate the partitioning of N2 fixed during pod development and to determine why the better N status of plants inoculated by strain 110 did not result in increased yields.

Biochemical Characterization of Fast- and Slow-Growing Rhizobia That Nodulate Soybeans

Fast-growing, acid-producing soybean rhizobia were examined to determine their biochemical relatedness to each other, to typical slow-growing Rhizobium japonicum strains, and to other fast-growing species of Rhizobium. Although both the fast- and slow-growing soybean rhizobia were positive for catalase, urease, oxidase, nitrate reductase, and penicillinase, the fast-growing strains grouped with other fast-growing species of Rhizobium in that they tolerated 2% NaCl, were capable of growth at pH 9.5, utilized a large variety of carbohydrates (notably disaccharides), and produced serum zones in litmus milk. In addition, these fast-growing strains were similar to other fast-growing species of Rhizobium in that they produced appreciable levels of β-galactosidase and nicotinamide adenine dinucleotide phosphate-linked 6-phosphogluconate dehydrogenase but had no detectable hydrogenase activity. The fast-growing soybean rhizobia share symbiotic host specificity with Bradyrhizobium japonicum, but appear to be related biochemically to the other fast-growing species of Rhizobium.

Effect of NaCl Salinity on the Growth and the Nitrogen Status of Nodulated Cowpea (Vigna sinensis L.) and Mung Bean (Phaseolus aureus L.)

Summary Plants of V. sinensis and P. aureus were grown in the presence of five levels of NaCl ranging from 10 to 100 mM. V. sinensis grew better than control plants in the presence of NaCl even up to 75 mM, while P. aureus showed a decline in its growth even at 10 mM NaCl concentration. Nodulation of V. sinensis by Rhizobium cowpea strain no. 3200 occurred up to a salt concentration of 100 mM NaCl. When P. aureus was infected with Rhizobium japonicum strain no. G49, nodulation did not occur in the plants growing with more than 25 mM NaCl. Successful nodulation of both species in the presence of NaCl was associated with some kind of salt tolerance either of the host plant or of the Rhizobium partner. Cowpea rhizobium strain no. 3200 was found to tolerate the NaCl salinity better than R. japonicum strain no. G49. Total nitrogenase activity estimated by the amount of N fixed during the growing period was found to be positively correlated with NaClext application up to 75 mM in V. sinensis. However, this result was difficult to correlate with the ARA of the nodules measured at harvest. In contrast, salt application had a negative effect on N fixation by P. aureus.

Metabolism of 14C-Labeled Photosynthate and Distribution of Enzymes of Glucose Metabolism in Soybean Nodules

The metabolism of translocated photosynthate by soybean (Glycine max L. Merr.) nodules was investigated by (14)CO(2)-labeling studies and analysis of nodule enzymes. Plants were exposed to (14)CO(2) for 30 minutes, followed by (12)CO(2) for up to 5 hours. The largest amount of radioactivity in nodules was recovered in neutral sugars at all sampling times. The organic acid fraction of the cytosol was labeled rapidly. Although cyclitols and malonate were found in high concentrations in the nodules, they accumulated less than 10% of the radioactivity in the neutral and acidic fractions, respectively. Phosphate esters were found to contain very low levels of total label, which prohibited analysis of the radioactivity in individual compounds. The whole nodule-labeling patterns suggested the utilization of photosynthate for the generation of organic acids (principally malate) and amino acids (principally glutamate).The radioactivity in bacteroids as a percentage of total nodule label increased slightly with time, while the percentage in the cytosol fraction declined. The labeling patterns for the cytosol were essentially the same as whole nodule-labeling patterns, and they suggest a degradation of carbohydrates for the production of organic acids and amino acids. When it was found that most of the radioactivity in bacteroids was in sugars, the enzymes of glucose metabolism were surveyed. Bacteroids from nodules formed by Rhizobium japonicum strain 110 or strain 138 lacked activity for phosphofructokinase and NADP-dependent 6-phosphogluconate dehydrogenase, key enzymes of glycolysis and the oxidative pentose-phosphate pathways. Enzymes of the glycolytic and pentose phosphate pathways were found in the cytosol fraction.In three experiments, bacteroids contained about 10 to 30% of the total radioactivity in nodules 2 to 5 hours after pulse-labeling of plants, and 60 to 65% of the radioactivity in bacteroids was in the neutral sugar fraction at all sampling times. This strongly suggests some absorption and metabolism of sugars by bacteroids in spite of the lack of key enzymes. Bacteroids did possess enzymes for the formation of hexose phosphates from glucose or fructose. Radioactivity in alpha,alpha-trehalose in bacteroids increased until, after 5 hours, trehalose was a major labeled compound in bacteroids. Thus, trehalose synthesis may be a major fate of sugars entering bacteroids.

Mutants of Rhizobium japonicum with Increased Hydrogenase Activity

Some strains of Rhizobium japonicum can use hydrogen as an energy source for growth under microaerophilic conditions. Mutant strains have been selected that use hydrogen in the presence of high partial pressures of oxygen. The mutants contain more hydrogenase than the parent strain, both as free-living cells and as bacteroids in nitrogen-fixing soybean root nodules.

Influence of several methods for rhizobial inoculation on nodulation and yield of soybeans

Two field experiments were planted using different techniques for inoculation of soybeans. Three cultivers each belonging to a group of growth habit were inoculated by four methods in the first experiment. In the second one, two strains ofRhizobium japonicum (USDA 110 and 122) inoculated by other four methods, were compared on two indeterminate varieties, (Amsoy-71 and Williams).