Japonicum Strains Research Articles

Synthetic studies on the lipooligosaccharide Nod Bj-IV (C 18:1, Fuc, Gro) produced by Bradyrhizobium japonicum strain USDA61

Synthetic studies on the lipooligosaccharide Nod Bj-IV (C 18:1, Fuc, Gro) produced by Bradyrhizobium japonicum strain USDA61

Genotype x Bradyrhizobium japonicum strain interactions in dinitrogen fixation and agronomic traits of soybean (Glycine max L. Merr.)

Bradyrhizobium japonicum strain G49 has been the only inoculum used in French soils. Soybean (Glycine max L. Merr.) cultivars were selected and tested according to their performances with this rhizobial strain. The aim of the present study was to determine the consequences of strain substitution on N2 fixation abilities of various genotypes. Three genotypes and cultivar Weber, in combination with B. japonicum strain G49 or SMGS1, were cultivated in pots and tested for nitrogenase activity under differing nitrogen nutrition conditions. The reliability of ARA (acetylene reduction activity) measurement for assessing symbiotic nitrogen fixation under the experimental conditions used was checked. Genotypic variability for symbiotic fixation activity was observed with each strain under soil culture conditions; important genotype x strain interactions were also involved. These results were corroborated for the protein yield and other yield component performances of the various genotype-strain associations. Thus, in France, the replacement of strain G49 with another one might result in the alteration of the relative agronomic performances of the soybean cultivars, since N2 fixation is considered as a major factor of soybean productivity.

Identification and inter-relationship analysis of Bradyrhizobium japonicum strains by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD)

Genomic DNA of 13 Bradyrhizobium japonicum strains was prepared and analysed by restriction fragment length polymorphism (RFLP) with nif and nod probes, and by random amplified polymorphic DNA (RAPD) with 11 primers of arbitrary nucleotide sequence. Polymorphism was observed in both analyses. The RFLP and RAPD banding patterns of different strains were used to calculate genetic divergence and to construct phylogenetic trees, allowing studies on the relationships between the strains. RFLP with nif and nod probes permitted the separation of the strains into two divergent groups, whereas RAPD separated them into four main groups. RAPD allowed closely related strains to be distinguished.

Two differentially regulated nitrate reductases required for nitrate-dependent, microaerobic growth of Bradyrhizobium japonicum

Native PAGE of Triton x-100-solubilized membranes from Bradyrhizobium japonicum strain PJ17 grown microaerobically (2% O2, v/v) in defined nitrate-containing medium resolved two catalytically active nitrate reductase (NR) species with apparent molecular masses of 160 kDa (NRI) and 200 kDa (NRII). NRI and NRII were also found in membranes from cells of strain PJ17 that were first grown in defined medium with glutamate and further incubated microaerobically in the presence of 5 mmol/l KNO3. However, only NRI was detected in cell membranes of strain PJ17 when nitrate was omitted from the microaerobic incubation medium. Four mutants unable to grow at low O2 tension in the presence of nitrate were isolated after transposon Tn5 mutagenesis. Membranes from mutants GRF110 and GRF116 showed mainly NRI, while the other two mutants, GRF3 and GRF4, expressed mostly NRII. These results indicate that the ability of B. japonicum PJ17 to grow under microaerobic conditions depends upon the presence of two membrane-bound NR enzymes whose synthesis seem to be independently induced by microaerobiosis (NRI) or by both microaerobiosis and nitrate (NRII).

Differences among Rhizobium meliloti and Bradyrhizobium japonicum strains in tolerance to desiccation and storage at different relative humidities

The behaviour of Rhizobium meliloti and Bradyrhizobium japonicum (three strains of each) during storage in atmospheres of controlled relative humidities (r.h.) offrom 3 to 83.5% at 30°C in the presence of air were investigated. Whatever the r.h. values imposed, the desiccation step did not significantly affect the survival rates except for B. japonicum G49. However, differences in behaviours and tolerances against protracted storage at the five r.h. values tested were noted between rhizobia and bradyrhizobia. Rhizobium meliloti strains were found more susceptible to storage at 3 or 83.5% r.h. than B. japonicum strains. Whatever the r.h. values, cell populations of B. japonicum declined at a rather constant rate and no surviving cells were observed after 100 days of storage. On the contrary, two step decreases were noted for R. meliloti stored at ⩽ 67.8% r.h. and appreciable survival rates were still obtained after 100 days of storage at 22, 43.6 or 67.8% r.h. Based on scanning electron microscope examinations, it was found that drying by itself did not morphologically affect brady(rhizobia) cells. The combined processes of drying and rehydration led to minor increases in cell volumes (for cells stored at 83.5% r.h.) and appearance of numerous granules and strands of exopolymers (for cells stored at 3% r.h.). After storage in their dried states, loss of infectiveness was observed for B. japonicum cells whereas R. meliloti cells maintained the same degree of symbiotic infectivity.

The identification of Bradyrhizobium japonicum strains isolated from Italian soils

Pyrolysis mass spectrometry (PYMS) and PCR using arbitrary primers were used to characterize strains of Bradyrhizobium japonicum isolated from fields in northern Italy. The combination of techniques allowed us to identify bacteria derived from inoculants and to demonstrate that information about the nature of one inoculant was incorrect. PYMS also indicated that the derivatives from one inoculant formed two distinct populations: one like the parent strain and the other altered phenotypically.

Evaluation of the Symbiotic Properties of Rhizobium fredii in European Soils

Summary The symbiotic properties of different Rhizobium fredii strains were evaluated in a controlled environment growth chamber, greenhouse, and field conditions. R. fredii strains HH 102-2 and HH 103 showed to be as good soybean inoculants as Bradyrhizobium japonicum USDA 110 in three years (1987, 1988 and 1992) of field experiments in Spanish soils. Thus, it is concluded that these two R. fredii strains are valid soybean inoculants in Spanish soils which, as all European soils, are devoid of indigenous B. japonicum and R. fredii strains. Competition experiments in field conditions (1987) showed that R. fredii strains HH102-2 and HH 103-2 were more competitive than B. japonicum USDA 110-1 to nodulate soybean cv. Williams. Results of competition studies in greenhouse conditions using acid (pH 4.9), neutral (pH 6.6) and alkaline (pH 8.1) soils showed that in the acid soil B. japonicum strains USDA 110 and 3-15-b3 totally outcompeted R. fredii strain HH 103 to nodulate on cv. Williams. Conversely, in the alkaline soil the fast-growing strain occupied more than 80% of the nodules. These results indicate that the soil pH could be an important factor in determining the competitive success of R. fredii and B. japonicum for nodulating soybeans.

Nitrogenase activity is required for the activation of iron‐stress response in iron‐inefficient T203 soybean

Abstract Iron (Fe)‐stress response and symbiotic nitrogen (N2) fixation are chemical reduction processes within the roots which interact to the extent that nodulated, Fe‐stressed, Fe‐inefficient soybean initiates Fe‐stress response, but similar nonnodulated soybean does not. The objective of this study was to examine the relationship between nitrogenase activity and Fe‐stress response in the Fe‐inefficient T203 soybean by inoculating plants with effective or ineffective strains of Bradyrhizobium japonicum on either side of split‐root systems. Half the roots of each plant were either inoculated with effective or ineffective strains or left uninoculated. Nodules formed on roots inoculated with the ineffective B. japonicum strain, SM5, were similar in number and shape to those on roots inoculated with the effective strain, 61A209, but roots inoculated with SM5 were free of nitrogenase activity. Hydrogen ion and reductant were not released from uninoculated roots or roots inoculated with SM5, but were release...

Nodulation, N 2 fixation and dry matter yield of soybean [ Glycine max (L.) merrill] inoculated with effective and ineffective Bradyrhizobium japonicum strains

Many soils harbour rhizobia of varying effectiveness, and this could influence the outcome of seed inoculation with selected rhizobial strains. A greenhouse study was made to compare the effect of inoculating an effective Bradyrhizobium japonicum strain onto seed (s) or into the bulk soil (b) with treatments containing an additional inoculation (with the same strain, or an ineffective B. japonicum strain) onto seed or into soil. Nodulation, N 2 fixation and growth of soybean were measured, using the 15N isotope dilution method to quantify the N fixed. The method of inoculation had significant effects on nodule distribution along soybean roots, nodule number and fresh weight, and N 2 fixation. In general, soil inoculation resulted in more nodules being formed, more uniform distribution of nodules on the root, and greater nitrogen fixation. The influence of the ineffective strain (in combinations containing both strains) on nodulation and N 2 fixation depended on where each or both strains were inoculated. Generally, highest N 2 fixation was achieved by inoculating the effective strain into soil. N 2 fixation by seed-inoculated effective strain was significantly depressed in the presence of the soil-inoculated ineffective strain (Es + Ib) but not when both strains were inoculated onto seed (Es + Is), or when both strains were inoculated together into soil (Eb + Ib). The trend for %Ndfa was generally similar to total nitrogen fixed. Our results therefore indicate that there can still be substantial N 2 fixation in legumes by highly effective strains even in the presence of less effective strains if the effective strains are fairly well distributed in soil.

Nodulation efficiency of Bradyrhizobium japonicum strains with genotypes of soybean varying in the ability to restrict nodulation

Competition from existing soil rhizobia has limited the benefits from nitrogen fixation for soybean grown in the American Midwest. A strategy being considered to overcome this problem is the use of varieties that are restricted in nodulation with soil strains, but nodulate normally with inoculant bradyrhizobia. In this study we examine the efficiency in nodulation of strains of Bradyrhizobium japonicum that have been reported as restricted in nodulation with specific genotypes of soybean, using a root-tip marking procedure in growth pouches. When B. japonicum USDA110 was applied to the soybean cultivars Hardee and Fiskeby V at the rate of 3.50 × 104 cells/pouch, more than 75% of the plants were nodulated above the root-tip mark, and average uppermost nodule position was above the root-tip mark. By contrast, when this strain was applied in similar concentration to the soybean cultivar Peking, few plants developed nodules above the root-tip mark, and the average position of the uppermost taproot nodule was nearly 30 mm below this mark. Nodulation was improved at higher rates of inoculation, but even when 3.50 × 106 cells were applied to each pouch, less than 50% of the plants were nodulated above the root-tip mark. Bradyrhizobium japonicum strain CB1809 (=USDA136) was also efficient in nodulation with cv. Fiskeby V, but with cv. Hardee, less than 65% of plants were nodulated above the root-tip mark, irrespective of inoculation rate. Because restriction of nodulation with the strains initially tested was not absolute, we examined the patterns of nodulation obtained following the inoculation of two restriction hosts, Peking and PI371607. In pure culture, serogroup USDA110 strains failed to induce significant taproot nodulation of cv. Peking in Leonard jars, but did induce lateral root nodulation. However, in a glasshouse experiment contrasting soil- and seed-applied inoculant, lateral-root nodulation of the restriction host PI371607 by USDA123 was not significant.Key words: Glycine max, competition, restriction, nodulation.

Variability among Soybean Genotypes in Response to Nodulation by a Rhizobitoxine‐Producing Strain of Bradyrhizobia

AbstractNodulation by rhizobitoxine‐producing (RT+) strains of Bradyrhizobium elkanii (syn. Bradyrhizobium japonicum; DNA homology group II) has been shown to reduce shoot growth, N2 fixation, and seed yield of ‘Forrest’ soybean (Glycine max [L.] Merr.). We conducted greenhouse and field experiments to assess the frequency of these negative responses among selected soybean cultivars. In the greenhouse, 38 cultivars representing a range of yield potentials, maturity groups, and growth habits were inoculated with B. elkanii strain USDA 94 (RT+) or B. japonicum strain USDA 110 (RT−). Large differences in tolerance to nodulation by RT+ bradyrhizobia were noted, as estimated visually and by comparing shoot weights (40 d after planting) of plants inoculated with the two strains. For the field study, two tolerant (Stafford and SS FFR 565), two moderately sensitive (Pharaoh and Williams 82), and two highly sensitive cultivars (Forrest and Hutcheson) were chosen. Seeds sown to vermiculite under greenhouse conditions were inoculated with either USDA 94 or a suspension of soil taken from the study site containing the indigenous bradyrhizobia. After 23 d, the nodulated seedlings were transplanted to 1.8‐m2 field microplots. Shoots were harvested at the R7 growth stage and analyzed for various response parameters. Only Forrest, Hutcheson, and Pharaoh were significantly affected by inoculation with USDA 94, which decreased seed yield, pod number, vegetative growth, and the amount of N2 fixed. These results support the hypothesis that significant genotypic variability exists in the response of soybeans to nodulation by RT+ bradyrhizobia. The greenhouse screening procedure can provide useful predictions of the field response of tolerant and highly sensitive cultivars, but may not be accurate for moderately sensitive cultivars.

Effects of Nitrate on Accumulation of Trehalose and other Carbohydrates and on Trehalase Activity in Soybean Root Nodules

Soybean ( Glycine max cv. Maple Arrow) plants were infected with Bradyrhizobium japonicum (strain 61-A-101), grown in sterilized Leonard jars, exposed to various amounts of nitrate either from the beginning or after completion of nodulation. The presence of 5 mM and more nitrate during nodulation caused a considerable reduction of the number and biomass of nodules per plant, of nitrogenase activity per nodule fresh weight. The carbohydrate content of nodules was determined on a dry weight basis. The level of the disaccharide trehalose, produced by the microsymbiont, was 50% lower in nodules formed in the presence of 20 mM nitrate than in control nodules formed in its absence. With regard to the non-structural carbohydrates produced by the plant, nodules formed in the presence of high amounts of nitrate contained about 75 % less starch but three- to fourfold higher levels of sucrose and pinitol than control nodules. Sucrose was the most abundant non-structural carbohydrate in nodules formed in the presence of 20 mM nitrate, accounting for 4–5 % of the dry weight. When plants with fully established nodules, grown in the absence of nitrate, were shifted to 20 mM nitrate, the levels of trehalose and starch decreased over a period of 3 weeks while the level of sucrose increased, until the carbohydrate levels attained similar values as found in nodules established in the presence of nitrate. The activity of trehalase, an enzyme known to be induced in nodules, was about 75% lower in nodules formed in the presence of nitrate than in control nodules. However, trehalase activity did not change in established nodules during a 3-week exposure to 20 mM nitrate. Similarly, the number of colony- forming bacteria recovered from the nodules and the activities of endochitinase and endoglucanase, two plant defense hydrolases, were not affected during a 3-week exposure to nitrate.

The effects of low root‐zone temperature stress on two soybean (Glycine max) genotypes when combined with Bradyrhizobium strains of varying geographic origin

In areas with short growing seasons, poor early vegetative growth of soybean (Glycine max [L.] Merr.) is often attributed to the restrictive effect of cool soil conditions on nodulation and N2‐fixation by this subtropical grain legume. However, there are few studies regarding potential genetic variability of soybean and Bradyrhizobium japonicum genotypes for nodulation at cool root‐zone temperatures (RZT). Experiments were conducted to (1) test for a threshold temperature for low RZT inhibition of soybean nodulation and (2) ascertain whether this threshold temperature response depends mainly on the micro‐ or macrosymbiont. In experiment 1 soybean seedlings (Glycine max [L.] Merr. cv. Maple Arrow) were inoculated with 1 ml of a log phase culture of B. japonicum strain 532C, H8 or H15 (the latter two strains were isolated from cold soils of Hokkaido, northern Japan) and maintained at either 16, 17.5, 19 or 25°C RZT. In experiment 2 seedlings of cv. Maple Arrow and a cold‐tolerant Evans isoline were combined with strain 532C and two Hokkaido strains (H5, H30) at both 19 and 25°C RZT. Results indicated that N2‐fixation at 44 days after inoculation was substantially reduced (30–40%) by RZT as high as 19°C, due to development of less nodule mass and to a delay in the onset of N2‐fixation and a small decrease in the number of nodules formed. However, the number of nodules formed was sharply reduced and the time required for the first appearance of nodules was significantly delayed below an RZT of 17.5°C. Differences between cultivars for nodulation and N accumulation were apparent at 25°C, but were abolished by growth at 19°C, indicating that, in spite of differences in growth potential between the cultivars under optimum RZT, both cultivars were equally limited by low RZT. Differences between B. japonicum strains were consistent across temperatures and were largely attributable to higher rates of specific nodule activity recorded for strain 532C, which seemed well adapted to low RZT. These results suggest that the host plant mediates the sensitivity of N2‐fixation under low RZT and that inoculation with B. japonicum strains from cold environments is unlikely to enhance soybean N2‐fixation under cool soil conditions.

Microscopic characterization of early nodulation events in a non-nodulating soybean mutant (NN5) and lack of response to high inoculum dose

The microscopic events leading to nodulation in normally nodulating soybean [Glycine max (L.) Merr.] genotypes, and the effects of Bradyrhizobium strain and inoculum dose on nodulation, were examined in the NN5 non-nodulating mutant derived from cv. Williams. The NN5 mutant possesses the recessive genes ris and rJ 6 . Bradyrhizobium japonicum strain USDA 110 cells attached normally to the root surface of NN5, many in a polar manner as in its wild-type parent, but failed to induce root hair curling and sub-epidermal cell division in the root. Co-culturing NN5 and Williams did not modify nodulation of either genotype

Nodulation of field-grown soybean in non-inoculated soils: multi-site comparison of two Bradyrhizobium japonicum strains and sensitivity to environmental factors

A comparison of two strains of Bradyrhizobium japonicum was conducted in field experiments from 1986 to 1989, in locations where soybean was cropped for the first time. Eighty-five comparisons on early nodulation and grain yield were carried out in 45 field trials in 30 different locations throughout France. Comparisons were differentiated according to inoculum origin, inoculation technique, climatic conditions and soil type. There was a significant difference between the two strains G49 and SMGS 1 in nodule number at soybean vegetative stage V3, whereas average grain yields were similar. Nevertheless, marked variability between locations was observed. Differences in nodulation were mainly due to low nodulation with strain G49. Correspondence analysis defined essential environmental factors responsible for differences in nodulation between the two strains : April average temperature, May rainfall, soil pH and origin of inoculum. Cluster analysis defined groups with similar behaviour. Nodulation with strain SMGS 1 was less sensitive to environmental factors, whereas nodulation with strain G49 was particulary limited by drought and in calcareous soil. The ability of strain SMGS 1 to produce nodules regardless of the environmental conditions is a property which could prove to be useful in strain selection.

Numerical Taxonomy and DNA Relatedness of Tropical Rhizobia Isolated from Hainan Province, China

A total of 63 strains of rhizobia isolated from Hainan Province, a tropical region of the People’s Republic of China, and 27 representative strains belonging to the genera Rhizobium, Brady rhizobium, and Agrobacterium were compared by performing numerical taxonomy, DNA hybridization, and DNA base composition analysis to determine the relationships among these rhizobia. The results indicated that the strains isolated from Hainan Province fell into two distinct phena, the slowly growing rhizobia and the fast-growing rhizobia. The slowly growing rhizobia, which formed three subphena that seemed to be three subspecies, are Bradyrhizobium japonicum strains. The fast-growing strains belong to the genus Rhizobium and might be further divided into three specific groups. Sometimes both slowly growing rhizobia and fast-growing rhizobia were isolated from host plants belonging to the same genus or species or even from the same nodule. There was no correlation between hosts and the distribution of rhizobia in the subphena. Isolates obtained from members of the same host genus or species fell into different groups or subgroups.

Trehalose and trehalase in root nodules from various legumes

Nitrogen‐fixing (effective) nodules from various legume‐Rhizobium combinations were analyzed for trehalose and other soluble carbohydrates using gas chromatography and for trehalase activity using biochemical assays. Whereas the bacterial disaccharide trehalose was present only in the minority of the nodules, trehalase activity was found in all of them. Extracts from determinate nodules had a higher trehalase activity than extracts from indeterminate nodules. More detailed studies were done on soybean nodules formed in interactions with two effective and 5 ineffective Bradyrhizobium japonicum strains. Only in effective soybean nodules colonized by the strain 61‐A‐101 was trehalose a major soluble carbohydrate. Irrespective of the wildtype strains used. effective soybean nodules contained about 10 nkat trehalase g−1 fresh weight, whereas the ineffective nodules colonized by mutant strains derived from these wildtype strains contained 2 to 30 times less trehalase. However, a clear correlation between trehalose content and trehalase activity could not be established.

Composition and Distribution of Adenylates in Soybean (Glycine max L.) Nodule Tissue.

Adenylates (ATP, ADP, and AMP) may play a central role in the regulation of the O2-limited C and N metabolism of soybean nodules. To be able to interpret measurements of adenylate levels in whole nodules and to appreciate the significance of observed changes in adenylates associated with changes in O2-limited metabolism, methods were developed for measuring in vivo levels of adenylate pools in the cortex, plant central zone, and bacteroid fractions of soybean (Glycine max L. Merr cv Maple Arrow x Bradyrhizobium japonicum strain USDA 16) nodules. Intact nodulated roots were either frozen in situ by flushing with prechilled Freon-113(-156[deg]C) or by rapidly (<1 s) uprooting plants and plunging them into liquid N2. The adenylate energy charge (AEC = [ATP + 0.5 x ADP]/[ATP + ADP + AMP]) of whole-nodule tissue (0.65 [plus or minus] 0.01, n = 4) was low compared to that of subtending roots (0.80 [plus or minus] 0.03, n = 4), a finding indicative of hypoxic metabolism in nodules. The cortex and central zone tissues were dissected apart in lyophilized nodules, and AEC values were 0.84 [plus or minus] 0.04 and 0.61 [plus or minus] 0.03, respectively. Although the total adenylate pool in the lyophilized nodules was only 41% of that measured in hydrated tissues, the AEC values were similar, and the lyophilized nodules were assumed to provide useful material for assessing adenylate distribution. The nodule cortex contained 4.4% of whole-nodule adenylates, with 95.6% being located in the central zone. Aqueous fractionation of bacteroids from the plant fraction of whole nodules and the use of marker enzymes or compounds to correct for recovery of bacteroids and cross-contamination of the bacteroid and plant fractions resulted in estimates that 36.2% of the total adenylate pool was in bacteroids, and 59.4% was in the plant fraction of the central zone. These are the first quantitative assessments of adenylate distribution in the plant and bacteroid fractions of legume nodules. These estimates were combined with theoretical calculations of rates of ATP consumption in the cortex (9.5 nmol g-1 fresh weight of nodule s-1), plant central zone (38 nmol g-1 fresh weight of nodule s-1), and bacteroids (62 nmol g-1 fresh weight of nodule s-1) of soybean nodules to estimate the time constants for turnover of the total adenylate pool and the ATP pool within each nodule fraction. The low values for time constant (1.6-5.8 s for total adenylate, 0.9-2.5 s for ATP only) in each fraction reflect the high metabolic activity of soybean nodules and provide a background for further studies of the role of adenylates in O2-limited nodule metabolism.

NolMNO genes of Bradyrhizobium japonicum are co-transcribed with nodYABCSUIJ, and nolO is involved in the synthesis of the lipo-oligosaccharide nodulation signals.

A host-inducible lacZ fusion was mapped down-stream of the nodYABCSUIJ operon in Bradyrhizobium japonicum strain USDA110. Sequencing of this region identified three novel genes, nolMNO. RNA dot blot analysis showed that nolO transcription is nodD1-dependent and that a polar mutation in nodS, located 5 kilobases upstream of nolO, blocks the transcription of nolO. Coupled with the host-inducible nature of nolO expression, these results indicate that nolMNO are part of a 9-kilobase operon, nodYABCSUIJnolMNO. The lipo-oligosaccharide nodulation signals produced by strains SL67 (nolO-) and SL65(nolNO-) were purified, and their chemical structures were determined. In addition to the wild-type signal molecules, both mutants produced modified compounds that are not produced by the parent strain USDA110. The most prevalent difference observed was the absence of the 2-O-methylfucosyl residue from the mutant structures. In addition, metabolites were found in which the N-acetylglucosamine residue at the reducing end was glycosidically linked to glycerol. These alterations in the profiles of nodulation signals produced by strains SL67 and SL65 were accompanied by reduced nodulation efficiency on all hosts tested.

A 23S rRNA Targeted Specific Hybridization Probe for Bradyrhizobium japonicum

Summary The complete nucleotide sequences of 23S rRNA genes from Bradyrhizobium japonicum DSM 30131 and from closely related Rhodopseudomonas palustris DSM 126 were determined. A specific hybridization probe for Bradyrhizobium japonicum was designed and tested. The probe hybridized to nucleic acids from all Bradyrhizobium japonicum strains tested and did not react with nucleic acids from other reference bacteria.