Enhanced competitiveness of a Bradyrhizobium japonicum mutant strain improved for nodulation and nitrogen fixation

Bradyrhizobium japonicum strain TA-11NOD⁺, with altered indole biosynthesis, exhibited enhanced nodulation and nitrogen fixation on soybean in previous greenhouse studies. In this study, field experiments were conducted at Upper Marlboro, Maryland, in the summers of 1988 and 1993. In 1988, the site used was essentially free of soybean-nodulating bacteria and seed yield in plots inoculated with either I-110ARS or TA-11NOD⁺ was significantly higher by 12 or 20%, respectively, than that of the uninoculated controls. The 1993 site had an indigenous soil population (about 10⁴ cells g^-1) of symbiotically ineffective soybean-nodulating bacteria. Nevertheless, six-week-old Morgan soybean plants inoculated with strain TA-11NOD⁺ had 44% more nodules and exhibited 50% more nitrogen fixation by acetylene reduction when compared with plants that received the parental strain I-110ARS. Nodule occupancy, as determined using genetic markers for rifampicin and streptomycin resistance, was significantly higher for strain TA-11NOD⁺ than for strain I-110ARS. Overall, for the two years and the two soybean genotypes, the yield obtained with TA-11NOD⁺ was 6% higher than that obtained with I-110ARS. Competition experiments were conducted in the greenhouse and strain TA-11NOD⁺ was significantly more competitive than strain I-110ARS in competition with strains USDA 6 or USDA 438.     

Regulation of nodulation in the soybean-Rhizobium symbiosis : strain and cultivar variability

Double inoculation (15 h apart) of the soybean cultivar Williams with Bradyrhizobium japonicum I-110ARS reveals a rapid regulatory plant response that inhibits nodulation of distal portions of the primary root (M Pierce, WD Bauer 1984 Plant Physiol 73: 286-290). Only living, homologous rhizobia elicit the response. We conducted similar double inoculation experiments to test the hypothesis that this is a universal phenomenon in soybean symbioses. We investigated interactions of the cultivar McCall with the slow-growing strain Bradyrhizobium sp. 3185 (=3G4b16) and strains of the fast-growing soybean symbiont, Rhizobium fredii (USDA191 [Nod⁺ on McCall] and USDA257 [Nod⁻ on McCall]). Nodulation was not detectably inhibited when USDA257 was included in various combinations with an inoculum of USDA191. Strain USDA257 cohabited nodules with strain USDA191 when plants were inoculated sequentially with both strains, but USDA257 did not nodulate McCall when a sterile culture filtrate of USDA191 was added to USDA257 inoculum. There was only a slight inhibition of nodulation of distal portions of the primary root in double inoculation experiments with McCall and strain 3185. Because these results were unexpected, we repeated the experiments with Williams and strain I-110ARS. The response was similar to that observed in the McCall × 3185 interaction. Regulation of nodulation on the primary root thus appears to be variable and depend on strain X cultivar interactions.     

Influence of 5-Methyltryptophan-Resistant Bradyrhizobium japonicum on Soybean Root Nodule Indole-3-Acetic Acid Content

Bradyrhizobium japonicum mutants resistant to 5-methyltryptophan were isolated. Some of these mutants were found to accumulate indole-3-acetic acid (IAA) and tryptophan in culture. In greenhouse studies, nodules from control plants inoculated with wild-type bradyrhizobia contained 0.04, 0.10, and 0.58 μg of free, ester-linked, and peptidyl IAA g (fresh weight) of nodules⁻¹, respectively. Nodules from plants inoculated with 5-methyltryptophan-resistant bradyrhizobia contained 0.94, 1.30, and 10.6 μg of free, ester-linked, and peptidyl IAA g (fresh weight) of nodules⁻¹, respectively. This manyfold increase in nodule IAA content indicates that the Bradyrhizobium inoculum can have a considerable influence on the endogenous IAA level of the nodule. Further, these data imply that much of the IAA that accumulated in the high-IAA-containing nodules was of bacterial rather than plant origin. These high-IAA-producing 5-methyltryptophan-resistant bacteria were poor symbiotic nitrogen fixers. Plants inoculated with these bacteria had a lower nodule mass and fixed less nitrogen per gram of nodule than did plants inoculated with wild-type bacteria.     

Influence ofGlycine max nodulation on the persistence in soil of a genetically markedBradyrhizobium japonicum strain

Since competition with indigenous strains limits nodule occupancy by bacteria applied to seeds, the ecology of Bradyrhizobium inoculum strains used for soybean is of concern. A genetically marked strain,B. japonicum I-110 ARS, was directly enumerated from soil on selective medium. A clear long-term positive influence of even limitedGlycine max nodulation was shown by comparisons of population densities obtained with or without plant removal prior to nodule senescence in the first year and with an incompatible as well as a compatible soybean variety after 5 years.     

Competitive Ability and Efficiency in Nodule Formation of Strains of Bradyrhizobium japonicum

In the American Midwest, superior N₂-fixing inoculant strains of Bradyrhizobium japonicum consistently fail to produce the majority of nodules on the roots of field-grown soybean. Poor nodulation by inoculant strains is partly due to their inability to stay abreast of the expanding soybean root system in numbers sufficient for them to be competitive with indigenous bradyrhizobia. However, certain strains are noncompetitive even when numerical dominance is not a factor. In this study, we tested the hypothesis that the nodule occupancy achieved by strains is related to their nodule-forming efficiency. The nodulation characteristics and competitiveness of nine strains of B. japonicum were compared at both 20 and 30°C. The root tip marking technique was used, with the nodule-forming efficiency of each strain estimated from the average position of the uppermost nodule and the number of nodules formed above the root tip mark. The competitiveness of the nine strains relative to B. japonicum USDA 110 was determined by using immunofluorescence to identify nodule occupants. The strains differed significantly in competitiveness with USDA 110 and in nodulation characteristics, strains that were poor competitors usually proving to be inferior in both the average position of the uppermost root nodule and the number of nodules formed above the root tip mark. Thus, competitiveness was correlated with both the average position of the uppermost nodule (r = 0.5; P = 0.036) and the number of nodules formed above the root tip mark (r = 0.64; P = 0.005), while the position of the uppermost nodule was also correlated to the percentage of plants nodulated above the root tip mark (r = 0.81; P < 0.001) and the percentage of plants nodulated on the taproot (r = 0.67; P = 0.002).     

Influence of the Bradyrhizobium japonicum Hydrogenase on the Growth of Glycine and Vigna Species

The effect of the Bradyrhizobium japonicum hydrogenase on nitrogen fixation was evaluated by comparing the growth of Vigna and Glycine species inoculated with a Hup mutant and its Hup⁺ revertant. In all experiments, the growth of plants inoculated with the strain without hydrogenase was at least equal to the growth of the strain with hydrogenase. For Glycine usuriensis and Glycine max cv. Hodgson in liquid culture, the growth was higher with the Hup⁻ strain. It is possible that reduced rates of nitrogen fixation in the presence of hydrogenase are due to O₂ depletion caused by the hydrogen oxidizing, since the oxygen pressure in the air appears to be a limiting factor of symbiotic nitrogen fixation in the soybean.     

Positive role of nodulation on the establishment ofRhizobium japonicum in subsequent crops of soybean

The influence of soybean nodulation on the establishment ofRhizobium japonicum inRhizobium-free soil was examined. Seeds of nodulating (Rj ₁) and nonnodulating (rj ₁) isolines of soybeans and four other crop species (cowpeas, mungbeans, corn, and alfalfa) were grown in field plots that were inoculated with a genetically marked strain ofRhizobium (strain I-110 ARS) and the following year nodulating soybeans were grown in these plots and were inoculated with a different genetically marked subline of the same strain (strain I-110 FN). The proportion of nodules containing strain I-110 ARS relative to strain I-110 FN was determined and interpreted as reflecting the relative numbers of the two genetically marked sublines in the soil. The results clearly demonstrate that nodulation with the specific host plant (soybeans) has a significant positive role in the establishment ofRhizobium inRhizobium-free soil and suggests that alfalfa plants diminish the establishment of soybean rhizobia in soil.     

Host recognition in the Rhizobium-soybean symbiosis: detection of a protein factor in soybean root exudate which is involved in the nodulation process

The mechanism of host-symbiont recognition in the soybean-Rhizobium symbiosis was investigated utilizing mutants of R. japonicum defective in nodulation. Soybeans were grown in clear plastic growth pouches allowing the identification of the area on the root most susceptible to Rhizobium nodulation; the area between the root tip (RT) and smallest emergent root hair (SERH). The location of nodules in relation to this developing zone is an indication of the rate of nodule initiation. Nodules were scored as to the distance from the RT mark made at the time of inoculation. Seventy-eight per cent of the plants nodulate above the RT mark when inoculated with the wild type R. japonicum strain 3I1b110 with the average distance of the uppermost nodule being approximately 2 millimeters above the RT mark. These data indicate that the wild type strain initiates nodulation rapidly within the RT-SERH zone following inoculation. However, inoculation with the slow-to-nodulate mutant strain HS111 resulted in 100% of the plants nodulating only below the RT mark with the average distance of the uppermost nodule being approximately 56 millimeters below the RT mark. Thus, mutant strain HS111 is defective in the ability to rapidly initiate infection leading to nodulation within the RT-SERH zone. The location of the nodules suggest that stain HS111 must `adapt' to the root environment before nodulation can occur. To test this, strain HS111 was incubated in soybean root exudate prior to inoculation. In this case, 68% of the plants nodulated above the RT mark with the average distance of the uppermost nodule being approximately 1 millimeter below the RT mark. Experiments indicated that the change in nodule initiation by strain HS111 brought about by incubation in soybean root exudate was due to a phenotypic, rather than a genotypic change. The half-time of root exudate incubation for strain HS111 necessary for optimal nodulation enhancement was less than 6 hours. Heat sensitivity and trypsin sensitivity of the nodulation enhancement factor(s) in soybean root exudate indicate a protein was involved in the reversal of the delay in nodulation by mutant strain HS111.     

The Influence of Lipopolysaccharides and Glucans from Two Rhizobium leguminosarum bv. viciae Strains on the Formation and Efficiency of Their Symbioses with Pea Plants

The influence of lipopolysaccharides (LPS), glucans, and their unseparated complexes on nodulation activity of rhizobia and efficiency of their symbioses with pea plants was studied in vegetation tests. Two Rhizobium leguminosarum bv. viciae strains which differed in their symbiotic properties were used: strain 31 (fix⁺, efficient, moderately virulent, and moderately competitive) and strain 248b (fix^–, inefficient, highly virulent, and highly competitive). Preparations of LPS–glucan complex and the respective LPS from the highly virulent strain 248b increased the nodulation activity of both strains by 10–26%. Analogous preparations from a less virulent strain 31 did not have this ability. Unseparated LPS–glucan complexes from these strains increased the productivity of plants infected with the efficient strain by 18–23% but did not change it in plants inoculated with the other, inefficient strain. No significant influence of LPS preparations on the symbiosis productivity was observed. Glucans from both strains enhanced the nodulation ability of the highly virulent strain by 36–56%. In addition, treatment of pea plants with glucan from strain 248b increased nitrogen fixation by root nodules by 27% in plants inoculated with strain 31. In general, the formation and efficiency of the symbiosis of R. leguminosarum bv. viciae with pea plants was more influenced by preparations from strain 248b, highly virulent but deficient in nitrogen fixation, than by preparations from the nitrogen fixation–proficient but less virulent strain 31.     

Persistence of Rhizobium japonicum on the Soybean Seed Coat Under Controlled Temperature and Humidity

When Rhizobium japonicum strain 61A68 was added to surface-sterilized soybean (Glycine max) seed along with 12 different coating materials, a definite effect of temperature upon survival was observed both with and without coating materials. At a storage temperature of 15°C and 50 ± 5% relative humidity, from 0.9 to 14.1% of the original inoculum survived for 3 weeks. At 22.5°C, from 0.5 to 7.2% of the original inoculum survived. At 30°C, from 0.1 to 1.6% of the original inoculum survived. The data indicated that extremely large numbers of R. japonicum would have to be added to the seed to have numbers adequate for nodulation survive for 3 weeks of storage at ordinary temperatures.     

Effect of Inoculation and Leaf Litter Amendment on Establishment of Nodule-Forming Frankia Populations in Soil

High-N₂-fixing activities of Frankia populations in root nodules on Alnus glutinosa improve growth performance of the host plant. Therefore, the establishment of active, nodule-forming populations of Frankia in soil is desirable. In this study, we inoculated Frankia strains of Alnus host infection groups I, IIIa, and IV into soil already harboring indigenous populations of infection groups (IIIa, IIIb, and IV). Then we amended parts of the inoculated soil with leaf litter of A. glutinosa and kept these parts of soil without host plants for several weeks until they were spiked with [¹⁵N]NO₃ and planted with seedlings of A. glutinosa. After 4 months of growth, we analyzed plants for growth performance, nodule formation, specific Frankia populations in root nodules, and N₂ fixation rates. The results revealed that introduced Frankia strains incubated in soil for several weeks in the absence of plants remained infective and competitive for nodulation with the indigenous Frankia populations of the soil. Inoculation into and incubation in soil without host plants generally supported subsequent plant growth performance and increased the percentage of nitrogen acquired by the host plants through N₂ fixation from 33% on noninoculated, nonamended soils to 78% on inoculated, amended soils. Introduced Frankia strains representing Alnus host infection groups IIIa and IV competed with indigenous Frankia populations, whereas frankiae of group I were not found in any nodules. When grown in noninoculated, nonamended soil, A. glutinosa plants harbored Frankia populations of only group IIIa in root nodules. This group was reduced to 32% ± 23% (standard deviation) of the Frankia nodule populations when plants were grown in inoculated, nonamended soil. Under these conditions, the introduced Frankia strain of group IV was established in 51% ± 20% of the nodules. Leaf litter amendment during the initial incubation in soil without plants promoted nodulation by frankiae of group IV in both inoculated and noninoculated treatments. Grown in inoculated, amended soils, plants had significantly lower numbers of nodules infected by group IIIa (8% ± 6%) than by group IV (81% ± 11%). On plants grown in noninoculated, amended soil, the original Frankia root nodule population represented by group IIIa of the noninoculated, nonamended soil was entirely exchanged by a Frankia population belonging to group IV. The quantification of N₂ fixation rates by ¹⁵N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N₂-fixing capacity than populations belonging to group IIIa under the conditions applied. These results show that through inoculation or leaf litter amendment, Frankia populations with high specific N₂-fixing capacities can be established in soils. These populations remain infective on their host plants, successfully compete for nodule formation with other indigenous or inoculated Frankia populations, and thereby increase plant growth performance.     

Construction of a Symbiotically Effective Strain of Rhizobium leguminosarum bv. trifolii with Increased Nodulation Competitiveness

Genes involved in nodulation competitiveness (tfx) were inserted by marker exchange into the genome of the effective strain Rhizobium leguminosarum bv. trifolii TA1. Isogenic strains of TA1 were constructed which differed only in their ability to produce trifolitoxin, an antirhizobial peptide. Trifolitoxin production by the ineffective strain R. leguminosarum bv. trifolii T24 limited nodulation of clover roots by trifolitoxin-sensitive strains of R. leguminosarum bv. trifolii. The trifolitoxin-producing exconjugant TA1::10-15 was very competitive for nodulation on clover roots when coinoculated with a trifolitoxin-sensitive reference strain. The nonproducing exconjugant TA1::12-10 was not competitive for nodule occupancy when coinoculated with the reference strain. Tetracycline sensitivity and Southern analysis confirmed the loss of vector DNA in the exconjugants. Trifolitoxin production by TA1::10-15 was stable in the absence of selection pressure. Transfer of tfx to TA1 did not affect nodule number or nitrogenase activity. These experiments represent the first stable genetic transfer of genes involved in nodulation competitiveness to a symbiotically effective Rhizobium strain.     

Measurement of nitrogen fixation by soybean in the field using the ureide and natural N abundance methods

Nitrogen fixation by field-grown soybean (Glycine max [L.] Merrill) was assessed by the natural ¹⁵N abundance and ureide methods. The field sites (five) and genotypes (six, plus two levels of inoculation on Bragg) were chosen to provide a range of proportions of plant N derived from nitrogen fixation (P). Genotypes K466, K468, nts1007, and nts1116 and Davis were included on the basis of their reported tolerance of the suppressive effects of nitrate on nodulation and nitrogen fixation. Bragg was included as a `nitrate-sensitive' genotype. Seeds of all genotypes were inoculated at sowing with Bradyrhizobium japonicum CB1809 (USDA136). Amounts of nitrate in the soil profile (0-1.2 meter depth) at sowing ranged from 70 (site 3) to 278 kilograms per hectare (site 5), resulting in large effects on plant nodulation, on the δ¹⁵N values of nodulated plants, on the relative abundance of ureide-N in vacuum-extracted sap (VES) and stem extracts, and finally on the estimates of P. There was no relationship between amount of soil nitrate at sowing and the δ¹⁵N of the plant-available soil N. Correlation matrices of the measured and calculated parameters indicated generally weak correlations between crop growth (dry matter and N) and the parameters of symbiotic activity (nodule weight, δ¹⁵N, relative ureide-N); correlations were strong and highly significant between nodulation and the measures of nitrogen fixation (δ¹⁵N, relative ureide-N; r = 0.79-0.92). Estimates of P ranged between 0 and 68% (δ¹⁵N) and between 6 and 56% (ureide) and were highly correlated (r = 0.97). Results indicated that the ureide method can be used with confidence to assess P by field-grown crops of soybean.     

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.     

Rapid Colored-Nodule Assay for Assessing Root Exudate-Enhanced Competitiveness of Bradyrhizobium japonicum

The effects of root exudate (RE) treatment on nodule occupancy by Bradyrhizobium japonicum were investigated by a rapid colored-nodule assay, which is based on the observation that B. japonicum L-110 and its antibiotically marked derivatives form dark-red nodules on certain soybean (Glycine max) cultivars, whereas other strains form beige nodules. The efficacy of the assay was confirmed by direct immunofluorescence and by antibiotic platings of nodule bacteria. Both logarithmic- and stationary-phase cultures of the reference strain, L-110Nal, were used in paired-competition studies with RE-treated or untreated cells of seven challenge strains. On the basis of field and greenhouse competition studies, these strains were placed into three competitiveness groups: high (AN-11, AN-16aStrRif, and AN-6), intermediate (AN-3 and 122SR), and low (I-110ARS and AN-18). Seedlings of G. max cv. Centennial were inoculated with two ratios of challenge to reference strain, 1:1 and 1:9, and nodule occupancy was determined after the V4 to V5 stage of ontogeny. Two of the strains showed increased occupancy in response to RE treatment at the 1:1 inoculation ratio. Logarithmic- and stationary-phase cultures of AN-6 showed increased occupancy, from 22 to 38% (P < 0.10) and from 23 59 39% (P < 0.05), respectively. While the maximum increase for stationary-phase cultures of AN-16aStrRif was from 34 to 47% (P < 0.05), logarithmic-phase cultures failed to respond to RE treatment. The results of these studies indicate that RE treatment increases the nodule occupancy of some, but not all, B. japonicum strains and that the colored-nodule assay could be rapidly and reliably used to determine the competitive ability of B. japonicum.     

Ethylene Inhibitors Restore Nodulation to sym 5 Mutants of Pisum sativum L. cv Sparkle

The sym 5 mutants of pea, Pisum sativum L. cv Sparkle, do not differ in growth habit from their normal parent and nodulate poorly at a root temperature of 20°C. If inhibitors of ethylene formation or action (Co²⁺, aminoethoxyvinylglycine, or Ag⁺) are added to the substrate, nodulation of the sym 5 mutants is increased. Similar treatments of four other mutant sym lines do not restore nodulation. When Ag⁺ is added to the substrate from 4 days before to 4 days after inoculation with rhizobia, nodulation of sym 5 mutants is increased. The roots of the mutant need only be exposed to Ag⁺ for 4 hours to significantly increase nodule numbers. The content of free 1-aminocyclopropane-1-carboxylic acid and the production of ethylene in the lateral roots of sym 5 mutants do not differ from Sparkle.     

Influence of Azospirillum Strains on the Nodulation of Clovers by Rhizobium Strains

Mixed cultures of several Azospirillum and Rhizobium trifolii strains caused either an inhibition or stimulation of nodule formation on plant hosts as compared with nodulation of plants inoculated with R. trifolii alone. Azospirillum strains affected the nodulation process at a precise cell ratio (R. trifolii/Azospirillum cells) and time of inoculation. All Azospirillum strains used showed a variation in their ability to inhibit or enhance nodulation by R. trifolii strains. When nonviable cell preparations of Azospirillum strains were used for mixing experiments, no effect on nodulation was observed. A decrease in the effectiveness of normally Nod⁺ Fix⁺R. trifolii strains was observed when an Azospirillum strain caused an increase in nodule number.     

Stimulated nodulation of soybeans by Rhizobium japonicum mutant (B-14075) that catabolizes the conversion of tryptophan to indol-3yl-acetic acid

Nodulations of Glycine max L. seedlings by parental Rhizobium japonicum (USDA strain 26) and a tryptophan catabolic mutant (tan 4b, NRRL strain B-14075) were evaluated for effectiveness. Seedlings grown in plastic pouches were compared based on increases in root weight (wt.), nodule volume (vol.) and acetylene-reducing (AR) activity. The tan 4b mutant, which can produce extracellular indol-3yl-acetic (IAA) and indol-3yl-pyruvic (IPA) acids asymbiotically, enhanced nodulation significantly. Nodulation by strain 26 was most effective when a basal nutrient was supplemented with sucrose, CaCO₃, EDTA, nicotinic acid and glutamate. In contrast, effective nodulation by tan 4b did not require an exogenous glutamate (0.34 mM)-nicotinate (81 μM) combination. Like strain 26 inoculum supplemented with exogenous IAA (0.1 mM), taproot formation associated with unsupplemented tan 4b was inhibited by exposure to direct light.     

Legume-Rhizobium Interactions: Cowpea Root Exudate Elicits Faster Nodulation Response by Rhizobium Species

Preinfection events in legume-Rhizobium symbiosis were analyzed by studying the different nodulation behaviors of two rhizobial strains in cowpeas (Vigna sinensis). Log-phase cultures of Rhizobium sp. strain 1001, an isolate from the plant nodule, initiated host responses leading to infection within 2 h after inoculation, whereas log-phase cultures of Rhizobium sp. strain 32H1 took at least 7 h to trigger a discernible response. The delay observed with strain 32H1 could be eliminated by incubating the rhizobial suspension, before inoculation, for 4.5 h either in the cowpea rhizosphere/rhizoplane condition or in the root exudate of cowpea plants, grown without NH₄⁺ in the rooting medium. The delay could not be eliminated by incubating the rhizobial suspension in the rooting medium of plants grown in the presence of 5 mM NH₄⁺, indicating that there is a regulatory role of combined nitrogen in triggering preinfection events by the legume. The substance(s) in the root exudate which elicited the faster nodulation response by Rhizobium sp. strain 32H1 could be separated into a high-molecular-weight fraction by Sephadex G-100 gel filtration. The data support the notion that legume roots release substances that favor the development of rhizobial features essential for infection and nodulation.     

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (mol C₂H₄g^–1 nodule fr. wt. h^–1), were both affected by the added nitrate.