Partitioning of nitrogen from biological fixation and fertilizer in Phaseolus vulgaris

Nitrogen uptake, distribution and remobilization in the vegetative and reproductive parts of the plant were studied in bean (Phaseolus vulgaris L.) cultivars Negro Argel and Rio Tibagi inoculated with either Rhizobium strain C05 or 127 K-17. Greenhouse grown plants were supplied with 2.5 mg N (plant)⁻¹ day⁻¹ as KNO₃ or K¹⁵NO₃ and the relative contribution to total plant nitrogen of mineral and symbiotically fixed nitrogen was determined. Control plants included those entirely dependent on fixed nitrogen as well as uninoculated plants supplied with 10 mg N (plant)⁻¹ day⁻¹. No differences were observed between inoculated treatments in total nitrate reductase activity and in the amount of mineral nitrogen absorbed, but there were considerable differences in the contribution of fixed nitrogen. Nitrogen fixation supplied from 58 to 72% of the total nitrogen assimilated during the bean growth cycle and the symbiotic combinations fixed most of their nitrogen (66 to 78% of total nitrogen) after flowering. Maximum uptake of mineral nitrogen was in the 15-day-period between flowering and mid-podfill (47 to 58% of total mineral nitrogen). Nitrogen partitioning varied with Rhizobium strains, and inoculation with strain C05 increased the nitrogen harvest index of both cultivars. Applied mineral nitrogen had a variable effect and in cv. Negro Argel was more beneficial to vegetative growth, resulting in smaller nitrogen harvest indices. Seed yield was not increased by heavy nitrogen fertilization. In contrast, cv. Rio Tibagi always benefited from nitrogen applications. Among the various nitrogen sources supplying the grain, the most important one was the fixed nitrogen translocated directly from nodules or after a rapid transfer through leaves, representing from 60 to 64% of the total nitrogen incorporated into the seeds.     

Effects of water stress on N2 fixation and grain yield of Phaseolus vulgaris L.

Most dry bean production in Mexico is under non-irrigated conditions in the semi-arid highlands. One of the most limiting factors is insufficient moisture during the reproductive stage and sometimes during the vegetative stage. The objective of this experiment was to study the effect of drought on nodulation, N₂ fixation and grain yield of beans. The cultivars evaluated were: Flor de Mayo Bajio, Bayocel, Bat-477 and Honduras-35. The treatments were water stress treatments during vegetative or reproductive stage and a control of minimal stress. To measure N₂ fixation, ¹⁵N-labelled fertilizer was used. Data for soil moisture, nodule number and nodule dry weight, as well as, straw and grain yield and total N were taken. The results indicated that water stress during vegetative stage depressed nodulation temporarily, but after watering regularly plants not only recovered completely but were nodulated better than the control. Water stress during the reproductive stage depressed nodulation and after watering there was no recovery. Grain yield was not affected by water stress during vegetative stage but it was reduced when water stress was imposed during the reproductive stage. The percentage of N derived from fixation varied among cultivars but was not affected by water stress treatment. The highest N₂ fixation occurred in Bayocel and Bat-477 and the lowest in Honduras-35 and F.M. Bajio, although the amounts were not as low as in some other reports.     

Effects of high temperature on nodulation and nitrogen fixation by Phaseolus vulgaris L.

Screening of Rhizobium leguminosarum bv. phaseoli strains showed some that were able to nodulate common beans (Phaseolus vulgaris L.) at high temperatures (35 and 38°C/8 h/day). The nodulation ability was not related to the capability to grow or produce melanin-like pigment in culture media at high temperatures. However, nodules formed at high temperatures were ineffective and plants did not accumulate N in shoots. Two thermal shocks of 40°C/8 h/day at flowering time drastically decreased nitrogenase activity and nodule relative efficiency of plants otherwise grown at 28°C. Recovery of nitrogenase activity began only after seven days, when new nodules formed; total incorporation of N in tops did not recover for 2 weeks. Non-inoculated beans receiving mineral N were not affected by the thermal shock, and when growing continuously at 35 or 38°C had total N accumulated in shoots reduced by only 18%.     

Calcium and magnesium effects on symbiotic nitrogen fixation in the alfalfa (M. sativa) –Rhizobium meliloti system

An investigation of the roles of calcium and magnesium ions in symbiotic nitrogen fixation by legumes has shown that alfalfa plants ( Medicago sativa L. cv. Saranac and Apollo) deficient in either cation were poorly nodulated and retarded in growth on nitrogen-free media. This effect was reversed by supplementation with normal levels of these cations. After recovery, the calcium deficient seedlings showed continuing effects of early mineral deficiencies but recovered to 75% of the nitrogenase activity and had nearly the same yield as control plants. Magnesium deficient plants recovered nitrogenase activity to the same degree but grew to only about 50% of the weight of controls. Supplementation of non-deficient seedlings grown on N-free media with varying amounts of Ca²⁺ and Mg²⁺ resulted in the identification of an optimal ratio of calcium and magnesium near 2 when neither cation was growth limiting. A highly significant positive correlation was obtained between yield of dry matter and the fraction of total expressed nitrogenase activity that was actually available for dinitrogen reduction (nitrogen reducing equivalent). Bacteroids isolated from root nodules and freed of plant cytoplasmic components required high magnesium levels for maximal utilization of externally supplied ATP and dithionite. Ca⁺ was antagonistic to this activity but complemented Mg²⁺ in stimulating the respiration-supported nitrogenase activity of intact bacteroids which had been treated with a chelating agent. The effects of calcium and magnesium on the nitrogenase system of intact bacteroids may be due to binding of the Ca²⁺ ions to the bacteroid membrane.     

Rhizobium sp. strain ORS571 ammonium assimilation and nitrogen fixation.

Among rhizobia studied, Rhizobium sp. strain ORS571 alone grew unambiguously on N2 as sole N source. In ORS571 , only the glutamine synthetase (GS)-glutamate synthase ( GOGAT ) pathway assimilated ammonium. However, ORS571 exhibited two unique physiological aspects of this pathway: ORS571 had only GS I, whereas all other Rhizobiaceae studied had both GS I and GS II, and both NADPH- and NADH-dependent GOGAT activities were present. ORS571 GS-affected and NADPH- GOGAT -affected mutant strains were defective in both ammonium assimilation (Asm-) and N2 fixation (Nif-) in culture and in planta ; NADH- GOGAT mutants were Asm- but Nif+. "Bacteroid" GS activity was essentially nil, suggesting symbiotic ammonium export. Physiological studies on effects of glutamine, ammonium, methionine sulfoximine, and diazo-oxo-norleucine on nitrogenase induction in culture implied a regulatory role for the intracellular glutamine pool.     

Effect of Growth Irradiance on Photosynthesis and Transpiration inPhaseolus vulgaris L.

In comparison with primary leaves of French bean plants grown under a photon flux density of 100 μeinstein m-2 s-1 (LP), leaves grown under 400 μeinstein m-2 s-1 (HP) were thicker (contained 82 to 104% more dry matter per blade area), had 44 to 48% higher stomatal frequency, 18 to 26% more chlorophyll (a + b) per leaf area unit and 31 to 42% less chlorophyll (a + b) per dry matter unit, 41% higher photosynthetic and 38% higher transpiration rates at light saturation, 33% higher stomatal conductance and 40% higher Photosystem 2 (H2O → K3[Fe(CN)6]) activity of isolated chloroplasts. There were no significant differences in the Photosystem 1 (TMPD/Ascorbate → MV) activity per unit amount of chlorophyll. Higher growth irradiance increased the ratio of frequencies of stomata in the upper/lower epidermes.     

The effect of Rhizobium inoculation and nitrogen fertiliser on nitrogen fixation and seed yield of dry beans (Phaseolus vulgaris)

The potential benefit to be derived from seed inoculation of Phaseolus vulgaris beans with effective strains of Rhizobium phaseoli, was investigated in field experiments over three years on a site low in soil nitrogen and lacking indigenous effective strains of R. phaseoli. Inoculation with R. phaseoli (strain RCR 3644) produced significant increases in nodulation, nitrogenase activity and plant growth in all experiments. In trials in 1978 and 1979, with cv. Seafarer, inoculation, in the absence of nitrogen fertiliser doubled seed yields. In 1978, the seed yields from inoculated beans without nitrogen fertiliser (1–6 t/ha) were not significantly different from those obtained with uninoculated beans receiving the optimum nitrogen fertiliser treatment of 120 kg N/ha (1–75 t/ha). In 1979, with lower rainfall favouring more efficient utilisation of nitrogen fertiliser, inoculation gave seed yields (1–88 t/ha) equivalent to those obtained with 60 kg N/ha (1–70 t/ha) but significantly less than with 120 kg N/ha (2–88 t/ha). More precise estimates from nitrogen response curves showed that inoculation supplied the fertiliser equivalent of 105 and 70 kg N/ha in 1978 and 1979 respectively. In both years, significant benefits were also obtained by the combination of inoculation and nitrogen fertiliser. In a separate experiment in 1979, with four R. phaseoli strains inoculated onto eight bean cultivars, three were highly effective nitrogen fixers on all cultivars. Two strains (RCR 3644 and NVRS 963A) each increased mean yields, in the absence of nitrogen fertiliser, from 1–39 t/ha uninoculated to c. 2–5 t/ha inoculated whilst strain RCR 3622 was outstanding with a mean yield of 3-0 t/ha. An analysis of the nitrogen content of seed showed that gains from nitrogen fixation were 37–57 kg N/ha/growing cycle for the combination RCR 3644 with cv. Seafarer. However, 106 kg N/ha/growing cycle was recorded for the combination RCR 3622 and cv. Aurora.     

The effect of strA mutation on symbiotic nitrogen fixation by Rhizobium meliloti.

Studies on 3H-dihydrostreptomycin accumulation and binding to ribosomes showed that ineffective strain CMts17 carries strB type mutation changing its membrane permeability to the drug. Introduction of high level streptomycin resistance of strA type into strain CMts17 was correlated with acquisition of effectiveness and membrane permeability to the drug. This suggests that changes in membrane permeability, responsible for ineffectiveness of strain CMts17, can be reversed by strA mutation.     

Overexpression of the dctA gene in Rhizobium meliloti: effect on transport of C4 dicarboxylates and symbiotic nitrogen fixation.

Symbiotic nitrogen fixation may be limited by the transport of C4 dicarboxylates into bacteroids in the nodule for use as a carbon and energy source. In an attempt to increase dicarboxylate transport, a plasmid was constructed in which the Rhizobium meliloti structural transport gene dctA was fused to a tryptophan operon promoter from Salmonella typhimurium, trpPO. This resulted in a functional dctA gene that was no longer under the control of the dctBD regulatory genes, but the recombinant plasmid was found to be unstable in R. meliloti. To stably integrate the trpPO-dctA fusion, it was recloned into pBR325 and recombined into the R. meliloti exo megaplasmid in the dctABD region. The resultant strain showed constitutive dctA-specific mRNA synthesis which was about 5-fold higher than that found in fully induced wild-type cells. Uptake assays showed that [14C]succinate transport by the trpPO-dctA fusion strain was constitutive, and the transport rate was the same as that of induced control cells. Acetylene reduction assays indicated a significantly higher rate of nitrogen fixation in plants inoculated with the trpPO-dctA fusion strain compared with the control. Despite this apparent increase, the plants had the same top dry weights as those inoculated with control cells.     

Effect of Rhizobium sp. BARIRGm901 inoculation on nodulation,nitrogen fixation and yield of soybean (Glycine max) genotypes in gray terrace soil

Soybean plants require high amounts of nitrogen, which are mainly obtained from biological nitrogen fixation. A field experiment was conducted by soybean (Glycine max) genotypes, growing two varieties (Shohag and BARI Soybean6) and two advanced lines (MTD10 and BGM02026) of soybean with or without Rhizobium sp. BARIRGm901 inoculation. Soybean plants of all genotypes inoculated with Rhizobium sp. BARIRGm901 produced greater nodule numbers, nodule weight, shoot and root biomass, and plant height than non-inoculated plants. Similarly, inoculated plants showed enhanced activity of nitrogenase (NA) enzyme, contributing to higher nitrogen fixation and assimilation, compared to non-inoculated soybean plants in both years. Plants inoculated with Rhizobium sp. BARIRGm901 also showed higher pod, stover, and seed yield than non-inoculated plants. Therefore, Rhizobium sp. BARIRGm901 established an effective symbiotic relationship with a range of soybean genotypes and thus increased the nodulation, growth, and yield of soybean grown in gray terrace soils in Bangladesh.     

Selection for improved nitrogen fixation inGlycine max (L.) Merr. andPhaseolus vulgaris L.

Summary While symbiotic nitrogen (N₂) fixation byG. max andP. vulgaris reduces their need for combined N, N₂ fixation under field conditions is rarely maximized. This paper reviews constraints to N₂ fixation in these species, then examines the genetic variability recorded for traits affecting N₂ fixation and the further work needed in this area. It considers emerging programs for the improvement of N₂ fixation inG. max andP. vulgaris and pays particular attention to methodological considerations.Scientific Journal Series, Minnesota Agri. Exp. Station. No 14190.     

Induced genetic variability in Rhizobium leguminosarum for nitrogen fixation parameters in Vicia faba L.

Sumary The objective of this work was to know the behaviour and variability of Rhizobium leguminosarum after irradiation. The induced variation was tested under greenhouse conditions on the variety JV 3 of broad beans (Vicia faba) in six replications. Induced genetic variabilty was observed for strain, parent and mutant versus parent. Out of 24 irradiated strains, strain 93-32 performed better with a greater number of nodules and higher dry weight of nodules per plant and biological yield. Environment played an important role in the expression of characters observed. High heritability and genetic advance of these traits indicated that the nitrogen fixation ability of Rhizobium can easily be improved by selection.     

Effects of abscisic acid,cytokinins, and light on isoflavonoid phytoalexin accumulation inPhaseolus vulgaris L.

Cotyledons ofPhaseolus vulgaris L. contain small amounts of phaseollin and kievitone. Isolating the cotyledons from the plant does not alter phaseollin levels. Kievitone levels, however, although not affected in light-incubated cotyledons, increased rapidly in dark-incubated cotyledons. Abscisic acid (ABA) at 10^-4 M stimulated the accumulation of phaseollin in excised cotyledons in both light and darkness, whereas benzylaminopurine (BAP) increased these levels only in the light. The kievitone level was influenced by ABA and BAP only in dark-incubated cotyledons, i.e., inhibited at 10^-4 M. When excised cotyledons were treated with mercuric chloride, both phaseollin and kievitone accumulated rapidly in both light and darkness. The effect of ABA on these cotyledons was similar to that on non-treated cotyledons. The results demonstrate that the synthesis of the two phytoalexins is regulated by separate mechanisms and indicate that the phytoalexin composition is dependent on the physiological condition of the cotyledons. ABA and BAP may play a role in the resistance response of the plant.Abbreviations ABA abscisic acid - BAP benzylaminopurine     

Rhizobium meliloti genes required for C4-dicarboxylate transport and symbiotic nitrogen fixation are located on a megaplasmid.

A mutant of Rhizobium meliloti unable to transport C4 dicarboxylates (dct) was isolated after Tn5 mutagenesis. The mutant, 4F6, could not grow on aspartate or the tricarboxylic acid cycle intermediates succinate, fumarate, or malate. It produced symbiotically ineffective nodules on Medicago sativa in which bacteroids appeared normal, but the symbiotic zone was reduced and the plant cells contained numerous starch granules at their peripheries. Cosmids containing the dct region were obtained by selecting those which restored the ability of 4F6 to grow on succinate. The Tn5 insertion in 4F6 was found to be within a 5.9-kilobase (kb) EcoRI fragment common to the complementing cosmids. Site-specific Tn5-mutagenesis revealed dct genes in a segment of DNA about 4 kb in size extending from within the 5.9-kb EcoRI fragment into an adjacent 2.9-kb EcoRI fragment. The 4F6 mutation was found to be in a complementation group in which mutations yielded a Fix- phenotype, whereas other dct mutations in the region resulted in mutants which produced effective nodules in most, although not all, plant tests (partially Fix-). The dct region was found to be located on a megaplasmid known to carry genes required for exopolysaccharide production.     

Introduction of the Escherichia coli gdhA gene into Rhizobium phaseoli: effect on nitrogen fixation.

Rhizobium phaseoli lacks glutamate dehydrogenase (GDH) and assimilates ammonium by the glutamine synthetase-glutamate synthase pathway. A strain of R. phaseoli harboring the Escherichia coli GDH structural gene (gdhA) was constructed. GDH activity was expressed in R. phaseoli in the free-living state and in symbiosis. Nodules with bacteroids that expressed GDH activity had severe impairment of nitrogen fixation. Also, R. phaseoli cells that lost GDH activity and assimilated ammonium by the glutamine synthetase-glutamate synthase pathway preferentially nodulated Phaseolus vulgaris.     

Role of oxygen limitation and nitrate metabolism in the nitrate inhibition of nitrogen fixation by pea

The impact of nitrate (5–15 m M , 2 to 7 days) on nitrogenase activity and nodule-oxygen limitation was investigated in nodulated, 21-day-old plants of a near-isogenic nitrate reductase-deficient pea mutant (A3171) and its wild-type parent ( Pisum sativum L. cv. Juneau). Within 2 days, 10 or 15 m M nitrate, but not 5 m M nitrate, inhibited the apparent nitrogenase activity (measured as in situ hydrogen evolution from nodules of intact plants) of wild-type plants; none of these nitrate levels inhibited the apparent nitrogenase activity of A3171 plants. Nodule-oxygen limitation, measured as the ratio of total nitrogenase activity to potential nitrogenase activity, was increased in both wild-type and A3171 plants by all nitrate treatments. By 3 to 4 days the apparent nitrogenase activity of A3171 and wild-type plants supplied with 5 m M nitrate declined to 53 to 69% of control plants not receiving nitrate. By 6 to 7 days the apparent nitrogenase activity of A3171 plants was similar to the control value whereas that of the wild-type plants continued to decline. From 3 to 7 days, no significant differences in nodule-oxygen limitation were observed between the nitrate (5 m M ) and control treatments. The results are interpreted as evidence for separate mechanisms in the initial (O₂ limitation) and longer-term (nitrate metabolism) effects of nitrate on nitrogen fixation by effectively nodulated pea.     

Rhizobium strain effects on pea: The relation between nitrogen accumulation, phosphoenolpyruvate carboxylase activity in nodules and asparagine in root bleeding sap

Pisum sativum L. cv. Bodil was infected with various strains of Rhizobium leguminosarum (R501, 128c53, B155, 18a or 1044). The Rhizobium genotype influenced the activity of the plant enzyme phosphoenoipyruvate (PEP) carboxylase (EC 4.1.1.31), and the assimilation of fixed N in the root nodules. The specific activity of nodule PEP carboxylase was lowest in the symbioses, which accumulated the least total N (R501 and 128c53). The root bleeding sap of the less effective symbioses contained a lower proportion of asparagine and a higher proportion of glutamine than the more effective symbioses (B155,18a and 1044). The N yield of the symbioses was related neither to the net respiratory CO₂ evolution of the root system nor to the nitrogenase linked nodule respiration. The lower yielding symbioses accumulated a larger proportion of the fixed N in the nodules due to a higher proportion of total dry weight contained in the nodule tissue. However, the concentration of soluble protein in the nodules of the lower-yielding symbioses was lower than that recorded for the higher yileding symbioses. The effect of the Rhizobium strains on N yield was maintained at maturity, and reflected in seed yields.     

Effect of Rhizobium Sp. Inoculation on N2-Fixing and Photosynthetic Activities of Two Cowpea [Vigna unguiculata (L.) Walp.] Genotypes

Time course of symbiotic N₂-fixing and photosynthetic activities during vegetative growth from 30 d after plantation until pod set was measured in the CB5 and 7964 cowpea [Vigna unguiculata (L.) Walp.] genotypes of contrasting senescence traits. At emergence, seedlings were inoculated with a "non-cowpea miscellany" Rhizobium strain generally used to inoculate Cicer arietinum. Maximum N₂-fixing activity occurred in inoculated CB5 and 7964 plants about 54 and 68 d after plantation, respectively. A similar temporal shift of maximum was found for net photosynthetic rate (P _N), confirming a good coordination between the two processes. A higher P _N was found from the first measurements in inoculated plants of both genotypes as compared with uninoculated plants. Apparently, the maximum activity of both N₂-fixation and P _N was timed to occur at a particular stage of plant ontogeny correlating the high N supply with the high N demand by the plant. Rhizobium inoculation did not significantly affect partitioning coefficients of biomass to various plant organs but extended leaf longevity by about 10 d in the CB5 genotype, retarding thus the monocarpic senescence. This revised version was published online in June 2006 with corrections to the Cover Date.     

Bacterial-type ferredoxin genes in the nitrogen fixation regions of the cyanobacterium Anabaena sp. strain PCC 7120 and Rhizobium meliloti.

The nucleotide sequence of a region located downstream of the nifB gene, both in the cyanobacterium Anabaena sp. strain PCC 7120 and in Rhizobium meliloti, has been determined. This region contains a gene (fdxN) whose predicted polypeptide product strongly resembles typical bacterial ferredoxins. Cyanobacteria have not previously been shown to contain bacterial-type ferredoxins. The presence of this gene suggests that nitrogen-fixing cyanobacteria have at least four distinct ferredoxins.     

Localized and internal effect of nitrate on symbiotic dinitrogen fixation

Alfalfa (Medicago sativa L.) is a deeply rooted perennial legume which, under field conditions, may be exposed to varying NO₃^? concentrations with depth. Our objective was to characterize the effect of localized (deep vs shallow) exposure of alfalfa root systems to NO₃^? on symbiotic N₂ fixation and NO₃^?-N uptake. Cuttings of a single alfalfa plant were grown in vertical split root systems in a controlled environment chamber. The split root system was a rigid acrylic tube (5 cm diam. by 60 cm long) filled with silica sand and divided into upper and lower sections at the 30-cm depth by a 5-mm-thick wax layer. Roots penetrated the wax layer, but mixing of nutrient solutions between the sections was prevented. Nodulation was restricted to the upper section. The plants were subjected for 10 days to the following treatments: both sections of the split root system received nutrient solution containing either 0.5, 5.25, or 10 mM NO₃^?; the upper section received 0.5 mM NO₃^? while the lower section received 10 mM NO₃^?; or the upper section received 10 mM NO₃^? while the lower section received 0.5 mM NO₃^?. Increasing supply of NO₃^? in the nutrient solution to both sections resulted in higher NO₃^?-N uptake, lower nodule mass and lower specific nitrogenase activity. Although NO₃^?-N uptake did not differ, plants exposed to 10 mM NO₃^? for 10 days in the upper, nodulated section of the root system had a 20% lower nodule mass than plants exposed to the same NO₃^? concentration in the lower, non-nodulated section of the root system. Specific nitrogenase activity was not different between these two treatments. Therefore, we conclude that: (1) nodule mass was dependent on two factors, the amount of NO₃^?-N taken up and the concentration of NO₃^? within the nodulated root zone; and (2) specific nitrogenase activity was little affected by the concentration of NO₃^? surrounding the nodules, but was strongly inhibited by NO₃^?-N taken up.