N2-fixing Ability Research Articles

Field Comparison of Dinitrogen Fixation Determined with Nitrogen‐15‐Depleted and Nitrogen‐15‐Enriched Ammonium Sulfate in Selected Inbred Backcross Lines of Common Bean

The field determination of N2 fixation by 15N‐isotopic methods provides a direct and precise measure of N2 fixation. Since 15N‐enriched fertilizers are expensive, 15N‐depleted (NH4)2SO4 was used as a more economical, readily available alternative to determine N2 fixation in common bean, Phaseolus vulgaris L. The objectives of this study were to determine whether 15N‐depleted (NH4)2SO4 provided reliable estimates of relative differences in N2 fixation among bean lines comparable to those obtained using 15N‐enriched (NH4)2SO4, and whether nine previously selected progeny lines differed from their respective recurrent parents for N2 fixation ability. The nine progeny lines and three parents were grown in two randomized complete block field experiments that were treated identically except that each experiment received only one of the two 15N fertilizers. Lines were harvested at an early reproductive stage (R3) and maturity (R9), partitioned into plant parts, and the 15N and total N yield of the tissue determined. The two 15N fertilizers produced comparable differences among lines at the R9 stage based on significant genotypic rank correlations between the two experiments, and similar fertilizer use efficiency values (% FUE). Three of the five selected progeny lines from population 24 fixed significantly more N2 than their recurrent parent ‘Sanilac’. The four progeny lines from population 21 were not significantly different from or less effective in N2 fixation than their recurrent parent ‘Porrillo Sintetico’, therefore, either previous selection based on indirect measures of N2 fixation was not effective, or the two parents did not differ genetically for N2 fixation. In all progeny and parental lines, seed N2 fixed was positively rank correlated with plant N2 fixed, and the highest N2‐ fixing lines in both populations had more fixed N2 in the seeds than did low N2‐fixing lines.

Recovery of N2 Fixation in Soybean Following Insect-Induced Defoliation

Recovery of N2-fixing ability (measured by C2H2-C2H4 assay) of greenhouse grown soybean following defoliation by soybean looper, Pseudoplusia includens (Walker) (Lepidoptera: Noctuidae), was examined in two experiments. Experiment 1 examined nodulation, N2 fixation, and dry weight of plant parts of ‘Forrest’ soybean at 0, 2, and 4 wk post defoliation (WPD) (70% defoliation). Experiment 2 examined these same variables in ‘Davis’ soybean at 0, 1, 2, 3, 4, and 5 WPD (73%defoliation). In both experiments, leaf area, stem dry weight, and root dry weight of defoliated plants were greatly reduced at 0 WPD; they never recovered to the level of control plants. Although N2-fixing ability of defoliated plants was reduced 4.9-fold in Experiment 1 and 8.7-fold in Experiment 2 at 0 WPD, there was no significant difference in N2-fixing ability of defoliated and control plants in either experiment at 4 WPD. Recovery of N2-fixing ability was due to partial recovery of total nodule dry weight, combined with complete recovery of nodule specific activity.

Nutrient relations in coppiced black cottonwood and red alder

Two-year-old coppice of black cottonwood and red alder, grown in pure culture and in mixture, were compared using terminal twigs and leafless shoots harvested in the winter. Terminal twigs were taken with buds intact; they were about 15 cm long. Leafless shoot samples included all above-ground components. In pure culture, dry weights of the leafless shoots per plant were similar for the two species. In mixture with alder, however, weight of the cottonwood plants was enhanced and that of alder was reduced, but neither response was statistically significant. Nutrient concentration, content per plant, and utilization varied by the plant tissues analyzed, cultural treatment (purevs. mixed), and species. In general, nutrient concentrations were higher in the terminal twigs than in the leafless shoots of both species. Cultural treatment did not significantly affect nutrient concentration in cottonwood twigs or in the leafless shoots of either species. Concentrations of N and Fe were significantly higher and those of Mn were lower in twigs of mixed alder than in twigs of pure alder. Twigs of cottonwood were significantly higher than those of alder in concentration of P and Zn, and lower in N, Mn, and Cu. Compared with alder, cottonwood leafless shoots were significantly higher in concentration of Ca, but lower in N, S, Cu, and Mn. With few exceptions, nutrient content was highest in the shoots of the large plants of mixed cottonwood, intermediate in medium-sized pure cottonwood and pure alder, and lowest in the small mixed alder. Cottonwood was significantly more efficient than alder in use of N, S, and Cu, and less efficient in use of Ca. Some of the differences between cultural treatments and species may be associated directly or indirectly with the N2-fixing ability of red alder. Mixed culture of the two species appears promising because of the increased growth of cottonwood. Planted separately in pure culture, the choice between cottonwood and alder may be determined, in part, by the nutritional status of the soil where plantations are established.

Analysis of the Symbiotic Performance of Bradyrhizobium japonicum USDA 110 and Its Derivative I-110 and Discovery of a New Mannitol-Utilizing, Nitrogen-Fixing USDA 110 Derivative

Previously, Bradyrhizobium japonicum USDA 110 was shown to contain colony morphology variants which differed in nitrogen-fixing ability. Mannitol-utilizing derivatives L1-110 and L2-110 have been shown to be devoid of symbiotic nitrogen fixation ability, and non-mannitol-utilizing derivatives I-110 and S-110 have been shown to be efficient at nitrogen fixation. The objectives of this study were to determine the effect of media carbon sources on the symbiotic N(2)-fixing ability of strain USDA 110 and to compare the effectiveness of strain USDA 110 and derivative I-110. Based on acetylene reduction activity and the nitrogen content of 41-day-old soybean plants, neither derivative I-110 nor cultures of USDA 110 grown in media favoring non-mannitol-using derivatives had symbiotic nitrogen fixation that was statistically superior to that of cultures of USDA 110 grown in media favoring mannitol-using derivatives. In another experiment 200 individual nodules formed by strain USDA 110 grown in yeast extract gluconate were screened for colony morphology of occupying variant(s) and acetylene reduction activity. Nodules occupied by mannitol-using derivatives (large colony type on 0.1% yeast extract-0.05% K(2)HPO(4)-0.08% MgSO(4) . 7H(2)O-0.02% NaCl-0.001% FeCl(3) . 6H(2)O [pH 6.7] with 1% mannitol [YEM] plates) had a mean acetylene reduction activity equal to that of nodules occupied by non-mannitol-using derivatives (small colony type on YEM plates). A total of 20 large colonial derivatives and 10 small colonial derivatives (I-110-like) were isolated and purified by repeated culture in YEM and YEG (same as YEM except 1% gluconate instead of 1% mannitol) media, respectively, followed by dilution in solutions containing 0.05% Tween 40. After 25 days of growth, soybean plants inoculated with the large colony isolates had mean whole-plant acetylene reduction activity, whole-plant dry weight, and whole-plant nitrogen contents equal to or better than those of plants inoculated with either the small colony isolates (I-110-like) or the I-110 (non-mannitol-using) derivative. Hence, the existence of a mannitol-utilizing derivative that fixes nitrogen in a culture of strain USDA 110 obtained from the U.S. Department of Agriculture, Beltsville, Md., was established. This new USDA 110 derivative was designated as MN-110 because it was a mannitol-utilizing nitrogen-fixing USDA 110 derivative. This derivative was morphologically indistinguishable from the non-nitrogen-fixing derivative L2-110 found in cultures obtained earlier from the U.S. Department of Agriculture, Beltsville. DNA-DNA homology and restriction enzyme analyses indicated that MN-110 is genetically related to other USDA 110 derivatives that have been characterized previously.

Bidirectional Selection for Nitrogenase Activity and Shoot Dry Weight Among Late Generation Progenies of a Virginia x Spanish Peanut Cross1

Abstract Improvement of the host contribution to nitrogen fixation has been proposed as a method of increasing nitrogen fixation. Significant variability and generally high broad-sense heritability estimates (.60 ± .27 to .82 ± .26 for nitrogenase activity and .53 ± .29 to .85 ± .26 for shoot dry weight) have been reported for F2-derived families from a cross between the Virginia (Arachis hypogaea L. ssp. hypogaea var. hypogaea) cultivar NC 6 and the Spanish (ssp. fastigiata Waldron vulgaris Harz.) breeding line 922, indicating selection for increased nigtogen fixation should be effective in this population. Lines from this population were chosen randomly from F2-derived families selected for high and low nitrogenase activity and high and low shoot dry weight after evaluation at three dates and two locations in each of 2 years (F5 and F6 generations). This study's objectives were to evaluate the N2-fixing ability of the selected lines and to evaluate the association between plant growth habit and N2 fixation. Twenty-four lines in each of the four selection groups and the parents, NC 6 and 922, were evaluated at two sampling dates and two locations. Mean nitrogenase activity of lines selected for increased nitrogenase activity was significantly greater than the mean of the lines selected for low nitrogenase activity. Improved nitrogenase activity was associated with increased fruit weight. The fruit weight mean of the group selected for increased fruit weight. The fruit weight mean of the group selected for increased nitrogenase activity was 39% greater than the mean of the group selected for low nitrogenase activity. Mean shoot dry weight of lines selected for increased shoot dry weight was significantly greater than the mean of the lines selected for low shoot dry weight; however, the fruit weight means of these two groups did not differ. It was hypothesized that selection for increased N2 fixation in a population derived from a cross between Virginia and Spanish types would eliminate genotypes with Spanish growth habit. Groups selected for high nitrogenase activity and for high shoot dry weight had longer and wider leaflets, longer cotyledonary laterals and greater main stem height than did their respective low selection groups. However, these traits chosen to characterize plant growth habit were inadequate in discriminating parental growth habits. Consequently, the data neither substantiated nor refuted the hypothesis.

TEMPERATURE-SENSITIVE NODULATION AND N2 FIXATION OF Rhizobium leguminosarum BIOVAR Phaseoli STRAINS

Two strains of Rhizobium leguminosarum biovar phaseoli, both previously Nod+Fix+ and efficient in N2 fixation, now have altered symbiotic properties presumably through spontaneous mutation. Rhizobium leguminosarum biovar phaseoli strain RCR 3605 was Nod−Fix− in a cool growth temperature regime (14–23 °C) and Nod+Fix− in a warm regime (17–23 °C) on seven of nine bean cultivars studied. Rhizobium leguminosarum biovar phaseoli strain CIAT 57 was Nod+Fix− in both temperature regimes. CIAT 57 and RCR 3605 are synonyms for strains derived from the same parent strain but housed in different culture collections. The nodulation deficiency in CIAT 57 is presumably genetically predetermined in the bacterium rather than in the plant because no Nod+Fix+ phenotype was identified on nine bean cultivars tested. A control R. leguminosarum biovar phaseoli strain, RCR 3644, was efficient on all cultivars. In the warm temperature regime, the Nod+Fix− phenotype was observed with strain RCR 3605 only on some cultivars, suggesting that the host plant genome played a role in this ineffective symbiosis. It is likely that deleterious genetic mutations during prolonged storage may be altering the N2-fixing ability of these rhizobial strains and that commercial inoculants must be continually monitored for symbiotic performance of strains with a variety of host plant cultivars. Key words: Field bean, Rhizobium leguminosarum biovar phaseoli, N2 fixation

Further Studies on Free-living Nitrogen-Fixing Microorganisms, Other than Azotobacter, Isolated from Egyptian Soils and Waters

Summary Re-examination of the twenty organisms previously isolated by Abd-El-Malek et al. (1979), using the acetylene reduction test, showed that only one isolate, identified as Bacillus sp., had the ability for reducing acetylene. Screening several hundred fresh isolates from Egyptian soil and water samples for N2-fixing ability revealed that 115 organisms were capable of repeated growth on N-deficient medium, showing nitrogen gains by the Kjeldahl analysis. Only five isolates were able to reduce acetylene under aerobic conditions. Addition of 0.01 % Difco yeast extract was essential for growth stimulation and increasing the amount of nitrogen gains by these isolates. Amounts of nitrogen fixed ranged from 4.8 to 10.5 ppm and from 9.6 to 26.7 ppm in the N-deficient medium and the N-poor medium, respectively. On the basis of morphological, cultural, and biochemical characteristics, the N2-fixing cultures were identified as two strains of Bacillus (S15/b and S77/g), two strains of Pseudomonas (S44/b and W70/n), one strain of Klebsiella (S145/m), and one strain of Micrococcus (S171/h).

15N2 incorporation and acetylene reduction by Azospirillum isolated from rice roots and soils

Nitrogen fixation by strains of Azospirillum isolated from several rice soils and rice cultivars was investigated by15N2 incorporation and C2H2 reduction. C2H2 reducing ability markedly varied among the strains obtained from soils differing widely in their physico-chemical properties. Large variations in15N2 incorporation by Azospirillum isolated from the roots of several rice cultivars were also noticed. The present study reveals that rice cultivars harbour Azospirillum with differential N2-fixing ability and that plant genotype is of importance for optimal associations.

Dinitrogen fixation in pea beans (Phaseolus vulgaris) as affected by growth stage and temperature regime

Tolerance of beans (Phaseolus vulgaris L.) to low temperatures is essential for growth under Canadian growing conditions. At air temperatures typical of a southern Alberta growing season (10–23 °C), 'Aurora' fixed 433 mg N per plant, more than 'Sanilac' and 'Comtesse,' which averaged 307 mg N per plant, and 'Kentwood,' which fixed the least: 214 mg N per plant. At a higher temperature regime (15–23 °C), typical of a southern Ontario growing season, 'Aurora' again fixed the most N2 (456 mg N per plant), while the other three cultivars fixed an average of 370 mg N per plant. The amount of N2 fixed increased as the time to reach maximum acetylene (C2H2) reducing activity lengthened. This suggested that it may be advantageous to select beans that have a longer vegetative stage to prolong the time of active N2 fixation within the limits of acceptable season length to ensure maturity. 'Aurora' was not affected by the lower temperature regime, and although these temperatures delayed the initiation of N2 fixation by 21 days, 'Aurora' compensated by producing 26% greater nodule dry weight and 40% higher maximum C2H2-reducing activity than that observed at the higher temperature regime. Growth and N2 fixation of the other three cultivars were adversely affected by the lower temperature regime, not because of lower nodule numbers or mass but because of lower nodule activity. The good root growth of 'Aurora' at low temperatures may explain its superiority for N2 fixation at lower temperatures. C2H2 reduction and nodule dry weight were acceptable indicators of N2 fixation only during early plant growth when all nodules were new and uniformly active. The ideal time to select the bean cultivars for N2-fixing ability was just before the peak of maximum C2H2-reducing activity and corresponded to the stage from buds-near-breaking to anthesis for both temperature regimes. Molar ratios of C2H2 reduced to N2 fixed suggested that the relative efficiency of N2 fixation varied with temperature and growth stage but not with bean cultivar.

Reduction of cyclopropene as criterion of active-site homology between nitrogenase and its Fe–Mo cofactor

THE nitrogenase Fe–Mo cofactor (FeMo-co)1 is usually characterised by its unique ability to restore N2-fixing ability to extracts of certain nitrogenase-less mutants1,2 and by its spectral properties, some of which can be correlated with those of the FeMo protein itself3. The role of FeMo-co in nitrogenase function is not yet clear. Recently, it was reported that uncomplemented FeMo-co catalysed BH−4 reduction of C2H2 to C2H4 in a CO-inhibited, non-ATP-dependent process4. This was taken as evidence that FeMo-co is the active site for N2 reduction and binding in nitrogenase, although conversion of N2 to NH3 by cofactor-BH−4 mixtures was not observed4. C2H2 is readily reduced by many homogeneous and heterogeneous catalysts and can give only a single 2 e− reduction product, urging caution in exclusive reliance upon it as a model substrate5,6. The observation that nitrogenase catalyses reduction of cyclopropene to both propene and cyclopropane in a ∼2:1 ratio in vitro7 and also in vivo8 suggests a more demanding criterion8 for evaluation of FeMo-co catalysis, one which still avoids the difficult energetic requirements for N2 activation and reduction. This new criterion is strengthened by the fact that cyclopropene competitively inhibits N2 (ref. 9). We find that incubation of FeMo-co with a suitable ‘apoprotein’ extract yields a reconstituted FeMo-protein activity for reduction of cyclopropene to propene and cyclopropane. FeMo-co alone, however, only catalyses cyclopropane formation in the conditions described4 for C2H2 reduction. This result and other considerations suggest that active site homology between FeMo-co and nitrogenase has not yet been fully demonstrated on a function basis.

Methods for Growing Spirillum lipoferum and for Counting It in Pure Culture and in Association with Plants

Methods are described for growing Spirillum lipoferum in quantities sufficient to serve as inoculant in field trials of its associative N(2)-fixing ability with higher plants and as a source of cells for the preparation of nitrogenase, cytochromes, respiratory enzymes, etc. A heavy inoculum of S. lipoferum grown on NH(4) was transferred to a medium of minimal nitrogen content, and initial rapid growth at the expense of residual combined nitrogen was replaced later by slower growth on N(2). Conversion to N(2) fixation was prompt upon exhaustion of fixed nitrogen; growth on N(2) was most rapid at a pO(2) of 0.005 to 0.007 atm. Numbers of S. lipoferum can be estimated by diluting soil, crushed roots, or other material, and inoculating diluted samples into a stagnant semisolid medium. Development of a characteristic subsurface layer of organisms and demonstration the these organisms can reduce C(2)H(2) are presumptive evidence that they are S. lipoferum. With most-probable-number tables the observations can be converted to numbers of S. lipoferum in the samples. The most-probable-number method indicated that numbers of S. lipoferum may increase 100-fold or more in roots of maize removed from the plant and incubated for 24 h at 30 degrees C at a pO(2) initially adjusted to 0.01 atm.