C2H2 Reduction Research Articles

Nitrogen Fixation on the Tropical Volcano, La Soufriere (Guadeloupe): I. A Survey of Nitrogen Fixation by Blue-Green Algal Microepiphytes and Lichen Endophytes

Results of a survey of nitrogen-fixing blue-green algal epiand endophytes growing in the cloud-shroud climate on the tropical volcano La Soufriere (Guadeloupe, French West Indies) are reported. Microepiphyllae on Clusia mangle leaves increased with leaf age, and rates of C2H2 reduction were 47 times higher during cloud-shroud conditions than during solar shocks. Phyllosphere rates of C2H2 reduction for Norantea were about 0.76-2.22 7) moles/cm2/hr for the oldest leaves, and epicaulous rates of C2H2 reduction for Tibouchina were the highest measured in plants tested. The Hapalosiphon flexuosus rate of C2H2 reduction was 1.26 7) moles/ gdw/hr at pH 4.5 on Sphagnum. Endophytic blue-green algae in the lichens Stereocaulon ramulosum and Cora pavonia were 37.6 and 3966 -j moles/gdw/hr, respectively. THE VEGETATION NEAR MOUNTAIN SUMMITS in the Lesser Antilles is stunted, and the climate is wetter and colder than at lower elevations. In the Greater Antilles, similar plant associations occur at lower altitudes. The dwarf plant associations occurring at high elevations (approximately 1000 m) have been termed dwarf forests, cloud forests, and mossy forests (Holdridge 1967, Howard 1970, Ewell & Whitmore 1973). The predominant aspect of climate is an almost permanent layer of cumulus clouds formed by upsweeping moist air (Odum et al. 1970). Research results reported here were collected on the tropical volcano La Soufriere located in the Parc Naturel on Guadeloupe (16?40'N, 60?10'W), Lesser Antilles, French West Indies. La Soufriere erupted in the years 1590, 1635, 1797, 1836-1837, 1892, and 1976-1977 (Howard et al. 1980). Studies of the vegetation by Henri Stehle (1935, 1979) described pre-eruption plant associations on La Soufriere. The ecotone at 1100 m, between the tropical rain forest below and the stunted vegetation above, is composed of the shrub C. mangle, which encircles the volcano. Above this Clusia ecotone, termed Clusietum guadelupense by Stehle (zone, La foret rabougrie), are cliff faces dominated by Sphagnum and bromeliads (zone, Les savanes d'altitude). The windblown summit (1456 m) has a flora of low-growing mosses, ferns, club mosses, and bromeliads. The flora has been described in detail by Stehle (1935, 1979), Howard et al. (1980), and Sastre (1978). New radial fractures opened on the southeast flank of the dome of La Soufriere in 1956 and reopened in July 1976, emitting H2S, mud, and boulders. Activity continued until 1 March 1977 (Howard et al. 1980). This eruption divided the pre-eruptive flora into discreet posteruption zones (Fig. 1). The south and southwest portions were destroyed, and the southeastern Col de l'Echelle, after receiving phreatic material, was a desert (Howard et al. 1980). The vegetation of the western flank was somewhat damaged by H2S emissions, but the northern flank was untouched (Sastre et al. 1983). Recovery of vegetation after the eruption has been described by Sastre (1978), Sastre et al. (1983), Blanchard et al. (1984), and Blanchard (1985). The cloud-shroud regime (sensu Odum et al. 1970) above 1100 m on La Soufriere is characterized by nearly constant cloud cover, low photon flux densities between 90 and 585 ,uEm2/sec PAR (photosynthetic active radiation, 400-700 nm), heavy rainfall (10 m/yr) and cool temperatures (17?C). This zone may begin below 1000 m as in Puerto Rico, or above 2000 m as in New Guinea (Leigh 1975). The prevalent cloud-shroud climate of La Soufriere was sporadically punctuated by events of intense insolation. Between 18 July 1984 and 20 May 1985, there were 12 completely clear days and 13 partly clear days (for 2-6 hr). These rare insolation shocks had photon flux densities from 1620 to 2070 ,uEm2/sec PAR and air temperatures of approximately 24?C, with higher temperatures inside sunlit plants (pers. obs.). Persistent fog cover lowers photon flux density, which in turn lowers leaf and air temperatures. Low water vapor deficits may depress both growth in some species and promote colonization by the poikilohydric algae, lichens, and mosses (Weaver et al. 1973, Grubb 1977, LaBastille I Received 27 October 1985, revision accepted 18 June 1986. 194 BIOTROPICA 19(3): 194-1-99 1987 This content downloaded from 157.55.39.112 on Wed, 07 Sep 2016 04:59:22 UTC All use subject to http://about.jstor.org/terms

Variation inRhizobium leguminosarum response to short term application of NH4NO3 to nodulatedPisum sativum L.

This study was conducted to determine the effect of short term application of NH4NO3 on nodule function and to determine whether the rhizobial isolate used was a significant factor in this effect. Pea plants were inoculated with 10 differentRhizobium leguminosarum isolates and grown for 3 weeks in N-free medium before addition of 0, 1, 2 or 5 mM NH4NO3 for 2 to 7 days. Acetylene reduction and leghemoglobin content decreased with increasing exposure time to NH4NO3 and with increasing concentration of NH4NO3. NH4+ and NO3− depletion from the nutrient medium were assayed in plants exposed to 5 mM NH4NO3 and mean uptake rates were similar for each ion. There were significant differences among isolates in the rate of decrease of C2H2 reduction with increasing NH4NO3 concentration (C2H2 reduction responsiveness to NH4NO3) 4 and 7 days after addition of NH4NO3 but no differences after 2 days of exposure to NH4NO3. There were significant differences among isolates in NH4+ depletion from the nutrient medium but these differences were not correlated with the differences observed in C2H2 reduction. Ranking of the isolates for C2H2 reduction responsiveness to NH4NO3 applied to plants with nodules was different from that obtained when NH4NO3 was applied at seeding. Isolates with varying sensitivity to NH4NO3 may be useful tools for determining the mechanisms responsible for inhibition of symbiotic N2 fixation by combined nitrogen.

Sensitivity of selected Frankia isolates from Casuarina, Allocasuarina and North American host plants to sodium chloride

Three experiments examined the effects of NaCl concentrations 0 to 500 mM on the growth of isolates of Frankia from Casuarinaceae and selected North American host plants. Four Casuarina isolates grew well in defined medium (pyruvate‐BAP) but not in a yeast extract medium. Conversely the non‐Casuarina isolates preferred the yeast‐extract medium, although two of them grew in the defined medium. When grown in their preferred medium, the Casuarina isolates were little affected by NaCl concentrations up to 200 mM but did not grow at 500 mM. The non‐Casuarina isolates, with the exception of an isolate from Purshia tridentata. were severely affected above 50 mM NaCl.Nitrogenase activity (C2H2 reduction) by the non‐Casuarina isolates could not be detected in low‐N medium although protein determinations indicated that a low level of nitrogen fixation had occurred. All four Casuarina isolates showed nitrogenase activity in culture, up to 200 mM NaCl, although at that concentration of NaCl, growth was affected more than that of cultures in N‐supplemented medium. All four strains showed a marked increase in nitrogenase activity up to 72 h after the addition of C2H2, with the magnitude of the effect and their subsequent behaviour being strain dependent.The results indicate that the isolates of Frankia from Casuarina and Allocasuarina, and that from Purshia tridentata, are more tolerant of NaCl than isolates from species not normally growing under sodic conditions. They provide optimism that these strains could successfully establish in saline soils if introduced with species of host plants tolerant to these soils.

The acetylene reduction assay inactivates root nodule uptake hydrogenase in some actinorhizal plants

Actinorhizal nodules do not usually evolve H2 due to the action of an uptake hydrogenase. We have found that nodules of several Frankia symbioses evolved large amounts of H2 gas when returned to air following exposure to 10 kPa C2HT2 during an acetylene reduction assay. Increased H2 evolution in air persisted for several days when intact root systems of Alnus incana (L.) Moench (inoculated with Frankia UGL 011101) were treated with 10 kPa C.H2 for 1 h. Full recovery of uptake hydrogenase activity required 4 to 8 days. Studies with crude homogenates of nodules of the same plants showed that hydrogenase (measured amperometrically with phenazine metho‐sulfate as electron acceptor) was directly affected, since activity in treated nodules was only 10% of that in untreated nodules. A survey of actinorhizal symbioses revealed variation in the effect of an acetylene reduction assay on hydrogen metabolism. Nodules of three species, including Alnus rubra Bong, inoculated with Frankia HFPArD. showed complete inactivation of hydrogenase. H2 evolution in air was 25% of the C2H2 reduction rate and H, evolution in Ar/O2 was equal to the QH2 reduction rate. Two symbioses, Ceanothus americanus L. (soil inoculant) and Batista glomerata Baill. (soil inoculant) showed no change following an acetylene reduction assay. A third group of symbioses showed an intermediate response.

Nitrate Utilization and Dinitrogen Fixation (Acetylene Reduction) by Nitrate Reductase‐Defecient Mutants of Pea1

Three NO−3 reductase pea (Pisum sativum L.) mutants and the ‘Juneau’ control were evaluated for the ability to absorb and accumulate NO−3 from solution, and for growth and N2 fixation (C2H2 reduction) in the presence and absence of NO−3. In short‐term (20 h) hydroponic studies, the mutants absorbed NO−3 at rates similar to Juneau, but accumulated more NO−3 than Juneau in both shoots and roots. Seedlings of Juneau assimilated 63%, while seedlings of mutants assimilated <35% of absorbed NO−3. In long‐term studies with plants grown in vermiculite in the presence of 15 mM NO−3, dry weights of mutants A300 and A317 were <50% of Juneau. However, growth of mutant A334, which had only 6 to 8% of the NO−3 reductase activity of Juneau, was equal to the control. Growth of mutant A300 was not as rapid as the other genotypes when N2 fixation or NH+4 was the only N source. All three mutants and Juneau exhibited good levels of C2H2 reduction activity and growth in the absence of mineral N. Also, the NO−3 reductase mutations did not lessen the inhibition of N2 fixation by NO−3 and the cause of the inhibition remains unclear.

Cultivar and Rhizobium strain effect on nitrogen fixation and transport inPhaseolus vulgaris L.

The effects of Rhizobium strain and its interaction with plant cultivar were examined in glasshouse-grownPhaseolus vulgaris in two experiments where the physiological attributes defining the symbiotic efficiency were determined. Strains of Rhizobium significantly affected nodulation, rates of N accumulation, partitioning of N within the mature shoot and remobilizaton of the N stored in the vegetative organs to the seeds. The most efficient symbiosis (strain CO5 with Negro Argel), in comparison with the least efficient symbiosis (strain 127 K-17 with Venezuela-350) showed higher rates of C2H2 reduction from flowering to mid pod fill stage, evolved less hydrogen from nodules and showed higher rates of N transport as well as higher percentages of ureide-N in the xylem sap. At maturity, the best cultivar/strain association exceeded the total N accumulated in the seed and the harvest index of the poorest symbiosis in 88% and 20%, respectively. The other symbiotic combinations were intermediate in all characteristics. Nitrogen accumulation in plant shoot showed highly significant correlation with acetylene reduction rates, nodule relative efficiency, total N transport in the xylem sap and percentage of N transported as ureides.

Nonsymbiotic Dinitrogen Fixation in No-till and Conventional Wheat-fallow Systems1

The objective of this study was to determine if differences in nonsymbiotic N, fixation could account for the positive.N budgets in the plant-soil system of no-till soils as compared with balanced or negative budgets under conventional tillage conditions (stubble mulch and plow) in a winter wheat (Triticum aestivum L.) crop-fallow rotation. This study was conducted on a Duroc silt loam (fine silty, mixed, mesic, Pachic Haplustolls) in western Nebraska from May 1982 to September 1983. Nonsymbiotic N2 fixation was estimated using the C2H2 reduction assay on intact soil cores from plow, stubble mulch, no-till, and native sod management practices. Nitrogen fixation potentials of no-till soils were twofold greater than those of plowed soils and were positively correlated with higher soil-water contents for reduced tillage. The amount of N, fixed during one cycle of the wheat-fallow rotation, however, as calculated from the C2H2 reduction assay, was 0.33 kg N ha ' yr~', which falls far short of the positive budget of 26 kg N ha~' yr~' found in the no-till soil. Lower incubation temperatures, which simulated soil temperatures of tillage systems in the field, resulted in an even lower fixation than at 25°C. Additional Index Words: N budget, C2H2 reduction, plow, stubble mulch, Triticum aestivum L. Lamb, J.A., J.W. Doran, and G.A. Peterson. 1987. Nonsymbiotic dinitrogen fixation in no-till and conventional wheat-fallow system. Soil Sci. Soc. Am. J. 51:356-361. A STUDY OF CULTIVATION in relation to soil N losses ./A. from a virgin grassland soil was made at the High Plains Agric. Lab., Sidney, NE (Lamb et al., 1985). A N budget for the soil and plant system was made after 12 yr of cultivation under three different tillage systems. Results revealed that the no-till system had a positive N budget while the plow and stubble mulch budgets were balanced. The objective of this study was 1 Contribution of the Dep. of Agronomy, Univ. of Nebraska-Lincoln. Published as Paper no. 8141, J. Series, Nebr. Agric. Exp. Stn., Lincoln, NE 68583. The research was conducted under Project no. 12-73. Received 6 May 1986. 2 Former Graduate Assistant now Assistant Professor, Northwest Exp. Stn., Univ. of Minnesota, Crookston, MN 56716; Soil Scientist, USDA-ARS, Dep. of Agronomy, Univ. Nebraska-Lincoln; and Professor, Dep. of Agronomy, Colorado State Univ. Fort Collins, respectively. to determine if nonsymbiotic N2 fixation potential would account for the additional N in the no-till system. Moore (1966) reported that many organisms—Azotobacter, Clostridium, other aerobic and anaerobic bacteria, and cyanobacteria—are capable of fixing atmospheric N2 under field conditions. Pederson et al. (1978) reported that only one of 109 western Nebraska soils cropped to winter wheat demonstrated a potential to fix N2. Paul et al. (1971) found that cultivated wheat (Triticum aestivum L.)-fallow soils in Canada fixed only small quantities of N2 nonsymbiotically. The grassland areas they studied only fixed approximately 1 kg N2 ha~' yr~' and were considered systems in equilibrium where N losses and gains were small. Virgin soil fixed a larger amount than cultivated soil subjected to a wide range of cropping conditions. Steyn and Delwiche (1970) studied nonsymbiotic N2 fixation in California soils with the C2H2 reduction technique. They reported N2 fixation values from 2.1 to 4.8 kg ha~' yr^. The lowest N2 fixing soils were in native grassland. Fallowed soil and soil cropped to wheat fixed 3.5 and 40 kg N2 ha~' yr., respectively. All of their measurements were made in the laboratory on disturbed soil cores. Soil moisture and available soluble energy sources were identified as major factors limiting N2 fixation. Jurgensen (1973) concluded that the extent of nonsymbiotic N2 fixation in cultivated soils was small, but long-term N gains may be significant where crop residue was not removed. In a study conducted in the 1979 to 1980 season, Roper (1983) reported a greater amount of nonsymbiotic N2 fixation in a straw-incorporated treatment as compared to incorporation of a treatment where the straw was burned before cultivation. Evidence in the literature (Steyn and Delwiche, 1970; Moore, 1966; Jurgensen, 1973), points to the possibility that a no-till system, where crop residue is left on the soil surface, may have a significant potential for nonsymbiotic N2 fixation. Furthermore, the surface residues under no-till also increase the soil moisture content compared to conventional tillage systems such as stubble mulch and plow. This creates a more favorable environment for N2 fixation. No-till LAMB ET AL.: NONSYMBIOTIC DINITROGEN FIXATION 357 systems could therefore have enough nonsymbiotic N2 fixation to result in positive N budget as compared to N budgets for conventional systems. MATERIALS AND METHODS

Acetylene Reduction by Bacteroids Isolated from Stem Nodules of Sesbania rostrata. Specific Role of Lactate as an Energy-yielding Substrate

Bacteroids isolated from stem nodules of Sesbania were able to reduce C2H2 when glucose or succinate was the energy-yielding substrate. To be efficient, glucose always required low O2 concentrations, both with free O2 and with O2 delivered by leghaemoglobin extracted from French-bean nodules. Lactate detected in the stem nodule cytosol supported C2H2 reduction by bacteroid preparations, but the O2 tensions required for optimal activity were always higher than those defined for glucose, as predicted by the model of Bergersen & Trinchant (J. theor. Biol. 115, 93–102, 1985). In contrast, lactate was only present at very low concentrations in the cytosol of French-bean nodules and was unable to support C2H2 reduction by bacteroids isolated from these nodules. Lactate dehydrogenase (EC 1.1.1.27) extracted from stem nodule bacteroids and free-living cells of Rhizobium sp. (Sesbania) exhibited a higher affinity for lactate than did the enzyme from Rhizobium meliloti, R. leguminosarum biovar phaseoli or Bradyrhizobium and their symbiotic forms. Lactate appears to be a specific energy-yielding substrate supporting N2 fixation by Sesbania bacteroids and could play a major role when fermentative processes take place in the nodules.

Effects of Sulfur Dioxide on Nitrogen Fixation, Carbon Partitioning, and Yield Components in Snapbean

AbstractThe air pollutant SO2 is known to affect plant biochemistry and physiology, although very little is known about its effects on N2‐fixation in legumes. This study sought to determine if N2‐fixation, C partitioning, and plant productivity of snapbean (Phaseolus vulgaris L.) were affected under short‐term, low‐level SO2 exposures. Plants were exposed, 29 d after planting (7 d before anthesis), to 0, 18, and 36 µmol SO2 m−3 for 4 h d−1 for 5 d in a fumigation chamber. On the last day of SO2 treatment, plants were also exposed to 14CO2 to determine changes in C partitioning patterns. At these concentrations, there was no visible damage to plant tissue and no significant changes in dry weight or yield components. Only the 36 µmol SO2 m−3 treatment reduced C2H2 reduction rates, but recovery to near control rates occurred within 24 h after SO2 removal. Leaves of plants treated with 18 µmol SO2 m−3 exported more of their total assimilated 14C than control plants, while those treated with 36 µmol SO2 m−3 retained greater amounts. Retention of 14C at the 36 µmol SO2 m−3 level may account for the inhibition of C2H2‐reduction because of less photosynthate arriving at the root nodules. These data suggest that SO2 levels that do not cause visible injury may interfere with C metabolism and transport in snapbean.

The biota and environment of fumaroles on Mt Melbourne, northern Victoria Land

The aim of this investigation was to provide a general description of the biota and environment of fumarolic ground close to the summit (2733 m) of Mt Melbourne. Heat flow through the ground was examined and analyses made of the physico-chemical properties of the soil. Bryophyte vegetation comprisedCampylopus pyriformis (K.F. Schultz) Brid. andCephaloziella exiliflora (Tayl.) Steph. The former is a new antarctic record. These grew as scattered and confluent cushions where surface ground temperatures ranged between 14°–31°C. Algae were epiphytic on bryophytes and formed crusts over most other areas of warm ground. Six species of cyanophytes and five unicellular chlorophytes are described. The majority were mesophilic butMastigocladus laminosus showed a strong preference for soil temperatures >30°C. A loosely lichenised chlorophyte was also encountered. The only soil fauna was a testate amoeba,Corythion dibium. Heterotrophic microorganisms included thermotolerant fungi, actinomycetes and bacteria as well as typical mesophilic microflora. Nitrogen fixation, analysed using both C2H2 reduction and15N2 enrichment, occured in bryophyte cushions and surface algal crusts containingM. laminosus and in sub-surface samples lacking cyanophytes. The biota are compared with those found at other thermal localities in Antarctica and the dispersal of propagules to these areas is discussed.

Variation in N2 fixation, N and P contents of mycorrhizalVigna unguiculata in relation to the progressive development of extraradical hyphae ofGlomus mosseae

Vigna unguiculata cv. 58–185 grown in a sterile Dek soil was inoculated withRhizobium sp. orRhizobium sp. plusGlomus mosseae. Response of the host plant to the treatments was estimated by periodic measurements of shoot and nodule dry weights, N2 fixation (C2H2 reduction activity) and N and P contents up to the 50th day of the growth cycle. It was only 45 days after planting that shoot dry weight of dually inoculated plants differed significantly from that of plants inoculated withRhizobium sp. alone. Nodule dry weight and N2 fixation of dually inoculated plants were significantly higher than those of plants inoculated withRhizobium sp. alone from day 20 after planting, but there was no significant difference in N content (%). During the first 20 days, shoot P content (%) of both sets of plants decreased progressively, P content of dually inoculated plants being lower than that of the others. Later, P content of dually inoculated plants increased rapidly whereas P content of the other plants remained constant. Increase in nodule dry weight, N2 fixation and P content of dually inoculated plants corresponded to the onset of the development of the extra-radical hyphae ofGlomus mosseae. In the rhizosphere.

Physiological and Genetic Characterization of a Diazotrophic Pseudomonad

A soil isolate, 4B, which had been previously assigned to the genus Pseudomonas and shown to be capable of reducing C2H2 with simple phenolic compounds as sole carbon source, was further characterized in comparison with two other diazotrophs which were identified as pseudomonads. The DNA base composition of 4B was 60.2 mol% G+C. Plasmid DNA was not detected in alkaline SDS lysates of 4B by agarose gel electrophoresis. Comparable maximum C2H2 reduction activities in 4B were observed under microaerobic conditions (pO2 about 0.003 atm) with either 28 mM-glucose or 5 mM-protocatechuate as carbon source. N2 fixation was confirmed by the cellular incorporation of 15N2 in cultures of 4B grown in N-free medium. Extensive biochemical tests, including the carbon utilization pattern, demonstrated that 4B was closely related to Pseudomonas delafieldii (ATCC 17505) although the latter did not fix N2. 4B had metabolic patterns different from the two other strains reported to be diazotrophic pseudomonads; all three contained DNA homologous to the nif HDK genes of Klebsiella pneumoniae M5A1.

Autotrophic Growth and Nitrogen Fixation in Derxia gummosa

Summary: Initial attempts to grow N2-fixing Derxia gummosa autotrophically on H2, CO2, O2 and N2 in a closed system yielded variable results. Poor growth was found to be due to rapid O2 depletion and the requirement for an agar surface. In a closed system, C2H2 reduction assays could not be carried out due to complete consumption of H2. Hence a flow-through culturing technique was developed to supply gases at a constant partial pressure and to perform C2H2 reduction assays in a continuous flow system. Hydrogenase of autotrophic D. gummosa was not inhibited by C2H2, even at 0.5 atm, and the K m of hydrogenase for H2 was approximately 0.15 atm. The effects of O2 and H2 on C2H2 reduction were examined, using the flow-through assay system. The rate of C2H2 reduction decreased below 0.074 atm H2, suggesting that ATP and reductant supply were limiting the nitrogenase activity.

Regulation of nitrogen fixation by nitrite and glutamine in Derxia gummosa

Nitrogenase activity of cells of Derxia gummosa (30 h growth in cultures without combined nitrogen) was not inhibited on adding nitrate. However, on adding either azaserine or methionine sulfoximine (MSX) with nitrate to these cells, nitrogenase (C2H2 reduction) was inhibited because nitrite accumulated in the reaction mixtures. Nitrite inhibition of the in vivo C2H2 reduction had a Ki value of 16 μM. Both ammonia and glutamine inhibited N2 fixation (C2H2 reduction) in intact cells and in those treated with toluene. This inhibition by ammonia was relieved by methionine sulfoximine but not by glutamine. Azaserine enhanced the inhibition of nitrogenase produced by either ammonia or glutamine, since these treatments resulted in an accumulation of glutamine.

Regulation of nitrogen fixation by nitrite and glutamine inDerxia gummosa

Nitrogenase activity of cells of Derxia gummosa (30 h growth in cultures without combined nitrogen) was not inhibited on adding nitrate. However, on adding either azaserine or methionine sulfoximine (MSX) with nitrate to these cells, nitrogenase (C2H2 reduction) was inhibited because nitrite accumulated in the reaction mixtures. Nitrite inhibition of the in vivo C2H2 reduction had a Ki value of 16 μM. Both ammonia and glutamine inhibited N2 fixation (C2H2 reduction) in intact cells and in those treated with toluene. This inhibition by ammonia was relieved by methionine sulfoximine but not by glutamine. Azaserine enhanced the inhibition of nitrogenase produced by either ammonia or glutamine, since these treatments resulted in an accumulation of glutamine.

Effect of the herbicide glyphosate on nitrification, denitrification, and acetylene reduction in soil

The effect of glyphosate on N2 fixation, denitrification, and nitrification in an agricultural soil was investigated. Effects of the pure herbicide and commercial formulation, Roundup+ (Monsanto Company), were compared in soil under aerobic and anaerobic conditions. Anaerobic C2H2 reduction was inhibited by high herbicide levels. Denitrification in non-amended soil was either unaffected (N2O reduction) or stimulated (NOinf3sup−reduction); in glucose-amended soil, N2O reduction was inhibited and NO3-reduction unaffected by both glyphosate and Roundup. Roundup caused greater stimulation of N2O reduction than pure glyphosate; no other significant formulation effects were observed. Nitrification was inhibited by the two formulations. Ammonium oxidation were both influenced. Pure glyphosate was more inhibitory than Roundup. No toxicity to any of these activities should be seen at recommended field application rates of the herbicide.

Acetylene reduction, H2 evolution and 15N2 fixation in the Alnus incana-Frankia symbiosis

Acetylene reduction, (15)N2 reduction and H2 evolution were measured in root systems of intact plants of grey alder (Alnus incana (L.) Moench) in symbiosis with Frankia. The ratios of C2H2: (15)N2 were compared with C2H2:N2 ratios calculated from C2H2 reduction and H2 evolution, and with C2H2:N2 ratios calculated from accumulated C2H4 production and nitrogen content. It was possible to calculate C2H2:N2 ratios from C2H2 reduction and H2 evolution because this source of Frankia did not show any hydrogenase activity. The ratios obtained using the different methods ranged from 2.72 to 4.42, but these values were not significantly different. It was also shown that enriched (15)N could be detected in the shoot after a 1-h incubation of the root-system. It is concluded that the measurement of H2 evolution in combination with C2H2 reduction represents a nondestructive assay for nitrogen fixation in a Frankia symbiosis which shows no detectable hydrogenase activity.

Symbiotic and Free‐living Denitrification by Bradyrhizobium japonicum

AbstractDenitrification by Bradyrhizobium japonicum (formerly Rhizobium japonicum) was characterized in two strains, USDA‐110 and USDA‐23, to determine the extent of denitrifying enzyme expression during symbiotic growth with soybean [Glycine max (L.) Merr.]. The rhizobia grew in anaerobic batch cultures, depleting NO3‐ and producing N2O in the presence of C2H2. Specific denitrifying activities of resting cells of rhizobia were compared with those of the common soil denitrifier Pseudomonas fluorescens, and were found to range from 14 to 18% of the P. fluorescens activity. Bacteroid specific activities were less than activities of free‐living rhizobial cells, though the ratio depended on the growth stage of the cells. Denitrification was observed in detached whole nodules containing either of the strains, but not in nodules formed by a NO3‐ reductase mutant of B. japonicum 110. Denitrification rates of nodules were unaffected by the presence of external O2, though the rates were limited by the external NO3‐ concentration. Denitrification by intact, nodulated soybean plants under aerobic conditions was observed. In the presence of NO3‐ denitrification and N2 fixation (C2H2 reduction) occurred simultaneously. The estimated ratio of N denitrified to N fixed ranged from 0 to 0.3, depending on assay conditions. The observations suggest that denitrification in soybean nodules in the field can be a continuous process, independent of soil aeration. However, it is likely to be slow relative to N2 fixation under most conditions.

A comparison of inhibition of French‐bean and soybean nitrogen fixation by nitrate, 1% oxygen or direct assimilate deprivation

Inhibition by NO−3 of acetylene reduction in bean (Phaseolus vulgaris L. cv. Contender) and soybean (Glycine max L. cv. Amsoy 71) was measured in parallel with nodule carbohydrate and nitrate metabolism. In bean the onset of inhibition of C2H2 reduction (6 h) coincided with decreased import of assimilates and a lowering of carbohydrate pools (sucrose, glucose and starch). Nitrate reductase (EC 1.6.6.1) activity was induced in all plant organs after 3 h but no nitrite was detected in the nodules. In soybean, nodule carbohydrate concentrations and import of assimilates into the nodules increased markedly between 6 to 24 h after supply of nitrate when the nitrogenase (EC 1.7.99.2) was progressively inhibited. High nitrate reductase activity was observed in the nodules and nitrites accumulated because of insufficient nitrite reductase activity. The nitrate‐induced inhibition of nitrogenase was compared with the inhibition observed with low oxygen around the roots (1% O2) or with direct assimilate deprivation (girdling or decapitation). Soybean and bean appeared equally sensitive to these treatments as regards to acetylene reduction. The results are discussed in relation to the current hypotheses explaining nitrate‐induced inhibition of dinitrogen fixation: assimilate deprivation or nitrite poisoning. Present data are in favour of the first for bean and of the second for soybean.

Aspects of nitrogen fixation and denitrification byAzospirillum

Model experiments were performed to investigate the nitrogen fixation (C2H2 reduction) and denitrification (N2O formation) capabilities ofAzospirillum spp. in association with wheat. Plants and bacteria were grown together for a week and then assayed for activities. This association performed C2H2 reduction or N2O formation, depending on the concentrations of nitrate and oxygen in the vessels. Both activities depended on theAzospirillum strains used. The newly isolatedAzospirillum amazonense strains Y1 and Y6 showed significant C2H2 reduction and low N2O formation in association with wheat under the conditions employed and are possibly useful in practice. A cell-free preparation fromAzospirillum brasilense Sp 7 possessed a cytochrome cd type dissimilatory nitrite reductase.