Effects of oxygen partial pressure and combined nitrogen on N2-fixation (C2H2) associated withZea mays and other gramineous species

Summary The objectives of this investigation were to determine the effects of oxygen partial pressure (pO₂) and combined nitrogen (NH ₄ ⁺ ) on rates of acetylene reduction (AR) associated with roots of intact corn, sorghum, and pearl millet plants. Soil-grown plants were carefully removed from soil and incubated hydroponically with the root system enclosed in a plastic cylinder; the tops were left exposed to ambient conditions. Oxygen concentrations around the root systems were controlled by sparging the nutrient solution with known quantities of O₂ in N₂. Ammonium nitrogen was added to the nutrient solution following establishment of AR rates to determine its effect on rates of N₂-fixation (AR). Substantial AR rates (0.1–1.5 mol C₂H₄ g dry wt^–1 h^–1) were associated with roots exposed to 0–2% O₂ (v/v) (0.0–2.02 kPa) in N₂ following at 12–24 h period of exposure to the reduced oxygen tension. Root systems exposed to air failed to demonstrate AR while those exposed to 100% N₂ showed lower activity than those at reduced pO₂ values. Addition of NH ₄ ⁺ (10–20 g N ml^–1 of nutrient solution) reduced AR by 75–90% within 24 h after addition. Oxygen uptake by roots exposed to low pO₂ was substantially reduced.     

Effect of seed inoculation,mycorrhizal infection and organic amendment on wheat growth

Summary The effect of seed inoculation withAzotobacter spp. orAzospirillum spp., and garbage amendment (0.5%), on the growth of wheat was studied in a field experiment under sub-tropical conditions. Two levels of N fertilizer were applied, the usual field rate (150 kg N ha^–1) and half this amount. Tillering of plants, dry matter contents and nitrogenase activity were determined 30, 60 and 90 days after sowing. At the end of the experimental period, spore numbers and percentage of mycorrhizal infection were observed in the rhizosphere and root systems of plants. Straw and grain yields were also determined. The results of this study showed that seed inoculation and/or organic amendment stimulated plant growth, nitrogenase activity and mycorrhizal infection. This was more noticeable withAzotobacter than withAzospirillum. Inoculation withAzotobacter together with 1/2 N dose and organic amendment was the most effective application (19.75 and 10.70 t ha^–1 were recorded for straw and grain yield, respectively).     

Nitrogen fixation and respiration by root nodules ofAlnus rubra Bong.: Effects of temperature and oxygen concentration

Summary Using a root nodule cuvette and a continuous flow gas exchange system, we simultaneously measured the rates of carbon dioxide evolution, oxygen uptake and acetylene reduction by nodules ofAlnus rubra. This system allowed us to measure the respiration rates of single nodules and to determine the effects of oxygen concentration and temperature on the energy cost of nitrogen fixation. Energy cost was virtually unchanged (2.8–3.5 moles of carbon dioxide or oxygen per mole of ethylene) from 16 to 26°C (pO₂=20 kPa) while respiration and nitrogenase activity were highly temperature dependent. At temperatures below 16°C, nitrogenase activity decreased more than did respiration and as a result, energy cost rose sharply. Acetylene reduction ceased below 8°C. Inhibition of nitrogenase activity at low temperatures was rapidly reversed upon return to higher temperatures. At high temperatures (above 30°C) nitrogenase activity declined irreversibly, while respiration and energy cost increased.Energy cost was nearly unchanged at oxygen partial pressures of 5 to 20 kPa (temperature of 20°C). Respiration and nitrogenase activity were strongly correlated with oxygen tension. Below 5 kPa, acetylene reduction and oxygen uptake decreased sharply while production of carbon dioxide increased, indicating fermentation. Fermentation alone was unable to support nitrogenase activity. Acetylene reduction was independent of oxygen concentration from 15 to 30 kPa. Nitrogenase activity decreased and energy cost rose above 30 kPa until nearly complete inactivation of nitrogenase at 70–80 kPa. Activity declined gradually, such that acetylene reduction at a constant oxygen concentration was stable, but showed further inactivation when oxygen concentration was once again increased. Alder nodules appear to consist of a large number of compartments that differ in the degree to which nitrogenase is protected from excess oxygen.Supported by United States Department of Agriculture Grant 78-59-2252-0-1-005-1     

Response of the Endophytic Diazotroph Gluconacetobacter diazotrophicus on Solid Media to Changes in Atmospheric Partial O2 Pressure

Gluconacetobacter diazotrophicus is an N₂-fixing endophyte isolated from sugarcane. G. diazotrophicus was grown on solid medium at atmospheric partial O₂ pressures (pO₂) of 10, 20, and 30 kPa for 5 to 6 days. Using a flowthrough gas exchange system, nitrogenase activity and respiration rate were then measured at a range of atmospheric pO₂ (5 to 60 kPa). Nitrogenase activity was measured by H₂ evolution in N₂-O₂ and in Ar-O₂, and respiration rate was measured by CO₂ evolution in N₂-O₂. To validate the use of H₂ production as an assay for nitrogenase activity, a non-N₂-fixing (Nif⁻) mutant of G. diazotrophicus was tested and found to have a low rate of uptake hydrogenase (Hup⁺) activity (0.016± 0.009 μmol of H₂ 10¹⁰ cells⁻¹ h⁻¹) when incubated in an atmosphere enriched in H₂. However, Hup⁺ activity was not detectable under the normal assay conditions used in our experiments. G. diazotrophicus fixed nitrogen at all atmospheric pO₂ tested. However, when the assay atmospheric pO₂ was below the level at which the colonies had been grown, nitrogenase activity was decreased. Optimal atmospheric pO₂ for nitrogenase activity was 0 to 20 kPa above the pO₂ at which the bacteria had been grown. As atmospheric pO₂ was increased in 10-kPa steps to the highest levels (40 to 60 kPa), nitrogenase activity decreased in a stepwise manner. Despite the decrease in nitrogenase activity as atmospheric pO₂ was increased, respiration rate increased marginally. A large single-step increase in atmospheric pO₂ from 20 to 60 kPa caused a rapid 84% decrease in nitrogenase activity. However, upon returning to 20 kPa of O₂, 80% of nitrogenase activity was recovered within 10 min, indicating a “switch-off/switch-on” O₂ protection mechanism of nitrogenase activity. Our study demonstrates that colonies of G. diazotrophicus can fix N₂ at a wide range of atmospheric pO₂ and can adapt to maintain nitrogenase activity in response to both long-term and short-term changes in atmospheric pO₂.     

The influence of shading on associative N2 fixation

Summary The effect of reduced solar radiation on associative N₂-fixation and plant parameters was studied in three field experiments (1978–80). Gahi-3 pearl millet (Pennisetum americanum (L.) K. Monch.) field plots were shaded with saran shade cloth that reduced solar radiation by 50% and 75%. Acetylene reduction activity (ARA) was reduced by shading in one of the three experiments. The two non-responding experiments were conducted on a wall-drained, low-activity site (ARA means ranging 17–68 n moles ethylene core^–1 h^–1), the responding experiment was conducted on a poorly drained, high-ARA site.Shading affected the plants drastically, reducing fresh weight and dry matter yields up to 46% (50% shade) and 57% (75% shade). Shading also reduced dry matter percentage from 19.6 (no shade) to 15.3 (75% shade) and increased nitrogen content from 0.6% (no shade) to 1.53% (75% shading). However, shading did not affect protein yield. Inoculation withAzospirillum brasilense had no measurable effect on yield or acetylene reduction in the first two experiments.In the third experiment, shading reduced mean ARA of inoculated plots over 100% but had no significant effect on control plots. Inoculation significantly increased ARA in the nonshaded plots but not in shaded plots. Acetylene reduction activity was high, with means ranging between 208 and 465 n moles ethylene evolved core^–1 h^–1. Soil moisture and millet growth stage also affected acetylene reduction activity.     

Nodule structure and nitrogenase activity ofCoriaria arborea in response to varying pO2

When excised root nodules ofCoriaria arborea are assayed for nitrogenase activity at various pO₂ they show a broad optimum between 20 and 40 kPa O₂, with some evidence for adaptation. Continuous flow assays of nodulated root systems of intact plants indicate that Coriaria shows an acetylene induced decline in nitrogenase activity. When root systems were subject to step changes in pO₂ nitrogenase activity responded with a steep decline followed by a slower rise in activity both at lower and higher than ambient pO₂. Thus Coriaria nodules are able to adapt rapidly to oxygen levels well above and well below ambient. Measurement of nodule diffusion resistance showed that the adaptation is accompanied by rapid increase in resistance at above ambient pO₂ and decrease in resistance at below ambient pO₂. Plants grown with root systems at pO₂ from 5–40 kPa O₂ did not differ in growth or nodulation. The anatomy of Coriaria nodules shows they have a dense periderm which encircles the nodule and also closely invests the infected zone. The periderm is both thicker and more heavily suberised in nodules grown at high pO₂ than at low pO₂. Vacuum infiltration of India ink indicates that oxygen diffusion is entirely through the lenticel and via a small gap adjacent to the stele.     

Effects of organic substrates and chloramphenicol or nalidixic acid on acetylene reduction associated with roots of intact maize and sorghum plants

To study the role or organic substrate availability as a factor limiting associative N₂-fixation we measured acetylene reduction (AR) associated with roots of intact maize and sorghum plants before and after adding organic substrates to the nutrient solution in a hydroponic system. Chloramphenicol (Cam) or nalidixic acid (NA) was added along with the substrate to determine whether bacterial protein synthesis or cell replication was necessary to support increased AR following amendment. The grasses were grown in pots in a greenhouse or on a light bench for 4–6 weeks, and then brought into the laboratory to measure AR. Intact plants were separated from soil and transferred into plastic cylinders containing an N-free nutrient solution. The roots were isolated from the shoots by a silicone rubber seal and exposed to oxygen concentrations of 0–10 kPa. Rates of AR were measured before and after adding 0.01–0.10% (w/v) carbon as glucose, malate, succinate, ethanol, acetate, glutarate, propionate, or resorcinol. Only resorcinol and ethanol failed to substantially increase AR activity. Rates of AR increased by 1.5-to 2-fold within 2h and by 5-to 15-fold after 24h. Cam and NA prevented the stimulation of AR by glucose, but neither inhibitor caused AR associated with unamended plants to decrease. We conclude that the highly variable rates of AR that have been reported for associative symbioses, even under well-controlled conditions were governed to a large extent by the amount and type of organic substrates exuded by the roots. Proliferation of diazotrophs appeared to be necessary to increase root-associated AR activity but not to maintain a constant level of activity.     

Effect ofAzospirillum inoculation on nitrogen fixation and growth of several winter legumes

Summary Inoculation of naturally nodulatedPisum sativum L. (garden pea) withAzospirillum in the greenhouse caused a significant increase in nodule numbers above controls. Field inoculation of garden peas in the winter 1981–1982 andCicer arietinum L. (chick pea), in winter 1982–1983, withAzospirillum one week after plant emergence, produced a significant increase in seed yield, but did not affect plant dry matter yield. ForVicia sativa L. (vetch) grown in soil in the greenhouse and in the field for forage, winter 1980–1981, inoculation significantly increased dry matter yield, %N, N-content, and acetylene reduction (nitrogen fixation) activity. InHedysarum coronarium L. (sulla clover), winter 1981–1982, inoculated with both its specificRhizobium (by the slurry method) andAzospirillum, 7 days after emergence, there was an increase in acetylene reduction above controls inoculated withRhizobium alone. These results suggest that it is possible, under conditions tested in this work, to increase nodulation, nitrogen fixation, and crop yields of winter legumes by inoculation withAzospirillum.     

Nitrogenase Activity (Acetylene Reduction) of Root-Associated, Cold-Climate Azospirillum, Enterobacter, Klebsiella, and Pseudomonas Species During Growth on Various Carbon Sources and at Various Partial Pressures of Oxygen

A comprehensive view of the diazotrophic bacterial flora of plants requires that attention be paid to the appropriate carbon and oxygen requirements during isolation of the bacteria. Twenty compounds (monosaccharides, disaccharides, polyols, and organic acids) were therefore examined as carbon and energy sources for nitrogenase activity in semisolid stab cultures at pO₂ values of 0.21, 0.02, and ≤0.002 with 12 strains of diazotrophic root-associated bacteria. With the facultatively anaerobic bacteria of the genera Klebsiella and Enterobacter, the best substrate was sucrose, followed by fructose and mannitol, whereas among the organic acids, only malic and fumaric acids supported any activity. With the obligately aerobic bacteria of the genera Azospirillum and Pseudomonas, disaccharides were not utilized for nitrogen fixation, but several organic acids were accepted in addition to monosaccharides and polyols; malate and glucose were the best substrates. The patterns of the carbon sources utilized for nitrogen fixation were coherent within the species, with the exception of one Klebsiella pneumoniae and one Enterobacter agglomerans strain, both isolated from the same individual grass plant, which were unable to utilize lactose. Anaerobic conditions (pO₂ value of ≤0.002) were required for maximum nitrogenase activity with the facultatively anaerobic bacteria, with the exception of one strain of E. agglomerans, which required atmospheric oxygen (pO₂ value of 0.21). Also, the obligately aerobic diazotrophs required atmospheric oxygen for maximum nitrogenase activity. The maximum specific nitrogenase activities (expressed as micromoles of C₂H₄ · milligram of bacterial protein⁻¹ · hour⁻¹) noted during the exponential growth phase of the bacteria were the following: 2.68 with Azospirillum lipoferum on malate, 2.41 with K. pneumoniae and 1.58 with E. agglomerans on sucrose, and 0.95 with Pseudomonas sp. on malate.     

Oxygen limitation of N2 fixation in stem-girdled and nitrate-treated soybean

The effects of increasing rhizosphere pO₂on nitrogenase activity and nodule resistance to O₂diffusion were investigated in soybean plants [Glycine max (L.) Merr. cv. Harosoy 63] in which nitrogenase (EC 1.7.99.2) activities were inhibited by (a) removal of the phloem tissue at the base of the stem (stem girdling), (b) exposure of roots to 10 mM NO₃over 5 days (NO₃-treated), or (c) partial inactivation of nitrogenase activity by an exposure of nodulated roots to 100 kPa O₂(O₂-inhibitcd). In control plants and in plants which had been treated with 100 kPa O₂, increasing rhizosphere O₂concentrations in 10 kPa increments from 20 to 70 kPa did not alter the steady-state nitrogenase activity. In contrast, in plants in which nitrogenase activities were depressed by stem girdling or by exposure to NO₃, increasing rhizosphere pO₂resulted in a recovery of 57 or 67%, respectively, of the initial, depressed rates of nitrogenase activity. This suggests that the nitrogenase activity of stem-girdled and NO₃-treated soybeans was O₂-limited. For each treatment, theoretical resistance values for O₂diffusion into nodules were estimated from measured rates of CO₂exchange, assuming a respiratory quotient of 1.1 and 0 kPa of O₂in the infected cells. At an external partial pressure of 20 kPa O₂, the stem-girdled and NO₃^--treated plants displayed resistance values which were 4 to 8.6 times higher than those in the nodules of the control plants. In control and O₂-inhibited plants, increases in pO₂from 20 to 70 kPa in 10 kPa increments resulted in a 2.5- to 3.9-fold increase in diffusion resistance to O₂, and had little effect on either respiration or nitrogenase activity. In contrast, in stem-girdled and NO₃^--treated plants, increases in external pO₂had little effect on diffusion resistance to O₂, but resulted in a 2.3- to 3.2-fold increase in nodule respiration and nitrogenase activity. These results are consistent with stem-girdling and NO₃^--inhibition treatments limiting phloem supply to nodules causing an increase in diffusion resistance to O₂at 20 kPa and an apparent insensitivity of diffusion resistance to increases in external pO₂.     

Seasonal variation in rates of heterotrophic nitrogen fixation (acetylene reduction) in Zostera noltii meadows and uncolonised sediments of the Bassin d'Arcachon, south-west France

Nitrogen fixation (acetylene reduction) rates were measured over an annual cycle in meadows of the seagrass Z. noltii and uncolonised sediments of the Bassin d'Arcachon, south-west France, using both slurry and whole core techniques. Measured rates using the slurry technique in Z. noltii colonised sediments were consistently higher than those determined in isolated cores. This was probably due to the release of labile organic carbon sources during preparation of the slurries. Thus, in colonised sediments the whole core technique may provide a more accurate estimate of in situ activity. Acetylene reduction rates measured by the whole core technique in colonised sediments were 1.8 to 4-fold greater, dependent upon the season, in the light compared with those measured in the dark, indicating that organic carbon released by the plant roots during photosynthesis was an important factor regulating nitrogen fixation. In contrast acetylene reduction rates in uncolonised sediments were independent of light.Addition of sodium molybdate, a specific inhibitor of sulphate reduction inhibited acetylene reduction activity in Z. noltii colonised sediments by > 80% as measured by both slurry and whole core techniques irrespective of the light regime, throughout the year inferring that sulphate reducing bacteria (SRB) were the dominant component of the nitrogen fixing microflora. A mutualistic relationship between Z. noltii and nitrogen fixing SRB in the rhizosphere, based on the exchange of organic carbon and fixed nitrogen is proposed. In uncolonised sediments sodium molybdate initially severely inhibited acetylene reduction rates, but the level of this inhibition declined over the course of the year. These data indicate that the nitrogen fixing SRB associated with the Zostera roots and rhizomes were progressively replaced by an aerobic population of nitrogen fixers associated with the decomposition of this recalcitrant high C:N ratio organic matter.Acetylene and sulphate reduction rates in the seagrass beds showed distinct summer maxima which correlated with a reduced availability of NH ₄ ⁺ in the sediment and the growth cycle of Z. noltii in the Bassin. Overall, these data indicate that acetylene reduction (nitrogen fixation) activity in the rhizosphere of Z. noltii was regulated both by release of organic carbon from the plant roots and maintenance of low ammonium concentrations in the root zone due to efficient ammonium assimilation.Nitrogen fixation rates determined from acetylene reduction rates measured by the whole core technique ranged from 0.1 to 7.3 mg N m^–2 d^–1 in the Z. noltii beds and between 0.02 and 3.7 mg N m^–2 d^–1 in uncolonised sediments, dependent upon the season. Nitrogen fixation in the rhizosphere of Z. noltii was calculated to contribute between 0.4 and 1.1 g N m^–2 y^–1 or between 6.3 and 12% of the annual fixed nitrogen requirement of the plants. Heterotrophic nitrogen fixation therefore represents a substantial local input of fixed nitrogen to the sediments of this shallow coastal lagoon and contributes to the overall productivity of Z. noltii in this ecosystem.     

Root Nodule Enzymes of Ammonia Assimilation in Alfalfa (Medicago sativa L.) : DEVELOPMENTAL PATTERNS AND RESPONSE TO APPLIED NITROGEN

Nitrogenase-dependent acetylene reduction activity of glasshouse-grown alfalfa (Medicago sativa L.) decreased rapidly in response both to harvesting (80% shoot removal) and applied NO₃⁻ at 40 and 80 kilograms N per hectare. Acetylene reduction activity of harvested plants grown on 0 kilogram N per hectare began to recover by day 15 as shoot regrowth became significant. In contrast, acetylene reduction activity of all plants treated with 80 kilograms NO₃⁻-N per hectare and harvested plants treated with 40 kilograms NO₃⁻-N per hectare remained low for the duration of the experiment. Acetylene reduction of unharvested alfalfa treated with 40 kilograms N per hectare declined to an intermediate level and appeared to recover slightly by day 15. Changes in N₂-fixing capacity were accompanied by similar changes in levels of nodule soluble protein.     

Nitrogenase activity in the papyrus swamps of Lake Naivasha,Kenya

Nitrogenase activity (acetylene reduction activity) was found to occur universally in the Cyperus papyrus swamp in Lake Naivasha. Low rates of acetylene reduction activity (0.9–104.9 nmol C₂H₄ g d.wt. roots^-1 h^-1) were associated with excised roots of C. papyrus but higher rates of activity (89.0–280.4 nmol C₂H₄ g d.wt. roots^-1 h^-1) were associated with intact root systems of the plant. It was estimated that nitrogen fixation associated with young roots alone could supply about 26% of the nitrogen requirements of growing papyrus plants. Acetylene reduction activity in the lake bottom sediments was generally low and associated with adjacent papyrus stands. Plate counts of putative aerobic and facultatively anaerobic N₂-fixing bacteria associated with papyrus roots showed the presence of high numbers of diazotrophs (5.4 × 10⁶ CFU g d.wt. roots^-1). Fewer numbers of N₂-fixing bacteria were detected in the sediments (1.9 × 10³-3.2 × 10⁴ CFU g d.wt. sediment^-1).     

Mechanisms of salt tolerance and interactive effects of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> inoculation on maize cultivars grown under salt stress conditions

The present work has been performed to study the growth and metabolic activities of two maize cultivars (cv. 323 and cv. 324) which are shown to have different tolerances to salt stress and to determine the effects of inoculation with Azospirillum spp. Along with identifying the mechanisms of maize salt tolerance and the role of Azospirillum (growth promoting rhizobacteria) in elevating salinity stress conditions is examined Maize cv. 323 was the most sensitive to salinity, while cultivar 324 was the most resistant of the 12 maize cultivars tested. Cultivars differences were apparent with certain growth criteria as well as related metabolic activities. The lack of a negative response to increasing NaCl concentration for water content, dry matter yield and leaf area of cv. 324 up to a concentration of – 0.6 MPa indicated salt tolerance. While for cv. 323 there was a marked inhibitory effect of salinity on growth. In the tolerant cv. 324, soluble and total saccharides, soluble protein in shoots and total protein in roots increased with salinity stress. The sensitivity of cv. 323 however was associated with depletion in saccharides and proteins. Proline accumulation was higher and detected earlier at a lower salinity concentration in the salt sensitive cv. 323 comapred to the salt tolerant cv. 324. When salt stressed maize was inoculated with Azospirillum, proline concentration declined significantly. The present study showed, in general, that the concentration of most amino acid increased on exposure to NaCl as well as when inoculated with Azospirillum. The relatively high salt tolerance of cv. 324, compared with cv. 323 was associated with a significantly high K⁺/Na⁺ ratio. Azospirillum inoculation markedly altered the selectivity of Na⁺, K⁺ and Ca⁺⁺ especially in the salt sensitive cultivar cv. 323. Azospirillum restricted Na⁺ uptake and enhanced the uptake of K⁺ and Ca⁺⁺ in cv. 323. A sharp reduction in the activity of nitrate reductase and nitrogenase in shoots and roots of both cultivars was induced by salinity stress. This reduction in NR and NA activity was highly significant at all salinity concentrations. Azospirillum inoculation stimulated NR and nitrogenase activity in both shoots and roots of both cultivars. The differential effect of Azospirillum inoculation on maize cv. 323 and cv. 324 illustrates the different sensitivity of these two cultivars to stress, but still does not provide any clues as to the key events leading to this difference.     

Respiration and nitrogenase activity in nodules ofCasuarina cunninghamiana and cultures ofFrankia sp. HFP020203: Effects of temperature and partial pressure of O2

The effects of time after exposure to acetylene and of nodule excision were examined using a flow-through system. After a transient depression in the rate of acetylene reduction that began about 1.5 min after exposure to acetylene, the rate recovered to 98% of the initial maximum value after 40 min. After nodule excision the rate stabilized to 90% of the initial maximum value observed in the intact plant.Excised nodules, measured at 6-min intervals in a closed system, with frequent changes of the gas mixture, were used for the remaining experiments. Acetylene reduction by the nodules increased rapidly as temperature was increased between 6 and 26°C. Between 26 and 36°C there was relatively little effect of temperature on acetylene reduction.Nodules and cultures ofFrankia were compared with respect to the effect of temperature and pO₂ (partial pressure of oxygen) on oxygen uptake. Cultures ofFrankia were grown on a nitrogen-free medium at either 0.3 kPa O₂ (vesicles absent) or 20 kPa O₂ (vesicles present). Oxygen uptake by nodules (vesicles absent) and by vesicle-containing cultures was strongly dependent on pO₂ at values below 20 kPa. This suggests the presence of a barrier to oxygen diffusion. Oxygen uptake was dependent on temperature as well as on pO₂, but the Q₁₀ was much larger for the cultures than for the nodules. This suggests that vesicles or related structures are not the source of the diffusion barrier in Casuarina nodules. Respiration by cultures ofFrankia lacking vesicles became O₂-saturated at low pO₂ values. Thus these cultures did not have a significant diffusion barrier. From these results it is concluded that nodules ofCasuarina cunninghamiana have a barrier to oxygen diffusion supplied by the host tissue and not byFrankia.     

The biology ofAzospirillum-sugarcane association II. Ultrastructure

Summary Tissue cultures of sugarcane support abundant growth ofAzospirillum brasilense (SP 7). Visible after 1–2 weeks as a white or pink slime, this growth reaches 2×10⁸ bacteria/mm² on the surface of callus. Growth of the bacterium is strictly extracellular in viable callus, and instances of intracellular growth result from rupture of the cell wall during senescence of callus tissue. A significant proportion of the bacterial population on callus is pleomorphic. Varying the nitrogen source in the nutrient medium caused no obvious effect on callus cell structure. The presence of the bacterium caused structural alterations in callus cells which did not inhibit overall growth of the bacterium. Growth of callus as tight groups of cells lacking intercellular spaces may be important for the establishment of a long-term association withAzospirillum. The interface of bacteria and live callus tissue is at the surface of tight cell groups. Browning of the surface cell layers of these groups in the presence ofAzospirillum is not of the rapid nature known for hypersensitivity reactions. Rather, this production of phenolics appears to be due to the accumulation of extracellular bacterial metabolites. The ultrastructure of this and other callus reactions is described. As evidenced by organogenesis, the associated cultures have remained viable for at least 18–20 months.Florida Agricultural Experiment Station Journal Series No. 1695.     

Denitrification and nitrogen fixation by Azospirillum

A model system is described where Azospirillum and germinated wheat seeds were grown in association for a week and then assayed for nitrogen fixation (C₂H₂-reduction) and denitrification (N₂O-formation) activities. The association performed C₂H₂-reduction and N₂O-formation under microaerobic conditions. Both activities were measurable after already 3–5 h of incubation with substantial rates and were strictly dependent on the presence of both plants and bacteria. During the week of the growth of the association, the bacteria had lived exclusively from the carbon compounds supplied by the roots of the plants. C₂H₂-reduction activity by the association was more or less the same with all the Azospirillum brasilense strains, but lower with A. lipoferum and with the A. amazonense strains tested. Two nitrogenase negative mutants of Azospirillum brasilense showed virtually no activity in the association. C₂H₂-reduction activity was strongly dependent on the growth temperature of the association. Denitrification (N₂O-formation) was high also at higher temperatures and at pH-values in the medium around 7.8 but not at neutrality and was strictly dependent on nitrate. The Azospirillum strain used strongly determined the rate of the N₂O-formation in the association. It is suggested that Azospirillum may be beneficial to crops particularly under tropical conditions.Dedicated to Professor Dr. Gerhart Drews, Freiburg, on the occasion of his 60th birthday     

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

Nitrogen fixation associated with non-legumes in agriculture

Summary This review examines the nitrogen cycle in upland agricultural situations where nonlegume N₂-fixation is likely to be important for crop growth. Evidence for associative fixation is adduced from accumulation of N in the top 15 cm soil under grasses, from N balances for crop production obtained from both pot and field experiments, in tropical and temperate environments, measurements of nitrogen (C₂H₂ reduction) activity, uptake of¹⁵N₂ by plants and¹⁵N isotope dilution. Factors influencing the activity such as the provision of carbon substrate by the plant and the efficiency of its utilisation by the bacteria, plant cultivar, soil moisture and N levels, and inoculation with N₂-fixing bacteria are discussed. Crop responses to inoculation withAzospirillum are detailed. The breakdown of crop residues, particularly straw, can support large levels of N₂-fixation. Cyanobacteria as crusts on the soil surface also fix nitrogen actively in many environments. Fixation by the nodulated, non-legume treesCasuarina andParasponia has beneficial effects in some cropping systems in Asia. I conclude that nonlegume N₂-fixation makes a significant contribution to the production of some major cereal crops in both temperate and tropical environments.     

N2 Fixation (C2H2 reduction) by epiphylls on coffee,Coffea arabica

Nitrogen fixation (C₂H₂ reduction) by epiphylls on coffee,Coffea arabica, grown in sites with different degrees of shade, was determined. Coffee leaves with nitrogen-fixing epiphylls were found in all sites in approximately equal numbers. Rates of C₂H₂ reduction were similar for all sites and throughout the year, averaging 3.21 nmoles C₂H₂ reduced leaf with epiphylls^–1 day^–1. Apparently, neither rates of activity nor abundance of leaves with nitrogen-fixing epiphylls is related to the degree of shade in a site. No correlation was found between percent epiphyll cover and the presence or magnitude of nitrogen-fixing activity. Calculated annual fixation by epiphylls on coffee was low, ranging from 0.7 g N₂ ha^–1 year^–1 for the shadeless site to 1.4 g N₂ ha^–1 year^–1 for the site withIngajinicuil shade trees. These results suggest that epiphyll fixation is not an important source of nitrogen for the coffee ecosystem studied.