Diurnal variation in algal acetylene reduction (nitrogen fixation) in situ

Diurnal variation in algal nitrogen fixation was studied in Lake Mendota, Wisconsin, during the summers of 1971 to 1973. Approximately two-thirds of the daily acetylene reduction in the surface decimeter occurred before noon. The decline in acetylene reduction (nmoles/liter·hr) near midday was partially because the algae relocated themselves at greater depths. However, acetylene reducing activity (nmoles per A₆₆₃ unit chlorophyll a per hour) also decreased as midday approached. Occasionally algae would resurface near the end of the day. On average, acetylene reduction (nmoles per liter per hour) was maximum at about 0900 Central standard time in the top decimeter, and acetylene reduction between 0830 and 0930 Central standard time represented 13% of the total daily acetylene reduction. Furthermore, acetylene reduction in the top decimeter, on average, represented 3.6% of the total acetylene reduction in the column. Calculation of the contribution by nitrogen fixation to a lake's fixed nitrogen budget is discussed.     

Pathogenicity of the rice root nematode,Hirschmanniella oryzae to rice plants in relation to nematode reproduction,plant growth,grain yield and biochemical changes

Influence of different inoculum levels of 0, 10, 100, 1000 and 10,000 individuals of Hirschmanniella oryzae on nematode reproduction and plant growth of rice cv. Giza171 and biochemical changes of infected plants was studied under screen-house conditions. Rate of nematode build up (P_f/P_i) was negatively correlated with the progressive increase in nematode inoculum levels. The percentage reduction in growth parameters, rice grain yield and the amount of total and reducing sugars were markedly affected showing a negative correlation with the tested inocula. The conspicuous reductions of plant growth, yield and total and reducing sugar contents were obtained by using 1000 and 10,000 nematodes per pot. The inoculum level of 1000 nematodes per pot was identified as critical population at which control programme must be started.     

Pathogenicity of root-knot nematode,Meloidogyne incognita on Lens culinaris (Medik.)

Effects of the root-knot nematode (Meloidogyne incognita) on lentil (Lens culinaris) were studied under greenhouse conditions. The plants were inoculated with 250, 500, 1000, 2000 and 4000 J₂ per plant. Plant growth, yield, nodulation, seed weight, chlorophyll, nitrogen, phosphorus and potassium, (NPK) contents, as compared to control, were found decreased in all the nematode infected plants. The extent of reduction increased with an increase in inoculum levels. The reductions were significant at 500 J₂ and at higher inoculum levels, i.e. 1000, 2000 and 4000 J₂ per pot over the control. An increase in inoculum level caused enhancement in galling, egg mass production and nematode population. At higher inoculum levels, the population of the nematode in the root as well as in the soil increased to a greater magnitude than at lower inoculum levels. On the contrary, reproduction factor (RF) and rate of population increase (RPI) decreased with increasing inoculum levels.     

Acetylene reduction (nitrogen fixation) associated with corn inoculated with Spirillum.

Sorghum and corn breeding lines were grown in soil in field and greenhouse experiments with and without an inoculum of N2-fixing in Spirillum strains from Brazil. Estimated rates of N2 fixation associated with field-grown corn and sorghum plants were less than 4 g of N2/ha per day. The mean estimated N2-fixation rates determined on segments of roots from corn inoculated with Spirillum and grown in the greenhouse at 24 to 27 degrees C were 15 g of N2/ha per day (16 inbreds), 25 g of N2/ha per day (six hybrids), and 165 g of N2/ha per day for one hybird which was heavily inoculated. The corresponding mean rates determined from measurements of in situ cultures of the same series of corn plants (i.e., 16 inbreds, six hybrids, and one heavily inoculated hybrid) were 0.4, 2.3, and 1.1 g of N2/ha per day, respectively. Lower rates of C2H2 reduction were associated with control corn cultures which had been treated with autoclaved Spirillum than with cultures inoculated with live Spirillum. No C2H2 reduction was detected in plant cultures treated with ammonium nitrate. Numbers of nitrogen-fixing bacteria on excised roots of corn plants increased an average of about 30-fold during an overnight preincubation period, and as a result acetylene reduction assays of root samples after preincubation failed to serve as a valid basis for estimating N2 fixation by corn in pot cultures. Plants grown without added nitrogen either with or without inoculum exhibited severe symptoms of nitrogen deficiency and in most cases produced significantly less dry weight than those supplied with fixed nitrogen. Although substantial rates of C2H2 reduction by excised corn roots were observed after preincubation under limited oxygen, the yield and nitrogen content of inoculated plants and the C2H2-reduction rates by inoculated pot cultures of corn, in situ, provided no evidence of appreciable N2 fixation.     

Comparison of N(2) Fixation and Yields in Cajanus cajan between Hydrogenase-Positive and Hydrogenase-Negative Rhizobia by In Situ Acetylene Reduction Assays and Direct N Partitioning

Pigeon peas [Cajanus cajan (L.) Millsp.] were grown in soil columns containing ¹⁵N-enriched organic matter. Seasonal N₂ fixation activity was determined by periodically assaying plants for reduction of C₂H₂. N₂ fixation rose sharply from the first assay period at 51 days after planting to a peak of activity between floral initiation and fruit set. N₂ fixation (acetylene reduction) activity dropped concomitantly with pod maturation but recovered after pod harvests. Analysis of ¹⁵N content of plant shoots revealed that approximately 91 to 94% of plant N was derived from N₂ fixation. The effect of inoculation with hydrogenase-positive and hydrogenase-negative rhizobia was examined. Pigeon peas inoculated with strain P132 (hydrogenase-positive) yielded significantly more total shoot N than other inoculated or uninoculated treatments. However, two other hydrogenase-positive strains did not yield significantly more total shoot N than a hydrogenase-negative strain. The extent of nodulation by inoculum strains compared to indigenous rhizobia was determined by typing nodules according to intrinsic antibiotic resistance of the inoculum strains. The inoculum strains were detected in almost all typed nodules of inoculated plants.

Gas samples were taken from soil columns several times during the growth cycle of the plants. H₂ was never detected, even in columns containing pigeon peas inoculated with hydrogenase-negative rhizobia. This was attributed to H₂ consumption by soil bacteria. Estimation of N₂ fixation by acetylene reduction activity was closest to the direct ¹⁵N method when ethylene concentrations in the gas headspace (between the column lid and soil surface) were extrapolated to include the soil pore space as opposed solely to measurement in the headspace. There was an 8-fold difference between the two acetylene reduction assay methods of estimation. Based on a planting density of 15,000 plants per hectare, the direct ¹⁵N fixation rates ranged from 67 (noninoculated) to 134 kilograms per hectare, while grain yields ranged from 540 to 825 kilograms per hectare. Grain yields were not increased with N fertilizer.

     

Nodule growth and nitrogen fixation of <Emphasis Type="Italic">Calopogonium mucunoides</Emphasis> L. show low sensitivity to nitrate

It is well established that nitrate is a potent inhibitor of nodulation and nitrogen fixation in legumes. The objective of this study was to demonstrate the relative insensitivity of these processes to nitrate with Calopogonium mucunoides, a tropical South American perennial legume, native to the cerrado (savannah) region. It was found that nodule number was reduced by about half in the presence of high levels of nitrate (15 mM) but nodule growth (total nodule mass per plant) and nitrogen fixation (acetylene reduction activity and xylem sap ureide levels) were not affected. Other sources of N (ammonium and urea) were also without effect at these concentrations. At even higher concentrations (30 mM), nitrate did promote significant inhibition (ca. 50%) of acetylene reduction activity, but no significant reduction in xylem sap ureides was found. The extraordinary insensitivity of nodulation and N₂ fixation of C. mucunoides to nitrate suggests that this species should be useful in studies aimed at elucidating the mechanisms of nitrate inhibition of these processes.     

Biological Nitrogen Fixation is not a Major Contributor to the Nitrogen Demand of a Commercially Grown South African Sugarcane Cultivar

It has previously been reported that endophytic diazotrophic bacteria contribute significantly to the nitrogen budgets of some graminaceous species. In this study the contribution of biological nitrogen fixation to the N-budget of a South African sugarcane cultivar was evaluated using ¹⁵N natural abundance, acetylene reduction and ¹⁵N incorporation. Plants were also screened for the presence of endophytic diazotrophic bacteria using acetylene reduction and nifH-gene targeted PCR with the pure bacterial strains. ¹⁵N natural abundance studies on field-grown sugarcane indicated that the plants did not rely extensively on biological nitrogen fixation. Furthermore, no evidence was found for significant N₂-fixation or nitrogenase activity in field-grown or glasshouse-grown plants using ¹⁵N incorporation measurements and acetylene reduction assays. Seven endophytic bacterial strains were isolated from glasshouse-grown and field-grown plants and cultured on N-free medium. The diazotrophic character of these seven strains could not be confirmed using acetylene reduction and PCR screening for nifH. Thus, although biological nitrogen fixation may occur in South African sugarcane varieties, the contribution of this N-source in the tested cultivar was not significant.     

Effect of methabenzthiazuron on growth and nitrogenase activity in Vicia faba

The effects of the herbicide methabenzthiazuron (175 and 220 g ha^-1) on vegetative and reproductive growth, nodulation and nitrogenase activity of Vicia faba were studied in the field under Mediterranean conditions. Nitrogenase activity of excised nodules was estimated using the acetylene reduction assay four times during the developmental period. Leaf area index, dry weight and nitrogen content of the different parts of the plants were measured. Methabenzthiazuron-treated plants showed an increase in nodulation, nitrogenase activity and vegetative growth at early pod fill. Methabenzthiazuron also caused an increase in leaf N content and fruits. These were transient effects found during early and mid pot fill. Nevertheless, plants treated with these sublethal doses of herbicide improved seed production and nitrogen content of seeds at harvest time. The stimulatory effect of methabenzthiazuron on N₂ fixation and vegetative growth seems not be related with the transient stimulatory effect on photosynthetic capacity, also caused by the herbicide, since the stimulatory effect on N₂ fixation was apparent during pod fill, when photosynthetic capacity declined and was not modified by methabenzthiazuron.     

Contribution of nitrogen fixation to nitrogen nutrition in an alpine sedge community (Caricetum curvulae)

Summary In situ acetylene reduction assays (ARA) were carried out over two growing seasons at 2550 m in the upper alpine zone of the Tyrolean Central Alps of Austria. For comparative purposes, some Fabaceae species introduced into the upper alpine zone from lower elevation (2000 m) were subjected to ARA. At the end of the growing season the potted plants were transferred to the laboratory where their acetylene reducing activities were measured again. In situ nitrogenase activity is very low. The highest values were found in association with Leucanthemopsis alpina and Veronica bellidioides (150 and 217 nmol ethylene 24 h^-1 per pot respectively). Higher levels of activity were detected in pots transferred to the laboratory (maximum value 750 nmol ethylene 24 h^-1 per pot; assay temperature about 12°C higher than in the field) and in the Fabaceae transferred to the upper alpine zone (14×10³ nmol ethylene 24 h^-1 per pot of Trifolium badium and T. pallescens). Maximum nitrogen input in the field is in the range of 8 mg m^-2 a^-1. Therefore, under natural circumstances biological nitrogen fixation contributes only very small amounts of nitrogen to this alpine vegetation system, the process being inhibited by low soil temperatures. Possible alternative sources and patterns of N acquisition are discussed in relation to the overall nitrogen economics of plants of the upper alpine zone.     

Effect of Rotylenchulus reniformis on Yield and Nitrogen,Potassium, Phosphorus and Amino Acid Content of Seed of Glycine max

Soybean cultivars varied in their response and tolerance to low initial Rotylenchulus reniforrnis populations of 10,000 nematodes/3.8 liters of soil, but a high initial population of 25,000 consistently reduced yields on resistant and susceptible cultivars by an average of 33.1%. At the 10,000 nematode inoculum level, dry seed yields of Hood decreased while those of Pickett increased significantly. Generally, total phosphorus decreased 11.1 and 11.5% and potassium increased 5.9 and 4.5% in seeds harvested from plants receiving initial inoculum levels of 5000 and 10,000 nematodes/pot, respectively. Little change in the total nitrogen content in seed was noted. Leucine content of seeds from infected plants was slightly less than from noninfected plants.     

Symbiotic Effectiveness and Host-Strain Interactions of Rhizobium fredii USDA 191 on Different Soybean Cultivars

Nodulation, acetylene reduction activity, dry matter accumulation, and total nitrogen accumulation by nodulated plants growing in a nitrogen-free culture system were used to compare the symbiotic effectiveness of the fast-growing Rhizobium fredii USDA 191 with that of the slow-growing Bradyrhizobium japonicum USDA 110 in symbiosis with five soybean (Glycine max (L.) Merr.) cultivars. Measurement of the amount of nitrogen accumulated during a 20-day period of vegetative growth (28 to 48 days after transplanting) showed that USDA 110 fixed 3.7, 39.1, 4.6, and 57.3 times more N₂ than did USDA 191 with cultivars Pickett 71, Harosoy 63, Lee, and Ransom as host plants, respectively. With the unimproved Peking cultivar as the host plant, USDA 191 fixed 3.3 times more N₂ than did the USDA 110 during the 20-day period. The superior N₂ fixation capability of USDA 110 with the four North American cultivars as hosts resulted primarily from higher nitrogenase activity per unit nodule mass (specific acetylene reduction activity) and higher nodule mass per plant. The higher N₂-fixation capability of USDA 191 with the Peking cultivar as host resulted primarily from higher nodule mass per plant, which was associated with higher nodule numbers. There was significant variation in the N₂-fixation capabilities of the four North American cultivar-USDA 191 symbioses. Pickett 71 and Lee cultivars fixed significantly more N₂ in symbiosis with USDA 191 than did the Harosoy 63 and Ransom cultivars. This quantitative variation in N₂-fixation capability suggests that the total incompatibility (effectiveness of nodulation and efficiency of N₂ fixation) of host soybean plants and R. fredii strains is regulated by more than one host plant gene. These results indicate that it would not be prudent to introduce R. fredii strains into North American agricultural systems until more efficient N₂-fixing symbioses between North American cultivars and these fast-growing strains can be developed. When inoculum containing equal numbers of USDA 191 and of strain USDA 110 was applied to the unimproved Peking cultivar in Perlite pot culture, 85% of the 160 nodules tested were occupied by USDA 191. With Lee and Ransom cultivars, 99 and 85% of 140 and 96 nodules tested, respectively, were occupied by USDA 110.     

Nitrogen Fixation and Nitrogen Assimilation in a Temperate Saline Ecosystem

As nitrogen is known to be a limiting factor for plant growth, we were interested in the relationship between soil microbial activity and the nitrogen assimilation of 5 different halophytes from 4 saline sites near the lake “Neusiedlersee”, Austria. The following were studied between May and October 1985: nitrogen fixation (¹⁵N₂ and acetylene reduction): N-mineralization; several soil characteristics and in vivo nitrate reductase activity of roots and shoots of these plants. NO^?₃, org. N- and carboxylate contents of both roots and shoots, as well as the effect of NO^?₃-fertilization on the amounts of these substances, were determined on plants growing in the field during a 3-day period in September 1985. Fertilization led to a decrease in acetylene reduction activity at most sites, and an increase in the nitrate reductase activity of the shoots of all plants. Overall, carboxylate and organic nitrogen contents of these halophytes did not change in response to fertilization. Only in the roots of Aster tripolium and Atriplex hastata was there a marked increase in the nitrate reductase activity in response to fertilization. Species growing at the same site, such as Plantago maritima and Lepidium crassifolium showed contrasting levels of assimilatory activity. Apparent low rates of ammonification and nitrification were detected in soils from the 4 sites. The results are discussed in relation to the nitrogen and carbon economies of the microorganisms and plants.     

Lack of effect of toxic aluminium concentrations on nitrogen fixation by nodulatedStylosanthes species

Summary Effects of three solution aluminium concentrations (0, 25, and 100M) on nitrogen fixation by well-nodulated plants ofStylosanthes hamata, Stylosanthes humilis andStylosanthes scabra are reported. Plants were inoculated with Rhizobium CB756 and grown for 21 days in an aluminium-free nutrient solution at pH 5.3 before imposition of the aluminium treatments.Nitrogen fixation was measured both by the increase in total nitrogen content of the plants and acetylene reduction in roots of plants harvested at 10 and 20 days after imposition of the aluminium treatments. Solution aluminium concentrations as high as 100M, had no detrimental effect on nitrogen fixation in any species.     

CO(2) Exchange and Dinitrogen Fixation of Subterranean Clover in Response to Light Level

Small swards of nodulated subterranean clover plants were grown in pots to a common dry weight under controlled conditions. The rooting medium was a porous calcined clay. All mineral nutrients except nitrogen were supplied daily in solution. Pots then were placed in an assimilation chamber for 3 days for the measurement of net CO₂ exchange at light levels ranging from 0.1 to 2.0 millieinsteins per square meter per second. N₂-fixation (acetylene reduction) of each pot was measured subsequently. H₂-evolution and N₂-fixation were measured for similar treatments in separate experiments using smaller pots.     

Limitation of acetylene reduction (nitrogen fixation) by photosynthesis in soybean having low water potentials

The role of photosynthesis and transpiration in the desiccation-induced inhibition of acetylene reduction (nitrogen fixation) was investigated in soybean (Glycine max [L.] Merr. var. Beeson) using an apparatus that permitted simultaneous measurements of acetylene reduction, net photosynthesis, and transpiration. The inhibition of acetylene reduction caused by low water potentials and their aftereffects could be reproduced by depriving shoots of atmospheric CO₂ even though the soil remained at water potentials that should have favored rapid acetylene reduction. The inhibition of acetylene reduction at low water potentials could be partially reversed by exposing the shoots to high CO₂ concentrations. When transpiration was varied independently of photosynthesis and dark respiration in plants having high water potentials, no effects on acetylene reduction could be observed. There was no correlation between transpiration and acetylene reduction in the CO₂ experiments. Therefore, the correlation that was observed between transpiration and acetylene reduction during desiccation was fortuitous. We conclude that the inhibition of shoot photosynthesis accounted for the inhibition of nodule acetylene reduction at low water potentials.     

Characteristics of nitrogen-fixingKlebsiella oxytoca isolated from wheat roots

Summary Of 45 fermentative gram negative bacterial isolates examined from wheat roots, three were capable of fixing atmospheric nitrogen as determined by the acetylene reduction technique and by protein contents of cells. A gram negative non-motile facultatively anaerobic bacterial strain capable of N₂ fixation was identified asKlebsiella oxytoca ZMK-2.Optimal growth and N₂ fixation occurred at pH 6.5. The optimum temperatures for growth under anaerobic conditions ranged between 30°–37°C. Acetylene reduction by intact cells was strikingly inhibited by 0.1 atm. or greater partial pressure of O₂. Furthermore, the accumulation of H₂ in the gas phase over cultures ofKlebsiella oxytoca ZMK-2 at partial pressures greater than 0.02 atm. resulted in a striking inhibition in the rate of C₂H₂ reduction. The addition of suspensions of eitherKlebsiella oxytoca ZMK-2 orAzotobacter vinelandii or a mixed culture of these two organisms to axenic cultures of wheat plants produced no significant increase in plant growth as measured by plant dry weight or nitrogen content of plants.     

N2 Fixation by Azospirillum brasilense and Its Incorporation into Host Setaria italica

Growth and nitrogen fixation were followed during the life cycle of Setaria italica (foxtail millet) inoculated with Azospirillum brasilense in controlled-environment growth chambers. The plants were fertilized at seeding with a limiting amount of combined nitrogen and maintained with an N-free mineral solution. During maturation of the plants, substantial nitrogenase activity, measured by acetylene reduction, developed in the rhizosphere, with total fixation estimated to be equivalent to 20% of the N in the inoculated plants. The peak of this activity coincided with depletion of soluble nitrogen from the system, which in turn was reflected by a sharp decrease in the nitrate reductase activity of the leaves. A. brasilense was found in association with the root populations at 8 × 10⁷ cells per gram of dry weight. An increase in shoot growth occurred at this time, but no significant increase in total plant nitrogen could be demonstrated. ¹⁵N₂ enrichment experiments confirmed that fixation was occurring, but only about 5% of the nitrogen fixed by A. brasilense was incorporated into the plants within 3 weeks. There was thus no evidence of direct bacterium-to-plant transport of fixed nitrogen, but rather a slow transfer suggesting the gradual death of bacteria and subsequent mineralization of their nitrogen, at least under growth-room conditions.     

Diurnal and seasonal patterns of nitrogen fixation in analnus hirsuta plantation of central Korea

We used an acetylene reduction assay to measure rates of nitrogen fixation on a 38-year-oldAlnus hirsuta plantation in central Korea. The diurnal pattern of acetylene reduction changed significantly during May, August, and October, typically varying by 3-fold throughout the course of the day. Maximum rates occurred at 3 p.m. in May and October, but at 6 p.m. in August. Increasing trends were evident during the early growing season, with sustained high rates from mid-May through late September; July had the highest rates, averaging 7.2 μmole g^-1 dry nodule h^-1. The average nodule biomass for this plantation was 220 kg ha ’. Rates of acetylene reduction were related to soil temperature, but not to soil moisture content. Combining these nodule biomass calculations with seasonal average acetylene reduction rates yielded an estimate of current annual nitrogen fixation of 60 kg N ha^-1 for the plantation. This rate of annual nitrogen addition was very large in relation to the yearly nitrogen requirements of coniferous and deciduous forests in central Korea.     

Nitrogen fixation by the hydrogen-oxidizing bacterium Alcaligenes latus

The aerobic hydrogen-oxidizing bacterium Alcaligenes latus represented by three strains was found to be able to grow with dinitrogen as the sole nitrogen source: The doubling time of total (Kjeldahl) nitrogen during growth on glucose at 30°C under an atmosphere containing 2% (v/v) oxygen in dinitrogen amounted to 39 h, while that in the presence of ammonium was 3 h. Nitrogen fixation did apparently not occur under air. During diazotrophic growth the cells accumulated up to 75% (w/dry weight) poly--hydroxybutyric acid. The efficiency of nitrogen fixation varied between 10 and 15 mg N per g glucose utilized. The specific nitrogenase activity measured in the acetylene reduction assay amounted to 5–17 nmol C₂H₄ formed per min and mg protein.     

Hydroponic growth and the nondestructive assay for dinitrogen fixation

Hydroponic growth medium must be well buffered if it is to support sustained plant growth. Although 1.0 millimolar phosphate is commonly used as a buffer for hydroponic growth media, at that concentration it is generally toxic to a soybean plant that derives its nitrogen solely from dinitrogen fixation. On the other hand, we show that 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid, pK_a 6.1, has excellent buffering capacity, and it neither interferes with nor contributes nutritionally to soybean plant growth. Furthermore, it neither impedes nodulation nor the assay of dinitrogen fixation. Hence, soybean plants grown hydroponically on a medium supplemented with 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid and 0.1 millimolar phosphate achieve an excellent rate of growth and, in the absence of added fixed nitrogen, attain a very high rate of dinitrogen fixation. Combining the concept of hydroponic growth and the sensitive acetylene reduction technique, we have devised a simple, rapid, reproducible assay procedure whereby the rate of dinitrogen fixation by individual plants can be measured throughout the lifetime of those plants. The rate of dinitrogen fixation as measured by the nondestructive acetylene reduction procedure is shown to be approximately equal to the rate of total plant nitrogen accumulation as measured by Kjeldahl analysis. Because of the simplicity of the procedure, one investigator can readily assay 50 plants individually per day.