Nitrogen fixation (acetylene reduction) associated with roots of winter wheat and sorghum in Nebraska

Root segments and root-soil cores (6.5-cm diameter) from fields and nurseries of winter wheat and sorghum were tested for N2 fixation by using the acetylene reduction assay. Wheat samples (approximately 1,200) from 109 sites generally had low or no activity (0 to 3.1 nmol of C2H4 produced per h per g [dry weight] of root segments), even after 24 h of incubation. However, a commercial field of Scout 66, located in western Nebraska, exhibited appreciable activity (290 nmol of C2H4 produced per h per g [dry weight] of root segments). Of 400 sorghum lines and crosses, grain sorghums (i.e., CK-60A, Wheatland A, B517, and NP-16) generally exhibited higher nitrogenase activity than forage sorghums or winter wheats. CK-60A, a male sterile grain sorghum, was sampled at four locations and had the most consistent activity of 24 to 1,100 nmol of C2H4 produced per h per core. The maximum rate extrapolated to 2.5 g of N per hectare per day. Numerous N2-fixing bacterial isolates were obtained from wheat and sorghum roots that exhibited high nitrogenase activity. Most isolates were members of the Enterobacteriacae, i.e., Klebsiella pneumoniae, Enterobacter cloacae, and Erwinia herbicola.     

Nitrogen fixation (acetylene reduction) associated with duckweed (lemnaceae) mats

Duckweed (Lemnaceae) mats in Texas and Florida were investigated, using the acetylene reduction assay, to determine whether nitrogen fixation occurred in these floating aquatic macrophyte communities. N(2)-fixing microorganisms were enumerated by plating or most-probable-number techniques, using appropriate N-free media. Results of the investigations indicated that substantial N(2)-fixation (C(2)H(2)) was associated with duckweed mats in Texas and Florida. Acetylene reduction values ranged from 1 to 18 mumol of C(2)H(4) g (dry weight) day for samples incubated aerobically in light. Dark N(2) fixation was always two- to fivefold lower. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (7 to 10 muM) reduced acetylene reduction to levels intermediate between light and dark incubation. Acetylene reduction was generally greatest for samples incubated anaerobically in the light. It was estimated that 15 to 20% of the N requirement of the duckweed could be supplied through biological nitrogen fixation. N(2)-fixing heterotrophic bacteria (10 cells g [wet weight] and cyanobacteria (10 propagules g [wet weight] were associated with the duckweed mats. Azotobacter sp. was not detected in these investigations. One diazotrophic isolate was classified as Klebsiella.     

Nitrogen fixation (acetylene reduction) inAquaspirillum magnetotacticum

Aquaspirillum magnetotacticum strain MS-1 and two nonmagnetic mutants derived from it reduced C₂H₂ microaerobically but not anaerobically even with NO₃ ^?. This organism apparently is not capable of NO₃ ^?-dependent nitrogen fixation. Cells ofA. magnetotacticum reduced C₂H₂ at rates comparable to those ofAzospirillum lipoferum grown under similar conditions, but much lower than that ofAzotobacter vinelandii grown aerobically. Cells ofA. magnetotacticum in anaerobic cultures lacking NO₃ ^? did not reduce C₂H₂ until O₂ was introduced. Optimum rates of C₂H₂ reduction byA. magnetotacticum were obtained at 200 Pa O₂. C₂H₂ reduction was inhibited by more than 1 kPa O₂ or 0.2 mM NO₃ ^? or NH₄ ⁺. These results suggest thatA. magnetotacticum fixes N₂ only under microaerobic, N-limited conditions.     

Nitrogen fixation (acetylene reduction) on a coral reef

Nitrogen fixation rates associated with various substrates on a fringing reef at Eilat, Red Sea, were estimated by in situ acetylene reduction. High rates of acctylene reduction were associated with bare substrates, such as sand and dead coral skeletons. Low rates of acetylene reduction were associated with substrates covered by macroalgae or living coral tissue. Estimates of nitrogen fixation in various reef zones, based on these measurements, indicate that the sand-covered lagoon is responsible for more than 70% of the fixation in the reef. Consequently, the lagoon may serve as an important source of nitrogen for the coral reef community.     

Biological dinitrogen fixation (acetylene reduction) associated with Florida mangroves

Biological dinitrogen fixation in mangrove communities of the Tampa Bay region of South Florida was investigated using the acetylene reduction technique. Low rates of acetylene reduction (0.01 to 1.84 nmol of C(2)H(4)/g [wet weight] per h) were associated with plant-free sediments, while plant-associated sediments gave rise to slightly higher rates. Activity in sediments increased greatly upon the addition of various carbon sources, indicating an energy limitation for nitrogenase (C(2)H(2)) activity. In situ determinations of dinitrogen fixation in sediments also indicated low rates and exhibited a similar response to glucose amendment. Litter from the green macroalga, Ulva spp., mangrove leaves, and sea grass also gave rise to significant rates of acetylene reduction.Higher rates of nitrogenase activity (15 to 53 nmol of C(2)H(4)/g [wet weight] per h were associated with washed excised roots of three Florida mangrove species [Rhizophora mangle L., Avicennia germinans (L) Stern, and Laguncularia racemosa Gaertn.] as well as with isolated root systems of intact plants (11 to 58 mug of N/g [dry weight] per h). Following a short lag period, root-associated activity was linear and did not exhibit a marked response to glucose amendment. It appears that dinitrogen-fixing bacteria in the mangrove rhizoplane are able to use root exudates and/or sloughed cell debris as energy sources for dinitrogen fixation.     

Nitrogen fixation (acetylene reduction) associated with communities of heterocystous and non-heterocystous blue-green algae in mangrove forests of Sinai

Summary High rates of nitrogen fixation (acetylene reduction) are associated with communities of heterocystous and non-heterocystous blue-green algae, which are widespread and abundant in the coastal mangrove forests of the Sinai Peninsula.Heterocystous forms, particularly representatives of the Rivulariaceae, grow in aerobic environments, where nitrogenase activity may be limited by the availability of nutrients such as Fe and PO₄–P. Desiccated communities of Scytonema sp. reduce acetylene within ten minutes of wetting by tidal sea water. Communities dominated by the non-heterocystous Hydrocoleus sp., Hyella balani, Lyngbya aestuarii, Phormidium sp. and Schizothrix sp., occur in close contact with anaerobic sediments and reduce acetylene in the dark as well as in the light.Nitrogen fixation in all these communities is light dependant and may be supplemented by an alternative source of reductant in the dark. The indications are that nitrogen fixation by these communities of blue-green algae, makes a significant contribution to the overall nitrogen input of the mangrove ecosystem.     

Nitrogen fixation (acetylene reduction) and cross inoculation in 12 Prosopis (mesquite) species

The leguminous tree mesquite (Prosopis spp) exists on millions of hectares of semi-arid regions of the world. No whole plant acetylene reductions for mesquite have been reported in the literature and nodulation has only been reported for three of the forty-four species. We report greenhouse studies in which 12Prosopis species representing African and North and South American germplasm (1) became nodulated when inoculated with rhizobia strain isolated from a North American mesquite, (2) grew on a nitrogen free nutrient media, (3) reduced acetylene to ethylene, and (4) had a positive significant correlation between the acetylene reduction rates and above ground dry matter. The capability of mesquite to fix nitrogen must now be considered firmly established.     

Nitrogen fixation associated with roots of Kallar grass (Leptochloa fusca L. Kunth)

Summary An ecological survey was made to measure the N₂-fixing activity in the rhizosphere of a salt-tolerant grass,Leptochloa fusca (L.) Kunth. Samples were obtained every month at two sites over a period of one year. Soil cores, unwashed, washed and surface-sterilized roots were subjected to acetylene reduction assay (ARA). ARA values up to 50 nmoles h^–1 for soil cores, 1095 nmoles g dry root^–1 h^–1 for unwashed roots, 4929 nmoles g dry root^–1 h^–1 for washed roots and 2494 nmoles g dry root^–1 h^–1 for surface-sterilized roots were observed but for most samples the range was 1–200 nmoles g dry root^–1 h^–1. A lag period of 5–7 h was observed before the onset of N₂-fixing activity by excised roots and O₂ levels had no effect on this lag. Values for roots incubated without preincubation were similar to those for unwashed preincubated roots. Activity was highest in September, October and November when the temperature is not very high and photosynthetic activity is reasonably good. N₂-fixing-bacteria were counted on the same samples by plate count and MPN methods, the latter being estimated on the basis of ARA and pellicle formation. Fairly high numbers of bacteria (10⁴–10⁷) were recorded in the histoplane fraction which indicates the presence of diazotrophs in the inner cells of grass roots.     

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.     

Nitrogen assimilation in field-grown winter wheat: Direct measurements of nitrate reduction in roots using15N

Summary Nitrate fertiliser labelled with¹⁵N was applied to a field grown crop of winter wheat. Uptake and assimilation of fertiliser nitrate was studied by monitoring the appearance of labelled nitrate and labelled amino acids in the xylem sap. Shortly after applying¹⁵N-nitrate to the soil about 30 per cent of recently absorbed¹⁵N was in the reduced form, indicating that roots of cereal crops can make a substantial contribution in reducing nitrate. Seasonal changes in crop growth andin vivo NRA are also described.     

Nitrogen fixation inpara-nodules of wheat roots by introduced free-living diazotrophs

Nitrogen-fixation (C₂H₂-reduction) was demonstrated in wheat root nodules (p-nodules) induced by 2,4-dichlorophenoxyacetate (2,4-D) and inoculated withA. brasilense. By lowering the O₂ tension it was possible to distinguish the nitrogenase activity of bacteria located within thep-nodule of the wheat root system from that in the rhizosphere. Using cytological evidence, nitrogenase activity was attributed mainly to be coming from the bacteria within thep-nodule. It was also shown that the host plant was able to supply the necessary substrate required for the bacterial N₂-fixation (C₂H₂-reduction) within thep-nodules.     

Dark anaerobic nitrogen fixation (acetylene reduction) in the cyanobacterium Oscillatoria sp.

The filamentous, non-heterocytous, nitrogen-fixing cyanobacterium Oscillatoria sp. strain 23 (Oldenburg) showed cycling of acetylene reduction in light-dark cycles. Under aerobic conditions nitrogenase activity is exclusively present during the dark period. However, if anaerobic conditions were applied during the dark period, two activity maxima were observed. A relatively small activity peak occurred during the first few hours of the dark period and a high peak as soon as the light was switched on. A low activity remained during the second half of the dark period. This pattern of acetylene reduction in Oscillatoria agrees well with the field data on nitrogen fixation [Stal, L.J. and Krumbein, W.E. (1984), Mar. Biol. 82, 217–224].     

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.     

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.     

Dinitrogen (15N2) fixation and acetylene reduction in free-living strains ofRhizobium

Dinitrogen (¹⁵N₂) fixation of four free-livingRhizobium strains ranged from 0.8 to 2.3 μmol/mg biomass N. Parallel-grown cultures liberated 4–8 μmol hydrogen and reduced 12–23 μmol acetylene, giving a mean ratio of reduced acetylene-to-fixed¹⁵N₂ of 12. This ratio contrasts with lower values others have observed for asymbiotic diazotrophs.     

Nitrogen fixation (Acetylene reduction) by free-livingRhizobium meliloti

Evidence is presented thatRhizobium meliloti is able to fix nitrogen (as scored by acetylene reduction) while growing in the absence of a plant host. The highest nitrogen fixation rate was obtained when bacteria were grown in defined liquid medium under normal atmosphere in sealed tubes. The nitrogen fixation rates obtained are of the same order as those reported for other rhizobia.     

Absence of nitrogen fixation (acetylene reduction) by procaryotes in nectar of Banksias

Summary An earlier hypothesis that blue-green algae in the nectar ofBanksia telmatiaea contribute to the nitrogen economy of the host by fixing N₂ was tested. Field and laboratory experiments failed to demonstrate C₂H₄ production in C₂H₂-treated containers over extended periods. Soil N was not higher at the end of the flowering season and plants in which flower heads were removed prior to nectar production did not contain less N than the controls.     

Nitrogen fixation associated with grasses in Oregon.

Nitrogen fixation associated with both natural grasslands and grain crops of Oregon was studied using the acetylene-reduction assay. A number of the grasses collected has some acetylene-reducing activity. Agrostis tenuis Sibth. had substantially greater activity than any of the other species, with a mean rate estimated at 37 g N2 fixed per hectare per day. Assuming 100 days of activity, about 3 kg of N2 would be fixed per hectare per year. This quantity of nitrogen may be important in the maintenance of this species under natural conditions. Nitrogen-fixing microorganisms were isolated from the root surfaces of some of the grasses. Cultures of Bacillus macerans, Bacillus polymyxa, and Enterobacter cloacoa were isolated from wheat roots as were two cultures which have not been assigned a specific taxonomic classification. Strains of N2-fixing Bacillus species and Gram-negative aerobic bacteria were isolated from Festuca and Agrostis.