Rates Of C2H2 Reduction Research Articles

Nitrogen fixation in subarctic streams influenced by beaver (Castor canadensis)

Nitrogen fixation was measured in four subarctic streams substantially modified by beaver (Castor canadensis) in Quebec. Acetylene-ethylene (C2H2 → C2H4) reduction techniques were used during the 1982 ice-free period (May–October) to estimate nitrogen fixation by microorganisms colonizing wood and sediment. Mean seasonal fixation rates were low and patchy, ranging from zero to 2.3 × 10−3 µmol C2H4 · cm−2 · h−1 for wood, and from zero to 7.0 × 10−3 µmol C2H4 · g AFDM−1 · h−1 for sediment; 77% of all wood and 63% of all sediment measurements showed no C2H2 reduction. Nonparametric statistical tests were unable to show a significant difference (p > 0.05) in C2H2 reduction rates between or within sites for wood species or by sediment depth.

The effect of bacterial strain and temperature changes on the nitrogenase activity of Lotus pedunculatus root nodules

After 40 days of growth at 25°C, Lotus pedunculatus cav., cv. Maku plants infected with Rhizobium loti strain NZP2037 displayed similar relative growth rates but had twice the nodule mass and only one third the whole plant dry weight of plants infected with Bradyrhizobium sp. (Lotus) strain CC814s. In the NZP2037 symbiosis, the rate of CO2 evolution (per g dry weight of nodulated root) was 1.6 times as high as that in the CC814s symbiosis while the rate of C2H2 reduction (per g dry weight of nodule) was only 48% of that in the CC814s symbiosis. Studies of the effect of short term temperature changes on the gas exchange characteristics (CO2 and H2 evolution, C2H2 reduction) of these symbioses revealed wide differences in the optima for C2H2 reduction. Nodules infected with NZP2037 displayed maximal C2H2 reduction rates [157 μmol (g dry weight nodule)−1 h−1] at 12°C, whereas nodules infected with CC814s were optimal at 30°C [208 μmol (g dry weight nodule)−1 h−1]. These short term studies suggested that differences in temperature optima for N2 may have partially accounted for the poorer effectivity, at 25°C, of strain NZP2037 when compared with strain CC‐814s.The relative efficiency [RE = 1 – (H2 evolution/C2H2 reduction)] of N2 fixation varied widely with temperature in the two symbioses, but there was a general trend toward higher RE with lower temperatures. The ratio of CO2 evolution: C2H2 reduction (mol/mol) in nodulated roots infected with CC814s was constant (ca 10 CO2/C2H2) between 5°C and 30°C, whereas in plants infected with NZP2037 it reached a minimal value of 3.3 CO2/C2H2 at 10°C and was 19 CO2/C2H2 at the growing temperature (25°C).

Characteristics and nitrogenase activity (acetylene reduction) of microaerophilic bacteria from roots of aquatic angiosperms

Three strains of microaerophilic, N2-fixing bacteria isolated from Phalaris arundinacea L, Polygonum amphibium L, and Phragmites australis (Cav.) Trin. were tentatively identified as Azospirillum or Campylobacter. N2 fixation was studied by the C2H2 reduction and Kjeldahl digestion methods. Starter nitrogen-amended, semisolid agar mannitol medium supported higher growth and C2H2 reduction rates than similar media containing sugars or organic acids. The isolates reduced C2H2 maximally at between 25 and 27 °C, and at pH values between 5.6 and 7.3 compared with an optimum pH of 5.5 for C2H2 reduction by their habitat plant organs incubated under similar conditions. Isolates tolerated elevated levels of [Formula: see text] with concentrations of up to 57 μM enhancing C2H2 reduction and growth. C2H2-reducing phosphorus-starved cells responded to added [Formula: see text] after a lag (2.5–4 h).

Low root temperatures and nitrogenase activity in soybean

A continuously flowing open gas exchange system was used to monitor the rates of CO2 evolution, C2H2 reduction, and H2 evolution from intact nodulated soybean ('Harosoy 63') roots, while root temperatures were dropped from the growing temperature (25 °C) to 10 °C at the rate of 5 °C/h. Four Rhizobium japonicum inoculants were used, three of which (type S (Nitragin Co.), USDA 16, USDA 35) displayed net H2 evolution while the fourth (USDA 110) did not evolve H2. Between 25 and 15 °C, values for Q10 in the H2-evolving symbioses ranged from 2.0 to 2.7 for CO2 evolution. 1.3 to 2.4 for C2H2 reduction, and 3.2 to 3.7 for H2 evolution. Nodulated roots of USDA 110 displayed the highest Q10 values for both CO2 evolution (Q10 = 2.9) and C2H2 reduction (Q10 = 15.2). The temperature profiles of these gas exchange measurements were used to calculate both the relative efficiency (RE = 1 − (H2 evolution/C2H2 reduction)) of N2 fixation and the ratio between CO2 evolution and C2H2 reduction at temperatures between 10 and 25 °C. The effect of short-term changes on the CO2/C2H2 ratio varied with symbiotic association. In the type-S symbiosis, long-term (96 h) treatments at low root temperature (9.5 °C) decreased the amount of CO2 evolved per C2H2 reduced to 35% of the value obtained at 25 °C. This study did not determine whether these observed changes were associated with N2 fixation or growth and maintenance of the nodulated root. In all H2-evolving symbioses, the RE increased from ca. 0.6 to 0.9 in response to a temperature drop from 25 to 10 °C. Studies of net H2 uptake and 3H2 exchange at 25 and 15 °C indicated that the observed changes in relative efficiency were due to variations in electron allocation by nitrogenase rather than a relative increase in uptake hydrogenase activity. The increase in RE above 0.75 suggested that lower temperatures may have altered the minimal nitrogenase electron allocation to less than one H2 per N2 fixed. This observed increase in RE with lower temperatures indicated that, in the symbioses studied, H2 evolution may have provided a buffer which permitted the maintenance of high levels of N2 fixation during short-term or diurnal fluctuations in soil temperature.

Acetylene Reduction in an Avicennia marina Community in Southern Australia

Rates of C2H2 reduction associated with decomposing leaf litter of Avicennia marina (white mangrove) reached 469 nmol C2H4 g-1 h-1 in air after 21 days of decomposition. Maximum rates of C2H2 reduction measured in sea water were 328 nmol C2H4 g-1 h-1 for leaf litter confined in mesh bags and 352 nmol C2H4 g-1 h-1 for unconfined leaf litter; the rates in sea water were always, and often substantially, lower than rates in air. Rates of decomposition and C2H2 reduction were less in leaf litter confined in mesh bags than in unconfined litter. Decomposing dead fibrous roots and live roots showed average rates of C2H2 reduction in interstitial water of only 9.3 and 3.5 nmol C2H4 g-1 h-1, respectively. During decomposition of leaf litter, the concentration of nitrogen increased; 41-64% of this increase was estimated to be the direct result of N2 fixation. No change in nitrogen concentrations occurred in decomposing dead roots or in live roots. N2 fixation associated with decomposing leaf litter and dead roots, and with live roots and surficial sediments, is thought to supply about 40% of the nitrogen required annually by the Avicennia marina trees. This estimate is relatively low in comparison with data for other marine wetlands, and it follows from the low rates of C2H2 reduction associated with the root system and the sediments.

Role of Acacia Spp. In Nutrient Balance and Cycling in Regenerating Eucalyptus regnans F. Muell. Forests. II. Field Studies of Acetylene Reduction

Rates of C2H2 reduction by nodules of Acacia dealbata growing in 2-year-old Eucalyptus regnans regeneration were determined at monthly intervals for 1 year under field conditions. Intact soil cores containing roots and nodules showed maximum values of 7.1 μmol h-1 g-1 fresh weight of nodules with a mean value of 4.4 μmol h-1 g-1 fresh weight. Nodule mass was clearly related to climatic conditions, being greatest when both temperature and moisture levels were favourable, and was probably depressed during the sampling period by the prevailing severe drought. From the measured rates of C2H2 reduction, estimated N2 fixation was 12-32 kg ha-1 year-1. Rates of N2 fixation as high as this confirm the role of acacias as fast-growing pioneer species important in maintaining the nitrogen pool of the forest ecosystem.

Effects of soil salinity and mineral nitrogen on the acetylene reduction activity of Trifolium subterraneum

Subterranean clover (Trifolium subrerraneum L.) was grown under controlled environmental conditions in pots containing a variety of soils. Non-destructive assays were used to measure rates of acetylene (C2H2) reduction by the plants. Soil mineral nitrogen (N) status and, to a lesser extent, soil salinity were found to be the most important soil properties relating to the development of C2H2reduction activity in subterranean clover. Application of KNO3 to pots resulted in an immediate decrease in the rate of C2H2reduction. A linear relationship was found between the loss in C,H, reduction activity and the amount of N applied up to 40 mg N kg-1 soil. Addition of salt (NaCl) to pots caused an immediate decrease in C2H2 reduction activity, followed by a gradual recovery. A linear relationship was found between the loss in C2H2 reduction activity and the level of soil salinity induced by salt addition. The loss in C2H2reduction \vas reflected in the lower total N content of the plants when soil salinity levels exceeded 4 mS cm-1.

Variation in ability of Rhizobium leguminosarum isolates to fix dinitrogen symbiotically in the presence of ammonium nitrate

To assess the variation in symbiotic effectiveness of N2-fixing Rhizobium leguminosarum isolates in the presence of NH4NO3, peas were inoculated with 38 isolates and grown for 4 weeks with weekly additions of 2 mM NH4NO3. Acetylene reduction was inhibited relative to a N-free control in all isolates but the inhibition varied from 60 to 100%. There were also significant differences (p = 0.05) between isolates with varying inhibition of C2H2 reduction in H2 uptake, H2 evolution, and leghemoglobin content. Ten isolates were selected for further study of the importance of NH4NO3 concentration (0, 1,2, and 5 mM) and plant growth stage (3,4, and 5 weeks) to characters associated with N2 fixation and plant growth. Plant age, NH4NO3 treatment, and isolate were all significant sources of variation (p ≤ 0.01) for each character measured but interactions between these factors were also significant. There were significant differences between isolates in the rate of decrease in C2H2 reduction with increasing NH4NO3 concentration (C2H2 reduction responsiveness to NH4NO3) and this responsiveness term was correlated with C2H2 reduction rates at 2 and 5 mM NH4NO3 (p = 0.01). Acetylene reduction rates were correlated with shoot dry weights at 0.1, and 2 mM NH4NO3 (p = 0.001). These results suggest the possibility of enhancing N2 fixation in peas through selection of rhizobia with maximum effectiveness at low levels of combined nitrogen.

Dinitrogen fixation by a mature Ceanothus velutinus (Dougl.) stand in the Western Oregon Cascades

Dinitrogen fixation was measured in a 17-year-old snowbrush, Ceanothus velutinus var. velutinus (Dougl.), stand in the Western Oregon Cascades. Diurnal and seasonal rates of nitrogenase activity were measured in the field using the C2H2 reduction technique. Snowbrush had a total biomass, estimated with equations developed, of 42 680 kg∙ha−1, including 750 kg∙ha−1 of nodule biomass. Snowbrush fixed N2 for approximately 240 days annually. Except during precipitation events or periods of low xylem pressure potentials, C2H2 reduction rates in the summer and fall were significantly correlated with soil temperature (R2 = 0.93**, n = 6). A diurnal variation in nitrogenase activity also was measured. The annual N2 fixation rate was estimated at approximately 101 kg N∙ha−1 (C2H2). Sustained periods of precipitation suppressed nitrogenase activity and reduced the estimate by about 19%. The annual N2 fixation rate is higher than previously reported for other mature stands and primarily is attributed to the maintenance of a large nodule biomass.

High endogenous nitrogenase activity in isolated heterocysts of Anabaena sp. strain CA after nitrogen starvation.

Metabolically active heterocysts were isolated from a mutant of Anabaena sp. strain CA with fragile vegetative cells. Heterocysts isolated from cultures grown in 1% CO2 in air reduced C2H2 at 57 and 10 nmol of C2H2 per mg (dry weight) per min under H2 and Ar, respectively. However, if whole filaments were sparged with 1% CO2 in 99% Ar for 12 h before heterocyst isolation, these heterocysts showed C2H2 reduction rates of 83 nmol of C2H4 per mg (dry weight) per min under either H2 or Ar, or 40% the activity of whole filaments grown in 1% CO2 in air. Heterocysts isolated from cultures sparged with 100% Ar or 1% CO2 in 99% N2 had the same C2H2 reduction pattern as heterocysts from cultures grown in 1% CO2 in air, i.e., low activity under Ar and high activity under H2. Labeling of whole filaments incubated with NaH14CO3 for 12 h under 1% CO2 in air or 1% CO2 in 99% Ar resulted in a twofold higher accumulation of 14C-labeled compounds in vegetative cells and heterocysts of Ar-incubated cells. Our results suggest that during incubation under 1% CO2 in 99% Ar, presumably a nitrogen starvation condition, continuing photosynthetic fixation of CO2 leads to accumulation of material(s) in the heterocysts that supports a high, persistent endogenous rate of C2H2 reduction. This material appears to be, in part, glycogen.

Nitrogen fixation by white clover (Trifolium repens) in grasslands on soils contaminated with cadmium, lead and zinc

SummaryThe nitrogenase activity (potential nitrogen fixation) of a mixed white clover‐grass sward growing on a range of soils containing up to 216 μg g−1 Cd, 30 000μg g−1 Pb and 20 000 μg g−1 Zn was determined using the acetylene‐reduction assay. The plants were incubated in situ using an intact soil‐core technique. Little change in the rate of C2H2 reduction was observed during the daylight hours although a marked seasonal fluctuation was found, the maximum activity during the spring and declining to 20% of this by the autumn. Some reductions of nodule and plant size, and in nitrogenase activity, was observed in the most heavily contaminated sites. These effects were small; the plants and nodules otherwise appeared healthy. The potential for nitrogen fixation was equally high in all the sites at up to 80 g N ha−1 h−1 in the spring.

Effects of pea (Pisum sativum) genotypes and Rhizobium leguminosarum strains on N2(C2H2) fixation and growth

Factors affecting the efficiency of selection for increased N2(C2H2) fixation in Pisum sativum L. were examined. Ten genotypes, previously selected for variability in fixation rates when inoculated with commercial (mixed strain) Rhizobium leguminosarum, were examined using pure strain (128C52 and 10004) inocula. Strains and genotypes both affected fixation rates, but genotype × strain interaction was also found. Increases in relative growth rate (RGR) were associated with increases in fixation across strains but not with increases in fixation across genotypes. These differences in the response to changes of strain and genotype were further studied over a 4-week period using genotypes specifically selected for high or low rates of C2H2 reduction with both strains. Strain 128C52 and the genotypes selected for high rates of C2H2 reduction produced greater maximum rates of C2H2 reduction than their low counterparts, but the strains also differed in the time at which fixation began. Plants inoculated with strain 128C52 showed significant rates 3–4 days before those inoculated with strain 10004. This earlier availability of nitrogen in the former may have alleviated the early nitrogen starvation and hence increased RGR. All differences in fixation rate were related to nodulation. Although early nodulation and fixation are apparently important to plant growth under N-free conditions, the expression of genotype or strain variability might be reduced by combined N in the field owing to its inhibitory effect on fixation.

Isolation and characterization of heterocysts from Anabaena sp. strain CA

A comparative study has been made on the pigment composition and nitrogenase activity of whole filaments and isolated beterocysts from a mutant strain of Anabaena CA. The whole cell absorption spectra of intact filaments and isolated heterocysts showed close resemblance especially between 550–700 nm region. On a quantitative basis the chlorophyll a content was found almost equal between the vegetative cell and heterocyst but the c-phycocyanin content in the heterocyst was about 1/2 that of the vegetative cell. The purification of the phycobiliprotein on DEAE-cellulose showed the presence of c-phycocyanin (γmax 615 nm) and allophycocyanin (γmax 645 nm, shoulder 620 nm). Isolated heterocysts under H2 showed acetylene reduction rates of 57 nmol C2H4/mg dry wt·min (342 μmol C2H4/mg chl a·h), whereas intact filaments reduced at the rate of 18 nmol C2H4/mg dry wt·min (108 μmol C2H4/mg chl a·h). This rate accounts for 30% recovery of nitrogenase activity in isolated heterocysts compared to whole filaments. The activity was strictly light dependent and was linear under H2 for more than 3 h. Addition of as little as 5% H2 under argon stimulated the C2H2 reductionseveral fold. The acetylene reduction (nitrogenase activity) also showed tolerance to 5% added O2 either under H2 or argon. The results suggest that the heterocyst of Anabaena CA-V is different in some characteristics (viz., higher endogenous C2H2 reduction rate, prolonged activity and higher levels of phycobiliproteins) than those reported in other Anabaena species.

Host Plant and Rhizobium Effects on Acetylene Reduction in Alfalfa during Regrowth 1

The effects of host plant and Rhizobium strain variation on apparent N2 fixation (C2H2 reduction) and total nonstructural carbohydrate (TNC) of the root system during regrowth of alfalfa (Medicago sativa L.) were studied to determine the prevalence of the known decline in C2H2-reduction activity following shoot harvest. Harvest at 10% bloom in ramets of a ‘Dohfari’ clone inoculated with Rhizobium meliloti strain 102F28 decreased shoot carbon exchange rate (CER) 99% immediately, but C2H2 reduction declined only 75% in the following 3 days. Thereafter, CER and C2H2 reduction increased in parallel during shoot regeneration. Those data provided circumstantial evidence that carbohydrates stored in roots or root nodules were used to maintain low rates of C2H2 reduction during the intial period of regrowth. In experiments with seedlings of 10 alfalfa cultivars inoculated with R. meliloti 102F28 and in trials with Dohfari and ‘Vernal’ clones inoculated with one of 10 different R. meliloti strains, shoot harvest produced a significant (p ≤0.001) decline in C2H2 reduction within 3 days to values ranging from 4-30% of the original rate. Root system TNC generally declined over the same period, but in only two of the 30 host-symbiont combinations examined was that change significant (p ≤0.05). No significant correlation was observed between C2H2-reduction rate and root system TNC in the same plants at harvest (day 0) or on day 3 in any host-symbiont combination. Results from this study suggest that although nonstructural carbohydrates are consumed in alfalfa roots during defoliation, the), are not used to maintain maximum (day 0) rates of N2 fixation during the early stages of regrowth in widely different host-symbiont combinations. This evidence is consistent with the concept that the recovery of N2 fixation following shoot harvest depends upon the regrowth of new shoots.

Nitrate reduction and nitrogenase activity in excised corn roots

Root segments obtained from field-grown corn plants were divided into small sections and assayed for acetylene (C2H2) reduction. After overnight preincubation, C2H2 reduction started immediately and linear rates were observed for several hours. Addition of NO3− (1 mM) delayed the start of C2H2 reduction but linear rates followed thereafter. A similar pattern was observed with excised roots inoculated with Azospirillum lipoferum.Addition of malate did not eliminate the NO3− effect. Inoculation with A. lipoferum resulted in relatively large rates of C2H2 reduction and a shorter lag than normal (4 to 8 h). A transient accumulation of NO2− accompanied the disappearance of NO3− and C2H2 reduction started only after all the NO2− had been reduced. A very sizable fraction of the reduced NO3− and NO2− was accounted for as N2O accumulation. Thus, the results provided support for the in situ operation of both N2 fixation and denitrification in A. lipoferum – corn root associations and it is proposed that mature corn roots are able to provide sufficient carbon to support both processes.

The Azolla-Anabaena azollae Relationship

Visible absorption spectra are presented for the Azolla caroliniana Willd.-Anabaena azollae Strass. association and the individual partners. Although absorption by the phycobiliproteins of the endophytic cyanobacterium clearly complements the absorption by the fern pigments, their contribution to the absorption spectrum of the association is effectively concealed by the preponderance of the Azolla pigments. Action spectra for nitrogenase-catalyzed C(2)H(2) reduction in both the Azolla-Anabaena association and the endophytic Anabaena demonstrate that quanta absorbed by the phycobiliproteins is as effective as that absorbed by chlorophyll a in driving this photosystem I-linked process. Under anaerobic conditions, the inhibition of photosystem II activity by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron did not selectively decrease the relative quantum yields in the region of phycobiliprotein absorption. At the well-below saturating light intensities used for the action spectra studies, the absolute rates of C(2)H(2) reduction were increased uniformly via respiratory-linked processes under aerobic conditions. The occurrence of phycobiliproteins in heterocysts of the endophytic Anabaena was demonstrated using fluorescence microscopy of intact filaments. Fluorescence micrographs of Anabaena cylindrica filaments are presented for comparison.

Enzymes of Purine Biosynthesis and Catabolism in Glycine max

During the period examined from 12 to 63 days after planting, the ureides, allantoin and allantoic acid, were the predominant nitrogenous solutes in the xylem exudate of soybeans (Glycine max [L.]) growing solely on symbiotically fixed nitrogen, accounting for approximately 60% and greater than 95% of the total nitrogen in the xylem exudate before and after the onset of active nitrogen fixation, respectively. For plants between 18 and 49 days of age, the apparent rate of ureide export estimated from concentrations of ureides in xylem exudate collected over a period of one hour was closely related to the rate of nitrogen fixation estimated from measurements of C(2)H(2) reduction by nodulated root systems. After this time, the apparent rate of ureide export per plant continued to increase, reaching a maximum value at day 63 of 12 micromoles per plant per hour, even though the rate of C(2)H(2) reduction per plant declined approximately four-fold. The most probable pathway for the biosynthesis of ureides involves the catabolism of purines. The levels of phosphoribosylpyrophosphate (PRPP) synthetase, which catalyzes the formation of the PRPP required for purine synthesis, increased in parallel with the rates of nitrogen fixation (C(2)H(2)) from day 18 reaching a maximum value of 13.9 micromoles per plant per hour at day 49, and then both activities declined rapidly. During the period of active nitrogen fixation the ratio of PRPP synthesis estimated from measurements of PRPP synthetase activity in cell-free extracts to the apparent rate of ureide export was between 1 and 2. The activities of the enzymes of purine catabolism, xanthine dehydrogenase, uricase, and allantoinase, increased in parallel with the increases in nodule mass and the export of ureides with maximum activities of 13, 119, and 79 micromoles per plant per hour, corresponding with apparent rates of ureide export in the range of 9.5 to 11.9 micromoles per plant per hour. These results demonstrate that there is a close association between nitrogen fixation, PRPP synthetase activity, and ureide export in soybeans and support the proposal that recently-fixed nitrogen is utilized in the de novo synthesis of purines which are subsequently catabolized to produce the ureides.

The isolation of effective and ineffective mutants of cowpea rhizobium

A strain of cowpea Rhizobium was mutagenised and two ineffective mutants, M1 and M2, and an effective mutant, M3, were isolated. M1 produced more, but smaller nodules than the wild-type; these nodules lacked leghaemoglobin. M2 and the parental strain had similar nodulation characteristics, both forming large pink nodules. Plants inoculated with M3, nodulated earlier, produced more nodules (58%), had increased dry weights (26%) and the excised roots expressed greater acetylene (C2H2) reducing activity (39%) than plants inoculated with the wild-type. When competing with an indigenous population of effective rhizobia for nodule sites, M3 produced a higher proportion of the nodules (70–80%) than the parental strain (40–53%). M3 and the parental strain exhibited comparable rates of asymbiotic C2H2 reduction when grown on a defined medium, whereas M1 and M2 were inactive. The symbiotic properties of the mutants were unchanged after their reisolation following plant passage.

Nodulation and N2-fixation byInga jinicuil, a woody legume in coffee plantations

Nodule biomass and yearly C2H2 reduction rates are reported forInga jinicuil, a leguminous tree used for shade in Mexican coffee plantations. Annual fixation by this species approximates 35 kg ha−1; which, when compared to nitrogen additions from fertilizers, represents an important nitrogen input to the coffee ecosystem.

Comparing Time Course Profiles of Immediate Acetylene Reduction by Grasses and Legumes

The time course profiles of C(2)H(2) reduction by intact Scirpus olneyi (bulrush), Oryza sativa (rice) and Spartina alterniflora (cordgrass) with roots in atmospheres of N(2) and 30-day-old Glycine max (soybean) in air were all immediately linear. This is the first report of immediately linear rates of C(2)H(2) reduction by grass roots removed from soil. The immediately linear profile of C(2)H(2) reduction by soil-free grass roots was achieved by preventing contact between the roots and air. Roots of soybeans and S. olneyi receiving pretreatments of O(2) above normal environmental levels for 15 min before assay exhibited a short delay in C(2)H(2) reduction. These initially nonlinear rates of C(2)H(2) reduction are attributable to transient O(2) inhibition of nitrogenase. Initial nonlinear rates of C(2)H(2) reduction were also observed with immature soybean plants and with intact plant assays of O. sativa and S. olneyi in which C(2)H(2) was injected into cylinders surrounding the plant tops. These results indicate that, apart from O(2) inhibition of nitrogenase, the diffusion of C(2)H(2) and C(2)H(4) between the nitrogen-fixing sites and the sampling ports may cause initial nonlinear rates of C(2)H(2) reduction. We conclude that in situ plant-associated nitrogenase activity should result in immediate reduction of C(2)H(2) and that linear rates are observed when the proper assay conditions are used. Our data suggest that nitrogen fixation is closely associated with the roots of S. olneyi, O. sativa, and S. alterniflora growing in salt marsh sediment.