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1.
Excised roots of Spartina alterniflora Loisel. and corn reduced acetylene in air without the previously reported period of zero activity lasting 8 to 18 hours. The profiles of acetylene-dependent ethylene accumulation by excised roots and intact plants of S. alterniflora were similar. No significant change in the number of bacteria associated with the roots was detectable during the assay. Most of the nitrogenase activity was detected in the roots and rhizomes of the plants. The salt marsh sediment also was capable of reducing acetylene. Additional damage to roots by washing and cutting increased the rate of acetylene reduction with samples incubated in air. Low concentrations of nitrate significantly inhibited the nitrogenase activity associated with the sediment and excised roots, but not with intact plants. Rates of acetylene reduction by excised corn roots were low. Oxidation and endogenous production of ethylene in the absence of acetylene were negligible. Measurements made with excised grass roots as described probably reflect the occurrence and magnitude of nitrogenase activity associated with the plants in the field. 相似文献
2.
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 C 2H 4 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 C 2H 4 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 N 2-fixing bacteria associated with papyrus roots showed the presence of high numbers of diazotrophs (5.4 × 10 6 CFU g d.wt. roots -1). Fewer numbers of N 2-fixing bacteria were detected in the sediments (1.9 × 10 3-3.2 × 10 4 CFU g d.wt. sediment -1). 相似文献
3.
Summary Aerobic and anaerobic N 2-fixing bacteria developed in the rhizosphere of barley seedlings and exhibited N 2ase activity when seedlings were grown in sterilized sand-nutrient cultures containing low levels of combined nitrogen. The
source of the N 2-fixing bacteria appeared to be the seed. Average daily rates up to 0.9 μmoles C 2H 4 h −1 g −1 dry root tissue were measured, but the intensity of the activity was affected by moisture levels and concentration of combined
N in the rhizosphere. Removal and washing of the roots did not remove the activity, and roots remained active even after surface-sterilization.
An unidentified aerobic N 2-fixing bacterium was isolated from the rhizoplane of active barley roots. Inoculation of barley seedlings with the aerobic
N 2-fixing bacterium enhanced N 2ase activity of excised roots 10-fold, with average rates of 0.9, 1.1 and 1.3 μmoles h −1 g −1 dry root assayed under pO 2 of 0.01, 0.02 and 0.04 atm respectively. The aerobic N 2-fixing bacterium also exhibited N 2ase activity when inoculated into the rhizosphere of oat, rice and wheat seedlings. Microscopic observations of sterilized
live and stained barley roots suggest that the aerobic N 2-fixing bacterium is an endophyte which infects root tissue and metamorphoses into vesicle-like structures. 相似文献
4.
Summary Heterotrophic dinitrogen fixation in root associations of successional stages of the tropical mangrove plant community at
the Ganges river estuary in India was investigated by excised-root acetylene reduction assay, and enumeration and identification
of diazotrophic bacteria from sediment, root and tidal water samples. High to very high rates of nitrogenase activity (64–130
nmol C 2H 4/g dry root/h) were associated with washed excised roots of seven common early-successional mangrove species at the inundated
swamps. Declining, late-successional mangroves at the occasionally inundated ridges had considerably lower values and the
“declined” mangroves and other non-littoral species at embankment protected highlands had very low to insignificant values
of root nitrogenase activity. Total and inorganic nitrogen contents of the mangrove sediments were low and were positively
related to the stages of physiographic succession. Plant-associated sediments of particularly the old formation swamps had
very high C/N ratios. Nine isolates of nitrogen-fixing bacteria belonging to all known O 2 response groups were distinguished from a large population of diazotrophs associated with roots of mangroves and other associate
plant species of the community. The isolates differed with respect to their N 2-fixation efficiency and halotolerance in pure culture. There was no specificity of any of the bacterial isolates to any of
the plant species of the community but a higher number of efficient isolates were seen to be associated with mangroves at
the swampy succession. Sediment-free tidal water also contained a large population of microaerophilic and anaerobic N 2-fixing bacteria. 相似文献
5.
A heterotrophic semisolid medium was used with two sensitive assay methods, C 2H 2 reduction and O 2-dependent tritium uptake, to determine nitrogenase and hydrogenase activities, respectively. Organisms known to be positive for both activities showed hydrogenase activity in both the presence and absence of 1% C 2H 2, and thus, it was possible to test a single culture for both activities. Hydrogen uptake activity was detected for the first time in N 2-fixing strains of Pseudomonas stutzeri. The method was then applied to the most-probable-number method of counting N 2-fixing and H 2-oxidizing bacteria in some natural systems. The numbers of H 2-oxidizing diazotrophs were considerably higher in soil surrounding nodules of white beans than they were in the other systems tested. This observation is consistent with reports that the rhizosphere may be an important ecological niche for H 2 transformation. 相似文献
6.
The time course profiles of C 2H 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 2H 2 reduction by grass roots removed from soil. The immediately linear profile of C 2H 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 2H 2 reduction. These initially nonlinear rates of C 2H 2 reduction are attributable to transient O 2 inhibition of nitrogenase. Initial nonlinear rates of C 2H 2 reduction were also observed with immature soybean plants and with intact plant assays of O. sativa and S. olneyi in which C 2H 2 was injected into cylinders surrounding the plant tops. These results indicate that, apart from O 2 inhibition of nitrogenase, the diffusion of C 2H 2 and C 2H 4 between the nitrogen-fixing sites and the sampling ports may cause initial nonlinear rates of C 2H 2 reduction. We conclude that in situ plant-associated nitrogenase activity should result in immediate reduction of C 2H 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. 相似文献
7.
The N 2-fixing legume nodule requires O 2 for ATP production; however, the O 2 sensitivity of nitrogenase dictates a requirement for a low pO 2 inside the nodule. The effects of long term exposures to various pO 2s on N 2[ C2H2] fixation were evaluated with intact soybean ( Glycine max [L.] Merr., var. Wye) plants. Continuous exposure of their rhizosphere to a pO 2 of 0.06 atmospheres initially reduced nitrogenase activity by 37 to 45% with restoration of original activity in 4 to 24 hours and with no further change in tests up to 95 hours; continuous exposure to 0.02 atmosphere of O 2 initially reduced nitrogenase activity 72%, with only partial recovery by 95 hours. Similar exposures to a pO 2 of 0.32 atmospheres had little effect on N 2[ C2H2] fixation; a pO 2 of 0.89 atmospheres initially reduced nitrogenase activity by 98% with restoration to only 14 to 24% of that of the ambient O 2 controls by 95 hours. Re-exposure to ambient pO 2 of plants adapted to nonambient pO 2s reduced N 2[ C2H2] fixation to similar magnitudes as the reductions which occurred upon initial exposure to variant pO 2 conditions, and a time period was required to readapt to ambient O 2. It is concluded that the N 2[ C2H2]-fixing system of intact soybean plants is able to adapt to a wide range of external pO 2s as probably occur in soil. We postulate that this occurs through an undefined mechanism which enables the nodule to maintain an internal pO 2 optimal for nitrogenase activity. 相似文献
8.
Soluble root N concentrations of corn, sorghum, pearl millet, rice, wild rice, and soybeans were determined and related to measurements of nitrogenase activity and changes in availability of combined N to plants. In corn, sorghum, and pearl millet, applications of fertilizer N increased soluble root N concentrations, but root-associated nitrogenase activity was negligible in control and treated plants. Applications of NH 4NO 3 to rice increased the water soluble root N concentrations and inhibited root-associated nitrogenase activity. In wild rice, root-associated nitrogenase activity was absent during vegetative growth and developed at the reproductive growth stage. The soluble root N concentration decreased progressively as wild rice grew indicating that the availability of combined N in the root environment declined. Therefore, development of nitrogenase activity in wild rice is associated with the change in availability of combined N in the root environment. The development of nitrogenase activity in wild rice was probably not due to colonization of roots by N 2-fixing bacteria because most probable numbers of recovery did not significantly vary throughout the plants' growth cycle. In field-grown soybeans with or without fertilizer N application, we also observed a relationship between a decrease in soluble root N concentration and the development of nitrogenase activity. 相似文献
9.
Numbers and possible locations of N 2-fixing bacteria were investigated in roots of Spartina alterniflora Loisel, which support nitrogenase activity in the undisturbed native habitat. N 2-fixing bacteria were recovered in cultures both from S. alterniflora roots and from the surrounding sediment, and they formed a greater proportion of the bacteria recovered from root homogenates than from salt-marsh sediment. N 2-fixing bacteria were recovered in high numbers from the rhizoplane of S. alterniflora after roots were treated with 1 or 5% chloramine-T for 1 h or with 1% NaOCl for 1 or 2 h. Immersing S. alterniflora roots in 5% NaOCl for 1 h was more effective in distinguishing bacteria inside the roots since this treatment nearly eliminated N 2-fixing bacteria recoverable from the rhizoplane, although high numbers of N 2-fixing bacteria were recovered from homogenates of roots treated with 5% NaOCl for 1 h. However, this treatment was less effective with roots of Zea mays L. (Funks G4646) and Sorghum bicolor (L.) Moench (CK-60 A), indicating that techniques to surface sterilize roots should be evaluated for different plants. Bacteria were observed by light and electron microscopy inter- and intracellularly in the cortex and in the aerenchyma of S. alterniflora roots. This study clearly shows that bacteria, including N 2 fixers, colonize the interior of roots of S. alterniflora growing in a Chesapeake Bay, Maryland, salt marsh. 相似文献
10.
N 2(C 2H 2)-fixing bacteria were isolated from the rhizosphere of various cereals and forage grasses grown in the greenhouse and from the rhizosphere of field-grown wheat in Sweden. All 46 isolates from the greenhouse plants lost their nitrogenase activity during purification. By imposing a stronger selection pressure, we obtained pure isolates with nitrogenase activity from field-grown wheat. Some isolates were identified as Enterobacter agglomerans and Bacillus polymyxa, but several bacteria of uncertain taxonomy also occurred. One of the isolates grew and reduced acetylene only in the presence of other bacteria or certain vitamins. Species of Azotobacter or Azospirillum could not be isolated from the rhizosphere of any of the cereals and forage grasses studied. 相似文献
11.
Rhizobium japonicum 122 DES bacteroids from soybean nodules possess an active H 2-oxidizing system that recycles all of the H 2 lost through nitrogenase-dependent H 2 evolution. The addition of 72 μM H 2 to suspensions of bacteroids increased O 2 uptake 300% and the rate of C 2H 2 reduction 300 to 500%. The optimal partial pressure of O 2 was increased, and the partial pressure of O 2 range for C 2H 2 reduction was extended by adding H 2. A supply of succinate to bacteroids resulted in effects similar to those obtained by adding H 2. Both H 2 and succinate provided respiratory protection for the N 2-fixing system in bacteroids. The oxidation of H 2 by bacteroids increased the steady-state pool of ATP by 20 to 40%. In the presence of 50 mM iodoacetate, which caused much greater inhibition of endogenous respiration than of H 2 oxidation, the addition of H 2 increased the steady-state pool of ATP in bacteroids by 500%. Inhibitor evidence and an absolute requirement for O 2 indicated that the H 2-stimulated ATP synthesis occurred through oxidative phosphorylation. In the presence of 50 mM iodoacetate, H 2-dependent ATP synthesis occurred at a rate sufficient to support nitrogenase activity. The addition of H 2 to H 2 uptake-negative strains of R. japonicum had no effect on ATP formation or C 2H 2 reduction. It is concluded that the H 2-oxidizing system in H 2 uptake-positive bacteroids benefits the N 2-fixing process by providing respiratory protection of the O 2-labile nitrogenase proteins and generating ATP to support maximal rates of C 2H 2 reduction by oxidation of the H 2 produced from the nitrogenase system. 相似文献
12.
Nitrogenase (EC 1.7.99.2) activity in pea ( Pisum savitum) nodules formed after infection with Rhizobium leguminosarum (lacking uptake hydrogenase) was measured as acetylene reduction, H 2 evolution in air and H 2 evolution in Ar:O 2. With detached roots the relative efficiency, calculated from acetylene reduction, showed a decrease (from 55 to below 0%) with increasing temperature. With excised nodules and isolated bacteroids similar results were obtained. However, the relative efficiency calculated from H 2 evolution in Ar:O 2 was unaffected by temperature. Measurements on both excised nodules and isolated bacteroids showed a marked difference between acetylene reduction and H 2 evolution in Ar:O 2 with increased temperature, indicating that either acetylene reduction or H 2 evolution in Ar:O 2 are inadequate measures of nitrogenase activity at higher temperature. 相似文献
13.
High rates of acetylene (C 2H 2) reduction (nitrogenase activity) were observed in woodroom effluent from a neutral sulfite semi-chemical mill under aerobic (up to 644 nmol of C 2H 4 produced per ml per h) and under anaerobic (up to 135 nmol of C 2H 4 produced per ml per h) conditions. Pasteurized effluent developed C 2H 2 reduction activity when incubated under anaerobic but not under aerobic conditions. Activities were increased by addition of 0.5 to 3.0% glucose or xylose. Enrichment and enumeration studies showed that N 2-fixing Azotobacter and Klebsiella were abundant, and N 2-fixing Bacillus was present. Of 129 isolates of Klebsiella from pulp mills, lakes, rivers, and drainage and sewage systems, 32% possessed nitrogen-fixing ability. 相似文献
14.
The methodology, characteristics and application of the sensitive C 2H 2-C 2H 4 assay for N 2 fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N 2ase-catalyzed reduction of C 2H 2 to C 2H 4, gas chromatographic isolation of C 2H 2 and C 2H 4, and quantitative measurement with a H 2-flame analyzer. As little as 1 μμmole C 2H 4 can be detected, providing a sensitivity 10 3-fold greater than is possible with 15N analysis. A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C2H2-reducing activity in the field. Applications to field samples included an evaluation of N2 fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N2 fixation rate of 30 to 33 kg N2 fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N2 fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N2 fixing activity of soybeans. The validity of measuring N2 fixation in terms of C2H2 reduction was established through extensive comparisons of these activities using defined systems, including purified N2ase preparations and pure cultures of N2-fixing bacteria. With this assay it now becomes possible and practicable to conduct comprehensive surveys of N2 fixation, to make detailed comparisons among different N2-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N2 fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N2 fixation reaction. 相似文献
15.
Summary Previous investigations indicated some forage grass roots in Texas are heavily colonized with N 2-fixing bacteria. The most numerous N 2-fixing bacteria were in the genera Klebsiella and Enterobacter. In the present investigation inoculation experiments were
conducted using 18 isolates of these bacteria to determine if a N 2-fixing association could be established between the bacteria and the grasses Cynodon dactylon and Panicum coloratum. Plants were grown in soil for approximately 5 months in a greenhouse and were measured periodically for dry matter, nitrogen
accumulation, and acetylene reduction activity. Results of the investigation indicated that 25% of the plant-soil systems
were active in acetylene reduction and the activity was high enough to indicate agronomically significant quantities of N 2 were being fixed (>8kg N ha −1). However, plant systems extrapolated to fix>8 kg N ha −1 contained less nitrogen and accumulated less dry matter than plants less active in acetylene reduction. Inocula could not
be re-isolated from healthy grass roots indicating that the N 2-fixing activity may have not have been closely assiciated with plant roots. Future research is needed to determine factors
limiting colonization of grass roots. 相似文献
16.
A technique for C 2H 2-reduction assay on intact plants of Alnus incana (L.) Moench was evaluated. Cloned plants were grown, in pots, on fine gravel. During assay only the pot was inserted into a Perspex incubation chamber of simple construction. The incubation volume was rather small, plants with various shoot heights could be used, and the shoot was not exposed to the C 2H 4 produced. Intact plants showed high and constant C 2H 2-reduction rates during several hours of incubation. In comparison, excised nodulated roots conventionally incubated in test tubes showed low and decreasing rates, due to removal of the photo-synthesizing shoot and injury to the root nodules when drawn from the pot. Repeated nitrogenase activity assays on the same intact individual plants did not affect growth. The technique thus proved useful in studies. where repeated nitrogenase activity measurements are important. 相似文献
17.
Soybean ( Glycine max [L.] Merr. cv Davis) was grown in a split-root growth system designed to maintain control of the root atmosphere. Two experiments were conducted to examine how 80% Ar:20% O 2 (Ar:O 2) and air (Air) atmospheres affected N assimilation (NH 4NO 3 and N 2 fixation) and the partitioning of photosynthate to roots and nodules. Application of NH 4NO 3 to nonnodulated half-root systems enhanced root growth and root respiration at the site of application. A second experiment applied Ar:O 2 or air to the two sides of nodulated soybean half-root systems for 11 days in the following combinations: (a) Air to both sides (Air/Air); (b) Air to one side, Ar:O 2 to the other (Air/Ar:O 2), and (c) Ar:O 2 to both sides (Ar:O 2/Ar:O 2). Results indicated that dry matter and current photosynthate ( 14C) were selectively partitioned to nodules and roots where N 2 was available. Both root and nodule growth on the Air side of Air/Ar:O 2 plants was significantly greater than the Ar:O 2 side. The relative partitioning of carbon and current photosynthate between roots and nodules on a half-root system was also affected by N 2 availability. The Ar:O 2 sides partitioned relatively more current photosynthate to roots (57%) than nodules (43%), while N 2-fixing root systems partitioned 36 and 64% of the carbon to roots and nodules, respectively. The Ar:O 2 atmosphere decreased root and nodule respiration by 80% and nitrogenase activity by 85% compared to half-root systems in Air while specific nitrogenase activity of nodules in Ar:O 2 was 50% of nodules supplied Air. Results indicated that nitrogen assimilation, whether from N 2 fixation or inorganic sources, had a localized effect on root development. Nodule development accounted for the major decrease in total photosynthate partitioning to non-N 2-fixing nodules. Soybean compensates for ineffective nodulation by controlling the flux of carbon to ineffective nodules and their associated roots. 相似文献
18.
Nitrogenase (EC 1.7.99.2) activity (acetylene reduction) and nitrogen fixation ( 15N 2 fixation) were measured in cyanobacteria freshly isolated from the coralloid roots of Macrozamia riedlei (Fisch. ex Gaud.) Gardn. Light and gas phase oxygen concentration had marked interactive effects on activity, with higher (up to 100-fold) rates of acetylene reduction and 15N 2 fixation in light. The relationship between ethylene formation and N 2-fixation varied in the freshly isolated cyanobacteria from 4 to 7 nanomoles of C 2H 4 per nanomole 15N 2. Intact coralloid roots, incubated in darkness and ambient air, showed a value of 4.3. Maximum rates of nitrogenase activity occurred at about 0.6% O 2 in light, while in darkness there was a broad optimum around 5 to 8% O 2. Inhibition of nitrogenase, in light, by pO 2 above 0.6% was irreversible. Measurements of light-dependent O 2 evolution and 14CO 2 fixation indicated negligible photosynthetic electron transport involving photosystem II and, on the basis of inhibitor studies, the stimulatory effect of light was attributed to cyclic photophos-phorylation. Nitrogenase activity of free-living culture of an isolate from Macrozamia ( Nostoc PCC 73102) was only slightly inhibited by O 2 levels above 6% O 2 and the inhibition was reversible. These cells showed rates of light-dependent O 2 evolution and 14CO 2 fixation which were 100- to 200-fold higher than those by the freshly isolated symbiont. Furthermore, nitrogenase activity was dependent on both photosynthetic electron transport and photophosphorylation. These data indicate that cyanobacteria within cycad coralloid roots are differentiated specifically for symbiotic functioning in a microaerobic environment. Specializations include a high heterocyst frequency, enhanced permeability to O 2, and a direct dependence on the cycad for substrates to support nitrogenase activity. 相似文献
19.
The carbon cost of nitrogenase activity was investigated to determine symbiotic efficiency of the actinorhizal root nodule symbiosis between the woody perennial Alnus incana and the soil bacterium Frankia. Respiration (CO 2 production) and nitrogenase activity (H 2 production) by intact nodulated root systems were continuously recorded in short-term assays in an open-flow gas exchange system. The assays were conducted in N 2:O 2, thus under N 2-fixing conditions, in all experiments except for one. This avoided the declines in nitrogenase activity and respiration due to N 2 deprivation that occur in acetylene reduction assays and during extended Ar:O 2 exposures in H 2 assays. Two approaches were used: (i) direct estimation of root and nodule respiration by removing nodules, and (ii) decreasing the partial pressure of O 2 from 21 to 15% to use the strong relationship between respiration and nitrogenase activity to calculate CO 2/H 2. The electron allocation of nitrogenase was determined to be 0.6 and used to convert the results into moles of CO 2 produced per 2e – transferred by nitrogenase to reduction of N 2. The results ranged from 2.6 to 3.4mol CO 2 produced per 2e –. Carbon cost expressed as gC produced per gN reduced ranged from 4.5 to 5.8. The result for this actinorhizal tree symbiosis is in the low range of estimates for N 2-fixing actinorhizal symbioses and crop legumes. Methodology and comparisons of root nodule physiology among actinorhizal and legume plants are discussed. 相似文献
20.
Isolated soybean ( Glycine max [L.] Merr. cv Wilkin) bacteroids have O 2-dependent nitrogenase activity which is strongly inhibited by supraoptimal O 2 concentrations. Oxygen-inhibited nitrogenase activity is recovered by addition of 10 millimolar sodium succinate or by lowering the O 2 concentration. Brief treatment of roots of intact soybean plants with 1.0 atmosphere O2 reduces nitrogenase activity (C2H2). There is a rapid partial recovery of activity within 2 to 3 hours, and a slower return to near normal levels by 36 hours. The drop and recovery of nitrogenase activity is accompanied by a parallel drop and increase in root respiration. There is a direct relationship between the change in respiration and the change in acetylene reduction following O2 treatment. The O2-mediated changes in nitrogenase activity and root respiration are not affected by the planting medium. The ratio of the change in respiration to the change in nitrogenase activity was the same in 13 soybean cultivars. 相似文献
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