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1.
The determination of N2-fixation in grasses by the excised root assay is evaluated. Prolonged “pre-incubation” of excised sorghum roots caused the development of increased rates of N2-ase activity. The results indicated that damage of N2-ase by O2 is unlikely to cause a prolonged delay before C2H2 reduction. Washing the collected roots with distilled water to prevent dessication and “pre-incubating” them at 30°C caused proliferation of the N2-fixing microflora and synthesis of N2-ase. Growth of the N2-fixing microflora also occurred under C2H2 during the inactive period. The length of time before N2-ase activity commenced was probably related to the initial concentration of available combined N. The results indicate that C2H2 reduction measurements with the excised grass roots tested overestimate the rates of N2-fixation. The possible causes for the prolonged period before detection of N2-ase activity by excised grass roots are discussed.  相似文献   

2.
The composition of the microflora, N2-fixing bacteria particularly, in different soils cultivated with wheat in Egypt was investigated in some samples collected from the fields after applying the agricultural practices recommended for wheat cultivation and just before sowing. The influence of carbon sources, mineral nitrogen and water regimes on potential dinitrogen fixation (acetylene reduction assay) in soils was investigated. The bacterial population densities including-N2-fixing organisms were related to a number of environmental factors such as organic matter content. Among diazotrophs, Azotobacter spp. and Azospirillum spp. were encountered in higher densities in comparison with clostridia. Unamended soils showed a lower acetylene-reducing activity (0.5–61.5 nmoles C2H4 g?1 h?1). Addition of glucose (1% w/w) greatly enhanced such activity being the highest (86.9–2846.5 nmoles C2H4 g?1 h?1) in the clay soil with the highest organic carbon content (1.42%). Glucose amendment had no significant influence on acetylene reduction in the saline soil. N2-fixation in barley straw-amended (1%) soils was not much higher than in unamended soils. Concentrations of up to 70 ppm ammonium-nitrogen depressed N2-fixation in soils that received barley straw. Acetylene reduction in submerged soil increased after addition of cellulose. Non-flooded conditions favoured N2-fixation in the fertile clay soil amended with sucrose.  相似文献   

3.
Summary Acetylene reduction activity by Azospirillum brasilense, either free-living in soils or associated with wheat roots, was determined in a sterilised root environment at controlled levels of O2 tension and with different concentrations of mineral N. In an unplanted, inoculated soil nitrogenase activity remained low, at approximately 40 nmol C2H4 h-1 per 2kg fresh soil, increasing to 300 nmol C2H4 h-1 when malic acid was added as a C source via a dialyse tubing system. The N2 fixation by A. brasilense in the rhizosphere of an actively growing plant was much less sensitive to the repressing influence of free O2 than the free-living bacteria were. An optimum nitrogenase activity was observed at 10 kPa O2, with a relatively high level of activity remaining even at an O2 concentration of 20 kPa. Both NO inf3 sup- and NH inf4 sup+ repressed nitrogenase activity, which was less pronounced in the presence than in the absence of plants. The highest survival rates of inoculated A. brasilense and the highest rates of acetylene reduction were found in plants treated with azospirilli immediately after seedling emergence. Plants inoculated at a later stage of growth showed a lower bacterial density in the rhizosphere and, as a consequence, a lower N2-fixing potential. Subsequent inoculations with A. brasilense during plant development did not increase root colonisation and did not stimulate the associated acetylene reduction. By using the 15N dilution method, the affect of inoculation with A. brasilense in terms of plant N was calculated as 0.067 mg N2 fixed per plant, i.e., 3.3% of the N in the root and 1.6% in the plant shoot were of atmospheric origin. This 15N dilution was comparable to that seen in plants inoculated with non-N2-fixing Psudomonas fluorescens.  相似文献   

4.
A genotype effect on associative (rhizosphere) N2-fixation was observed with two cultivars of Sorghum bicolor (nutans) with a maximum rate of 8 μmol C2H4 · h?1 · plant?1 in one genotype compared to 0.9 μmol in the other. Characteristics of the high fixing genotype were a reduced transpiration rate, a lower number of stomata and increased root exudate production per gram root dry weight with higher concentration of dicarboxylic acids. The bacterial rhizosphere composition revealed a three times higher number of N2-fixing bacteria, a tenfold reduction of actinomycetes and a threefold reduction of Arthrobacter associated with the high fixing cultivar compared to the low fixing genotype. From these and other plant rhizospheres two new nitrogen fixing bacteria, Pseudomonas stutzeri and Erwinia herbicola, were characterized. With the N2-fixing bacteria Azospirillum brasilense and Klebsiella pneumoniae an enhancement of specific nitrogenase activity by aromatic compounds, for example phenolics, the herbicide alachlor and the insecticide carbofuran was demonstrated. An oscillating nitrogenase activity in Azospirillum brasilense under microaerobic conditions was found, resulting from an encystation and deencystation under those conditions. Experiments with wheat roots demonstrated that reduced oxygen tensions, essential for a maximum rhizosphere N2-fixation, reduced root growth significantly and altered the N-metabolism of the roots.  相似文献   

5.
The N2-ase activities of field-grown Brazilian grasses were measured with C2H2 reduction by soil cores containing the plants. C2H2 and C2H4 were observed to diffuse at similar rates through soil and equilibrated across the Brazilian soil in 3 h, but could take up to 30 h or more with some British soils. The diurnal fluctuation in the rates of N2-ase activity by Brachiaria mutica and Sorghum vulgare were similar and the variation in rate was correlated with soil temperature. Estimates of N2-fixation by measurement of C2H2 reduction by soil cores ranged from 14.7 to 51.4 g N ha?1 day?1 and were much lower than with “pre-incubated” excised roots from the cores or taken directly from the field. The merits of the soil core and the “pre-incubated” excised root assays are discussed. ft1|Present address: U.S. Department of Agriculture, Science and Education Administration, Agricultural Research, Northeastern Region, Room 309, Building 001, Beltsville, MD 20705, U.S.A.  相似文献   

6.
Sustainable cropping systems rely on a minimum of external inputs. In these systems N is largely acquired in animal manures and leguminous green manures. Little is known of how these organic forms of N fertilizer influence the presence and activity of free-living N2-fixing bacteria. High concentrations of inorganic N in soil inhibit N2-fixation in cyanobacteria and Azotobacter spp. It is likely that manure and fertilizer applications would result in concentrations of inorganic N capable of inhibiting N2 fixation and, ultimately, the presence of these organisms. We investigated the effect of synthetic and organic N fertilizer sources on the populations and N2-fixation potential of free-living N2-fixing bacteria in the Farming Systems Trial at the Rodale Research Institute. Field plots received the following N treatments prior to corn (Zea mays L.) production: (1) Legume rotations and green manures supplying about 165 kg N ha-1; (2) beef cattle manure applied at a rate of 220 kg N ha-1 (plus 60 kg N ha-1 from 1994 hay plow-down); or (3) fertilizer N (urea and NH4NO3) applied at a rate of 145 kg N ha-1. Soil samples were collected at two depths from corn plots four times during the growing season, and analyzed for soil moisture, soil pH, numbers of N2-fixing cyanobacteria and Azotobacter spp., extractable NH inf4 sup+ and NO inf3 sup- , and potentially mineralizable N. Soil samples collected in mid-July were analyzed for nitrogenase activity (by C2H2 reduction) and total C and N. Populations of Azotobacter spp. and cyanobacteria were influenced only slightly by treatment; however, cyanobacteria species composition was notably influenced by treatment. Nitrogenase activity in surface soils was greatest in legume-N plots and in subsurface plots levels were greatest in fertilizer-N plots. Populations and activity of free-living N-fixing bacteria appeared to be somewhat reduced in all plots as a result of low soil pH levels and high concentrations of inorganic N across all treatments. Annual applications of N to all plots resulted in high levels of potentially mineralizable N that in turn may have reduced non-symbiotic N2-fixation in all plots.  相似文献   

7.
Studies of the physiology of the Spirillum lipoferum recognized as the major organism responsible for N2-fixation in the roots of Digitaria decumbens cv transvala were performed in order to improve the methods of culture and help to explain the physiology of this N2-fixing grass bacterial association.Methods for isolation, purification and N2-fixation assays are described. Acetylene concentrations used for N2-ase activity measurements should be at least 12%. the Vmax of cultures in the log phase being at a pC2H2 of 0.12 atm and the apparent Km 0.022 atm. Optimal temperatures for N2-dependent growth are between 32 and 40°C. and little N2-fixation is observed below 24°C. At 42°C the N2-ase is inactivated. When cultures grown at 28 or 36°C are transferred to lower temperatures nitrogenase activity declines rapidly. One hour after transfer to 17°C activity is about half that before transfer and is maintained at this level for at least 8 h. After transfer to 10°C activity ceases after 1 h. Growth is very pH dependent, optimal growth on N2 occurring only between pH 6.8 and 7.8. Nitrogen fixation below pH 5.5 and above 8.0 is less than one-quarter of the optimal. No N2-fixation occurs in the absence of O2 and maximal N2-dependent growth is reached at 1.5% O2 in the gas mixture bubbled through liquid cultures.In contrast to previous reports, several sugars including glucose can be used by the Spirillum for N2-fixation, but only when small amounts of starter nitrogen or organic acids are added to the medium. Efficiencies of N2-fixation on malate and glucose are similar and about 60% of that of cells incorporating NH+4-N. Efficiency of NO-3 incorporation is 74% of that of NH+4-N grown cultures. High observed efficiencies (52 mg N2 fixed g?1 malate or glucose) are attributed to carbon limited growth at optimum or O2 limited conditions, both facilitated by slow diffusion rates through the semi-solid agar medium used.  相似文献   

8.
The influence of soil pH, redox potential and added organic matter on N2-fixation by Azospirillum was studied. Application of rice straw to alluvial, laterite and acid-sulphate Pokkali soils under submerged conditions enhanced the population of N2-fixing Azospirillum spp. An acid-sulphate saline soil of extremely low pH (3.2) harboured Azospirillum spp with appreciable N2-fixing activity. Enrichment cultures originating from soils with low pH (<4.0) possessed lower N2-fixing activity compared to cultures from soils with higher pH values (upto 6.6). Azospirillum cultures from soils that had undergone prolonged waterlogging showed lower N2-fixing activity than cultures isolated from soils submerged for a few days. A relationship was shown between the in vitro N2-fixing activity of Azospirillum cultures and the redox status of the soil samples; activity was high when the soil redox potential was between ?50 to ?150mV. The results show that the N2-fixing activity of Azospirillum cultures is governed by fluctuations in soil redox potential, pH and organic matter.  相似文献   

9.
Bacteria associated with roots of grasses from Florida, Ecuador and Venezuela were isolated and their N2-fixing ability was demonstrated by C2H2 reduction assay. The bacterial isolates have been classified as Azospirillum brasilense (formerly Spirillum lipoferum). These N2-fixing isolates have been compared with several Brazilian strains. Fluorescent antibody (FA) techniques were used to assist identifying isolates of N2-fixing bacteria from grass roots. Tests with antisera prepared against four strains of Azospirillum were used to define serological groups. Antigen-antibody specificity was demonstrated using both Azotobacter and Azospirillum antisera against known species of other soil microorganisms and numerous unidentified soil bacteria. Several applications of the FA technique are suggested to identify N2-fixing bacteria associated with grass roots.  相似文献   

10.
Summary This study is an attempt to describe the dominant N2-fixing microflora associated with the roots of wetland rice. Rice cultivar Giza 171 was grown in a phytotron on two alluvial Egyptian soils for 8 days, a stage when the nitrogenase activity of undisturbed plants reached a level of 245 × 10–6 mol C2H4 h–1 g–1 dry weight of leaf. The roots and rhizosphere soils were then used for counting and isolating dominant diazotrophs. Counts and initial enrichment steps were carried out on a selective medium made of an axenic rice plantlet, the spermosphere model, incubated under 1 % acetylene. The counts were very high, exceeding 108 bacteria g–1 dry weight of rhizosphere soil. Enterobacteriaceae were dominant; most isolates were Enterobacter cloacae belonging to different biotypes in the two soils. Enterobacter agglomerans, Citrobacter freundii and Klebsiella planticola were also present as members of the dominant microflora. Azospirillum brasilense and Azospirillum lipoferum were present as well, but less abundant.  相似文献   

11.
In order to study the establishment of a spermosphere, the C2H2 reduction activity of N2-fixing bacteria isolated from river sand was examined in a simulated spermosphere in the river sand which contained sucrose, an amino acid mixture, and CN- released from plant seeds. The sand incubated with 10-10 to 10-9 mol CN- 30 g sand-1 exhibited higher C2H2 reduction activity than that without CN-. The change in the most probable number of N2 fixers with increasing quantities of CN- roughly corresponded to that in C2H2 reduction activity. However, the most probable number of non-N2-fixing bacteria decreased except for CN--tolerant ones. Both C2H2 reduction activity and proliferation of the N2 fixers isolated on a modified Burk's medium were almost similar to those in the bacteria in the sand. In contrast, the proliferation of some nonfixers decreased with an increasing CN- concentration. C2H2 reduction activity of N2 fixers cultured in combination with non-fixers exhibited a clear peak at 10-7 M CN- as for C2H2 reduction activity in the sand. We therefore speculate that cyanide evolved from seeds during a pregermination period may suppress the growth of general bacteria, but may promote the proliferation of N2 fixers, thus contributing to the establishment of a spermosphere.  相似文献   

12.
An extensive survey of native conifers (Dacrydium, Podocarpus, Libocedrus, Phyllocladus and Agathis spp.) showed that C2H2 reduction was often associated with their mycorrhizal short roots (‘nodules’). C2H2 reduction was associated with roots only if it also occurred in surrounding soil, but it could be found in soils and not in the root region. Over 50 per cent of the C2H2 reduction activity could be removed by washing roots in distilled water while complete loss of activity occurred when they were surface sterilized with hypochlorite solution. C2H2 reduction may also be associated with long roots of podocarps and roots of non-podocarp species. Fermenting and humifying horizons of forest soils showed much greater rates of C2H2 reduction than either mineral soil or roots. Results suggest that previous claims for nitrogen fixation by podocarp roots can be attributed to nitrogen fixation by bacteria in the root region rather than by endophytic organisms.  相似文献   

13.
A comprehensive report of the acetylene reduction assay for measurement of N2 fixation is presented. The objective is to facilitate the effective use and identify some potential limitations of the method. The report is based on more than 200 accounts of the use of this technique in 15 countries during the last 5 years. Methods of measurement of N2 fixation are compared. Nomenclature, e.g., N2[C2H2] fixed, is introduced to identify values of N2 fixation determined by C2H2-C2H2 assay. The biochemical basis of the assay is described along with relevant characteristics including Km, C2H2/N2 conversion factor, and specific N2[C2H2]-fixing activities obtained with various systems. Effects of combined nitrogen, temperature, light, pO2, N2, pC2H2 and water on activity are summarized. Available methods for sample preparation, assay chamber, gas phase, assay condition, termination of reaction, C2H4 analysis and expression of results are compared. The many uses of the C2H2-C2H4 assay for investigations of the biochemistry of nitrogenase and physiology of N2-fixing organisms, definition of N2-fixing organisms and measurement of field N2 fixation by legume, non-legume, soil, marine, rhizosphere, phylloplane and mammalian samples are tabulated.  相似文献   

14.
Acetylene reduction activities (ARAs) of soils and rice plants during rice-growing season were monitored in temperate region in northeast China. This activity was significantly higher in rhizosphere soil than that in inter-row soil after rice seedlings were transplanted. The ARA was high for most of growing season, suggesting that the native N2-fixing bacteria responded to rice roots very quickly. Sixteen strains of free-living N2-fixing bacteria were isolated from three different soils. The ARAs of these strains were correlated with the averaged soil ARAs, suggesting that the isolated strains were likely the active flora responsive to rice roots. The strains were inoculated by soaking seedling roots into the liquid culture for 2 h, and the seedlings were transplanted into pots. Most strains tested did not show any growth-promoting effects except Azotobacter armeniacus and Azotobacter nigricans, which showed growth-promoting effects only at late rice growth stage and only when inoculated in combination but not separately. Present data indicated the promising future applications of these two strains in combination in the region, but further research is needed to understand the underlying mechanisms.  相似文献   

15.
Acetylene reduction by non-symbiotic, heterotrophic micro-organisms in a range of soils containing different concentrations of heavy metals was determined using intact soil cores. The suitability of this method for the soils used in this investigation was established. Samples were collected seasonally, and were incubated under standard conditions (darkness: 15°). Mean values of metal concentrations in the soil (μg g?1) were: Cd: 1–200; Pb: 60–8000; Zn: 70–26000, Cu: 20–40. Rates of acetylene reduction were generally low, from 2800 to 50000 nmol C2H4, m?2 day?1. Assuming a 3:1 ratio of C2H2 reduction to N2 fixation, this represents a rate of 0.3 to 5.0 g N fixed ha?1 day?1 in the surface 150 mm of soil. No consistent effect of heavy metal concentration was found. The most important factors determining activity were soil moisture content and possibly inorganic nitrogen concentration. It thus appears that the bacteria in polluted soils are capable of adapting to potentially toxic concentrations of heavy metals, or that these metals are present in the soils tested in unavailable or non-toxic forms.  相似文献   

16.
Interactions between the N2-fixing bacterium Azospirillum brasilense and the mycorrhizal fungus Glomus mosseae were studied in relation to their effects on the growth and nutrition of Zea mays (C4) and Lolium perenne (C3) plants. Although roots from plants inoculated with Azospirillum exhibited C2H2 reduction activity no significant effect of inoculation on N concentration in the plant shoots was found. With non-mycorrhizal plants, inoculation with Azospirillum resulted in increased dry matter production at the first harvest compared to the effect achieved by supplying N as fertilizer, but this trend was reversed at the last harvest. However, with mycorrhizal maize plants, Azospirillum, which stimulated the development of VA mycorrhiza, was still effective in improving plant growth and nutrient uptake at the last harvest. Azospirillum and N behaved similarly in enhancing the growth and nutrition of mycorrhizal maize. The dual inoculation of maize by Azospirillum and Glomus produced plants of a similar size, N content, and a higher P content, than those supplied with N and P.  相似文献   

17.
High rates of nitrogen fixation (acetylene reduction) were associated with Equisetum arvense L. and three other species of the same genus. Excised rhizomes and roots were assayed by incubation under ambient air and N2-enriched air (pO2 0.10atm), using the acetylene reduction method. There was a lag of about 1 day before high activity was observed under a pO2 of 0.10 atm, but under ambient air the lag was longer and varied from sample to sample. The duration of this lag usually coincided with that required for the establishment of a definite pO2 level (a threshold value) in the incubation atmosphere. The threshold pO2 value was about 0.15 atm for E. arvense, E. palustre, and E. hyemale, and 0.10 atm for E. ramosissimum. On incubation of the plant materials, acetylene reduction activity (ARA) increased gradually as the atmospheric pO2 decreased, and maximum ARA was observed under a definite pO2 value in the range from 0.04 to 0.01 atm.The highest value was 69nmol C2H44 formed g?1 fresh wt h?1 with a sample of E. arvense atpO2 0.03 atm. When the pO2 decreased below 0.01 atm, ARA decreased very rapidly and finally ceased. Several strains of N2-fixing bacteria were isolated from rhizomes and roots of E. arvense and all were classified as members of the family Enterobacteriaceae.  相似文献   

18.
Influence of mineral and symbiotic nitrogen nutrition on proton release of roots, phosphorusuptake and root development of red clover Red clover was cultivated in pots containing a loamy sand soil of low buffer capacity. Nitrogen supply was either NH4NO3 and Ca(NO3)2 in the mineral treatment or Rhizobium fixed N2 in the symbiotic treatment. During six cuts the plants decreased the pH of the soil from 7.6 to 6.0 in the mineral treatment and to 5.2 in the symbiotic treatment. Both treatments yielded the same amount of shoot dry matter per pot. The N2-fixing red clover produced more root fresh weight associated with larger root length, root surface, and root density per pot compared with plants grown with mineral nitrogen. Due to stronger soil acidification and better root growth N2-fixing red clover was able to exploit the rock phosphate component from a partially acidulated P-fertilizer to a higher degree than NH4NO3 or Ca(NO3)2 fed plants. The proton release of symbiotically grown plants could be accounted for by mineral cation excess (difference of cation and anion uptake) in shoots and roots by only 68%. It is therefore assumed that the excess of H+ released was accompanied by a release of anions.  相似文献   

19.
The conversion factor between C2H2 reduction and N2 fixation was studied in two soils. In one study small cores from (he two soils were drained to three water tensions: 0.20, 1.17 and 4.89 kPa. At each tension the N2ase activity was measured with both O.1 aim 15N2 and 0.1 aim C2H2. The conversion factor was different for the two soils. 1.0 and 3.1. respectively. The water content had no influence on the value of the conversion factor in this first study, in which the fixation corresponded to about 1mg N m?2 day ?1 at the depth 0–3 cm.In another study glucose was added to one of the soils to enhance the N2ase activity. The activity was measured at 75 and 100% water saturation with both 0.9 atm 15N2 and 0.1 atm C2 H2. At the lower water content the conversion factor was 2.6 and at water saturation the factor was 15.7. The fixation rates were high in this study. 98 mg N m?2 day?1 at the lower water content and 42 mg at water saturation.By theoretical calculations it was shown that the concentration of dissolved N2 restricted the rate of fixation in the water-saturated samples of the second study, thus giving the high conversion factor. The critical level of N2asc activity in water-saturated soil, above which the actual C2H2 to N2-ratio will be higher than usual, was estimated to about 10mg Nm 2day?1, under the experimental conditions used in these studies.  相似文献   

20.
Among the factors which may affect colonization of roots by soil bacteria is that of rhizosphere oxygen partial pressure (pO2). The oxygen concentration in the root zone influences both microbes and roots. Roots exposed to low pO2, as might occur during flooding and waterlogging of the soil, become more leaky and loss of soluble carbon increases. To determine whether periods of low pO2 increased root colonization by a genetically altered pseudomonad we inoculated 3- to 4-week-old maize plants, grown in soil and transferred to a hydroponic system or grown in fritted clay, with Pseudomonas putida PH6(L1019)(lacZY+) following exposure of the roots to air or cylinder N2. Numbers of heterotrophs and the marked pseudomonad were determined by dilution plating. Low pO2 generally increased the numbers of bacteria associated with roots exposed to the treatments in solution or in undisturbed fritted clay rooting medium. Under low pO2 in a hydroponic system, roots of intact maize plants tended also to have higher soluble organic C and hexose (anthrone-detectable sugars) than roots exposed to air. The effect of low pO2 was most pronounced in the fritted clay where low pO2 favored colonization by the marked strain; numbers were 3- to 96-fold greater than those on roots flushed with air but accounted for only 0.06–0.61% of the total population. Roots exposed to low pO2 tended to accumulate more C. Results suggest that in the fritted clay, the pseudomonad was able to exploit the increased C supply and to achieve greater numbers on roots exposed to low pO2, whereas the dilution of carbon released from roots in the hydroponic apparatus did not allow for the same magnitude of increase on roots. Received: 2 December 1996  相似文献   

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