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.     

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.     

Characterization of Azospirillum Isolated from Nitrogen-Fixing Roots of Harvested Sorghum Plants

Root segments of harvested sorghum plants had acetylene reduction activity ranging from 11 to 61 nmol of ethylene formed per h per g (dry weight). Five strains of Azospirillum brasilense sp. nov. were isolated from root segments.     

Perfusion method for assaying microbial activities in sediments: applicability to studies of n(2) fixation by c(2)h(2) reduction

A perfusion method for assaying nitrogenase activity (acetylene reduction) in marine sediments was developed. The method was used to assay sediment cores from Spartina alterniflora (salt marsh), Zostera marina (sea grass), and Thalassia testudinum (sea grass) communities, and the results were compared with those of conventional sealed-flask assays. Rates of ethylene production increased progressively with time in the perfusion assays, reaching plateau values of 2 to 3 nmol . g of dry sediment . h by 10 to 20 h. Depletion of interstitial NH(4) was implicated in this stimulation of nitrogenase activity. Initial acetylene reduction rates determined by the perfusion assay of cores from the Spartina community ranged from 0.15 to 0.60 nmol of C(2)H(4) . g of dry sediment . h. These rates were similar to those for sediments assayed in sealed flasks without seawater when determined over linear periods of C(2)H(4) production. Initial values obtained by using the perfusion method were 0.66 nmol of C(2)H(4) . g of dry sediment . h for sediments from Zostera communities and 0.70 nmol of C(2)H(4) . g of dry sediment . h for sediments from Thalassia communities. In all cases, rates determined by simultaneous slurry assays were lower than those determined by the perfusion method.     

N(2) Fixation Associated with Decaying Leaves of the Red Mangrove (Rhizophora mangle)

N(2) (C(2)H(2)) fixation was associated with decaying leaves of Rhizophora mangle. The process was predominantly anaerobic, with about two-thirds of the nitrogenase activity being light dependent. Average N(2) fixation rates in the light were 11 mug of N per g (dry weight) per h for leaves that had decayed for 2 to 3 weeks. This nitrogen input is probably significant in the estuarine, detrital food chains linked to R. mangle.     

Dinitrogen fixation — acetylene reduction in soybeans during the reproductive growth period

In a greenhouse pot oulture experiment, a dinitrogen (N2) fixing — acetylene reduction activity profile was examined in detail as affected by plant age. Total [μmol C2H4 root-1 h-1] and speoifio nitrogenase [nmol C2H4 (mg nodule d. wt.)-1 min-1] activities peaked 63 days after sowing, near the end of flowering. The nitrogenase activities, nodule dry matter accumulation, top dry matter accumulation, and total nitrogen yield in the top dry matter were found to be highly correlated.     

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.     

Effect of substrate on metabolite production of Alternaria alternata.

Alternariol and alternariol monomethyl ether are commonly associated with weathered grain sorghum. Production of these metabolites and altenuene by isolates of Alternaria alternata was evaluated on various sterile grain substrates. At 35% moisture content and 25 C, metabolite yields were highest on rice, intermediate on sorghums, and lowest on wheat and yellow corn. Fourteen-to 21-day cultures on milled rice were best in terms of ease of metabolite recovery, even though yields were higher on 28-day cultures of rough and brown rice. Metabolite production was reduced when rice was supplemented with yeast extract or yeast extract plus Czapek-Dox broth.     

云南南亚热带北缘地区稻田亩产吨粮的种植模式及其生态生理学研究

1996～ 1 999年 ,在云南省蒙自县草坝镇 (南亚热带北缘 )进行了亩产吨粮的种植模式研究。通过对春、夏、秋、冬播玉米 ,早、中、晚稻以及冬小麦的系统研究 ,以地上部分总干物重、籽粒产量、总入射辐照量、叶面积持续期 (灌浆期 )、氮肥的利用率和生产力、水生产力为指标 ,对早稻—晚稻、早稻—秋玉米、早稻—冬小麦、春玉米—晚稻、夏玉米—冬小麦、中稻—冬小麦和中稻—冬玉米 7种复种模式作了比较 ,选出中稻—冬玉米为一年两熟亩产吨粮的最佳种植模式。     

Root-associated n(2) fixation (acetylene reduction) by enterobacteriaceae and azospirillum strains in cold-climate spodosols

N(2) fixation by bacteria in associative symbiosis with washed roots of 13 Poaceae and 8 other noncultivated plant species in Finland was demonstrated by the acetylene reduction method. The roots most active in C(2)H(2) reduction were those of Agrostis stolonifera, Calamagrostis lanceolata, Elytrigia repens, and Phalaris arundinacea, which produced 538 to 1,510 nmol of C(2)H(4).g (dry weight). h when incubated at pO(2) 0.04 with sucrose (pH 6.5), and 70 to 269 nmol of C(2)H(4). g (dry weight).h without an added energy source and unbuffered. Azospirillum lipferum, Enterobacter agglomerans, Klebsiella pneumoniae, and a Pseudomonas sp. were the acetylene-reducing organisms isolated. The results demonstrate the presence of N(2)-fixing organisms in associative symbiosis with plant roots found in a northern climatic region in acidic soils ranging down to pH 4.0.     

Nitrogen fixation associated with Suillus tomentosus tuberculate ectomycorrhizae on Pinus contorta var. latifolia

BACKGROUND AND AIMS: Tuberculate ectomycorrhizae are a unique form of ectomycorrhiza where densely packed clusters of mycorrhizal root tips are enveloped by a thick hyphal sheath to form a tubercle. The functional significance of such a unique structure has not previously been established. The purpose of the present study was to investigate and measure the potential nitrogenase activity associated with Suillus tomentosus/Pinus contorta tuberculate ectomycorrhizae in two stand ages, young and old, and across a range of nitrogen-poor soil conditions. METHODS: Short roots were compared with other mycorrhizae and non-mycorrhizal secondary roots using tuberculate ectomycorrhizae. Assessment of nitrogenase activity was determined and quantitative measurements were taken on tuberculate ectomycorrhizae in situ in a variety of different circumstances, by using an adaptation of the acetylene reduction assay. KEY RESULTS: Significant nitrogenase activity was measured associated with S. tomentosus/P. contorta tuberculate ectomycorrhizae whereas no nitrogenase activity was measured with non-tuberculate mycorrhizae or secondary roots without mycorrhizae. Average nitrogenase activity ranged from undetectable to 5696.7 nmol C2H4 g(-1) tubercle 24 h(-1). Maximum nitrogenase activity was 25,098.8 nmol C2H4 g(-1) tubercle 24 h(-1). Nitrogenase activity was significantly higher in young stands than in old stands of P. contorta. Season or some covariate also seemed to affect nitrogenase activity and there was suggestion of a site effect. CONCLUSIONS: Suillus tomentosus/P. contorta tuberculate ectomycorrhizae are sites of significant nitrogenase activity. The nitrogenase activity measured could be an important contribution to the nitrogen budget of P. contorta stands. Season and stand age affect levels of nitrogenase activity.     

Nitrogenase proteins from Gluconacetobacter diazotrophicus, a sugarcane-colonizing bacterium

Gluconacetobacter diazotrophicus Pal-5 grew well and expressed nitrogenase activity in the absence of NH4+ and at initial O2 concentrations greater than 5% in the culture atmosphere. G. diazotrophicus nitrogenase consisted of two components, Gd1 and Gd2, which were difficult to separate but were purified individually to homogeneity. Their compositions were very similar to those of Azotobacter vinelandii nitrogenase, however, all subunits were slightly smaller in size. The purified Gd1 protein contained a 12:1 Fe/Mo ratio as compared to 14:1 found for Av1 purified in parallel. Both Gd2 and Av2 contained 3.9 Fe atoms per molecule. Dithionite-reduced Gd1 exhibited EPR features at g=3.69, 3.96, and 4.16 compared with 3.64 and 4.27 for Av1. Gd2 gave an S=1/2 EPR signal identical to that of Av2. A Gd1 maximum specific activity of 1600 nmol H2 (min mg of protein)(-1) was obtained when complemented with either Gd2 or Av2, however, more Av2 was required. Gd2 had specific activities of 600 and 1100 nmol H2 (min mg protein)(-1) when complemented with Av1 and Gd1, respectively. The purified G. diazotrophicus nitrogenase exhibited a narrowed pH range for effective catalysis compared to the A. vinelandii nitrogenase, however, both exhibited maximum specific activity at about pH 7. The Gd-nitrogenase was more sensitive to ionic strength than the Av-nitrogenase.     

Nitrogen fixation by the hydrogen-oxidizing bacterium Alcaligenes latus

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

Eucaryote thermophily: role of lipids in the growth of Talaromyces thermophilus

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.     

Unicellular, aerobic nitrogen-fixing cyanobacteria of the genus Cyanothece.

Two marine, unicellular aerobic nitrogen-fixing cyanobacteria, Cyanothece strain BH63 and Cyanothece strain BH68, were isolated from the intertidal sands of the Texas Gulf coast in enrichment conditions designed to favor rapid growth. By cell morphology, ultrastructure, a GC content of 40%, and aerobic nitrogen fixation ability, these strains were assigned to the genus Cyanothece. These strains can use molecular nitrogen as the sole nitrogen source and are capable of photoheterotrophic growth in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and glycerol. The strains demonstrated a doubling time of 10 to 14 h in the presence of nitrate and 16 to 20 h under nitrogen-fixing conditions. Rapid growth of nitrogen-fixing cultures can be obtained in continuous light even when the cultures are continuously shaken or bubbled with air. Under 12-h alternating light and dark cycles, the aerobic nitrogenase activity was confined to the dark phase. The typical rates of aerobic nitrogenase activity in Cyanothece strains BH63 and BH68 were 1,140 and 1,097 nmol of C2H2 reduced per mg (dry weight) per h, respectively, and nitrogenase activity was stimulated twofold by light. Ultrastructural observations revealed that numerous inclusion granules formed between the photosynthetic membranes in cells grown under nitrogen-fixing conditions. These Cyanothece strains posses many characteristics that make them particularly attractive for a detailed analysis of the interaction of nitrogen fixation and photosynthesis in an aerobic diazotroph.     

播种量和施氮量对垄沟覆膜栽培冬小麦花后生理性状的影响

为协调冬小麦个体与群体间的关系,充分发挥旱作条件下垄沟栽培优势,以冬小麦品种小偃22为材料,采用二元二次正交旋转组合设计,通过田间试验研究了垄下集中施肥、垄上覆膜、膜际种植模式下播种量和施氮量对冬小麦花后生理性状的影响.结果表明:花后叶面积指数、旗叶叶绿素含量和净光合速率均随施氮量的增加而增加.灌浆前中期叶面积指数随播种量的增加呈先增后稳的趋势;灌浆后期叶面积指数随播种量的增加而降低.随播种量的增加,旗叶的叶绿素含量和净光合速率降低,单株产量呈先减少后增加的趋势.适宜的播种量可以协调个体与群体间的矛盾,而适量增施氮肥有利于花后小麦生理性状的改善和产量的提高.在供试条件下,小偃22在播种量112.5 kg hm-2与施氮量180 ～222 kg N·hm-2配置时,个体与群体的关系比较协调,花后叶面积指数较高,群体结构适宜,而且旗叶叶绿素含量、净光合速率和单茎产量较高,能获得高产.     

Control of dinitrogen fixation in ammonium-assimilating cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown at constant growth rate in a chemostat with different molar ratios of sucrose to ammonium (C/N) in the influent media. Both compounds were consumed at essentially the same ratios as were present in the influent media. At low (C/N)-ratios, the cultures were ammonium-limited. At increased (C/N)-ratio ammonium-assimilating cultures additionally began to fix dinitrogen. The (C/N)-ratio at which nitrogenase activity became measurable, increased when the ambient oxygen concentration was increased. Immunoblotting revealed the appearance of nitrogenase proteins when the activity became detectable. Nitrogenase activity as determined either by acetylene reduction or by total nitrogen fixation gave constant relative activities of 1:3.8 (mol of N₂ fixed per mol of acetylene reduced) under all sets of conditions used in this investigation. In spite of the oxygen dependent variation of the (C/N)-ratio, nitrogenase became active when the ammonium supply was less than about 14 nmol of ammonium per g of protein. This suggests that oxygen was not directly involved in the onset of dinitrogen fixation.     

NH(4)-Excreting Azospirillum brasilense Mutants Enhance the Nitrogen Supply of a Wheat Host

Spontaneous ethylenediamine-resistant mutants of Azospirillum brasilense were selected on the basis of their excretion of NH(4). Two mutants exhibited no repression of their nitrogenase enzyme systems in the presence of high (20 mM) concentrations of NH(4). The nitrogenase activities of these mutants on nitrogen-free minimal medium were two to three times higher than the nitrogenase activity of the wild type. The mutants excreted substantial amounts of ammonia when they were grown either under oxygen-limiting conditions (1 kPa of O(2)) or aerobically on nitrate or glutamate. The mutants grew well on glutamate as a sole nitrogen source but only poorly on NH(4)Cl. Both mutants failed to incorporate [C]methylamine. We demonstrated that nitrite ammonification occurs in the mutants. Wild-type A. brasilense, as well as the mutants, became established in the rhizospheres of axenically grown wheat plants at levels of > 10 cells per g of root. The rhizosphere acetylene reduction activity was highest in the preparations containing the mutants. When plants were grown on a nitrogen-free nutritional medium, both mutants were responsible for significant increases in root and shoot dry matter compared with wild-type-treated plants or with noninoculated controls. Total plant nitrogen accumulation increased as well. When they were exposed to a N(2)-enriched atmosphere, both A. brasilense mutants incorporated significantly higher amounts of N inside root and shoot material than the wild type did. The results of our nitrogen balance and N enrichment studies indicated that NH(4)-excreting A. brasilense strains potentially support the nitrogen supply of the host plants.     

Pectin decomposition and associated nitrogen fixation by mixed cultures of Azospirillum and Bacillus species.

Cocultures of different Azospirillum species with Bacillus polymyxa or Bacillus subtilis allow the efficient utilization of pectin as carbon and energy sources for nitrogen fixation. The nitrogenase activity obtained with cocultures was as high as 30-80 nmol C2H4 h-1 mL-1, a much higher value than that obtained with pure cultures of either Azospirillum (up to 13 nmol C2H4 h-1 mL-1) or B. polymyxa (up to 2 nmol C2H4 h-1 mL-1) alone. To establish to what extent each partner contributed to nitrogenase activity, acetylene reduction was assayed as a function of time and it was also measured on Azospirillum cultivated in the cultures filtrates of the Bacillus. The results suggested that the nitrogenase activity was mostly produced by Azospirillum. The nitrogenase activity occurred at the expense of the degradation and fermentation products of the pectin. The new pectinolytic species, Azospirillum irakense, utilized both degradation and fermentation products of pectin, whereas the nonpectinolytic strains (Azospirillum brasilense, Azospirillum lipoferum, Azospirillum amazonense) utilized only the fermentation products of pectin, including acetic and succinic acids. These cocultures can be considered as metabolic associations, where the Bacillus produces degradation and fermentation products of pectin, which can be used by Azospirillum species.     

Production of feed enzymes (phytase and plant cell wall hydrolyzing enzymes) by<Emphasis Type="Italic">Mucor indicus</Emphasis> MTCC 6333: Purification and characterization of phytase

The production of phytase and associated feed enzymes (phosphatase, xylanase, CMCase, alpha-amylase and beta-glucosidase) was determined in a thermotolerant fungus Mucor indicus MTCC 6333, isolated from composting soil. Solid-substrate culturing on wheat bran and optimizing other culture conditions (C and N sources, level of N, temperature, pH, culture age, inoculum level), increased the yield of phytase from 266 +/- 0.2 to 513 +/- 0.4 nkat/g substrate dry mass. The culture extract also contained 112, 194, 171, 396, and 333 nkat/g substrate of phosphatase, xylanase, CMCase, beta-glucosidase and alpha-amylase activities, respectively. Simple 2-step purification employing anion exchange and gel filtration chromatography resulted in 21.9-fold purified phytase. The optimum pH and temperature were pH 6.0 and 70 degrees C, respectively. The phytase was thermostable under acidic conditions, showing 82% residual activity after exposure to 60 degrees C at pH 3.0 and 5.0 for 2 h, and displayed broad substrate specificity. The Km was 200 nmol/L and v(lim) of 113 nmol/s per mg protein with dodecasodium phytate as substrate. In vitro feed trial with feed enzyme resulted in the release of 1.68 g inorganic P/kg of feed after 6 h of incubation at 37 degrees C.