A Quantitative Study of Fixation and Transfer of Nitrogen in Alnus

A quantitative study of the fixation of nitrogen and of itstransfer from the nodules to the remainder of the plant hasbeen made in Alnus plants during their first season of growth.Fixation per plant reached a maximum in late August but fellrapidly with the onset of autumn, while fixation per unit dryweight of nodule tissues was greatest in young nodules and wasof the same order as in nodulated legumes. Throughout the growthseason there was a steady transfer from the nodules of some90 per cent, of the nitrogen fixed. The rate of fixation relativeto the growth of the endophyte is much higher than in free-livingnitrogen-fixing organisms and is clearly governed by the nitrogenrequirements of the entire symbiotic system. These findingsare compatible with the view that the fixation process is extracellularto the endophyte.     

Hydrogen Reactions of Nodulated Leguminous Plants: II. Effects on Dry Matter Accumulation and Nitrogen Fixation

The interaction between the ATP-dependent evolution of H₂ catalyzed by nitrogenase and the oxidation of H₂ via a hydrogenase has been postulated to influence the efficiency of the N₂-fixing process in nodulated legumes. A comparative study using soybean (Glycine max L. Merr.) cv. Anoka inoculated with either Rhizobium japonicum strain USDA 31 or USDA 110 and cowpea (Vigna unguiculata L. Walp.) cv. Whippoorwill inoculated with Rhizobium strain 176A27 or 176A28 cultured on a N-free medium was conducted to address this question. Nodules from the Anoka cultivar inoculated with USDA 31 evolved H₂ in air and the H₂ produced accounted for about 30% of the energy transferred to the nitrogenase system during the period of active N₂ fixation. In contrast the same soybean cultivar inoculated with USDA 110 produced nodules with an active hydrogenase and consequently did not evolve H₂ in air. A comparison of Anoka soybeans inoculated with the two different strains of R. japonicum showed that mean rates of C₂H₂ reduction and O₂ consumption and mean mass of nodules taken at four times during vegetative growth were not significantly different.

When compared to Anoka inoculated with USDA 31, the same cultivar inoculated with USDA 110 showed increases in total dry matter, per cent nitrogen, and total N₂ fixed of 24, 7, and 31%, respectively. Cowpeas in symbiosis with the hydrogenase-producing strain 176A28 in comparison with the same cultivar inoculated with the H₂-evolving strain 176A27 produced increases in plant dry weight and total N₂ fixed of 11 and 15%, respectively. This apparent increase in the efficiency of N₂ fixation for nodulated legumes capable of reutilizing the H₂ evolved from nitrogenase is considered and it is concluded that provision of conclusive evidence of the role of the H₂-recycling process in N₂-fixing efficiency of legumes will require comparison of Rhizobium strains that are genetically identical with the exception of the presence of hydrogenase.

     

Isotopic Studies of Nitrogen Fixation in Non-Legume Root Nodules

Isotopic studies are presented on six of the eight recognizednon-legume nodule-forming genera of Angiosperms. Evidence hasbeen obtained of the occurrence of fixation of atmospheric nitrogenin the root nodules of Casuarina, Ceanothus, and Shepherdia,now examined isotopically for the first time. In further studiesof the nitrogen-fixing nodules of Hippopha and Alnus it is shownthat the fixation continues for a longer period after detachmentfrom the plant than is the case with legume nodules, and thatthe enrichment in ¹³N which they (and also detached Casuarinanodules) finally achieve after exposure to excess of the freeisotope considerably exceeds that shown by detached legume nodules.Fixation in detached Myrica nodules was not clearly affectedby reduction in oxygen supplied until the proportion of oxygenwas less than 5 per cent.     

Growth of Actinorhizal Plants as Influenced by the Form of N with Special Reference to Myrica gale and Alnus incana

Growth of two actinorhizal species was studied in relation tothe form of N supply in water culture. Non-nodulated bog myrtle(Myrica gale) and grey alder (Alnus incana) were grown withNH₄⁺, NH₄NO₃^– or NO₃^– (4 mol m^–3 N). A nodulatedseries of bog myrtle was also cultivated in N-free medium. Relative growth rate (RGR), utilization rate of N, and shoot/rootratio were highest for the two species with the N completelysupplied as NH₄⁺. In both species, nitrate was largely reducedin the roots and the presence of NO₃^– in combined-N supplyalways affected the RGR and N utilization rate negatively. BothN₂ fixation and complete NO₃^– nutrition represented conditionsof relative N-deficiency resulting in relatively low tissue-Nconcentrations and a greater allocation of dry mass to the roots.The physiological N status of nodulated M. gale plants was comparativelygood, as indicated by a normal nodule weight ratio and a relativelyhigh N₂-fixing rate per unit nodule mass. However, whole-plantN₂-fixing capacity remained relatively low in comparison withacquisition rates of N in combined-N plants. The anion charge from the nitrate reduction was largely directlyexcreted as an OH^– efflux. H ⁺ /N ratios generally agreedwith the theory. In comparison with NH₄⁺ nutrition, carboxylateconcentrations were higher in N₂-fixing M. gale plants and theH ⁺ /N ratio in nodulated plants was less than unity below thevalue for ammonium plants as previously found for other actinorhizalspecies. Therefore, NH₄⁺ should be an energetically more attractiveN source for actinorhizal plants than N₂. The results agree with commonly accepted views on energeticsof N uptake and assimilation in higher plants and support theconcept of a basically similar physiological behaviour betweennon-legumes and legumes. Key words: Actinorhizal symbioses, ammonium, H⁺/OH^– efflux, nitrate, N₂ fixation, NRA     

The Fixation of Nitrogen associated with the Root Nodules of Myrica gale L., with Special Reference to its pH Relation and Ecological Significance

The paper deals with the fixation of nitrogen by nodulated plantsof Myrica gale under experimental conditions, the fixation being,in the first year of development, of a magnitude comparableto that of legumes under similar conditions. The effect of acidityon the fixation has been investigated. The Myrica organism ismarkedly adapted to acid conditions. The observations are correlatedwith field data in Britain and help to explain the distributionand habits of the species.     

Nitrogen Fixation and Allantoin Formation in Soybean Plants

The activity of nitrogenase and the concentration of ammonia and allantoin (+ allantoic acid) in root nodules were measured throughout the growth period of soybean plants. Nitrogenase activity measured by acetylene reduction increased with plant growth and reached a maximum level at the flowering period. The level of ammonia and allantoin concentration in nodules was parallel with increased nitrogenase activity. At the late reproductive stage (pod-forming period), nitrogenase activity showed a marked decrease, but the ammonia and allantoin in the nodules remained at a constant level. Detached nodules from 56 day-old soybean plants were exposed to ¹⁵N₂ gas, and the distribution of ¹⁵N among nitrogen compounds was investigated. Enrichment of ¹⁵N in allantoin and allantoic acid reached a fairly high level after 90 min of nitrogen fixation; ca. 22% of ¹⁵N in acid-soluble nitrogen compounds was incorporated into allantoin + allantoic acid. In contrast, enrichment of ¹⁵N in amide nitrogen was relatively low. No significant ¹⁵N was detected in the RNA fraction. The data suggested that ureide formation in nitrogen-fixing root nodules did not take place through the breakdown of nucleic acids, but directly associated with the assimilating system of biologically fixed nitrogen.     

Effects of Nitrogen Nutrition on Leaf Expansion and Photosynthesis of Trifolium subterraneum L. II. Comparison between Nodulated Plants and Plants Supplied with Combined Nitrogen

Subterranean clover plants depending on symbiotic nitrogen fixationhad smaller leaf areas than control plants supplied with combinednitrogen in the nutrient solutions. There were no differencesin chlorophyll content per unit fresh weight of leaves or petioles,nor in net rates of carbon dioxide uptake per unit leaf areaat light intensities above 2000 fc and at carbon dioxide concentrationsabove 300 ppm. Dark respiration by the shoots of the nodulatedplants was considerably higher than for the controls. This couldhave been a direct result of nodule activity and is suggestedas a possible factor contributing to the slower growth of theseplants compared with the controls. A comparison of the nitrogen contents of shoots and roots showeda sub-optimal nitrogen status, particularly in the roots, ofthe nodulated plants. This is suggested as another factor contributingto the slower growth of the nodulated plants compared with thecontrols. The response patterns before and after the addition of combinednitrogen differed in a number of important respects from thosefound previously under conditions of a sub-optimal nitrogensupply in the nutrient solution outside the roots. These arebriefly discussed.     

Effects of Water, Aeration, and Salt Regime on Nitrogen Fixation in a Nodulated Legume--Definition of an Optimum Root Environment

Nitrogen assimilation of pea plants (Pisum sativum cv. Meteor)was studied in growth cabinets tinder a range of water, salt,and aeration regimes in the rooting medium. Treatments wereimposed in the period 14–30 d after germination usingseedlings already nodulated in an optimum root environment. Highest nitrogen fixation in water culture required a strengthof culture solution one fifth of that optimal for fixation insand culture. Fixation in water culture of optimum strengthwas significantly improved by continuous bubble aeration orby lowering the level of culture solution below the main zoneof nodulation. However, if supra-optimal concentrations of solutionwere used, fixation was markedly inhibited by lowering the solutionlevel, this being associated with an accumulation of high levelsof salts on exposed root and nodule surfaces. In – N (minus nitrogen) sand culture continuous waterloggingreduced nitrogen content to 40 per cent of that of non-stressedplants. In nitrate-fed plants waterlogging effects were lesssevere. Waterlogging decreased nodule tissue production anddecreased the specific activity of nitrogenase, as assayed byacetylene reduction. These effects were most marked three ormore cm below the sand surface. Watering on alternate days with free drainage at all times yieldedmaximum fixation in – N sand culture. Regimes increasingthe extent of waterlogging or drying out in comparison withthis optimum produced increasingly great decreases in nitrogenfixation. For equivalent reductions in total fixation, percentageN in dry matter was consistently lower in waterlogged than indroughted plants suggesting that excess water had the more specificeffect on symbiotic activity. Both forms of stress affectedparticularly the transport of nitrogen from root to shoot.     

Partitioning of Nitrogen Derived from N2 Fixation and Reserves in Nodulated Medicago sativa L. During Regrowth

Nodul{macron}ted alfalfa plants were grown hydroponically. Inorder to quantify N₂ fixation and remobilization of N reservesduring regrowth the plants were pulse-chase-labelled with ¹⁵N.Starch and ethanol-soluble sugar contents were analysed to examinechanges associated with those of N compounds. Shoot removalcaused a severe decline in N₂ fixation and starch reserves within6 d after cutting. The tap root was the major storage site formetabolizable carbohydrate compounds used for regrowth; initiallyits starch content decreased and after 14 d started to recoverreaching 50% of the initial value on day 24. Recovery of N₂fixation followed the same pattern as shoot regrowth. Afteran initial decline during the first 10 d following shoot removal,the N₂ fixation, leaf area and shoot dry weight increased sorapidly that their levels on day 24 exceeded initial values.Distribution of ¹⁵N within the plant clearly showed that a significantamount of endogenous nitrogen in the roots was used by regrowingshoots. The greatest use of N reserves (about 80% of N incrementin the regrowing shoot) occurred during the first 10 d and thencompensated for the low N₂ fixation. The distribution of N derivedeither from fixation or from reserves of source organs (taproots and lateral roots) clearly showed that shoots are thestronger sink for nitrogen during regrowth. In non-defoliatedplants, the tap roots and stems were weak sinks for N from reserves.By contrast, relative distribution within the plant of N assimilatedin nodules was unaffected by defoliation treatment. Key words: Medicago sativa L., N₂ fixation, N remobilization, N₂ partitioning, regrowth     

去叶对不同生长习性大豆固氮作用的影响

生殖生长期开始去叶后 ,(1)有限型、亚有限型和无限型 3种类型的大豆根瘤固氮酶活性都降低 ,而根瘤中酰脲含量则不同程度地增加 ;(2 )有限型大豆幼茎中酰脲含量明显增加 ,但亚有限型和无限型大豆变化不大 ;(3) 3种类型大豆幼茎中硝态氮含量增加明显     

Diversity of Frankia strains associated to Myrica gale in Western Europe: impact of host plant (Myrica vs. Alnus) and of edaphic factors

Myricaceae can be nodulated by a variety of Frankia strains isolated from other actinorhizal families. Consequently, the genus Myrica has been considered to have low specificity with respect to microsymbiont taxa. In contrast to controlled studies of Myrica infectious capacity, field studies in North America have indicated that M. gale symbionts belong to the genetic group of Alnus-infective strains. Myrica gale is the most widely distributed species in the genus so this study focused on describing the genetic diversity of M. gale-nodulating strains from 10 sites in Western Europe across a range of edaphic conditions. When possible, the specificity of M. gale-infective strains was compared with that of Alnus-infective strains from the same sites. Nodular strains from Belgium, France and Spain were characterized using PCR-RFLP of rrs gene and 16S-23S IGS. rrs-RFLP patterns showed a high level of homogeneity among European strains with one dominant genotype. IGS-RFLP patterns revealed the largest inter and intrasite diversity in France. In Belgium, Frankia strains were found to occur in two groups according to soil pH and organic matter characteristics of the sites. European M. gale-infective strains were genetically different from European Alnus and North American M. gale-infective strains indicating the possibility of different pathways of co-evolution among geographically isolated populations.     

A Host-specific, Nitrogen Fixation Mutant of Bradyrhizobium: Physiology on Three Host Plants

NC92 #284 is a transposon mutant of Bradyrhizobium sp. (Arachis)strain NC92 and has a host-specific fixation phenotype. It appearsto be ineffective on the host pigeonpea (90% reduction in shootN compared to that of the wild type), but partially effectiveon two other host plants, groundnut and siratro (50% and 20%reduction in shoot N compared to the wild type, respectively).To understand this phenomenon of host-specific fixation, thephysiological basis of the phenotypes was investigated. Host-dependentdifferences in symbiotic effectiveness were largely explainedby the degree to which nitrogenase activity was impaired inthe various #284 symbioses. Nodulation and the onset of nitrogenfixation were found to be delayed on all three hosts, but tothe greatest degree on pigeonpea. The specific activity of nitrogenaseper gram nodule was also reduced on all three hosts, again tothe greatest extent on pigeonpea. By contrast, the carbon costsand relative efficiencies of each symbiosis were similar tothe wild type. The results indicate that the host-specific fixationphenotype of #284 is reflected in a quantitative reduction inthe amount of N₂ fixed. Thus the phenotypes reflect the differentability of the three host plants to tolerate or support the#284 mutation, rather than a defect in a specific interactionbetween #284 and a particular host plant. Key words: Bradyrhizobium, nodules, nitrogenase activity, relative efficiency, Arachis hypogaea, Cajanus cajan, Macroptilium atropurpureum     

一种高效研究大豆根瘤共生固氮的营养液栽培体系

为建立一种既可高效结瘤固氮, 又具有一定产量的大豆(Glycine max)营养液栽培系统, 设计并进行了2个试验。首先在不同供氮条件下, 研究了接种根瘤菌对大豆的结瘤状况、固氮能力、生物量及产量的影响。结果表明, 供氮过高或过低, 均影响大豆生长、产量形成及根瘤固氮; 并且植物生长所需的最适供氮水平远高于生物固氮所需的最适供氮水平。此外, 大豆生物固氮活性最高的时期在生殖期第一期(R1期)之前。由此推断, 大豆R1期前, 供应较低的氮, 有利于根瘤形成及固氮; 而从R1期起, 应提高供氮水平, 以促进植物生长及产量的形成。在此基础上开展第2个试验, 对供氮条件进行了优化处理(即R1期前低氮供应、R1期开始中氮供应)。结果表明, 与持续供应高氮相比, 优化供氮处理不仅可获得较多固氮酶活性较高的大根瘤, 还能保持较好的生长、获得更高的百粒重及维持80%左右的产量。研究结果不仅可为高效研究大豆根瘤共生固氮提供营养液配方, 还可为大豆高产高效栽培提供试验依据。     

ARDRA对植物根瘤内共生放线菌Frankia多样性的研究

应用原核生物16SrDNA特异性引物rD1和fD1,通过ARDRA（amplified ribosomal DNA restriction analysis）法直接扩增自中国云南、东北地区赤杨属3种植物和沙棘属1种植物根瘤内FrankiaDNA,得到一长约1500bp的扩增产物,选用两种内切酶HaeⅢ、AfaⅠ联合对扩增产物进行酶切,得到稳定的酶切图谱,将所测48个感染赤杨的Frankia样本区分为3个不同的组,所测43个感染沙棘的Frankia样本区分为3个不同的组,显示根瘤内Frankia存在丰富的遗传多样性。     

Nitrogen Fixation in the Phyllosphere of Tropical Plants: Occurrence of Phyllosphere Nitrogen-Fixing Micro-organisms in Eastern India and their Utility for the Growth and Nitrogen Nutrition of Host Plants

Of the 560 leaf samples belonging to 259 species of green plantsexamined more than 50 per cent of the Angiosperms and 25 percent of the Pteridophytes and Gymnosperms revealed the presenceof N₂-fixing micro-organisms in their phyllosphere. Plants particularlyremarkable in this respect are orchids and several other epiphytes,Scindapsus officinalis, Ficus and cucurbits. Most of the isolatesappear to be biotypes of Klebsiella pneumoniae. The more activestrains fixed more than 5 mg N g^–1 glucose utilized andreduced more than 100 nmol C₂H₂ mg^–1 cell d. w h^–1. The efficacy of the phyllosphere N₂-fixing isolates for N-nutritionof host plants was studied by spraying suspensions of the culturesgrown on N-free media on rice and wheat seedlings. In IR-26rice or Sonalika and Janak wheat grown on soil in wooden flatsor earthenware pots, 22 per cent of the 161 cultures studiedcaused increased height and about three-quarters of the culturesenhanced dry weight by more than 50 per cent; chlorophyll andN-contents were enhanced more than 50 per cent by about halfand two-thirds of the cultures respectively. In N-free sandculture 26 of the 50 promising strains doubled N-content, and30 doubled dry weight of the tested plants. In some cases dryweight, number of grains per panicle, and 1000 grain weightwere increased by 300, 70–83 and 126–158 per centrespectively; N-content of straw and seed was increased three-or fourfold. In several cases the beneficial effects were foundto match closely the performance of plants receiving ammoniumsulphate. Nitrogen-fixing micro-organisms, nitrogen nutrition, phyllosphere, rice, tropical plants, wheat     

Glycine-Glomus-Rhizobium Symbiosis : VI. Photosynthesis in Nodulated, Mycorrhizal, or N- and P-Fertilized Soybean Plants

Soybean (Glycine max [L.] Merr. cv Hobbit) plants were grown in a growth chamber for 56 days in a phosphorus- and nitrogen-deficient soil and were colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd) Gerd. and Trappe and Rhizobium japonicum strain USDA 136, or by either organism alone, or by neither. Non-VAM plants received supplemental phosphorus and nonnodulated plants supplemental nitrogen to achieve the same rate of growth in all treatments. Plants of all four treatments had the same (P > 0.05) dry weights at harvest, but VAM plants had higher rates of CO₂ exchange (CER, P < 0.05) and lower leaf P concentrations (P < 0.01). Leaf nitrogen concentrations were lower in nodulated than in nitrogen-supplemented plants (P < 0.01) while starch concentrations were higher (P < 0.01). There was a significant negative relationship between nitrogen and starch (r = −0.989). Statistical evaluation of the data showed that some parameters (CER, leaf area and phosphorus content) were associated with phosphorus nutrition (or the presence of the VAM fungus), others (leaf fresh weight and root dry weight) with nitrogen nutrition (or the presence of Rhizobium), and some (leaf nitrogen and starch content) by both factors. The development of microsymbiont structures and nodule activity were significantly lower in the tripartite association than in plants colonized by one endophyte only. The findings suggest that endophyte effects go beyond those of simple nutrition and associated source-sink relationships.     

Symbiotic Nitrogen Fixation at the Late Stage of Nodule Formation in Lotus japonicus and Other Legume Plants

Lotus japonicus nodules are reviewed, and current research data on Nod-35 (uricase) gene, including that of Lotus japonicus as an example of a late nodulin gene are presented. Received 5 September 2000/ Accepted in revised form 2 October 2000     

Frankia and Alnus rubra Canopy Roots: An Assessment of Genetic Diversity, Propagule Availability, and Effects on Soil Nitrogen

The ecological importance of microbial symbioses in terrestrial soils is widely recognized, but their role in soils that accumulate in forest canopies is almost entirely unknown. To address this gap, this study investigated the Frankia–Alnus rubra symbiosis in canopy and forest floor roots at Olympic National Park, WA, USA. Sixteen mature A. rubra trees were surveyed and Frankia genetic diversity in canopy and forest floor nodules was assessed with sequence-based nifH analyses. A seedling bioassay experiment was conducted to determine Frankia propagule availability in canopy and forest floor soils. Total soil nitrogen from both environments was also quantified. Nodules were present in the canopies of nine of the 16 trees sampled. Across the study area, Frankia canopy and forest floor assemblages were similar, with both habitats containing the same two genotypes. The composition of forest floor and canopy genotypes on the same tree was not always identical, however, suggesting that dispersal was not a strictly local phenomenon. Frankia seedling colonization was similar in canopy soils regardless of the presence of nodules as well as in forest floor soils, indicating that dispersal was not likely to be a major limiting factor. The total soil nitrogen of canopy soils was higher than that of forest floor soils, but the presence of Frankia nodules in canopy soils did not significantly alter soil nitrogen levels. Overall, this study indicates that the Frankia–A. rubra symbiosis is similar in canopy and forest floor environments. Because canopy roots are exposed to different environmental conditions within very small spatial areas and because those areas can be easily manipulated (e.g., fertilizer or watering treatments), they present microbial ecologists with a unique arena to examine root–microbe interactions.     

An Examination of some Symbiotic Systems for Fixation of Nitrogen

By the use of ¹⁵N the occurrence of fixation of elemental nitrogenhas been demonstrated in the lichens Collema granosum and Leptogiumlichenoides (both of which contain Nostoc as a symbiont), andalso in the liverwort Blasia pusilla (which has Nostoc-containingcavities in the thallus). The circumstances indicate that thefixation in these instances should be attributed to the Nostoc. No evidence of fixation was obtained in similar isotopic testson the mycorrhizal roots of intact plants of Calluna vulgarisand of Pinus sylvestris, although these, more especially inthe case of Calluna, have been held by some previous authorsto be nitrogen-fixing.