Soil and environmental factors related to nodulation in Cowania and Purshia

Summary Nitrogen fixing trees and shrubs may be useful in revegetation efforts. The possibility that soil and environmental factors may influence a soil's capability to produce nodulated seedlings was explored.Purshia tridentata andCowania mexicana var. Stansburiana seedlings were grown in greenhouse trials using ten soils from native sites for each of the two genera. Treatments included a control and a six mmole nitrogen amendment as NH₄NO₃ for both surface and subsurface samples. Nodulation was often sparse for seedlings grown in surface collected samples. Although nodulation was usually better in subsoil samples, even some subsoils produced few or no nodules. Nitrogen additions inhibit nodulation and although soil nitrogen may be inhibitory in some unamended surface soils it is probably not a general cause of sparse nodulation. Nodule masses showed the same trends as nodule number but varied less with treatment and depth of soil source. Seedlings compensated for sparse nodulation with an increase in mass per nodule. Incidence of nodulation was related to some soil and environmental factors. Multiple regression analysis explained a substantial portion of nodulation variability. Soils from lower elevations with less precipitation did not produce well nodulated seedlings even in well watered greenhouse trials. Micronutrient cations, potassium, and phosphorus are positively correlated with nodulation incidence. The two genera were generally similar in nodulation responses to soil and environmental factors.Support for portions of this study was provided by the National Science Foundation (PCM-8204885). Any opinions, findings conclusions or recommendations expressed are those of the authors and do not necessarily reflect the views of the sponsor. Oregon Agricultural Experiment Station technical paper number 7293.     

Nodulation and biological nitrogen fixation of 80 soybean cultivars in symbiosis with indigenous rhizobia

Eighty soybean cultivars were assessed for their potential for nodulation and nitrogen fixation with indigenous rhizobia in a Nigerian soil. Seventy-six days after planting (DAP) 87%, 3% and 10% of the soybean cultivars had from 0 to 30, 31 to 60 and over 61 nodules/plant, respectively. Only 8% had a nodule dry weight of 600 to 1100 mg/plant. At 84 DAP the proportion of nitrogen derived from the atmosphere (Ndfa) ranged from 0 to 65% 16% of the cultivars derived 51 to 65% of their N₂ from the atmosphere. The diversity of soybean germplasm and the variation in nodulation and N₂ fixation permitted the selection of the five best cultivars in terms of their compatibility with indigenous rhizobia, % Ndfa and the amount of N₂ which they fixed.     

Temperature control of nitrogen fixation in Klebsiella pneumoniae

At growth temperatures above 37°C, Klebsiella pneumoniae does not grow in a medium containing N₂ or NO ₃ ^- as nitrogen sources. However, both the growth in the presence of other nitrogen sources as well as the in vitro nitrogenase activity are not affected at this temperature. The inability to fix N₂ at high temperature is due to the failure of the cells to synthesize nitrogenase and other nitrogen fixation (nif) gene encoded proteins. When cells grown under nitrogen fixing conditions at 30°C were shifted to 39°C, there was a rapid decrease of the rate of de novo biosynthesis of nitrogenase (component 1), nitrogenase reductase (component 2), and the nifJ gene product. There was no degradation of nitrogenase at the elevated temperature since preformed enzyme remained stable over a period of at least 3 h at 39°C. Thus, temperature seems to represent a third control system, besides NH ₄ ⁺ and O₂, governing the expression of nif genes of K. pneumoniae.     

Nodulation,nitrogen fixation and nitrogen uptake in pigeonpea (Cajanus cajan (L.) Millsp) of different maturity groups

Summary The seasonal patterns of nodulation, acetylene reduction, nitrogen uptake and nitrogen fixation were studies for 11 pigeonpea cultivars belonging to different maturity groups grown on an Alfisol at ICRISAT Center, Patancheru, India. In all cultivars the nodule number and mass increased to a maximum around 60–80 days after sowing and then declined. The nodule number and mass of medium- and late-maturing cultivars was greater than that of early-maturing cultivars. The nitrogenase activity per plant increased to 60 days after sowing and declined thereafter, with little activity at 100 days when the crop was flowering. At later stages of plant growth nodules formed down to 90 cm below the soil surface but those at greater depth appeared less active than those near the surface. All the 11 cultivars continued to accumulate dry matter until 140 days, with most biomass production by the late-maturing cultivars (up to 11 t ha⁻¹) and least by the early-maturing determinate cultivars (4 t ha⁻¹). Total nitrogen uptake ranged from 69 to 134 kg ha⁻¹. Nitrogen fixation by pigeonpea was estimated as the difference in total nitrogen uptake between pigeonpea and sorghum and could amount to 69 kg N ha⁻¹ per season, or half the total nitrogen uptake. Fixation by pigeonpea increased with crop duration, but there were differences within each maturity group. The limitations of the methods used for estimating N₂ fixation by pigeonpea are discussed. Submitted as J.A. No. 552 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT).     

Variation in magnesium and nitrogen content in seeds of antelope bitterbrush (Purshia tridentata, Rosaceae)

One-hundred and sixty-three seeds from 38 shrubs of antelope bitterbrush (Purshia tridentata) from four different sites were individually analyzed for N and Mg content. Seed nutrient content has previously been shown to affect seedling growth, competitive ability, and adult biomass and seed production, though estimations of nutrient concentration in seeds have always been based on bulk samples of more than one seed. The results from antelope bitterbrush show that individual seeds vary in N and Mg content (coefficient of variation = 15.9 and 10.1%, respectively), with most of the variation being attributable to seed size (over 70%). Larger seeds not only had greater absolute quantities of N and Mg, but also a greater concentration of N. Differences between seeds from the same shrub explained the second largest amount of variation (over 20%), while differences between shrubs and site of growth, though significant, accounted for much less of the variation (just over 5%). Soil N and Mg were not closely related to seed N or Mg at a site, suggesting that decisions on how much N or Mg to allocate to seeds are not entirely based on supply. Shrubs that were browsed most intensively the winter prior to seed production had seeds with higher concentrations of Mg. Differences in seed mineral content between shrubs suggest the possibility for natural selection to operate, though research to determine heritability of this character will be necessary to confirm how much of the observed variation is attributable to plasticity in seed filling.     

Nodulation and nitrogen fixation mutants of pea,Pisum sativum

Summary The 36 mutants which did not nodulate and 24 mutants which formed inefficient nodules with no or very low acetylene reduction activity were isolated among 86,000 M₂-seedlings of Finale pea, Pisum sativum L., after treatment with chemical mutagens. One mutant was found for approximately every 50 chlorophyll mutants. Most mutations were induced by ethyl methanesulfonate; some by diethyl sulfate, ethyl nitrosourea and acidified sodium azide. Putative mutants were selected as nitrogen deficient plants, yellowing from the bottom and up, when M₂ seedlings were grown in sand with a Rhizobium mixture and PK fertilizer. The mutants were verified in the M₃ generation by acetylene reduction assay on intact plants.     

Contribution of nitrogen fixation to nitrogen nutrition in an alpine sedge community (Caricetum curvulae)

Summary In situ acetylene reduction assays (ARA) were carried out over two growing seasons at 2550 m in the upper alpine zone of the Tyrolean Central Alps of Austria. For comparative purposes, some Fabaceae species introduced into the upper alpine zone from lower elevation (2000 m) were subjected to ARA. At the end of the growing season the potted plants were transferred to the laboratory where their acetylene reducing activities were measured again. In situ nitrogenase activity is very low. The highest values were found in association with Leucanthemopsis alpina and Veronica bellidioides (150 and 217 nmol ethylene 24 h^-1 per pot respectively). Higher levels of activity were detected in pots transferred to the laboratory (maximum value 750 nmol ethylene 24 h^-1 per pot; assay temperature about 12°C higher than in the field) and in the Fabaceae transferred to the upper alpine zone (14×10³ nmol ethylene 24 h^-1 per pot of Trifolium badium and T. pallescens). Maximum nitrogen input in the field is in the range of 8 mg m^-2 a^-1. Therefore, under natural circumstances biological nitrogen fixation contributes only very small amounts of nitrogen to this alpine vegetation system, the process being inhibited by low soil temperatures. Possible alternative sources and patterns of N acquisition are discussed in relation to the overall nitrogen economics of plants of the upper alpine zone.     

Nitrogen fixation and nitrogen metabolism in heliobacteria

Three species of anoxygenic phototrophic heliobacteria, Heliobacterium chlorum, Heliobacterium gestii, and Heliobacillus mobilis, were studied for comparative nitrogen-fixing abilities and regulation of nitrogenase. Significant nitrogenase activity (acetylene reduction) was detected in all species grown photoheterotrophically on N₂, although cells of H. mobilis consistently had higher nitrogenase activity than did cells of either H. chlorum or H. gestii. Nitrogen-fixing cultures of all three species of heliobacteria were subject to switch-off of nitrogenase activity by ammonia; glutamine also served to switch-off nitrogenase activity but only in cells of H. mobilis and H. gestii. Placing photosynthetically grown heliobacterial cultures in darkness also served to switch-off nitrogenase activity. Dark-mediated switch-off was complete in lactate-grown heliobacteria but in pyruvate-grown cells substantial rates of nitrogenase activity continued in darkness. In all heliobacteria examined ammonia was assimilated primarily through the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway although significant levels of alanine dehydrogenase were present in extracts of cells of H. gestii, but not in the other species. The results suggest that heliobacteria, like phototrophic purple bacteria, are active N₂-fixing bacteria and that despite their gram-positive phylogenetic roots, heliobacteria retain the capacity to control nitrogenase activity by a switch-off type of mechanism. Because of their ability to fix N₂ both photosynthetically and in darkness, it is possible that heliobacteria are significant contributors of fixed nitrogen in their paddy soil habitat.     

Nodulation and symbiotic nitrogen fixation of cowpea (Vigna unguiculata (L.) Walp)

Summary Rhizobium strains CIAT 301, CIAT 79 and SLM 602 were tested and found effective in the nodulation and nitrogen fixation of cowpea cv. MI-35 (Vigna unguiculata (L.) Walp) plants in growth chamber experiments. Fresh weight of nodules increased with plant age initially and stabilized in 20–30 days from planting, followed by a secondary flush of nodule growth after 30 days. Apparent nitrogen fixation per gram nodule fresh weight reached a maximum in 20–30 days after planting and then decreased, even though a flush of new nodules was produced.     

木麻黄共生固氮菌Frankia的分离鉴定及固氮效应

从浙江省短枝木麻黄(Casuarina equisetifolia)和细枝木麻黄(C.curninghamiana的根瘤中共分离获得14株共生菌株.形态观察表明,菌株具有分枝状菌丝、多腔孢囊、泡囊等典型的Frankia结构、16S rDNA测序结果表明,供试菌株均为Frankia,其中4株属于生理类群A,7株属于生理类群B,3株属于生理类群AB.固氮效应研究表明,菌株均具有固氨酶生物学活性,但菌株之间存在显著差异,其中菌株ZCN192固氨酶活性最强,可达2.897 μmol·mg-1·h-1,菌株ZCN199最低,固氮酶活性为0.056 μmol·mg-1·h-1.活体固氮试验显示,与阴性对照相比,供试菌株能显著提高苗高、地径和干重,且一般情况下,离体固氮酶活性强的菌株在活体接种时能获得更明显的固氮效应.     

Effect ofAzospirillum inoculation on nitrogen fixation and growth of several winter legumes

Summary Inoculation of naturally nodulatedPisum sativum L. (garden pea) withAzospirillum in the greenhouse caused a significant increase in nodule numbers above controls. Field inoculation of garden peas in the winter 1981–1982 andCicer arietinum L. (chick pea), in winter 1982–1983, withAzospirillum one week after plant emergence, produced a significant increase in seed yield, but did not affect plant dry matter yield. ForVicia sativa L. (vetch) grown in soil in the greenhouse and in the field for forage, winter 1980–1981, inoculation significantly increased dry matter yield, %N, N-content, and acetylene reduction (nitrogen fixation) activity. InHedysarum coronarium L. (sulla clover), winter 1981–1982, inoculated with both its specificRhizobium (by the slurry method) andAzospirillum, 7 days after emergence, there was an increase in acetylene reduction above controls inoculated withRhizobium alone. These results suggest that it is possible, under conditions tested in this work, to increase nodulation, nitrogen fixation, and crop yields of winter legumes by inoculation withAzospirillum.     

Delayed inoculation and starter nitrogen for enhancing early growth and nitrogen status ofLespedeza cuneata

Summary Two growth chamber experiments were conducted to determine the response ofLespedeza cuneata (Dumont) G. Don. (sericea lespedeza) to delayed inoculation and low levels of nitrogen fertilization. Nitrogen was supplied either as NH ₄ ⁺ or as NO ₃ ^– in solution. At 0.5 and 5.0 ppm nitrogen early growth and N₂(C₂H₂) fixation was inhibited by NH ₄ ⁺ and promoted by NO ₃ ^– . Inoculation at seeding did not negatively affect growth prior to the onset of N₂(C₂H₂) fixation. Delayed inoculation until the trifoliate stage thus did not increase growth or N₂ fixation during the first 40 days of growth. After 40 days, specific nitrogenase activity was highest for plants inoculated at the first trifoliate stage of growth. In contrast, growth and total shoot nitrogen accumulation were higher in plants inoculated at planting. The experimental results suggest that delaying inoculation is not a useful technique for improving early growth ofL. cuneata for surface mine reclamation.     

根瘤菌共生固氮能力的进化模式

根瘤菌-豆科植物共生固氮体系对农业的可持续性发展至关重要,也是研究原核与真核生物互利共生的模式体系之一。长期以来,根瘤菌共生固氮相关研究主要集中在结瘤因子与固氮酶合成及调控等少数关键基因,但仅获得这些关键基因却不能保证细菌获得结瘤固氮能力。随着比较和功能基因组学的快速发展和应用,越来越多的研究发现根瘤菌使用了很多系统发育分支特异的遗传机制与豆科植物建立有效的共生关系,进一步揭示了双方互利共生的复杂性。本综述总结了近年来比较基因组学、遗传学以及实验进化等方面的相关研究进展,在此基础上讨论根瘤菌共生固氮能力的进化模式。     

Nodulation and nitrogen fixation in extreme environments

Biological nitrogen fixation is a phenomenon occurring in all known ecosystems. Symbiotic nitrogen fixation is dependent on the host plant genotype, theRhizobium strain, and the interaction of these symbionts with the pedoclimatic factors and the environmental conditions. Extremes of pH affect nodulation by reducing the colonization of soil and the legume rhizosphere by rhizobia. Highly acidic soils (pH<4.0) frequently have low levels of phosphorus, calcium, and molybdenum and high concentrations of aluminium and manganese which are often toxic for both partners; nodulation is more affected than host-plant growth and nitrogen fixation. Highly alkaline soils (pH>8.0) tend to be high in sodium chloride, bicarbonate, and borate, and are often associated with high salinity which reduce nitrogen fixation. Nodulation and N-fixation are observed under a wide range of temperatures with optima between 20–30°C. Elevated temperatures may delay nodule initiation and development, and interfere with nodule structure and functioning in temperate Iegumes, whereas in tropical legumes nitrogen fixation efficiency is mainly affected. Furthermore, temperature changes affect the competitive ability ofRhizobium strains. Low temperatures reduce nodule formation and nitrogen fixation in temperate legumes; however, in the extreme environment of the high arctic, native legumes can nodulate and fix nitrogen at rates comparable to those observed with legumes in temperate climates, indicating that both the plants and their rhizobia have successfully adapted to arctic conditions. In addition to low temperatures, arctic legumes are exposed to a short growing season, a long photoperiod, low precipitation and low soil nitrogen levels. In this review, we present results on a number of structural and physiological characteristics which allow arctic legumes to function in extreme environments.     

Hydrogen metabolism and energy costs of nitrogen fixation

Abstract The high energy costs of biological nitrogen fixation are partly caused by hydrogen production during the reduction of dinitrogen to ammonia. Some nitrogen-fixing organisms can recycle the evolved hydrogen via a membrane-bound uptake hydrogenase. The energetic aspects of hydrogen metabolism and nitrogen fixation are discussed.
Studies on both isolated nitrogenase proteins and nitrogen-fixing chemostat cultures show that energy limitation will result in a high hydrogen production by nitrogenase. In plant- Rhizobium symbiosis, the supply of oxygen or photosynthetate is the limiting factor for nitrogen fixation. In both cases, nitrogen fixation is energy-limited, and it is concluded that a large amount of hydrogen is produced during nitrogen fixation in these symbioses.
Hydrogen reoxidation yields less energy than the oxidation of endogenous substrates, and therefore expression of hydrogenase under oxygen-limited conditions is energetically unfavourable. Moreover, hydrogen reoxidation can never completely regain the energy invested during hydrogen production. The controversial reports of the effect of hydrogen reoxidation on the efficiency of nitrogen fixation are being discussed.
The determination of the energy costs of nitrogen fixation (expressed as the amount of ATP needed to fix 1 mol of N₂) using chemostat cultures is described. Calculations show that the nitrogenase-catalysed hydrogen production has more influence on the efficiency of nitrogen fixation than the absence or presence of a hydrogen uptake system.     

Effect of inoculation ofAzospirillum lipoferum on nitrogen fixation in rhizosphere soil,their association with root,yield and nitrogen uptake by mustard (Brassica juncea)

Summary A microplot field experiment was conducted in the presence or absence of P and N application to evaluate the influence of the seed inoculation of mustard (cv. Baruna T₅₉) withAzospirillum lipoferum on N₂-fixation in rhizosphere, association of the bacteria with the roots and grain yield and N uptake. Inoculation significantly increased the N content in rhizosphere soil particularly at early stage (40 days) of plant growth, which was accompanied by the increased association of the bacteria (A. lipoferum) in rhizosphere soil, root surface washing and surface-sterilized macerated root. A significant increase in grain yield and N uptake was also observed due to inoculation. Application of P particularly at the 20 kg. ha^–1 level further enhanced the beneficial effect ofAzospirillum lipoferum inoculation, while N addition markedly reduced such an effect.     

Effect of ammonium or nitrate on nitrogen fixation by a terrestrial Blue-green alga

Summary The effects of ammonium or nitrate-nitrogen on biological nitrogen fixation by an algal crust are compared. Nitrate-nitrogen up to 3.0 moles N g^–1 sand/algal crust at 60% water holding capacity did not affect fixation, whereas an ammonium-nitrogen concentration of 0.2 moles N g^–1 crust markedly depressed fixation. Consequences of these differential effects are considered.     

A pellet method to demonstrate nitrogen fixation by free-living rhizobia

Summary A method is described to demonstrate nitrogen fixation by free-living Rhizobium cells. After aerobic growth in a nutrient solution, the bacteria are centrifuged. Acetylene reduction by the rhizobial cells in the pellet can be measured within a few days. Hydrogen gas frequently stimulates acetylene reduction.