The Development and Significance of the Root Nodules of Casaurina

Young plants of Casuarina cunninghamiana and of C. equisetifoliagrowing in water culture developed macroscopic nodules in 26to 35 days from inoculation, the nodulation being most successfulat pH near to neutrality and falling off much more rapidly atlower pH than in other non-legume nodule-forming genera, confirmingthe distinctiveness of the Casuarina organism. The roots springingfrom the nodule lobes are shown to be characterized by upwardgrowth and in this to resemble remarkably closely the correspondingroots of Myrica. Nodulated plants of Casuarina are able to growvigorously in culture solution free of combined nitrogen, showingthat fixation of atmospheric nitrogen occurs, amounting in C.cunninghamiana to 50 mg. per plant during 6 months of activegrowth. The evidence indicates that the fixation occurs in thenodules, and that these have exactly the same functional significanceas those of legumes.     

An Isotopic Study of the Fixation of Nitrogen associated with Nodulated Plants of Alnus, Myrica, and Hippophae

Nodulated plants of Alnus glutinosa, Myrica gale, and Hippophaërhamnoides, the root systems of which had been exposed to excessfree ¹⁵N, showed substantial enrichment in fixed ¹⁵N contentin all parts of the plant, but particularly in the root nodules.The data resemble closely those obtained by the present andprevious authors with legumes under comparable conditions, andsupport the conclusion already drawn from experiments of traditionaltype that the nodules of these non-legumes are similar to thoseof legumes in their function and relation to the rest of theplant. Nodulated plants of Alnus and Myrica continued to fixfree nitrogen concurrently with the uptake of combined nitrogenwhen the latter was supplied in the rooting medium in amountsunlikely to be exceeded in the field. Isotopic tests on detachednodules of one of the species (Alnus glutinosa) showed thatfixation continued, and though much reduced as compared withthat shown by attached nodules it considerably exceeded thatexperienced by the present and previous authors with detachedlegume nodules, on the basis of fixation per unit of total nodulenitrogen. It is probable that detached Alnus nodules presentconvenient material for the further study of various aspectsof the fixation process.     

The Nitrogen-Nutrition of Hippophae rhamnoides L.

It is shown that Hippophaë plants, inoculated at the six-leafstage with a suspension of crushed nodules, begin to developnodules in about two weeks and soon afterwards become able togrow satisfactorily in culture solution free of combined nitrogen,up to 79 mg. nitrogen per plant having been accumulated during7 months' active growth. It is concluded that the nodules fixatmospheric nitrogen, and that in its nitrogen nutrition theHippophaë plant shows a close similarity to the nodulatedlegume. It is pointed out that Hippophaë and the otherAngiospermous genera with nitrogen-fixing root nodules are allwoody plants or belong to a family which includes woody types.The possible significance of this is briefly discussed.     

The Physiology and Nitrogen-fixing Capability of Aquatically and Terrestrially Grown Neptunia plena: The Importance of Nodule Oxygen Supply

The aquatic legume Neptunia plena (L.) Benth. was grown in non-aeratedwater culture or vermiculite. Growth, nodulation, nitrogen fixationand nodule physiology were investigated. Over an 80-d period,plants grew and fixed nitrogen and carbon equally well in bothrooting media, although distribution of growth between plantparts varied. Total nodule dry weights and volumes were similarbut vermiculite-grown plants had three times as many (smaller)nodules than those grown in water. Oxygen diffusion resistanceof nodules exposed to 21% oxygen and 10% acetylene did not differsignificantly. Both treatments showed similar declines in rootrespiration and acetylene reduction activity (approx. 10%) whenroot systems were exposed to stepped decreases and increasesin rhizosphere oxygen concentration. However, nitrogenase activityof aquatically grown plants was irreversibly inhibited by rapidexposure of nodules to ambient air, whereas vermiculite-grownplants were unaffected. Aeration of water-cultured N. plenareduced stem length (but not mass) and number of nodules perplant. The concentration of nitrogen fixation by 163%. PossibleO₂ transport pathways from the shoot atmosphere to roots andnodules are discussed. Aquatic legume, diffusion resistance, Neptunia plena, nitrogen fixation, oxygen, root nodules     

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.     

Relationships between Acetylene Reduction Activity, Hydrogen Evolution and Nitrogen Fixation in Nodules of Acacia spp.: Experimental Background to Assaying Fixation by Acetylene Reduction under Field Conditions

Hansen, A. P., Pate, J. S. and Atkins, C. A. 1987. Relationshipsbetween acetylene reduction activity, hydrogen evolution andnitrogen fixation in nodules of Acacia spp.: Experimental backgroundto assaying fixation by acetylene reduction under field conditions.—J.exp. Bot. 38: 1–12 Glasshouse grown, symbiotically-dependent seedlings of Acaciaalata R.Br., .A. extensa Lindl., and A. pulchella R.Br. wereexamined for acetylene reduction in closed assay systems usingundisturbed potted plants, excavated whole plants, nodulatedroots or detached nodules. Nitrogenase activity declined sharplyover the first hour after exposure of detached nodules to acetylene(10% v/v in air), less steeply or not at all over a 3 h periodin assays involving attached nodules. Using detached nodules,rates of acetylene reduction, nitrogen (¹⁵N₂) fixation, andhydrogen evolution in air (¹⁵N₂) and acetylene-containing atmosphereswere measured in comparable 30 min assays. Total electron flowthrough nitrogenase in air was determined from rates of nitrogen(¹⁵N₂) fixation ( ? 3) plus hydrogen evolution, that in thepresence of acetylene from rates of acetylene reduction andhydrogen evolution in air: acetylene. Values for the ratio ofelectron flow in air: acetylene to that in air ranged from 0?43to 0?83 in A. pulcheila, from 0?44 to 0?66 in A. alala and from0?37 to 0?70 in A. extensa, indicating substantial inhibitionof electron flow through nitrogenase of detached nodules byacetylene. Relative efficiencies of nitrogenase functioningbased on hydrogen evolution and acetylene reduction were from0?15 to 0?79, those based on nitrogen (¹⁵N₂) fixation and hydrogenevolution from 0?53 to 0?87. Molar ratios of acetylene reducedto nitrogen (¹⁵N₂) fixed were 2?82 ? 0?24, 201 ? 0?15, and 1?91? 0?11 (?s.e.; n = 7) for A. pulcheila,A. extensa and A. alata respectively A standard 5–10 min acetylene reduction assay, conductedon freshly detached unwashed nodules in daytime (12.00–14.00h), was calibrated for field use by comparing total N accumulationof seedlings with estimated cumulative acetylene reduction overa 7-week period of glasshouse culture. Molar ratios for acetylenereduced: nitrogen fixed using this arbitrary method were 3?58for A. alata, 4?82 for A. extensa and 1?60 for A. pulchella.The significance of the data is discussed. Key words: Acacia spp, nitrogenase functioning     

Young Reproductive Structures Promote Nitrogen Fixation in Soya Bean

In many legumes the transition from the vegetative to the reproductivephase of development is associated with a marked increase inthe rate of symbiotic nitrogen fixation. In soya bean [Glycinemax (L.) Merr.), the removal of reproductive parts at differentstages of their development showed that the increase in nitrogenfixation rate was primarily due to the presence of flower buds.The increase in the fixation rate of intact reproductive plantswas accompanied by a rapid increase in the weight of noduleson lateral roots and it is suggested that these nodules areresponsible for much of the nitrogen fixation which occurs duringreproductive growth. Maintaining plants in the vegetative stateprovided evidence which suggests that it is the flower budsand not the flowering stimulus which are responsible for theincrease in fixation rate. The marked effects on vegetativegrowth of removing reproductive parts suggests that the mechanisminvolved in the promotion of nitrogen fixation may be hormonal. Glycine max (L.) Merr., soya bean, nitrogen fixation     

Assimilate Partitioning in Red and White Clover Either Dependent on N2 Fixation in Root Nodules or Utilizing Nitrate Nitrogen

During vegetative growth in controlled environments, the patternof distribution of ¹⁴C-labelled assimilates to shoot and root,and to the meristems of the shoot, was measured in red and whiteclover plants either wholly dependent on N₂ fixation in rootnodules or receiving abundant nitrate nitrogen but lacking nodules. In experiments where single leaves on the primary shoot wereexposed to ¹⁴CO₂, nodulated plants of both clovers generallyexported more of their labelled assimilates to root (+nodules),than equivalent plants utilizing nitrate nitrogen, and thiswas offset by reduced export to branches (red clover) or stolons(white clover). The intensity of these effects varied with experiment.The export of labelled assimilate to growing leaves at the terminalmeristem of the donor shoot was not influenced by source ofnitrogen. Internode elongation in the donor shoot utilized nolabelled assimilate. Whole plants of white clover exposed to ¹⁴CO₂ on seven occasionsover 32 days exhibited the same effect on export to root (+nodules),which increased slightly in intensity with increasing plantage. Nodulated plants had larger root: shoot ratios than theirequivalents utilizing nitrate nitrogen. Trifolium repens, Trifolium pratense, red clover, white clover, nitrogen fixation, nitrate utilization, assimilate partitioning     

The effect of ammonium nitrogen on fixation of elemental nitrogen inAlnus andMyrica

Summary 1. A substantial formation of nodules occurred on plants ofAlnus glutinosa andMyrica gale grown in water culture with different levels of ammonium nitrogen labelled with N¹⁵ present in the culture solution. The nodules tended to be fewer but larger than on plants in solution free of combined nitrogen.2. The nodules continued to fix atmospheric nitrogen despite the presence of ammonium nitrogen in the rooting medium, though fixation per unit weight of nodule tissues formed was somewhat lower than in nitrogen-free solution. Among other possible reasons this could have been due to a substitution of ammonium nitrogen for elemental nitrogen at the nitrogen-fixing centres of the nodule, but evidently this does not occur to any great extent.3. In Alnus but not in Myrica fixation per plant was considerably enhanced in the presence of a low level of ammonium nitrogen, owing to greater nodule development. At higher ammonium levels, in excess of the plants' requirements, fixation per plant was still of comparable order to that in nitrogen-free solution, but now only represented some 24 to 45 per cent of the total nitrogen accumulated by the plants.4. The results suggest that under field conditions some fixation of atmospheric nitrogen will always be associated with nodules present.     

Nitrogen Fixation, Nodule Numbers Per Unit Ground Area and Bioenergetics

The utilization of dinitrogen by legumes is influenced by thegenetic characteristics of both the plant and the Rhizobiumresident in root nodules. The number of nodules required tomeet the demand for nitrogen depends on the capacity of individualnodules to supply nitrogen and the availability of soil nitrogen.In this paper some simple equations are derived which enablethe number of nodules per unit ground area to be calculated.Further calculations are made of nodule mass, specific nodulerespiration and the work required to expand nodules againstthe impedance of the soil. Nitrogen fixation, nodules, respiration, bioenergetics     

Effectiveness of Lotus Root Nodules: III. EFFECT OF COMBINED NITROGEN ON NODULE EFFECTIVENESS AND FLAVOLAN SYNTHESIS IN PLANT ROOTS

When grown in a nutrient solution containing combined nitrogen(NH₄NO₃), Lotus pedunculatus and L. tenuis seedlings inoculatedwith a fast-growing strain of Rhizoblum (NZP2037) did neitherdevelop root nodules nor develop flavolans in their roots. Incontrast, the roots of nodulated seedlings growing in a nitrogen-freenutrient solution contained flavolans. Flavolan synthesis coincidedwith root nodule development on these plants. When added as a single dose, high concentrations of NH₄NO₃ (5and 10 mg N per plant) stimulated the growth of L. pedunculatusplants but suppressed nodulation and nitrogen fixation. In contrastthe continued supply of a low concentration of NH₄NO₃ (1?0 mgN d^–1 per plant) stimulated nitrogen fixation by up to500%. This large increase in nitrogen fixation was associatedwith a large increase in nodule fresh weight per plant, a doublingof nodule nitrogenase activity, and a lowering of the flavolancontent of the plant roots. The close relationship between nitrogendeficiency, nodule development, and flavolan synthesis in L.pedunculatus meant that it was not possible (by nitrogen pretreatmentof plants) to alter the ineffective nodule response of a Rhizobiumstrain (NZP2213) sensitive to the flavolan present in the rootsof this plant.     

Some effects of combined nitrogen on the nodule symbioses of Casuarina and Ceanothus

Summary 1. The effect of ammonium-nitrogen on the further growth and activity in fixation of nodules already present at the commencement has been studied inCasuarina cunninghamiana growing in water culture and inCeanothus velutinus var.laevigatus in Peralite culture. 2. In Casuarina, at a low level of ammonium-nitrogen nodule growth remained similar to that in plants in nitrogen-free solution, but was stimulated in Ceanothus. In both genera nodule growth was strongly retarded at higher levels. 3. Fixation of nitrogen fell continuously in Casuarina as the level of ammonium-nitrogen was increased, while that in Ceanothus was unaffected at a low level but markedly decreased at a higher one. These effects were compounded from the changes noted in 2 and a tendency for the efficiency of nodule tissues in fixation to fall in the presence of ammonium-nitrogen, though this was not always shown. 4. In both genera but especially in Casuarina the growth of plants entirely dependent on nodule nitrogen was inferior to that of plants additionally supplied with ammonium-nitrogen. The reasons are discussed. 5. The results indicate the effects likely to be produced in the field as the soil nitrogen level rises through the action of the nodules of these species.     

Fine Structure of Peanut Root Nodules Induced by Nod+Fix- Strains of Bradyrhizobium with Special Reference to Lipid Bodies

Ineffective nodules of peanut induced by two nod⁺fix^–strains of Bradyrhizobium sp. were compared with the ones inducedby nod⁺fix⁺ strain NC92. One of the fix^– strains, 639is a transconjugate Tn5 mutant of NC92, while the other, 7091,is an isolate from ICRISAT soil. Both induce small nodules lackingleghemoglobin and nitrogen-fixing activity. Ultrastructuralobservations revealed that the nodules of 639, have enlargedperibacteroid space and lack persistence of nodule function.The 7091-induced nodules showed impediment in bacteroid releaseand differentiation. In both the ineffective nodules large amountsof lipid bodies were found to accumulate several times in excess,compared to the effective NC92 nodules. The large lipid accumulationin absence of nitrogen fixation supports the hypothesis thatin peanut nodules lipid bodies are utilized as a supplementarysource of carbon and energy for nitrogen fixation. Peanut, lipid bodies, nitrogen fixation, nod⁺fix^– Bradyrhizobium, ultrastructure     

Effect of Source of Nitrogen on the Growth of Fiskeby Soya Bean: the Carbon Economy of Whole Plants

Fiskeby V soya bean was grown from seed germination to seedmaturation with two contrasting patterns of nitrogen metabolism:either wholly dependent on dinitrogen fixation, or with an abundantsupply of nitrate nitrogen, but lacking root nodules. The carbonand nitrogen economies of the plants were assessed at frequentintervals by measurements of photosynthesis, shoot and rootrespiration, and organic and inorganic nitrogen contents. Plantsfixing atmospheric nitrogen assimilated only 25–30 percent as much nitrogen as equivalent plants given nitrate nitrogen:c. 40 per cent of the nitrogen of ‘nitrate’ plantswas assimilated after dinitrogen fixation had ceased in ‘nodulated’plants. The rates of photosynthesis and respiration of the shootsof soya bean were not markedly affected by source of nitrogen;in contrast, the roots of ‘nodulated’ plants respiredtwice as rapidly during intense dinitrogen fixation as thoseof ‘nitrate’ plants. The magnitude of this respiratoryburden was calculated to increase the daily whole-plant respiratory loss of assimilate by 10–15 per cent over thatof plants receiving abundant nitrate. It is concluded that ‘nodulated’plants grew more slowly than ‘nitrate’ plants inthese experiments for at least two reasons: firstly, the symbioticassociation fixed insufficient nitrogen for optimum growth and,secondly, the assimila tion of the nitrogen which was fixedin the root nodules was more energy-demanding in terms of assimilatethan that of plants which assimilated nitrogen by reducing nitratein their leaves.     

Responses of N2 Fixation-linked Respiration to Host-plant Energy Status in White Clover Acclimated to a Controlled Environment

Ryle, G. J. A., Powell, C. E. and Gordon, A. J. 1988. Responsesof N₂ fixation-linked respiration to host-plant energy statusin white clover acclimated to a controlled environment.—J.exp. Bot. 39: 879–887. Single plants of white clover, acclimated to a controlled environmentand dependent for nitrogen on N₂ fixation in their root nodules,were darkened, defoliated or exposed to enhanced CO₂ levelsto establish the quantitative relationships between the photosynthesisof the host plant and the N₂ fixation metabolism of root nodules. The nodule respiration associated with N₂ fixation (FLR) declinedrapidly to 10–15% of its normal rate following plant darkeningearly in the photoperiod. Darkening at progressively later intervalsduring the photoperiod demonstrated a positive, apparently linearrelationship between duration of illumination and total FLRduring the photoperiod and the following night period. Completeor partial defoliation reduced FLR according to the leaf arearemoved: again, there was a strong positive correlation betweencurrent rate of photosynthesis, whether of defoliated or undefoliatedplants and the FLR of root nodules. Doubling the current rateof photosynthesis, by enhancing CO₂ levels around the shoots,promoted FLR within 1–2 h when plants were stressed bylack of light. However, enhanced CO₂ levels increased FLR onlyslowly over a period of several hours in plants entrained tothe normal growing conditions. It is concluded that, in these plants acclimated to a uniformand favourable controlled environment, the supply and utilizationof photosynthetic assimilate in N₂ fixation was finely balancedand quantitatively linked during a single diurnal period andthat nodule functioning was not depressed by lack of energysubstrate. Key words: White clover, N₂ fixation, photosynthesis.     

Oxygen Diffusion in Lupin Nodules: I.VISUALIZATION OF DIFFUSION BARRIER OPERATION

Root nodules of Lupinus albus (L.) cv. Multolupa were subjectedto short- and medium-term stresses by lowering rhizosphere temperaturefrom 25 to 16°C (2 h), detopping plants (3 h), darkeningplants (21 h) or exposing roots to 20 mol m^–3 KNO₃ for4 d. All experimental treatments produced increases in oxygendiffusion resistance, compared with control plants. These correlatedwith structural changes in the nodule cortex, which is describedin detail for the first time. The most noticeable change isthe occlusion of intercellular spaces by a glycoprotein whichwas identified using the monoclonal antibody MAC236. This glycoproteinwas also found surrounding bacteria in intercellular spacesof the cortex of control nodules. Key words: Oxygen diffusion resistance, glycoprotein, nodules, nitrogen fixation, Lupinus albus     

Defoliation in White Clover: Regrowth, Photosynthesis and N2 Fixation

Single plants of white clover, grown in a controlled environmentand dependent for nitrogen on fixation in their root nodules,were defoliated once by removing approximately half their shoottissue. Their regrowth was compared with the growth of comparableundefoliated plants. Two similar experiments were carried out:in the first, plants were defoliated at 2.5 g, and in the secondat 1.2 g total plant d. wt. Defoliation reduced rate of N₂ fixation by > 70 per cent,rate of photosynthesis by 83–96 per cent, and rate ofplant respiration by 30–40 per cent. Nodule weights initiallydeclined following defoliation as a result of loss of carbohydratesand other unidentified components. No immediate shedding ofnodules was observed but nodules on the most severely defoliatedplants exhibited accelerated senescence. The original rates of N₂ fixation were re-attained after 5–6or 9 d regrowth, with increase in plant size at defoliation.In general, the rate of recovery of N₂ fixation was relatedto the re-establishment and increase of the plant's photosyntheticcapacity. Throughout the growth of both defoliated and undefoliatedplants nodule respiration (metabolism) accounted for at least23 ± 2 per cent of gross photosynthesis. The unit ‘cost’of fixing N₂ in root nodules, in terms of photosynthate, appearedto be unaffected by defoliation, except perhaps for plants veryrecently defoliated. Similarly, the percentage nitrogen contentsof shoot, root and nodules of defoliated plants became adaptedwithin a few days to those characteristic of undefoliated plants. Trifolium repens, white clover, N₂ fixation, defoliation, photosynthesis, respiration     

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.     

Comparative Growth and Symbiotic Performance of Seedlings of Acacia spp. in Defined Pot Culture or as Natural Understorey Components of a Eucalypt Forest Ecosystem in S.W. Australia

Hansen, A. P. and Pate, J. S. 1987. Comparative growth and symbioticperformance of seedlings of Acacia spp. in defined pot cultureor as natural understorey components of a eucalypt forest ecosystemin S.W. Australia.—J. exp. Bot. 38: 13–25 Growth, nitrogen accumulation, nodulation and nitrogenase activity(C₂H₂ reduction activity) were monitored in a dense stand ofseedlings of Acacia pulchella R.Br. and A. alata R.Br. duringtwo growing seasons after fire at a jarrah (Eucalyptus marginataDonn ex Sm.) forest site at Illawarra, 35 km south-east of Perth.Nitrogen fixation, estimated by a previously calibrated C₂H₂reduction assay, was essentially restricted to the winter andspring months (July to October) and was estimated to contribute37% and 9% respectively of the total N accumulated by A. pulchelladuring the first and second seasons of growth. Comparable valuesfor A. alata were 29% and 2%. Comparisons with fully symbioticplants raised in a glasshouse in supposedly non-limiting growthconditions in minus nitrogen sand culture indicated that waterstress rather than high temperatures was responsible for lossof nodules, cessation of symbiotic activity and attenuationof growth during summer in the field. By 19 months glasshouseplants had gained 130–230 times the dry weight and accumulated110–160 times the total N of similarly aged, field grownplants. Growth, nodulation and N₂ fixation of symbiotically-dependentsand cultured plants of A. pulchella and A. alata respondedmarkedly to phosphate, and additions of water and nutrientsto 3-year plants of A. pulchella during a winter growing seasonin the field indicated that periods of reduced soil moistureand low availability of nutrients, particularly phosphorus,might limit symbiotic performance under natural conditions. Key words: Seasonal nitrogen fixation, Acacia spp     

The Cobalt Requirement of Non-legume Root Nodule Plants

As already shown for Alnus glutinosa, cobalt is found to beessential for the proper growth of nodulated plants of Casuarinacunninghamiana and Myrica gale in a nitrogen-free rooting medium.If cobalt is not supplied, the plants develop symptoms of nitrogendeficiency; under the conditions of the experiments such symptomsbecame pronounced during the second season of growth of theseperennial plants. No cobalt requirement could be detected innon-nodulated plants of Alnus and Myrica supplied with nitrateor ammonium-nitrogen, and this suggests that in nodulated plantsthe need for cobalt is confined to the nodules. Vitamin B₁₂analogues are shown to be present in the nodules in relativelylarge amounts when cobalt is supplied, their formation beingattributed to the endophytes, which may therefore require cobaltfor their growth. The great reduction in fixation of atmosphericnitrogen in cobalt-deficient nodules may be due to a retardedgrowth of the endophyte, though this is not the only possibility.The cobalt relation of these non-legumes appears to be basicallysimilar to that of legumes.