Plant-endophyte symbiosis in non-leguminous plants

Summary A wide taxonomic range of non-leguminous dicotyledonous plants bear root nodules and are able to fix atmospheric nitrogen. These plants belong to the orders Casuarinales, Myricales, Fagales, Rhamnales, Coriariales, and Rosales. Actinomycetes are involved in the root-nodule symbiosis. Nitrogen fixation is inhibited by hydrogen and carbon monoxide. Combined nitrogen depress nodule formation, but nitrogen fixation still occurs in the presence of combined nitrogen in the medium. In nitrogen-free medium Alnus plants fix in one season of 48 weeks 500 mg N per plant and Ceanothus plants 760 mg N per plant. Fixation by the other plant species is about of the same order. Field estimates showed that the nitrogen increase of the soil was about 61.5–157 kg N per ha per annum, depending on the age of the trees, under Alnus, 58.5 kg N per ha per annum under Casuarina, about 60 kg N per ha per annum under Ceanothus, 27–179 kg N per ha per annum underHippopha? rhamnoides, and about 61.5 kg N per ha per annum underDryas drummondii with someShepherdia spp. Non-leguminous root nodules belong to two types: coralloid root nodules and root nodules where the apex of each nodule lobe produces a negatively geotropic root. The primary infection occurs through the root hairs where a curling effect is observed. In the host cells the endophyte presents itself in three forms: hyphae, vesicles and bacteria-like cells. Vesicles are probably associated with nitrogen fixation, whereas the bacteria-like cells function in the endophyte's survival and dispersal. The endophyte is an obligate symbiont. TheAlnus glutinosa endophyte has been isolated and grownin vitro in root-nodule callus tissue. However, the isolated endophyte produces only ineffective root nodules in re-inoculation tests.     

The Carbon Balance of a Legume and the Functional Economy of its Root Nodules

Budgets for carbon and nitrogen in shoot, root, and nodulesof garden pea (Pisum sativum L.) are drawn up for a 9-d intervalin the life cycle, from data on nitrogen fixation, carbon accumulationin dry matter, respiratory output of plant organs, and organicsolute exchange between shoot and nodulated root. Of the carbon gained photosynthetically by the shoot from theatmosphere 26 per cent is incorporated directly into its drymatter, 32 per cent translocated to the nodules, and 42 percent to the supporting root. Of the nodules’ share, 5per cent is consumed in growth, 12 per cent in respiration,and 15 per cent returned to the shoot via the xylem, as aminocompounds generated in nitrogen fixation. Growth and respirationof the root utilize, respectively, 7 and 35 per cent. The respiratory efficiency of a nodulated root in terms of nitrogenfixation (5.9mg C per mg N₂-N fixed) is found to be very similarto that of an uninoculated root assimilating nitrate (6.2 mgC per mg NO₃-N reduced). The nodules require in growth, respiration,and export 4.1 mg C ( 10.3 mg carbohydrate) for each mg N whichthey fix. The nodules consume 3 ml O₂ for every 1 ml N₂ utilized in fixation. In exporting a milligram of fixed nitrogen the nodules requireat least 0.35 ml of water. Almost half of this requirement mightbe met by mass flow into the nodules via the phloem.     

Partitioning and Utilization of Carbon and Nitrogen in Nodulated Roots and Nodules of Chickpea (Cicer arietinum) Grown at Two Moisture Levels

The partitioning and utilization of carbon (C) and nitrogen(N) in nodulated roots and nodules of chickpea (Cicer arietinumL.) was studied at two moisture levels at 10-d intervals 40–140d after sowing (DAS). More C was used in respiration and lessin growth of nodulated roots and nodules under water stresscompared to controls during all growth stages except at theearly vegetative stage. Similarly, less nitrogen was investedin dry matter of both nodules and nodulated roots under stress,except during the vegetative stage where more nitrogen was used.Calculated over the entire growth period, as much as 14 and20% of the total nitrogen and 3 and 4% of the total carbon fixedby the plant was lost to the rooting medium under controlledand stressed conditions, respectively. The efficiency of nitrogen fixation with respect to net C utilizationwas maximal during seed filling under both control and stressconditions However, the efficiency of nitrogen fixation wasalways greater under drier conditions. Carbon, chickpea (Cicer arietinum L.), nitrogen, nitrogen fixation, partitioning     

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.     

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.     

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     

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     

Multi‐symbiotic systems: functional implications of the coexistence of grass–endophyte and legume–rhizobia symbioses

The coexistence of symbionts with different functional roles in co‐occurring plants is highly probable in terrestrial ecosystems. Analyses of how plants and microbes interact above‐ and belowground in multi‐symbiotic systems are key to understand community structure and ecosystem functioning. We performed an outdoor experiment in mesocosms to investigate the consequences of the interaction of a provider belowground symbiont of legumes (nitrogen‐fixing bacteria) and a protector aerial fungal symbiont of grasses (Epichloё endophyte) on nitrogen dynamics and aboveground net primary productivity. Four plants of Trifolium repens (Trifolium, a perennial legume) either inoculated or not with Rhizobium leguminosarum, grew surrounded by 16 plants of Lolium multiflorum (Lolium, an annual grass), with either low or high levels of the endophyte Neotyphodium occultans. After five months, we quantified the number of nodules in Trifolium roots, shoot biomass of both plant species, and the contribution of atmospheric nitrogen fixation vs. soil nitrogen uptake to above ground nitrogen in each plant species. The endophyte increased grass biomass production (+ 16%), and nitrogen uptake from the soil – the main source for the grass. Further, it reduced the nodulation of neighbour Trifolium plants (?50%). Notably, due to a compensatory increase in nitrogen fixation per nodule, this reduced neither its atmospheric nitrogen fixation – the main source of nitrogen for the legume – nor its biomass production, both of which were doubled by rhizobial inoculation. In consequence, the total amount of nitrogen in aboveground biomass and aboveground productivity were greatest in mesocosms with both symbionts (i.e. high rhizobia + high endophyte). These results show that, in spite of the deleterious effect of the endophyte on the establishment of the rhizobia–legume symbiosis, the coexistence of these symbionts, leading to additive effects on nitrogen capture and aboveground productivity, can generate complementarity on the functioning of multi‐symbiotic systems.     

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.     

NITROGEN FIXATION IN THE BAYBERRY (MYRICA PENSYLVANICA) AND ITS ROLE IN COASTAL SUCCESSION

The nodules on roots of Myrica pensylvanica (bayberry) contain a bacterial endophyte. By using the acetylene reduction technique these plant endophyte associations were shown to be capable of fixing nitrogen. As nodulation was plentiful and fixation vigorous, it is proposed that the success of M. pensylvanica as an early successional plant of dunes and impoverished coastal soils is due in part to the nitrogen-fixing capacity of its nodular association.     

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.     

Interrelationship Between Plant Developmental Stage, Plant Growth Rate, Nitrate Utilization and Nitrogen Fixation in Hydroponically Grown Soybean

The growth rate of the indeterminate soybean plant [Glycinemax (L.)Merr.] slows as it proceeds from vegetative phase intoreproductive growth. Yet, the well-nodulated plant acquiresmost of its nitrogen during reproductive growth. Thus, the interrelationshipbetween plant developmental stage and nitrogen fixation wasexamined. It is shown that, regardless of the age of the hydroponicallygrown soybean plant at the time of its inoculation with Bradyrhizobiumjaponicum, the highest rate of nitrogen fixation occurs duringthe pod-filling stage (R5). Nevertheless, maximum total nitrogenfixation is generally achieved when inoculation occurs at thefull-bloom stage (R2). It is shown, however, that flower budsand flowering are not responsible for the enhanced nodulationand nitrogen fixation. Rather, the data suggest that the onsetof rapid nodulation occurs soon after the initiation of thedevelopmentally programmed drop in foliar nitrate reductaseactivity. The ensuing increase in nitrogen fixation providesthe plant with much of its needed nitrogen and hence stimulatesplant mass accumulation during pod-fill. It is suggested thatnitrogen fixation enhances growth of the soybean plant by increasingits net photosynthetic efficiency during reproductive growthand by providing the needed nitrogen at the appropriate timefor maximum seed growth. Key words: Glycine max, nitrate, nitrogen fixation, nodulation     

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.     

The Development of Primary Root Nodules on Vicia faba L. Grown at Two Temperatures

The development of primary root nodules on the field bean (Viciafaba L.) grown at 10 and 18 °C was examined. The sequenceof anatomical changes observed was the same in both temperatureregimes. Nodules developed much more slowly at 10 °C andthe nodules were much larger when corresponding anatomical changesoccurred. Primary root nodules eventually ceased growth in bothtemperature regimes but ultimate nodule volume was nearly twiceas great as 10 °C as at 18 °C. The larger size did notcompensate for the lower specific nitrogenase activity of cold-grownnodules: nitrogen fixation (acetylene reduction) rates on awhole plant basis were much lower at 10 °C than at 18 °C.There was no difference in the total number of primary rootnodules in the two temperature regimes but their distributionwas biased towards the upper part of the root and the epicotylat 18 °C. Vicia faba L., field bean, nodulation, nitrogen fixation, temperature     

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.     

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     

Effects of the fungal endophyte, Neotyphodium lolii, on net photosynthesis and growth rates of perennial ryegrass (Lolium perenne) are independent of In Planta endophyte concentration

• Background and Aims Neotyphodium lolii is a fungal endophyteof perennial ryegrass (Lolium perenne), improving grass fitnessthrough production of bioactive alkaloids. Neotyphodium speciescan also affect growth and physiology of their host grasses(family Poaceae, sub-family Pooideae), but little is known aboutthe mechanisms. This study examined the effect of N. lolii onnet photosynthesis (P_n) and growth rates in ryegrass genotypesdiffering in endophyte concentration in all leaf tissues. • Methods Plants from two ryegrass genotypes, Nui D andNui UIV, infected with N. lolii (E+) differing approx. 2-foldin endophyte concentration or uninfected clones thereof (E–)were grown in a controlled environment. For each genotype xendophyte treatment, plant growth rates were assessed as tilleringand leaf extension rates, and the light response of P_n, darkrespiration and transpiration measured in leaves of young (30–45d old) and old (>90 d old) plants with a single-chamber openinfrared gas-exchange system. • Key Results Neotyphodium lolii affected CO₂-limited ratesof P_n, which were approx. 17 % lower in E+ than E– plants(P < 0·05) in the young plants. Apparent photon yieldand dark respiration were unaffected by the endophyte (P >0·05). Neotyphodium lolii also decreased transpiration(P < 0·05), but only in complete darkness. There wereno endophyte effects on P_n in the old plants (P > 0·05).E+ plants grew faster immediately after replanting (P < 0·05),but had approx. 10 % lower growth rates during mid-log growth(P < 0·05) than E– plants, but there was noeffect on final plant biomass (P > 0·05). The endophyteeffects on P_n and growth tended to be more pronounced in NuiUIV, despite having a lower endophyte concentration than NuiD. • Conclusions Neotyphodium lolii affects CO₂ fixation,but not light interception and photochemistry of P_n. The impactof N. lolii on plant growth and photosynthesis is independentof endophyte concentration in the plant, suggesting that theendophyte mycelium is not simply an energy drain to the plant.However, the endophyte effects on P_n and plant growth are stronglydependent on the plant growth phase.     

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.     

Comparative Response of Nodulated and Nitrogen-Supplied Cowpea (Vigna unguiculata (L.) Walp. var. Tuy) Plants to Infection by Cowpea Mosaic Virus

The effect of infection by the Cowpea Mosaic Virus (CpMV) onseveral parameters relevant to symbiotic nitrogen fixation wasdetermined in cowpea (Vigna unguiculata (L.) Walp. var. Tuy)plants nodulated with two strains of Rhizobium cowpea: IVIC–124and IVIC–38. Plants were virus-infected at the seedlingstage before Rhizobium inoculation. The effect of CpMV infectionon plant growth was analysed in nodulated and nitrogen-suppliedplants at 18, 25 and 35 d after germination. At all developmentalstages of nodulated plants CpMV infection caused a reductionof leaf chlorophyll content, leaf area, dry weight of shootsand roots, total nodule weight and nodule number. Most of thenodules from 18- and 25-d-old CpMV-infected plants did not exhibitleghaemoglobin pigmentation. CpMV infection delayed the onsetof nitrogenase activity in nodules of the rhizobial strain IVIC–124and the enzyme activity measured on a per plant basis was reducedin both strains at the first and second harvests. Significantnitrogenase activity was detected in 35-d-old infected plants.Some of the nodules of the rhizobial strain IVIC-124 and mostof the nodules from plants nodulated with the strain IVIC-38developed leghaemoglobin; however, the nodule-specific nitrogenaseactivity, estimated on a milligram nodule dry weight basis,was always higher in virus-infected plants, particularly in18-d-old CpMV-infected plants harbouring the IVIC–124strain. CpMV-infected nodules had a larger peribacteroidal space,a reduced number of peribacteroid units, a greater number ofbacteroids per unit, a lower number of vesicles and 88% lowertotal reducing sugar content. Starch accumulation was detectedin infected leaves of nodulated plants during the first harvest,while high levels of leaf reducing sugars and protein were presentat the second harvest. In healthy nodulated plants the rhizobialstrain IVIC–124 was shown to be more efficient than IVIC–38in promoting plant growth. However, the results indicate thatnodulation by rhizobial strain IVIC–124 and growth ofplants harbouring this strain were affected to a greater extentby virus infection. The effect of CpMV infection on leaf chlorophyllcontent, leaf area, carbohydrate level, leaf proteins and growthof nitrogen-supplied plants, as well as the symptoms inducedin the leaves, were less conspicuous than in nodulated plants. Key words: Cowpea, Rhizobium, virus infection, nodule untrastructure     

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