An ELISA Procedure for Quantification of Relative Amounts of Intercellular Glycoprotein in Legume Nodules

An enzyme-linked immunosorbant assay (ELISA) method based ona monoclonal antibody (MAC236) is described in which relativeamounts of an intercellular glycoprotein were quantified inextracts of whole legume nodules. This glycoprotein has recentlybeen shown to be an important component of the cortical oxygendiffusion barrier. The ELISA method is demonstrated on threeexamples of soybean (Glycine max L. Merr.) nodule systems whichhave been the subject of previously published investigations:(a), cv. Clarke inoculated with Bradyrhizobium japonicum RCR3442,nodulated root systems of which were subject to 10, 21 or 40%oxygen continuously for 28 d; (b), cv. Bragg and its supernodulatingmutant derivative (nts382) inoculated with Bradyrhizobium japonicumUSDA110; (c), cv. Clarke inoculated with Bradyrhizobium japonicumRCR3442 or RCR3407. ELISA results are related to oxygen diffusioncharacteristics defined in previous publications and show thatincreases in the amount of glycoprotein present correlated withincreases in supra-ambient (40%) levels of rhizosphere pO₂,in minimum gas diffusion resistance and in speed of diffusionbarrier response. Area data of component parts of nodule inner cortices suggestthat diffusion resistance control under sub-ambient (10%) oxygenlevels also involves cell expansion. The amount of MAC236 antigen in nodules is affected by bothhost plant genotype and rhizobial strain and the latter alsoappears to be involved in determining the morphological developmentof the nodule inner cortex.Copyright 1993, 1999 Academic Press Oxygen diffusion resistance, glycoprotein, nodules, Glycine max, ELISA     

A New Procedure for the Calculation of Oxygen Diffusion Resistance in Legume Nodules from Flow-through Gas Analysis Data

Plants of white lupin (Lupinus albus L cv Multolupa) and soyabean(Glycine max L cv Clarke) were grown in controlled-environmentcabinets, subjected to various stresses and their nodular nitrogenaseactivity and total root respiration measured When these measurementswere used to calculate nodular oxygen diffusion resistance,using a simplified equation for Fick's first law of diffusion,it was found that the apparent resistance of stressed nodulesincreased anomalously with decreases in external oxygen concentrationA new analysis procedure is proposed to alleviate this anomalyThis procedure also uses the simplified Fick's law equationbut includes a respiratory contribution to the total oxygenflux across the diffusion barrier which is not coupled to nitrogenaseactivity Also, resistance is modelled as an exponential functionof external oxygen concentration Use of this analysis procedureproduces realistic values for total resistance and providesa characterisation of this resistance into a minimum value andan adjustment factor for changes in external oxygen It is postulatedthat the additional respiration component represents the activityof nodule cortex cells involved in the diffusion barrier, particularlythat of vascular bundles Oxygen diffusion resistance, nodule, nitrogen fixation, respiration     

Direct Evidence for Changes in the Resistance of Legume Root Nodules to O2 Diffusion

Indirect evidence suggests that legumes can adjust rapidly theresistance of their root nodules to O₂ diffusion. Here we describeexperiments using O₂ specific micro-electrodes and dark fieldmicroscopy to study directly the operation of this diffusionbarrier. The O₂ concentration sensed by the electrode decreasedsharply in the region of the inner cortex and was less than1.0 mmol m^–3 throughout the infected tissue in nodulesof both pea (Pisum sativum) and french bean (Phaseolus vulgaris).In a number of experiments the ambient O₂ concentration wasincreased to 40% while the electrode tip was just inside theinner cortex. In 13 out of 21 cases the O₂ concentration atthis position either remained low and unchanged or increasedirreversibly to near ambient values. In the remaining casesthe O₂ concentration increased after 1 to 2.5 min and then decreasedto its former value. These results are ascribed to an increasein resistance of the barrier in response to increased O₂ fluxinto the nodule. It was shown microscopically that air spacesboth at the boundary between the infected zone and the innercortex, and within the infected zone started to disappear 3min after nodules were exposed to high ambient O₂ concentrationsand had disappeared completely after 8 min. These spaces werenot changed by exposure of the nodule for 10 min to either N₂or air. Key words: Oxygen, root nodules, air spaces     

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     

Oxygen, the NifA gene, Nodule Structure and the Initiation of Nitrogen Fixation

Both nitrogenase and the NifA gene, which controls the expressionof the nitrogenase enzyme, require microaerobic conditions inthe infected zone of a nodule. Such conditions depend on theability of the respiratory system to consume oxygen at approximatelythe maximum rate at which it can enter that region by diffusion.The balance between consumption and supply governs the minimumsize of a functional nodule. The nature of this balance is exploredin this paper using a model. It shows that it appears physicallyimpossible for legume root nodules, below a certain minimumsize, to fix nitrogen. Furthermore, experimental data are usedto suggest that the alternative oxidase respiratory systemsmay initially provide the large respiratory capacity requiredto create microaerobic conditions, suitable for the NifA geneand nitrogenase. Nitrogen fixation, NifA gene, diffusion resistance, oxygen     

Localization of carbonic anhydrase in legume nodules

Extracts of the central infected zone and the surrounding cortex of nodules from Lupinus angustifolius L., Vigna unguiculata L. (Walp), Pisum sativum L., Phaseolus vulgaris L., Vicia faba L. and Medicago sativa L. contained significant activities of carbonic anhydrase (CA). Immunoassay of extracts using antisera to a putative nodule CA (Msca1) cloned from M. sativa also indicated expression in both tissue types. Quantitative confocal microscopy using laser scanning imaging and a fluorescent CA‐specific probe (5‐dimethylaminonaphthalene‐1‐sulfonamide [DNSA]) localized expression to the infected cells in the central zone tissue and a narrow band of 2–3 files of cells in the cortical tissue that corresponded to the inner cortex. In the infected cells, the enzyme activity was distributed evenly in the cytosol, but in the inner cortical cells, it was restricted to the periphery – possibly to the plasma membrane or cell wall. The functions of CA in these two tissues are considered in relation to the carbon metabolism of nodules and the participation of the inner cortex in the regulation of gaseous diffusive resistance.     

Oxygen Diffusion in Lupin Nodules: II. MECHANISMS OF DIFFUSION BARRIER OPERATION

The oxygen diffusion resistance of Lupinus albus (L.) cv. Multoluparoot nodules was increased by subjection to short-term stresses;lowering rhizosphere temperature from 25 to 16 °C (2 h),detopping plants (3 h), darkening plants (21 h) or exposingroots to 20 mol m^–3 KN03 for 2, 4 or 6 d. Microscopicobservations and measurements showed that this resulted in thearea of open intercellular spaces within the inner cortex beingreduced due to both cell expansion and increased productionof an occluding glycoprotein. Electrophoretic and Western Blotanalysis using the monoclonal antibodies MAC236 and MAC265 showedtwo distinct glycoprotein antigens with molecular weights of240 and 135 kDa, respectively. Both antigens are localized withinintercellular spaces of the inner cortex. The amount of glycoproteinwas determined using either ELISA, with MAC265, or quantificationof immunolabelling with MAC236. This immunolabelling also localizedthe glycoprotein within globules adhering to the inside of theinner cortical cell walls. Key words: Oxygen diffusion resistance, glycoprotein, nodules, nitrogen fixation, Lupinus albus     

Morphological Alterations and Root Nodule Formation inAgrobacterium rhizogenes-mediated Transgenic Hairy Roots of Peanut (Arachis hypogaeaL.)

Transformed hairy roots were induced at the excised site ofthe epicotyl of dry mature seed of a Spanish type peanut(Arachishypogaea)cv. Java 13 2 weeks after inoculation with a wild typestrain ofAgrobacterium rhizogenes,MAFF-02-10266. Composite plantsconsisting of transformed roots with non-transformed shootswere cultured using pouches. Forty days after inoculation, thecomposite plant showed a root system with abundant root mass,more lateral branching and high fractal dimension compared tothe control. No differences were observed in production of rosette-typeroot hairs or the cross sectional structure between transformedand non-transformed roots. The inoculation ofBradyrhizobiumsp.A2R1 strain to the composite plants led to the induction oftransformed root nodules. These transformed root nodules showedproduction of leghaemoglobin in the bacterial zone and nitrogenaseactivity as assayed by C₂H₂reduction, and exhibited enlargementof the nodule cortex region andde novoroot formation from thenodule cortex.Copyright 1998 Annals of Botany Company Agrobacterium rhizogenes;Arachis hypogaeaL.; composite plant; peanut; transformation; root nodule.     

Gas diffusion pathway in nodules ofCasuarina cunninghamiana

The gas diffusion pathway in nodules was traced by vacuum infiltration with India ink or aniline blue and by electron microscopy. India ink infiltration was observed in the outermost and the innermost cortex in sliced nodules, but not in intact nodules. With aniline blue infiltration, it was observed that intercellular air spaces in the outermost and the innermost cortex were connected to those in nodule roots. No air spaces were in contact with walls of infected cells, although intercellular air spaces existed in some groups of uninfected cells within the infected zone. Infiltration with either India ink or aniline blue could not be observed in the infected zone in essentially all cases. Thus it is suggested that the discontinuity of the intercellular air spaces represents a major resistance to O₂ diffusion in nodules ofCasuarina cunninghamiana.     

Alterations in Apoplastic and Total Solute Concentrations in Soybean Nodules Resulting from Treatments Known to Affect Gas Diffusion

Ureide concentration in the cortical apoplast of soybean (Glycinemax(L.) Merr.) nodules increases rapidly in response to noduleexcision. The objective here was to determine if changes inapoplastic ureide may be related to the control of resistanceto gas diffusion which is thought to be localized in the nodulecortex. Following decapitation of shoots, nitrogenase activity(acetylene reduction) and ureide concentration in total noduleextracts declined over a period of several hours. Apoplasticureide concentration relative to total nodule ureide was elevatedunder these conditions, but the treatment effect was small comparedto non-decapitated controls. Decapitation also caused a significantdecline in the concentrations of sucrose, glucose, and D-pinitolin nodules. However, the decline in carbohydrates was similarin the nodule cortex and the nodule as a whole, suggesting thatthe carbohydrate changes are not related to a cortex-localizedmechanism. Non-invasive treatments involving increases or decreasesin oxygen concentration supplied to nodulated roots caused rapiddecreases in respiration of nodulated roots and in ureide concentrationin total nodule extracts, but did not cause major changes inapoplastic ureide concentrations. The combined results indicatethat apoplastic ureide is probably not involved in the regulationof resistance to gas diffusion. The rapid decline in noduleureide concentrations in response to changing oxygen supplydocuments the sensitivity of ureide synthesis and/or transportto alterations in nodule respiration and/or nitrogenase activity Key words: Glycine max, Pisum sativum, ureide, carbohydrates     

High pH Causes Disintegration of the Root Surface in Lupinus angustifolius L.

The effect of high pH on the morphology and anatomy of the rootsof lupin (Lupinus angustifolius L. cv. Yandee) and pea (Pisumsativum L. cv. Dundale) was examined in buffered solution. Themorphology and anatomy of lupin roots were markedly altered,and root growth was reduced by increasing solution pH from 5·2to 7·5, whereas pea roots were unaffected. In lupin roots,pH 7·5 caused disintegration of the root surface andimpaired root hair formation. Lupin roots grown at pH 7·5also had decreased cell lengths but increased cell diameterin both the epidermis and the cortex in comparison to rootsgrown at pH 5·2. High pH reduced cell volume greatlyin the epidermis, to a lesser extent in the outer cortex andnot at all in the inner cortex. It appears that in lupins, theprimary detrimental effects of growth at pH 7·5 is reducedlongitudinal growth of cells near the root surface with a consequentreduction in elongation of the cells in inner cortex.Copyright1993, 1999 Academic Press Lupinus angustifolius L., Pisum sativum L., high pH, root morphology, root anatomy     

Evidence that short-term regulation of nodule permeability does not occur in the inner cortex

Regulation of nodule permeability in response to short-term changes in environmental and physiological conditions is thought to occur by occlusion of intercellular spaces in the nodule inner cortex. To test this hypothesis, the permeability of legume nodules was altered by adapting them to either 20 or 80% O₂ over a 2.5-h period. The nodules were then rapidly frozen, cryo-planed and examined under cryo-scanning electron microscopy for differences in the number, area or shape factor of intercellular spaces. Comparisons were made between whole nodules and specific nodule zones (outer cortex, middle cortex, inner cortex and central zone) in each treatment. Gas analysis measurements indicated that nodules equilibrated at 20% O₂ had a 6.6-fold higher permeability than those equilibrated at 80% O₂ However, no significant differences were observed between pO₂ treatments in the number of open intercellular spaces, the cross-sectional area of those spaces, or the proportion of the tissue area present as open space in whole nodules or any nodule zone. Also, although nodules in both treatments possessed a boundary layer of tightly packed cells in the inner cortex, the total area of intercellular spaces between cells bordering this layer did not differ between treatments. Together these observations do not support the currently favored hypothesis that nodule permeability is regulated by opening or occlusion of intercellular spaces in the nodule inner cortex. Highly significant differences (P= 0.0006) were observed between O₂ treatments in the shape factor of the open intercellular spaces in all nodule zones. Nodules equilibrated at 80% O₂ had significantly more isodiametric spaces while those equilibrated at 20% O₂ had more long, narrow spaces. This observation suggests that the critical step in the regulation of nodule permeability to O₂ may be localized in the central, infected zone and involve changes in the ratio of the surface area of the intercellular space to the volume of the infected cell.     

The Effect of Irradiance on the Recovery of Soybean Nodules from Sodium Chloride-Induced Senescence

Soybean (Glycine max L. Merr) cv. Clarke plants inoculated withBradyrhizobium japonicum strain RCR3407 were grown either ina greenhouse with a low irradiance (200–400)µmolm^–2 s^–1) or in a controlled-environment growth cabinetwith a higher irradiance (600 µimol m^–2 s^–1).At 42 d plants were given a nitrogen-free nutrient solutioncontaining 50 mol m^–3 sodium chloride for 2 weeks andthen allowed to recover from salt-stress for a further 2 weeks. Salt treatment reduced plant growth by at least half in bothgrowth regimes, however, the controlled environment-grown (CEG)plants were five times larger than the greenhouse-grown (GG)plants in terms of dry weight and number/weight of nodules perplant, regardless of treatment. The structure of nodules, from both growth regimes, harvestedat the end of the 2 week salt-stress was similar to unstressedcontrol nodules. However, nodules harvested 1 week later fromboth CEG and GG plants had structural changes including degradationof bacteria in vacuoles around host cell nuclei, particularlyin the outer cell layers of the infected tissue. In addition,meristematic activity was seen in the cortex of some nodulesfrom GG plants. Young cells here contained infection threadsand newly-released bacteria. Nodules harvested 2 weeks after removal of the salt-stress fromCEG plants showed an apparent recovery from the stress. However,there was a very marked increase in the amount of starch inthe cortex which was not seen in equivalent GG nodules. In contrast,nodules from GG plants contained many vacuolate infected cellsand, consequently, a lowered bacteroid population. Further,meristematic activity was seen in a zone concentric to the infectedzone, newly-formed cells contained many large infection threadsand were interspersed with intercellular bacteria. The meristematicactivity increased the relative volume of cortical to infectedcells in these nodules. Growth conditions did not affect control nodule specific nitrogenaseactivity or oxygen diffusion resistance (R) and these parameterswere also not altered in CEG nodules exposed to salt plus the14 d recovery period. However, nitrogenase activity was greatlyreduced, and R increased by more than eight times in equivalentGG nodules exposed to salt plus recovery. It is hypothesized that the gross morphological changes werean attempt to counter salt toxicity and/or oxygen damage underconditions of reduced photosynthate supply to the nodules dueto the poor light levels in the greenhouse. However, soybeannodules supplied with adequate photosynthate were able to withstandand recover from long-term salt-stress with little alterationto their structural integrity. Key words: Soybean, sodium chloride, nitrogen fixation, light intensity, oxygen diffusion resistance     

Transport of Nitrate and Calcium into Legume Root Nodules

Nitrate transport into nodulated plants of soybean (Glycinemax), cowpea (Vigna unguiculata) and faba bean (Vicia faba)was investigated. Nitrate entering the root system of soybeandid not pass out of the vascular system into nodular tissuesin detectable quantities. On the other hand, nitrate could passfrom soil through the outer surface of nodules but did not penetratethe infected tissue. Similarly, nitrate was restricted to corticaltissues of cowpea and faba bean. Thus, nitrate cannot inhibitnitrogen fixation as a result of reduction to nitrite by nitratereductase within the bacteroid zone. These results are, however,consistent with an effect of nitrate on an oxygen diffusionresistance located in the nodule cortex. Unlike nitrate, measurable quantities of ⁴⁵calcium were transportedvia the xylem into infected and cortical tissues of soybeannodules: it also passed from the soil into the free space ofthe nodule cortex. Key words: Nitrate, legume nodules, calcium     

The Structure of Nitrogen Fixing Root Nodules on the Aquatic Mimosoid Legume Neptunia plena

Nodules of the aquatic mimosoid legume Neptunia plena (L.) Benth.were always found associated with roots but not stems. Theyappeared macroscopically 10 and 20 d after inoculation on plantsgrown hydroponically and in vermiculite, respectively. The developmentof nitrogen-fixing cells occurred in a series of stages notyet reported in legume nodule formation: initial infection wasapparently intercellular and rhizobia spread between cells andthrough intercellular spaces before penetrating individual hostcells by means of infection threads. Subsequently nodule developmentwas broadly similar to that described for indeterminate papilionoidnodules. The infection threads of Neptunia and pea nodules containeda matrix with a common epitope, which was, in Neptunia, extrudedfrom the infection thread at the point of bacterial release. The central tissue contained infected and interstitial cellsand was surrounded by a three-layered cortex and a phellem.Bounding the infected region was a layer two to three cellsthick with large, unoccluded intercellular spaces. Externalto this was a layer, one or more cells thick, in which the cellwalls were interlocked, reducing the number of radially orientedintercellular spaces. The outer layer, several cells thick,contained intercellular spaces many of which were occluded.These features did not vary with growth conditions in a waywhich might influence oxygen diffusion characteristics. However,the phellem of water-cultured nodules was much more aerenchymatousthan that of vermiculite-grown nodules. Aquatic legume, Neptunia plena, nitrogen fixation, oxygen, root nodules, Rhizobium     

Wavelength options for monitoring leghaemoglobin oxygenation gradients in intact legume root nodules

Nodule oximetry, based on spectrophotometric measurements ofleghaemoglobin (Lb) oxygenation in intact nodules, has providednumerous insights into legume nodule physiology. Fractionaloxygenation of Lb (FOL) has been monitored at various wavelengths,but comparisons among wavelengths have not been published previously.Changes in transmittance were monitored simultaneously at 660nm and either 560 or 580 nm as FOL was manipulated by changingthe O₂ concentration around nodules of Medicago sativa L. orLotus comiculatus L. Video microscopy at 580 nm was used togenerate two-dimensional maps of FOL gradients in intact nodules.In general, all three wavelengths gave similar results. Smalldiscrepancies between 660 and 580 nm, sometimes seen in noduleswith high O₂ permeability, may indicate interference by theferric Lb peak at 625 nm. A slightly longer wavelength, forexample 670 nm, might be preferable. No significant discrepanciesamong wavelengths were seen in nodules whose O₂ permeabilityhad been reduced by a 48 h exposure to 10 mM nitrate. Minorgradients in FOL were seen in nodules of M. sativa and Trifoliumrepens L. under air and steeper gradients could be induced byvarious treatments. The existence of these gradients indicatesat least some restriction of longrange O₂ diffusion within theinfected zone. The FOL maps do not have enough spatial resolutionto measure gradients within infected cells. Key words: Leghaernoglobin oxygenation, nodules, spectrophotometry, nodule oximetry     

Tracing the Path of Oxygen into Birdsfoot Trefoil and Alfalfa Nodules using Iodine Vapor

Although physiological control of nodule 0₂ permeability is an active area of research, the gas diffusion pathway between the atmosphere and the infected zone has not been firmly established. Previous studies have used infiltration of ink or dyes to identify points of entry, but such water-soluble tracers could give a misleading picture of gas diffusion pathways. We therefore used iodine vapor (and its reaction with starch) to trace gas-phase pathways into the infected zone of determinate birdsfoot trefoil (Lotus corniculatus) and indeterminate alfalfa (Medicago sativa) nodules. We also used histochemical methods to identify suberized or lignified layers that could act as barriers to gas diffusion. Birdsfoot trefoil nodules were surrounded by a suberized periderm, but nonsuberized cells and intercellular spaces were observed in the periderm between lenticels and their associated vascular bundles. Iodine entered birdsfoot trefoil nodules only through lenticels. The periderm appears to provide a significant barrier to gas diffusion. Although airspaces were rare in the nodule parenchyma (also referred to as the “inner cortex”), we found some evidence that a few air-filled pathways cross this secondary barrier, also in the vicinity of vascular bundles. Alfalfa nodules were cylindrically surrounded by a suberized endodermis which ended near the meristematic tip; iodine entered principally at the end of the endodermis near the meristem. Future research on physiological control of nodule O₂ permeability should concentrate on strategic “choke points”, associated with lenticels in determinate nodules, or in the zone proximal to the meristem in indeterminate nodules.     

Electron microscopic investigation of water occlusions in intercellular spaces in the inner cortex of lucerne nodules.

It is unclear to what extent oxygen diffusion pathways through the cortex of the nitrogen-fixing zone of indeterminate nodules are liquid filled and whether a blockage of these pathways is involved in varying nodule oxygen permeability to control nitrogenase activity. We examined the proportion of water-filled intercellular spaces of lucerne (Medicago sativa L.) nodules with cryo-scanning electron microscopy. This technique allows for direct observation of water accumulation. Thirty percent of all intercellular spaces in the inner cortex of lucerne nodules were liquid filled. Decreasing the nodule oxygen permeability by detopping of the plant or by increasing the rhizospheric oxygen partial pressure to 80 kPa had no statistically significant effect on the water distribution in the intercellular spaces. Therefore, the hypothesis of a continuous aqueous diffusion barrier in the inner cortex could not be supported. The abundance of glycoproteins in intercellular spaces of the inner cortex was investigated with immunoelectron microscopy. No alteration due to detopping or after increase of the rhizospheric oxygen partial pressure was observed. Therefore, our results do not support the hypothesis of a short-term regulation of oxygen permeability by blockage of diffusion pathways through morphological changes in the cortex region of the nitrogen-fixing zone of lucerne nodules.     

Time-course of changes involved in the operation of the oxygen diffusion barrier in white lupin nodules

Nodulated white lupins (Lupinus albus L. cv. Multolupa) weresubject to either darkening for 12 h, followed by 24 h recoveryin light, or to 50% O₂ for 30 min. For each treatment, noduleswere harvested at intervals for analysis by light and electronmicroscopy and determination of glycoprotein content using EnzymeLinked Immunosorbent Assays (ELISA). This allowed for an analysisof the sequence of events causing an increase in intercellularspace occlusion within the inner cortex. The temporal sequencein response to darkening appears to be: (1) an initial rapidincrease in the detectable levels of intracellular glycoprotein,due to either a state change or de novo synthesis, (2) a concomitantincrease in the volume of thickened cell walls, causing a reductionof intercellular space volume and (3) after 1–3 h a releaseof glycoprotein into the intercellular space network of theinner cortex, accompanied (and possibly spread) by the continuedconstriction of the spaces due to cell wall and cell contentexpansion. The results for exposure to 50% O₂ showed a similar,but much more rapid, sequence of events, operating within 15–30min. The main difference between the two sequences was the lackof expansion of thickened cell walls with increased pO₂. Also,it was possible to detect glycoprotein within cell walls followingexposure to 50% O₂ but not following darkening. These observationsare discussed in relation to proposed mechanisms for the operationof a variable oxygen diffusion barrier in legume nodules. Key words: Oxygen diffusion resistance, glycoprotein, nodules, Lupinus albus     

Development of Stem Nodules in a Tropical Forage Legume, Aeschynomene afraspera

Rhizobial infection occurred on the stem of Aeschynomene afrasperaat the site of emergence of adventitious root primordia. Rhizobiainvaded cortical cells at the base of the root primordium. Thefirst infected cell enlarged and collapsed after rhizobia hadmultiplied in large numbers. At this time, a meristematic zonewas initiated some distance beneath the first infected cell.Rhizobial penetration into the deeper cortex was by progressivecollapse of infected cells towards the meristematic zone; rhizobiaentering the cortical cells by invagination of the host cellwall. At the entry point, rhizobia were embedded in digitatecell wall material. These infection structures were restricted,always originating from the cell wall of an adjacent infectedcell. Soon after infection, the cell collapsed progressivelyforming infection strand-like structures which developed upto the meristematic zone. When infection had reached the meristematiczone, invaded host cells ceased to collapse but divided repeatedlyto form the nodule. Key words: Aeschynomene afraspera, stem nodulation