Aerial root nodules in the tropical legume,Pentaclethra macroloba

Summary Symbiotic nitrogen fixation in angiosperms normally occurs in buried root nodules and is severely inhibited in flooded soils. A few plant species, however, respond to flooding by forming nodules on stems, or, in one case, submerged roots with aerenchyma. We report here the novel occurrence of aerial rhizobial nodules attached to adventitious roots of the legume,Pentaclethra macroloba, in a lowland tropical rainforest swamp in Costa Rica. Swamp sapdings (1–10 cm diameter) support an average 12 g nodules dry weight per plant on roots 2–300 cm above water, and nodules remain in aerial positions at least 6 months. Collections from four swamp plants maintained linear activity rates (3–14 moles C₂H₄/g nodule dry weight/hr) throughout incubations for 6 and 13 hrs; excised nodule activity in most legumes declines after 1–2 hrs. Preliminary study of the anatomy and physiology suggest aerial nodules possess unusual features associated with tolerance to swamp conditions. High host tree abundance and nodulation in the swamp compared to upland sites indicate the aerial root symbiosis may contribute more fixed nitrogen to the local ecosystem than the more typical buried root symbiosis.     

Nodule Activity and Allocation of Photosynthate of Soybean during Recovery from Water Stress

Nodulated soybean plants (Glycine max [L.] Merr. cv Ransom) in a growth-chamber study were subjected to a leaf water potential (Ψ_w) of −2.0 megapascal during vegetative growth. Changes in nonstructural carbohydrate contents of leaves, stems, roots, and nodules, allocation of dry matter among plant parts, in situ specific nodule activity, and in situ canopy apparent photosynthetic rate were measured in stressed and nonstressed plants during a 7-day period following rewatering. Leaf and nodule Ψ_w also were determined. At the time of maximum stress, concentration of nonstructural carbohydrates had declined in leaves of stressed, relative to nonstressed, plants, and the concentration of nonstructural carbohydrates had increased in stems, roots, and nodules. Sucrose concentrations in roots and nodules of stressed plants were 1.5 and 3 times greater, respectively, than those of nonstressed plants. Within 12 hours after rewatering, leaf and nodule Ψ_w of stressed plants had returned to values of nonstressed plants. Canopy apparent photosynthesis and specific nodule activity of stressed plants recovered to levels for nonstressed plants within 2 days after rewatering. The elevated sucrose concentrations in roots and nodules of stressed plants also declined rapidly upon rehydration. The increase in sucrose concentration in nodules, as well as the increase of carbohydrates in roots and stems, during water stress and the rapid disappearance upon rewatering indicates that inhibition of carbohydrate utilization within the nodule may be associated with loss of nodule activity. Availability of carbohydrates within the nodules and from photosynthetic activity following rehydration of nodules may mediate the rate of recovery of N₂-fixation activity.     

Respiration and nitrogenase activity in nodules ofCasuarina cunninghamiana and cultures ofFrankia sp. HFP020203: Effects of temperature and partial pressure of O2

The effects of time after exposure to acetylene and of nodule excision were examined using a flow-through system. After a transient depression in the rate of acetylene reduction that began about 1.5 min after exposure to acetylene, the rate recovered to 98% of the initial maximum value after 40 min. After nodule excision the rate stabilized to 90% of the initial maximum value observed in the intact plant.Excised nodules, measured at 6-min intervals in a closed system, with frequent changes of the gas mixture, were used for the remaining experiments. Acetylene reduction by the nodules increased rapidly as temperature was increased between 6 and 26°C. Between 26 and 36°C there was relatively little effect of temperature on acetylene reduction.Nodules and cultures ofFrankia were compared with respect to the effect of temperature and pO₂ (partial pressure of oxygen) on oxygen uptake. Cultures ofFrankia were grown on a nitrogen-free medium at either 0.3 kPa O₂ (vesicles absent) or 20 kPa O₂ (vesicles present). Oxygen uptake by nodules (vesicles absent) and by vesicle-containing cultures was strongly dependent on pO₂ at values below 20 kPa. This suggests the presence of a barrier to oxygen diffusion. Oxygen uptake was dependent on temperature as well as on pO₂, but the Q₁₀ was much larger for the cultures than for the nodules. This suggests that vesicles or related structures are not the source of the diffusion barrier in Casuarina nodules. Respiration by cultures ofFrankia lacking vesicles became O₂-saturated at low pO₂ values. Thus these cultures did not have a significant diffusion barrier. From these results it is concluded that nodules ofCasuarina cunninghamiana have a barrier to oxygen diffusion supplied by the host tissue and not byFrankia.     

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.     

Nodulation of Glycine max by Six Bradyrhizobium japonicum Strains with Different Competitive Abilities

The root nodule locations of six Bradyrhizobium japonicum strains were examined to determine if there were any differences which might explain their varying competitiveness for nodule occupancy on Glycine max. When five strains were added to soybeans in plastic growth pouches in equal proportions with a reference strain (U.S. Department of Agriculture, strain 110), North Carolina strain 1028 and strain 110 were the most competitive for nodule occupancy, followed by U.S. Department of Agriculture strains 122, 76, and 31 and Brazil strain 587. Among all strains, nodule double occupancy was 17% at a high inoculum level (10⁷ CFU pouch⁻¹) and 2% at a low inoculum level (10⁴ CFU pouch⁻¹). The less competitive strains increased their nodule representation by an increase in the doubly occupied nodules at the high inoculum level. Among all strains, the number of taproot and lateral root nodules was inversely related at both the high and low inoculum levels (r = −0.62 and −0.69, respectively; P = 0.0001). This inverse relationship appeared to be a result of the plant host control of bacterial infection. Among each of the six strains, greater than 95% of the taproot nodules formed at the high inoculum density were located on 25% of the taproot length, the nodules centering on the position of the root tip at the time of inoculation. No differences among the six strains were observed in nodule initiation rates as measured by taproot nodule position. Taproot nodules were formed in the symbiosis before lateral root nodules. One of the poorly competitive strains (strain 76) occupied three times as many taproot nodules as lateral root nodules when competing with strain 110 (nodules were harvested from 4-week-old plants). Among these six wild-type strains of B. japonicum, competitive ability evidently is not related to nodule initiation rates.     

Spatial distribution and biomass of root nodules in a naturally regenerated stand of Alnus hirsuta (Turcz.) var. sibirica

To estimate nodule biomass of Alnus hirsuta var. sibirica, an N₂-fixing tree species, we examined the distribution pattern and size structure of nodules in a 17 to 18 year old stand naturally regenerated after disturbance by road construction in Japan. Nodules were harvested within 1 m from the outer edge of stems of plants with different sizes on four occasions from June to October. The diameter of the subtending root at the base of each nodule and nodule dry weight were measured in 20 cm increments outwards from the base of each stem. Horizontal distribution of nodules around each tree varied greatly among tree diameters at 1.3 m (dbh) within the even-aged stand. In particular, smaller individuals had a concentrated distribution of nodules close to the stem. Nodule abundance occurred further from the stems with increasing tree size. Nodule biomass within 1 m from the outer edge of individual stems increased with tree size ([nodule biomass] = 0.442 [dbh]^2.01, R ²?=?0.747, P?<?0.05). By using the relationship, nodule biomasses were estimated to be 84.1 kg ha^?1. These results suggest that it is necessary to take into account tree size and patterns of tree distribution in nodule biomass estimates.     

Transient responses of nitrogenase to acetylene and oxygen in actinorhizal nodules and cultured frankia

Nitrogenase activity in root nodules of four species of actinorhizal plants showed varying declines in response to exposure to acetylene (10% v/v). Gymnostoma papuanum (S. Moore) L. Johnson. and Casuarina equisetifolia L. nodules showed a small decline (5-15%) with little or no recovery over 15 minutes. Myrica gale L. nodules showed a sharp decline followed by a rapid return to peak activity. Alnus incana ssp. rugosa (Du Roi) Clausen. nodules usually showed varying degrees of decline followed by a slower return to peak or near-peak activity. We call these effects acetylene-induced transients. Rapid increases in oxygen tension also caused dramatic transient decreases in nitrogenase activity in all species. The magnitude of the transient decrease was related to the size of the O₂ partial pressure (pO₂) rise, to the proximity of the starting and ending oxygen tensions to the pO₂ optimum, and to the time for which the plant was exposed to the lower pO₂. Oxygen-induced transients, induced both by step jumps in pO₂ and by O₂ pulses, were also observed in cultures of Frankia. The effects seen in nodules are purely a response by the bacterium and not a nodule effect per se. Oxygen-induced nitrogenase transients in actinorhizal nodules from the plant genera tested here do not appear to be a result of changes in nodule diffusion resistance.     

Nitrogen Fixation (C(2)H(2) Reduction) by Broad Bean (Vicia faba L.) Nodules and Bacteroids under Water-Restricted Conditions

Water potentials of leaves and nodules of broad bean (Vicia faba L.) cultivated on a sandy mixture were linearly and highly (r² = 0.99) correlated throughout a water deprivation of plants. A decrease of 0.2 megapascal of the nodule water potential (Ψ_nod) induced an immediate 25% inhibition of the highest level of acetylene reduction of broad bean nodules attached to roots. This activity continued to be depressed when water stress increased, but the effect was less pronounced. Partial recovery of optimal C₂H₂ reduction capacity of mildly water stressed nodules (Ψ_nod = −1.2 megapascals) was possible by increasing the external O₂ partial pressure up to 60 kilopascals. The dense packing of the cortical cells of nodules may be responsible for the limitation of O₂ diffusion to the central tissue. Bacteroids isolated from broad bean nodules exhibited higher N₂ fixation activity with glucose than with succinate as an energy-yielding substrate. Bacteroids from stressed nodules appeared more sensitive to O₂, and their optimal activity declined with increasing nodule water deprivation. This effect could be partly due to decreased bacteroid respiration capacity with water stress. Water stress was also responsible for a decrease of the cytosolic protein content of the nodule and more specifically of leghemoglobin. The alteration of the bacteroid environment appears to contribute to the decline in N₂ fixation under water restricted conditions.     

Economy of Photosynthate Use in Nitrogen-fixing Legume Nodules: Observations on Two Contrasting Symbioses

The economy of C use by root nodules was examined in two symbioses, Vigna unguiculata (L.) Walp. (cv. Caloona):Rhizobium CB756 and Lupinus albus L. (cv. Ultra):Rhizobium WU425 over a 2-week period in early vegetative growth. Plants were grown in minus N water culture with cuvettes attached to the nodulated zone of their primary roots for collection of evolved CO₂ and H₂. Increments in total plant N and in C and N of nodules, and C:N weight ratios of xylem and phloem exudates were studied by periodic sampling from the plant populations. Itemized budgets were constructed for the partitioning and utilization of C in the two species. For each milligram N fixed and assimilated by the cowpea association, 1.54 ± 0.26 (standard error) milligrams C as CO₂ and negligible H₂ were evolved and 3.11 milligrams of translocated C utilized by the nodules. Comparable values for nodules of the lupin association were 3.64 ± 0.28 milligrams C as CO₂, 0.22 ± 0.05 milligrams H₂, and 6.58 milligrams C. More efficient use of C by cowpea nodules was due to a lesser requirement of C for synthesis of exported N compounds, a smaller allocation of C to nodule dry matter, and a lower evolution of CO₂. The activity of phosphoenolpyruvate carboxylase in nodule extracts and the rate of ¹⁴CO₂ fixation by detached nodules were greater for the cowpea symbiosis (0.56 ± 0.06 and 0.22 milligrams C as CO₂ fixed per gram fresh weight per hour, respectively) than for the lupin 0.06 ± 0.02 and 0.01 milligrams C as CO₂ fixed per gram fresh weight per hour. The significance of the data was discussed in relation to current information on theoretical costs of nitrogenase functioning and associated nodule processes.     

Carbon Dioxide Fixation by Lupin Root Nodules: I. Characterization, Association with Phosphoenolpyruvate Carboxylase, and Correlation with Nitrogen Fixation during Nodule Development

In vivo CO₂ fixation and in vitro phosphoenolpyruvate (PEP) carboxylase levels have been measured in lupin (Lupinus angustifolius L.) root nodules of various ages. Both activities were greater in nodule tissue than in either primary or secondary root tissue, and increased about 3-fold with the onset of N₂ fixation. PEP carboxylase activity was predominantly located in the bacteroid-containing zone of mature nodules, but purified bacteroids contained no activity. Partially purified PEP carboxylases from nodules, roots, and leaves were identical in a number of kinetic parameters. Both in vivo CO₂ fixation activity and in vitro PEP carboxylase activity were significantly correlated with nodule acetylene reduction activity during nodule development. The maximum rate of in vivo CO₂ fixation in mature nodules was 7.9 nmol hour⁻¹ mg fresh weight⁻¹, similar to rates of N₂ fixation and reported values for amino acid translocation.     

Characterization of four defense-related genes up-regulated in root nodules of Casuarina glauca

Actinorhizal plants are capable of high rates of nitrogen fixation, due to their capacity to establish a root-nodule symbiosis with N₂-fixing actinomycetes of the genus Frankia. Nodulation is an ontogenic process which requires a sequence of highly coordinated events. One of these mechanisms is the induction of defense-related events, whose precise role during nodulation is largely unknown. In order to contribute to the clarification of the involvement of defense-related genes during actinorhizal root-nodule symbiosis, we have analysed the differential expression of several genes with putative defense-related functions in Casuarina glauca nodules versus non-inoculated roots. Four genes encoding a chitinase (CgChi1), a glutathione S-transferase (CgGst), a hairpin-inducible protein (CgHin1) and a peroxidase (CgPox4) were found to be up-regulated in mature nodules compared to roots. In order to find out to which extend were the encoded proteins involved in nodule protection, development or both, gene regulation studies in response to SA and wounding as well as phylogenetic analysis of the protein sequences were performed. These were further characterized through expression studies after SA-treatment and wounding, and by phylogenetic analysis. We suggest that CgChi1 and CgGst are involved in defense or microsymbiont control and CgPox4 is involved in nodule development. For CgHin1 the question “defense, development or both” remains open.     

Mechanism of Nitrogenase Inhibition in Soybean Nodules : Pulse-Modulated Spectroscopy Indicates that Nitrogenase Activity Is Limited by O(2)

A novel, pulse-modulated spectroscopic system for measuring fractional leghemoglobin oxygenation and infected cell O₂ concentration (O_i) in intact attached nodules of soybean (Glycine max) is described. The system is noninvasive and uses a pulsed (1000 Hertz) light-emitting diode coupled to an optical fiber to illuminate the nodule with light at 660 nanometer. A second optical fiber receives a portion of the light reflected from the nodule and directs this to a photodiode. A lock-in amplifier measures only the signal from the photodiode which is in phase with the pulsed light from the light-emitting diode, and the voltage output from the amplifier, proportional to reflectance, is used to calculate fractional leghemoglobin oxygenation and the nanomolar concentration of free O₂ in the infected cells of the nodule (O_i). The system was used to show that inhibition of nitrogenase activity in soybean nodules by NO₃⁻ treatment, stem-girdling, continuous darkness, or nodule disturbance is caused by a reduction in O_i and limitation of respiration in support of nitrogenase activity. A plot of nitrogenase activity (measured as peak H₂ evolution in Ar:O₂) versus O_i for the various treatments was consistent with the concept that O_i limits in vivo nitrogenase activity in legume nodules under adverse conditions. The potential for using O_i to estimate nitrogenase activity in laboratory and field-grown legumes is discussed.     

Nitrate inhibition of legume nodule growth and activity : I. Long term studies with a continuous supply of nitrate

The synthesis and accumulation of nitrite has been suggested as a causative factor in the inhibition of legume nodules supplied with nitrate. Plants were grown in sand culture with a moderate level of nitrate (2.1 to 6.4 millimolar) supplied continuously from seed germination to 30 to 50 days after planting. In a comparison of nitrate treatments, a highly significant negative correlation between nitrite concentration in soybean (Glycine max [L.] Merr.) nodules and nodule fresh weight per shoot dry weight was found even when bacteroids lacked nitrate reductase (NR). However, in a comparison of two Rhizobium japonicum strains, there was only 12% as much nitrite in nodules formed by NR⁻R. japonicum as in nodules formed by NR⁺R. japonicum, and growth and acetylene reduction activity of both types of nodules was about equally inhibited. In a comparison of eight other NR⁺ and NR⁻R. japonicum strains, and a comparison of G. max, Phaseolus vulgaris, and Pisum sativum, the concentration of nitrite in nodules was unrelated to nodule weight per plant or to specific acetylene reduction activity. The very small concentration of nitrite found in P. vulgaris nodules (0.05 micrograms NO₂⁻-N per gram fresh weight) was probably below that required for the inhibition of nitrogenase based on published in vitro experiments, and yet the specific acetylene reduction activity was inhibited 83% by nitrate. The overall results do not support the idea that nitrite plays a role in the inhibition of nodule growth and nitrogenase activity by nitrate.     

Diurnal variation in the functioning of cowpea nodules

Nitrogenase (EC 1.7.99.2) activity of nodules of cowpea (Vigna unguiculata [L.] Walp), maintained under conditions of a 12-hour day at 30°C and 800 to 1,000 microeinsteins per square meter per second (photosynthetically active radiation) and a 12-hour night at 20°C, showed a marked diurnal variation with the total electron flux through the enzyme at night being 60% of that in the photoperiod. This diurnal pattern was, however, due to changes in hydrogen evolution. The rate of nitrogen fixation, measured by short-term ¹⁵N₂ assimilation or estimated from the difference in hydrogen evolution in air or Ar:O₂ (80:20; v/v), showed no diurnal variation. Carbon dioxide released from nodules showed a diurnal variation synchronized with that of nitrogenase functioning and, as a consequence, the apparent `respiratory cost' of nitrogen fixation in the photoperiod was almost double that at night (9.74 ± 0.38 versus 5.70 ± 0.90 moles CO₂ evolved per mole N₂ fixed). Separate carbon and nitrogen balances constructed for nodules during the photoperiod and dark period showed that, at night, nodule functioning required up to 40% less carbohydrate to achieve the same level of nitrogen fixation as during the photoperiod (2.4 versus 1.4 moles hexose per mole N₂ fixed).

Stored reserves of nonstructural carbohydrate of the nodule only partly satisfied the requirement for carbon at night, and fixation was dependent on continued import of translocated assimilates at all times. Measurements of the soluble nitrogen pools of the nodule together with ¹⁵N studies indicated that, both during the day and night, nitrogenous products of fixation were effectively translocated to all organs of the host plant despite low rates of transpiration at night. Reduced fluxes of water through the plant at night were apparently counteracted by increased concentration of nitrogen, especially as ureides, in the xylem stream.

     

Effects of carbohydrate on the internal oxygen concentration, oxygen uptake, and nitrogenase activity in detached pea nodules

The interaction between carbon substrates and O₂ and their effects on nitrogenase activity (C₂H₂) were examined in detached nodules of pea (Pisum sativum L. cv “Sparkle”). The internal O₂ concentration was estimated from the fractional oxygenation of leghemoglobin measured by reflectance spectroscopy. Lowering the endogenous carbohydrate content of nodules by excising the shoots 16 hours before nodule harvest or by incubating detached nodules at 100 kPa O₂ for 2 hours resulted in a 2- to 10-fold increase in internal O₂, and a decline in nitrogenase activity. Conversely, when detached nodules were supplied with 100 millimolar succinate, the internal O₂ was lowered. Nitrogenase activity was stimulated by succinate but only at high external O₂. Oxygen uptake increased linearly with external O₂ but was affected only slightly by the carbon treatments. The apparent diffusion resistance in the nodule cortex was similar in all of the treatments. Carbon substrates can thus affect nitrogenase activity indirectly by affecting the O₂ concentration within detached nodules.     

Phosphorus deprivation affects composition and spatial distribution of membrane lipids in legume nodules

In legumes, symbiotic nitrogen (N) fixation (SNF) occurs in specialized organs called nodules after successful interactions between legume hosts and rhizobia. In a nodule, N-fixing rhizobia are surrounded by symbiosome membranes, through which the exchange of nutrients and ammonium occurs between bacteria and the host legume. Phosphorus (P) is an essential macronutrient, and N₂-fixing legumes have a higher requirement for P than legumes grown on mineral N. As in the previous studies, in P deficiency, barrel medic (Medicago truncatula) plants had impaired SNF activity, reduced growth, and accumulated less phosphate in leaves, roots, and nodules compared with the plants grown in P sufficient conditions. Membrane lipids in M. truncatula tissues were assessed using electrospray ionization–mass spectrometry. Galactolipids were found to increase in P deficiency, with declines in phospholipids (PL), especially in leaves. Lower PL losses were found in roots and nodules. Subsequently, matrix-assisted laser desorption/ionization–mass spectrometry imaging was used to spatially map the distribution of the positively charged phosphatidylcholine (PC) species in nodules in both P-replete and P-deficient conditions. Our results reveal heterogeneous distribution of several PC species in nodules, with homogeneous distribution of other PC classes. In P poor conditions, some PC species distributions were observed to change. The results suggest that specific PC species may be differentially important in diverse nodule zones and cell types, and that membrane lipid remodeling during P stress is not uniform across the nodule.

ESI–MS and matrix-assisted laser desorption ionization–mass spectrometry imaging reveal alterations in Medicago truncatula nodules membrane lipid composition and spatial distribution in phosphorus deficiency.     

Nitrogen Assimilating Enzyme Activities and Enzyme Protein during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules

Effective (N₂-fixing) alfalfa (Medicago sativa L.) and plant-controlled ineffective (non-N₂-fixing) alfalfa recessive for the in₁ gene were compared to determine the effects of the in₁ gene on nodule development, acetylene reduction activity (ARA), and nodule enzymes associated with N assimilation and disease resistance. Effective nodule ARA reached a maximum before activities of glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), asparagine synthetase (AS), and phosphoenolpyruvate carboxylase (PEPC) peaked. Ineffective nodule ARA was only 5% of effective nodule ARA. Developmental profiles of GS, GOGAT, AAT, and PEPC activities were similar for effective and ineffective nodules, but activities in ineffective nodules were lower and declined earlier. Little AS activity was detected in developing ineffective nodules. Changes in GS, GOGAT, AAT, and PEPC activities in developing and senescent effective and ineffective nodules generally paralleled amounts of immunologically detectable enzyme polypeptides. Effective nodule GS, GOGAT, AAT, AS, and PEPC activities declined after defoliation. Activities of glutamate dehydrogenase, malate dehydrogenase, phenylalanine ammonia lyase, and caffeic acid-o-methyltransferase were unrelated to nodule effectiveness. Maximum expression of nodule N-assimilating enzymes appeared to require the continued presence of a product associated with effective bacteroids that was lacking in in₁ effective nodules.     

Nitrogenase activity, nodule respiration, and o(2) permeability following detopping of alfalfa and birdsfoot trefoil

Gas exchange measurements and noninvasive leghemoglobin (Lb) spectrophotometry (nodule oximetry) were used to monitor nodule responses to shoot removal in alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus). In each species, total nitrogenase activity, measured as H₂ evolution in Ar:O₂ (80:20), decreased to <50% of the initial rate within 1 hour after detopping, and net CO₂ production decreased to about 65% of the initial value. In a separate experiment in which nodule oximetry was used, nodule O₂ permeability decreased 50% within 5 hours in each species. A similar decrease in the O₂-saturated respiration rate (V_max) for the nodule central zone occurred within 5 hours in birdsfoot trefoil, but only after 24 hours in alfalfa. Lb concentration, also measured by oximetry, decreased after 48 to 72 hours. The decrease in permeability preceded the decrease in V_max in each species. V_max may depend mainly on carbohydrate availability in the nodule. If so, then the decrease in permeability could not have been triggered by decreasing carbohydrate availability. Both oximetry and gas exchange data were consistent with the hypothesis that, for the cultivars tested, carbohydrate availability decreased more rapidly in birdsfoot trefoil than in alfalfa nodules. Fractional Lb oxygenation (initially about 0.15) decreased during the first 24 hours after detopping but subsequently increased to >0.65 for a majority of nodules of each species. This increase could lead to O₂ inactivation of nitrogenase.     

Competitive Ability and Efficiency in Nodule Formation of Strains of Bradyrhizobium japonicum

In the American Midwest, superior N₂-fixing inoculant strains of Bradyrhizobium japonicum consistently fail to produce the majority of nodules on the roots of field-grown soybean. Poor nodulation by inoculant strains is partly due to their inability to stay abreast of the expanding soybean root system in numbers sufficient for them to be competitive with indigenous bradyrhizobia. However, certain strains are noncompetitive even when numerical dominance is not a factor. In this study, we tested the hypothesis that the nodule occupancy achieved by strains is related to their nodule-forming efficiency. The nodulation characteristics and competitiveness of nine strains of B. japonicum were compared at both 20 and 30°C. The root tip marking technique was used, with the nodule-forming efficiency of each strain estimated from the average position of the uppermost nodule and the number of nodules formed above the root tip mark. The competitiveness of the nine strains relative to B. japonicum USDA 110 was determined by using immunofluorescence to identify nodule occupants. The strains differed significantly in competitiveness with USDA 110 and in nodulation characteristics, strains that were poor competitors usually proving to be inferior in both the average position of the uppermost root nodule and the number of nodules formed above the root tip mark. Thus, competitiveness was correlated with both the average position of the uppermost nodule (r = 0.5; P = 0.036) and the number of nodules formed above the root tip mark (r = 0.64; P = 0.005), while the position of the uppermost nodule was also correlated to the percentage of plants nodulated above the root tip mark (r = 0.81; P < 0.001) and the percentage of plants nodulated on the taproot (r = 0.67; P = 0.002).