Consequences of Sporangial Development for Nodule Function in Root Nodules of Comptonia peregrina and Myrica gale

Frankia sp., the actinomycetous endophyte in nitrogen-fixing actinorhizal nodules, may differentiate two forms from its hyphae: vesicles and sporangia. In root nodules of Comptonia peregrina (L.) Coult. and Myrica gale L., sporangia may be either absent or present. Nitrogenase activity and symbiotic efficiency were contrasted in spore(+) and spore(−) nodules of these two host genera. Seedlings of C. peregrina nodulated with the spore(+) inoculum showed only 60% of the nitrogenase activity and 50% of the net size of their spore(−) counterparts after 12 weeks of culture. Measurements of acetylene reduction (i.e., nitrogenase activity) were coordinated with samplings of nodules for structural studies. Significant differences in acetylene reduction rates were discernible between spore(+) and spore(−) nodules commencing 4 weeks after nodulation, concomitant with the maturation of sporangia in the nodule. Spore(+) nodules ultimately reached less than half of the rate of nitrogenase activity of spore(−) nodules. Both types of nodules evolved only small amounts of molecular hydrogen, suggesting that both were equally efficient in recycling electrons lost to the reduction of hydrogen ions by nitrogenase. Respiratory cost of nitrogen fixation, expressed as the quotient of micromole CO₂ to micromole ethylene evolved by excised nodules, was significantly greater in spore(+) than in spore(−) nodules. M. gale spore(−) nodules showed variable effectivity, though all had low CO₂ to ethylene evolution ratios. M. gale spore(+) nodules resembled C. peregrina spore(+), with low effectivity and high respiratory cost for nitrogen fixation.     

The improvement and utilization in forestry of nitrogen fixation by actinorhizal plants with special reference toAlnus in Scotland

Summary Alnus species are used widely in Britain for land reclamation, forestry and other purposes. Rapid juvenile growth of the AmericanAlnus rubra makes it an attractive species for planting on N-deficient soils, particularly those of low organic content. In small plot trials, this species is nodulated by indigenous soil frankiae as effectively asAlnus glutinosa. Over a three year period both species return similar amounts of N to the ecosystem, estimated at up to 10–12 kg N ha^–1. Several strains ofFrankia have been isolated from local (Lennox Forest)A. rubra nodules. These differ morphologically and in their growth on different culture media, both from each other and fromA. glutinosa nodule isolates. AllAlnus isolates, however, have a total cellular fatty acid composition qualitatively similar to some other Group B frankiae. Glasshouse tests in N free culture suggest thatA. rubra nodules formed after inoculation of seedlings with American spore (–) isolates are three times more effective in N fixation than those inoculated with LennoxA. rubra spore (+) nodule homogenates. By contrast, the early growth of seedlings inoculated with spore (–)Frankia strains suggests at best a 35% improvement in N fixing activity over seedlings inoculated with LennoxA. rubra nodule isolates. Nevertheless, this improvement in activity, together with the better performance of seedlings inoculated with isolates compared with those treated with crushed nodule preparations, suggest that it would be worthwhile commercially to inoculate nursery stock with a spore (–)Frankia strain.     

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (mol C₂H₄g^–1 nodule fr. wt. h^–1), were both affected by the added nitrate.     

Isolation of infective and effective Frankia strains from root nodules of Alnus acuminata (Betulaceae)

Two Frankia strains were isolated from root nodules of Alnus acuminata collected in the Tucumano-oranense forest, Argentina. Monosporal cultures were obtained by plating a spore suspension of each strain and isolating a single colony. The strains (named AacI and AacIII) showed branched mycelia with polymorphic sporangia and NIR-vesicles. They differed in their ability to use carbon sources: the AacI strain grew well on pyruvate, while the AacIII strain grew on mineral medium supplemented with glucose or, alternatively, with sucrose. The two strains were sensitive to oleandomycin, erythromycin, kanamycin, penicillin G, streptomycin and chloramphenicol at 5 μg/ml. The AcIII strain exhibited a moderate resistance to rifampicin, ampicillin and vancomycin. The nitrogenase activity in vitro of the strains was significantly higher in basal medium without nitrogen than that determined in the presence of ammonium chloride. Both strains were infective on seedlings of Alnus glutinosa, inducing an approximately similar percentage of nodulated plants (80%), although strain AacIII produced a higher number of nodules per plant (≤15) than strain AacI (≤6). They were also effective for nitrogen fixation in planta, determined by the acetylene reduction assay. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nitrogenase activity of two genotypes of Acacia mangium as affected by phosphorus nutrition

The specific nodulation, nitrogenase activity (acetylene reduction) and budgets of carbon allocation to respiration by nodulated roots were examined in two provenances of Acacia mangium Willd. grown in a glasshouse for 17 weeks to investigate the effects of soil phosphorus and genotypes of the host plant on symbiotic nitrogen fixation. Application of phosphorus (0–80 mg P kg^-1 soil) increased specific nodulation (g nodule dry weight g^-1 plant dry weight) of provenance Ma11 by two-fold and the percentage of nodulated root respiration allocated to nitrogenase by 50%, but had no effect on specific activity of nitrogenase or specific respiration coupled with nitrogenase activity. Improved phosphorus nutrition increased the specific nitrogenase activity of provenance Ma9 by 2-fold, the percentage of nodulated root respiration allocated to nitrogenase, and specific nitrogenase-linked respiration by 50%, respectively, but had no effect on the specific nodulation. The percentage of respiration coupled with nitrogenase activity in nodulated root respiration by provenance Ma9 was 60–70% higher than that in provenance Ma11, regardless of phosphorus levels applied. At the optimal level of phosphorus addition (10 mg P kg^-1 soil), provenance Ma9 had a lower dry mass than provenance Ma11. This was accompanied by a lower nodulated root respiration and a higher percentage of nodulated root respiration allocated to nitrogenase activity in provenance Ma9.     

The regulation of nodulation,nitrogen fixation and ammonium assimilation under a carbohydrate shortage stress in the Discaria trinervis-Frankia symbiosis

N₂-fixation is sensitive to limitation in the availability of newly synthesised carbohydrates for the nodules. We decided to explore the response of the D. trinervis - Frankia symbiosis to a transient decrease in carbohydrate supply to nodules. Feedback inhibition of nodulation as well as nodule growth was not released by a 6-day dark stress in D. trinervis nodulated plants. However, nitrogen fixation and assimilation were affected by the imposed stress. Nitrogenase activity was totally inhibited after 4 days of darkness although high levels of nitrogenase components were still detected at this time. Degradation of FeMo and Fe nitrogenase subunits – both at similar rates – was observed after 6 days of dark stress, revealing the need for inactivation to precede enhancement of protein turnover. Glutamine synthetase (GS), malate dehydrogenase (MDH) and asparagine synthetase (AS) polypeptides were also degraded during the dark stress, although at a lower rate than nitrogenase. ARA and nitrogenase were totally recovered 8 days after resuming normal illumination. It seems that current nitrogenase activity and ammonium assimilation are not, or are only weakly linked with the feedback control of nodulation in D. trinervis. These observations give support to the persistence of an autoregulatory signal in mature nodules that is not sensitive to transient shortages of carbon supply and sustains the inhibition of nodulation in the transient absence of N₂ fixation.     

Nodule structure and nitrogenase activity ofCoriaria arborea in response to varying pO2

When excised root nodules ofCoriaria arborea are assayed for nitrogenase activity at various pO₂ they show a broad optimum between 20 and 40 kPa O₂, with some evidence for adaptation. Continuous flow assays of nodulated root systems of intact plants indicate that Coriaria shows an acetylene induced decline in nitrogenase activity. When root systems were subject to step changes in pO₂ nitrogenase activity responded with a steep decline followed by a slower rise in activity both at lower and higher than ambient pO₂. Thus Coriaria nodules are able to adapt rapidly to oxygen levels well above and well below ambient. Measurement of nodule diffusion resistance showed that the adaptation is accompanied by rapid increase in resistance at above ambient pO₂ and decrease in resistance at below ambient pO₂. Plants grown with root systems at pO₂ from 5–40 kPa O₂ did not differ in growth or nodulation. The anatomy of Coriaria nodules shows they have a dense periderm which encircles the nodule and also closely invests the infected zone. The periderm is both thicker and more heavily suberised in nodules grown at high pO₂ than at low pO₂. Vacuum infiltration of India ink indicates that oxygen diffusion is entirely through the lenticel and via a small gap adjacent to the stele.     

Ammonium assimilation in root nodules of actinorhizal Discaria trinervis. Regulation of enzyme activities and protein levels by the availability of macronutrients (N,P and C)

Asparagine was found to be the main N compound exported from Discaria trinervis nodules. Aspartate (Asp), glutamate (Glu), alanine (Ala) and serine (Ser) were also detected in root xylem sap, but at lower concentrations. A comparable picture is found in nodulated alfalfa. We hypothesized that a similar set of enzymes for Asn synthesis was present in D. trinervis nodules. We demonstrate the expression of most of the enzymes involved in the synthesis of Asn from NH⁺ ₄ and oxoacids, in nodules – but not in roots – of fully symbiotic D. trinervis. By complementation of enzyme assays (^A) and immunodetection (^I) we detected glutamane-synthetase (GS^{A, I}), Asp-aminotransferase (AAT^A), malate-dehydrogenase (MDH^{A, I}, at least two isoforms), Glu-dehydrogenase (GDH^A), Glu-synthase (GOGAT^I) and Asn-synthetase (AS^I). PEP-carboxylase (PEPC) activity was not detected. We previously shown that N acts as a negative regulator of nodulation and nodule growth, while P is a strong stimulator for nodule growth. We present data on the regulation of nodule N metabolism by altering, during 4 weeks, the availability of N, P and light in symbiotic D. trinervis. NH₄NO₃ (2 mM) induced inactivation and degradation of nodule GS, MDH and AS, but activation of GDH and AAT; the amount of nitrogenase components was not affected. A 10-fold increase in P supply did not greatly affect activity and amount of enzymes, suggesting that N metabolism is not P-limited in nodules. On the other hand, suppression of P supply induced an important reduction of nodule GS, GOGAT, MDH and AS protein levels, although nitrogenase was not affected. GDH was the only measured activity that was stimulated by limiting P supply. Shading plants did result in complete degradation of nitrogenase and partial degradation of GS, AS and nodule-specific MDH isoform, but GDH and AAT were activated. These results are discussed in connection with the regulation of nodulation and nodule growth in D. trinervis.     

木麻黄和桤木离体根瘤和立地土壤的固氮酶与N2O还原酶活性的研究

为了解非豆科固氮树种的固氮酶和N_2O还原酶(Nos)活性,采用乙炔还原法和乙炔抑制技术对细枝木麻黄(Casuarina cunninghamiana)和江南桤木(Alnus trabeculosa)离体根瘤及立地土壤的两种酶活性进行了研究。结果表明,离体根瘤只在厌氧条件下有固氮酶活性,在好氧条件下有Nos活性。根瘤区根际土和非根瘤区根际土的固氮酶活性在好氧条件大于厌氧条件,Nos活性只表现在厌氧条件下。在好氧条件下,根瘤区根际土和非根瘤区根际土的固氮酶活性无显著差异;根瘤区根际土的Nos活性显著大于非根瘤区根际土。除离体根瘤在好氧条件下不表现固氮酶活性外,细枝木麻黄和桤木的离体根瘤、根瘤区根际土和非根瘤区根际土的固氮酶活性均都大于Nos活性。好氧条件下根瘤区根际土的固氮酶活性与非根瘤区根际土的呈极显著正相关,而厌氧条件下根瘤的固氮酶活性与好氧条件下根瘤区根际土和非根瘤区根际土固氮酶活性、好氧条件下根瘤的Nos活性与厌氧条件下根瘤区根际土和非根瘤区根际土Nos活性均呈极显著负相关。这为研究弗兰克氏菌结瘤植物共生固氮体系对N2O汇强度的影响和调控奠定基础。     

Carbon cost of nitrogenase activity in Frankia–Alnus incana root nodules

The carbon cost of nitrogenase activity was investigated to determine symbiotic efficiency of the actinorhizal root nodule symbiosis between the woody perennial Alnus incana and the soil bacterium Frankia. Respiration (CO₂ production) and nitrogenase activity (H₂ production) by intact nodulated root systems were continuously recorded in short-term assays in an open-flow gas exchange system. The assays were conducted in N₂:O₂, thus under N₂-fixing conditions, in all experiments except for one. This avoided the declines in nitrogenase activity and respiration due to N₂ deprivation that occur in acetylene reduction assays and during extended Ar:O₂ exposures in H₂ assays. Two approaches were used: (i) direct estimation of root and nodule respiration by removing nodules, and (ii) decreasing the partial pressure of O₂ from 21 to 15% to use the strong relationship between respiration and nitrogenase activity to calculate CO₂/H₂. The electron allocation of nitrogenase was determined to be 0.6 and used to convert the results into moles of CO₂ produced per 2e^– transferred by nitrogenase to reduction of N₂. The results ranged from 2.6 to 3.4mol CO₂ produced per 2e^–. Carbon cost expressed as gC produced per gN reduced ranged from 4.5 to 5.8. The result for this actinorhizal tree symbiosis is in the low range of estimates for N₂-fixing actinorhizal symbioses and crop legumes. Methodology and comparisons of root nodule physiology among actinorhizal and legume plants are discussed.     

Dinitrogen fixation in a water-stressed Alnus clone is limited by host xerotolerance

The osmotolerance, rather than the halotolerance, of the endosymbiont predicted the xerotolerance of acetylene reduction by Alnus nodulated withFrankia ARgP5 ^AG . Cloned plants ofAlnus glutinosa (L.) Gaertn. AG8022-16 were subjected to water stress under controlled conditions in an environmental growth chamber. Transpiration, stomatal conductance, and leaf water potential had decreased after successive 10 day periods of moderate (75% of water demand) and severe (50% of water demand) water stress. After severe stress had wilted the plants, reducing leaf water potential to –2.10 MPa, nitrogenase activity had fallen to 2.51 M per plant per hour. The reported rapid turnover of nitrogenase implies thatFrankia mycelium was metabolically active at this low water potential, a water potential at which no Alnus-derivedFrankia has been reported active. Although ARgP5 ^AG was similar to other such strains in halotolerance (lower limitca.–1.25 MPa), the low water potential limit for growth with glucose (a non-assimilated osmoticum) wasca.–2.53 MPa. Nitrogenase activity was apparently more limited by host xerotolerance than by endophyte xerotolerance.Journal article J-5400 of the Oklahoma Agriculture Experiment Station, Oklahoma State University, Stillwater, OK 74078, USA.     

Nitrogen fixation and respiration by root nodules ofAlnus rubra Bong.: Effects of temperature and oxygen concentration

Summary Using a root nodule cuvette and a continuous flow gas exchange system, we simultaneously measured the rates of carbon dioxide evolution, oxygen uptake and acetylene reduction by nodules ofAlnus rubra. This system allowed us to measure the respiration rates of single nodules and to determine the effects of oxygen concentration and temperature on the energy cost of nitrogen fixation. Energy cost was virtually unchanged (2.8–3.5 moles of carbon dioxide or oxygen per mole of ethylene) from 16 to 26°C (pO₂=20 kPa) while respiration and nitrogenase activity were highly temperature dependent. At temperatures below 16°C, nitrogenase activity decreased more than did respiration and as a result, energy cost rose sharply. Acetylene reduction ceased below 8°C. Inhibition of nitrogenase activity at low temperatures was rapidly reversed upon return to higher temperatures. At high temperatures (above 30°C) nitrogenase activity declined irreversibly, while respiration and energy cost increased.Energy cost was nearly unchanged at oxygen partial pressures of 5 to 20 kPa (temperature of 20°C). Respiration and nitrogenase activity were strongly correlated with oxygen tension. Below 5 kPa, acetylene reduction and oxygen uptake decreased sharply while production of carbon dioxide increased, indicating fermentation. Fermentation alone was unable to support nitrogenase activity. Acetylene reduction was independent of oxygen concentration from 15 to 30 kPa. Nitrogenase activity decreased and energy cost rose above 30 kPa until nearly complete inactivation of nitrogenase at 70–80 kPa. Activity declined gradually, such that acetylene reduction at a constant oxygen concentration was stable, but showed further inactivation when oxygen concentration was once again increased. Alder nodules appear to consist of a large number of compartments that differ in the degree to which nitrogenase is protected from excess oxygen.Supported by United States Department of Agriculture Grant 78-59-2252-0-1-005-1     

Nitrogenase Inhibition in Nodules from Pea Plants Grown Under Salt Stress Occurs at the Physiological Level and can be Alleviated by B and Ca

In order to shed new light on the mechanisms of salt-mediated symbiotic N₂-fixation inhibition, the effect of salt stress (75 mM) on N₂-fixation in pea root nodules induced by R. leguminosarum was studied at the gene expression, protein production and enzymatic activity levels. Acetylene reduction assays for nitrogenase activity showed no activity in salt-stressed plants. To know whether salt inhibits N₂-fixing activity at a molecular or at a physiological level, expression of the nifH gene, encoding the nitrogenase reductase component of the nitrogenase enzyme was analyzed by RT-PCR analysis of total RNA extracted from nodulated roots. The nifH messenger RNA was present both in plants grown in the presence and absence of salt, although a reduction was observed in salt-stressed plants. Similar results were obtained for the immunodetection of the nitrogenase reductase protein in Western-blot assays, indicating that nitrogen fixation failed mainly at physiological level. Given that nutrient imbalance is a typical effect of salt stress in plants and that Fe is a prosthetic component of nitrogenase reductase and other proteins required by symbiotic N₂-fixation, as leghemoglobin, plants were analyzed for Fe contents by atomic absorption and the results confirmed that Fe levels were severely reduced in nodules developed in salt-stressed plants. In a previous papers (El-Hamdaoui et al., 2003b), we have shown that supplementing inoculated legumes with boron (B) and calcium (Ca) prevents nitrogen fixation decline under saline conditions stress. Analysis of salt-stressed nodules fed with extra B and Ca indicated that Fe content and nitrogenase activity was similar to that of non-stressed plants. These results indicate a linkage between Fe deprivation and salt-mediated failure of nitrogen fixation, which is prevented by B and Ca leading to increase of salt tolerance.     

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

Cinnamate toxicity expression on phenylalanine ammonia-lyase activity,germination and growth of cucumber (Cucumis sativis) seedlings

Low pH (5.2) decreased nodule number and acetylene reduction. Aluminium further depressed those parameters in theRhizobium leguminosarum-Pisum sativum associations examined. In the Al-treated plants nodule formation by strains 128C53 and 128C30 was not affected by 3 or 15 and 30 or 60 μM Al, respectively, as compared with the number of nodules on plants grown at pH 5.2 in the absence of Al. However, improved nodulation rates by those strains did not enhance plant dry weight or reduced nitrogen content. No differences in nitrogenase activity were found among strains of nodulating plants grown at the same aluminium level. These results suggest that Al-ions affected specifically nitrogenase activity and that this effect was primarily responsible for the reduction in plant growth.     

Growth and nitrogen fixation inAlnus glutinosa (L.) Gaertn. under carbon dioxide enrichment of the root atmosphere

The effects of aeration of the N-free rooting medium with elevated CO₂ on (a) acetylene reduction by perlite-grown plants and (b) N₂-fixation and long-term growth of nutrient solution-grown plants were determined for nodulatedAlnus glutinosa (L.) Gaertn. In the former experiments, roots of intact plants were incubated in acetylene in air in darkened glass jars for 3 hr, followed by a further 3 hr incubation period in air enriched with CO₂ (0–5%). During incubation, the CO₂ content of the jars increased by 0.17% per hour due to respiration of the root system, so that the CO₂ content at 3 hr was 0.5%. Additional enrichment of the rooting medium gas-phase with CO₂ equivalent to 1.1% and 1.75% CO₂ of the gas volume significantly increased nitrogenase activity (ethylene production) by 55% and 50% respectively, while enrichment with greater than 2.5% CO₂ decreased activity. In contrast, ethylene production by control plants, where CO₂ was not added to the assay jars, decreased by 8% over the assay period. In long-term growth experiments, nodulated roots of intactAlnus glutinosa plants were sealed into jars containing N-free nutrient solution (pH 6.3) and aerated with air, or air containing elevated levels of CO₂ (1.5% and 5%). Comparison of the appearance of CO₂-treated with air treated plants suggested that 1.5% CO₂ stimulated plant growth. However, at harvest after 5 or 6 weeks variability between plants masked the significance of differences in plant dry weight. A significant increase of 33% in total nitrogen of plants aerated with 1.5% CO₂, compared with air-treated plants, was demonstrated, broadly in line with the short-term increase in acetylene reducing activity observed following incubations with similar CO₂ concentrations. Shoot dry weight was not affected significantly by long-term exposure to 5% CO₂, the main effect on growth being a 20% reduction in dry weight of the root system, possibly through inhibition of root system respiration. However, in contrast to the inhibitory effects of high CO₂ on acetylene reduction there was no significant effect on the amounts of N₂ fixed.     

Augmentation of H2 photoproduction in Rhodopseudomonas palustris by N-heterocyclic aromatic compounds

Increases of 23- (5.6 mmol acetylene reduced mg dry wt^–1) and 16- (4 mmol acetylene reduced mg dry wt^–1) fold in nitrogenase activity and 12- (671 l H₂ mg dry wt^–1 h^–1) and 6- (349 l mg dry wt^–1 h^–1) fold in H₂ photoproduction in Rhodopseudomonas palustris JA1 over 24 h were achieved with pyrazine 2-carboxylate (3 mM) and 3-picoline (3 mM), respectively, and were higher than earlier reports of enhancement (1.5 to 5- fold) in biological H₂ production using various alternative methods.     

Nitrogen fixation byRubus ellipticus J. E. Smith

Summary Nitrogenase activity as assayed by acetylene reduction was observed in detachedRubus ellipticus J. E. Smith root nodules collected in the field and tested under ambient conditions. The nitrogenase activity was 8.4 moles C₂H₄.gfr. wt nodule^–1.h^–1 or 24.0 moles C₂H₄.g dry wt nodule^–1.h^–1 being at a rate comparable with that measured in some other non-legumes assayed in Java at the same time under similar conditions. Nodule morphology bore little resemblance to the root nodules of other non-leguminous plants and nodule structure was different from the other rosaceous examples.The endophyte inhabiting the root nodules was actinomycetal.     

N2-fixation in Erwinia herbicola

Four from 18 strains of Erwinia herbicola tested had nitrogenase activity and grew with N₂ as sole source of nitrogen under strict anaerobic conditions with a doubling time of 20–24 h. Nitrogenase activity started only 96–120 h after transfer to a special medium maintained under anaerobic conditions. A ten fold increase in protein per culture found after the maximum nitrogenase activity of 80–130 nmol C₂H₄. mg protein^-1·min^-1 was accompanied by a fall in pH of the medium (20 mM phosphate buffer and in 125 mM Tris-buffer) from pH 7.2 to 5.4 or less, but only to 6.8 in 100 mM phosphate buffer. In all cases we found a sharp curtailing of nitrogenase activity 48 h after the maximum. The bacteria utilized only 35–50% of the nitrogen fixed for growth. Erwinia herbicola strains differed from two strains of Enterobacter agglomerans in being unable to fix nitrogen on agar surfaces exposed to air. Specific nitrogenase activity in Erwinia herbicola is compared with data reported for other Enterobacteriaceae and is found to be higher than that reported for Klebsiella pneumoniae, Enterobacter cloacae or Citrobacter freundii.     

Competition between strains ofBradyrhizobium japonicum for nodulation of soybeans at different nitrogen fertilizer levels

Competitive abilities of 3 strains ofBradyrhizobium japonicum (E104, E109, E110) for nodulation of soybean (Glycine max) at increasing nitrogen fertilizer levels were studied. Dry weight of plants nodulated by strain E110 were depressed at 10 g N·m^–2, the highest fertilizer level, even when mixed with strain E109. Strain E104 alone or mixed with E109 increased dry matter production. Strain E110 formed many dually infected nodules with strain E104 present but not with strain E109. However, strain E104 formed nodules containing strain E109. Neither strain E110 or E109 produced bacteriocin, so the incompatibility of these two strains had to be due to another reason. Strain E104 successfully competes with strain E109 but not with E110 at 10 g N·m^–2. It is concluded that strain E110 dominates the symbiotic relationships even if other strains are also present in the nodules. However, at a high N-fertilizer level strain E110 decreases the plant yield in contrast to E104, which could be recommended as inoculant at increased levels of soluble soil-N.