Effects of solution pH and bicarbonate on the growth and nodulation of a range of grain legume species

Defining plant adaptation to soil conditions is critical for the successful introduction of grain legume species into farming systems. This nutrient solution study examined the effects of pH (4, 5, 6, 7 and 8) and bicarbonate (5 mM KHCO₃) on the growth and nodulation of 14 grain legume species supplied with N or reliant on N₂-fixation. Species includedPisum sativum L.,Cicer arietinum L.,Lens culinaris Med., and a range ofLupinus, Vicia andLathyrus species. Species differed greatly in response to solution pH. For both N-fertilized and N₂-fixing plants, shoot growth ofL. culinaris was very sensitive to low pH (pH < 7), whereas shoot growth ofLupinus angustifolius L. andLupinus albus L. was sensitive to higher pH (pH ≥ 6). Other species had a broader optimal pH range for growth when supplied with N, but were generally sensitive to low pH (pH < 7 forC. arietinum andVicia sativa L., pH < 6 forP. sativum, Vicia faba L.,Lathyrus sativus L. andLathyrus cicera L., and pH < 5 forVicia benghalensis L. andVicia narbonensis L.) when reliant on N₂-fixation. For these other species, symbiotic N₂-fixation appeared to be more sensitive than host plant growth to low pH. This finding was supported by lower nodule numbers and mass, and lower N concentrations in shoots of sensitive species at low pH relative to higher pH. ForL. culinaris, nodule numbers and mass were relatively unaffected by pH 5–8, N concentrations in shoots were high at low pH and plants developed symptoms relating to H⁺ toxicity at pH as high as 7. These results indicate that host plant growth ofL. culinaris is more sensitive to low pH than theRhizobium symbiosis. ForL. albus andL. angustifolius, both host plant growth and symbiotic N₂-fixation appeared to be equally sensitive to pH ≥ 6.Lupinus pilosus Murr. was more tolerant of high pH than the otherLupinus species. At pH 4, two genotypes ofC. arietinum had better early nodulation than other species.Vicia ervilia L. nodulated poorly at all levels of solution pH, indicating that the commercial Group E inoculum (Rhizobium leguminosurum bv.viceae SU303) may not be effective for this species in solution culture. Addition of bicarbonate decreased shoot growth, nodulation and N concentrations in shoots of most species. Early nodulation (nodule number) ofLathyrus ochrus (L.) DC was not affected by the bicarbonate treatment.     

The Influence of Foliar Applications of Urea on the Nodulation Pattern of Certain Leguminous Species

In greenhouse experiments with seven species of legumes, thespraying of urea on to the leaves was shown to affect nodulationadversely, without impairing the growth of the plants. In Phaseolusvulgaris, Vicia sativa, and Pisum sativum three-times-weeklysprays of 1 per cent. aqueous urea either prevented or markedlyreduced nodule development during the 8-week experimental period.In Medicago sativa and Trifolium pratense the urea treatmentresulted in a delay in nodulation so that numbers of nodulesat the first sampling (4 weeks) were reduced, while numbersat later samplings were higher since nodulation had been delayeduntil the root system was larger and provided a greater numberof potential nodule sites. In Trifolium hybridum and T. repensthe urea-treated plants showed reduced nodulation throughoutthe 6-week experimental period. In these experiments the advance effects on nodulation cannotbe due to high concentration of combined nitrogen in the rootingmedium, but it is suggested they derive from a high level ofnitrogen within the plant.     

Stem Nodulation in Legumes: Diversity,Mechanisms, and Unusual Characteristics

Rhizobia can establish a nitrogen-fixing symbiosis with plants of the Leguminosae family. They elicit on their host plant the formation of new organs, called nodules, which develop on the roots. A few aquatic legumes, however, can form nodules on their stem at dormant root primordia. The stem-nodulating legumes described so far are all members of the genera Aeschynomene, Sesbania, Neptunia, and Discolobium. Their rhizobial symbionts belong to four genera already described: Rhizobium, Bradyrhizobium, Sinorhizobium, and Azorhizobium. This review summarizes our current knowledge on most aspects of stem nodulation in legumes, the infection process and nodule development, the characterization and unusual features of the associated bacteria, and the molecular genetics of nodulation. Potential use as green manure in lowland rice of these stem-nodulating legumes, giving them agronomical importance, is also discussed.     

Evolving ideas of legume evolution and diversity: a taxonomic perspective on the occurrence of nodulation

Legumes evolved about 60 million years ago (Ma), and nodulation 58 Ma. Nonnodulation remains common in Caesalpinioideae, with smaller numbers in Mimosoideae and Papilionoideae. The first type of infection by bacteria may have been at junctions where lateral roots emerged, followed by formation of infection threads to confine bacteria and convey them to some cells in the developing nodule, where they were generally released into symbiosomes. Infection threads were a prerequisite for root-hair infection, a process better controlled by the host, leading to a higher degree of specificity between symbionts. An alternative process, dating from the same time and persisting in about 25% of legumes, did not involve infection threads, bacteria entering a few host cells, surrounded by an undefined matrix. These cells divided repeatedly to give uniform infected tissue, with bacteria released into symbiosomes. Such legumes may have less stringent control of nodulation processes, and are found mainly in tropical and warm temperate areas. In each type of nodule, meristems may or may not be retained, leading to indeterminate or determinate forms. Nodule morphology and structure are host-determined, but the effectiveness of nitrogen fixation is largely controlled by the bacterial symbionts, which vary greatly in genotypic and phenotypic characters.     

Co-inoculation with antibiotic-producing bacteria to increase colonization and nodulation by rhizobia

A study was conducted to determine whether colonization of legume roots and nodulation byRhizobium meliloti andBradyrhizobium japonicum could be enhanced by using inocula containing microorganisms that produce antibiotics suppressing soil or rhizosphere inhabitants but not the root-nodule bacteria. An antibiotic-producing strain of Pseudomonas and one of Bacillus were isolated, and mutants ofR. meliloti andB. japonicum sp. resistant to the antibiotics were used. The colonization of the alfalfa rhizosphere and nodulation byR. meliloti were enhanced by inoculation of soil withPseudomonas sp. in soil initially containing 2.7×10⁵ R. meliloti per g. The colonization of soybean roots byB. japonicum was enhanced by inoculating soil with three cell densities ofBacillus sp., and nodulation was stimulated byBacillus sp. added at two cell densities. In some tests, the dry weights of soybeans and seed yield increased as a result of these treatments, and co-inoculation with Bacillus also increased pod formation. Inoculation of seeds withBacillus sp. and the root-nodule bacterium enhanced nodulation of soybeans and alfalfa, but colonization byB. japonicum andR. meliloti was stimulated only during the early period of plant growth. Studies were also conducted withStreptomyces griseus and isolates ofR. meliloti andB. japonicum resistant to products of the actinomycete. Nodulation of alfalfa byR. meliloti was little or not affected by the actinomycete alone; however, both nodulation and colonization were enhanced if the soil was initially amended with chitin andS. griseus was also added. Chitin itself did not affectR. meliloti. Treatments of seeds with chitin orS. griseus alone did not enhance colonization of alfalfa roots byR. meliloti or soybean roots byB. japonicum, but the early colonization of the roots by both bacterial species was promoted if the seeds received both chitin andS. griseus; this treatment also increased nodulation and dry weights of alfalfa and soybeans and the N content of alfalfa. It is suggested that co-inoculation of legumes with antibiotic-producing microorganisms and root-nodule bacteria resistant to those antibiotics is a promising means of promoting nodulation and possibly nitrogen fixation.     

The effects of lime-pelleting and lime-superphosphate fertilizer on the growth of three annual legumes in an acid sandy soil

Summary Three pioneer pasture legume species,Trifolium subterraneum, Trifolium glomeratum andOrnithopus compressus, were grown in a sandy soil of pH 5.0. The growth and nodulation of each species was examined in the presence and absence of lime pelleting and with superphosphate and lime-superphosphate fertilizer. In each species tested, plant yield, nodule number and nitrogen recovery as maximal where lime pelleting and lime-super were applied together. Plant calcium indicated that the response to lime was due to changes in soil pH rather than to a calcium response. T. glomeratum was the species most responsive to lime application but its growth was at best only half that ofT. subterraneum andO. compressus, which produced equally under both favourable (pH 5.9) and unfavourable (pH 4.4) conditions created by fertilizer applications.     

Studies of an effective strain ofFrankia fromAllocasuarina lehmanniana of the Casuarinaceae

Summary Seedlings ofCasuarina spp. andAllocasuarina spp. were grown from seed in the greenhouse and inoculated with a nodule suspension fromC. equisetifolia. Plants ofCasuarina spp. nodulated regularly and were effective in nitrogen-fixation. Only one species ofAllocasuariona, A. lehmanniana formed root nodules. Using these plants as source of inoculum, the isolation of a newFrankia sp. HFPA11I1 (HFP022 801) was made and the strain was grown in pure culture.Frankia sp. HFPA11I1 grows well in a defined medium and shows typical morphological characteristics. In media lacking combined nitrogen, the filamentours bacterium forms terminal vesicles in abundance and differentiaties large intrahyphal or terminal sporangia containing numerous spores. This strain, used as inoculum, nodulates effectively seedlings ofC. equisietifolia andC. cunninghamiana, forming nodules with verically-growing nodule roots. Although effective in acetylene reduction, the endophyte within the nodules is filamentous and lacks veiscles. When used to inoculated seedlings ofA llocasuarina lehmanniana, Frankia sp. HFPA11I1 induces root nodules which are coralloid and lacking nodule roots. The nodules are effective in acetylene reduction and the filamentous hyphae ofFrankia within the nodule lobes lack vesicles. Effective nodulation inA. Lehmanniana depends upon environmental conditions of the seedlings and proceeds much more slowly than in Casuariana.     

Sensitivity of the common bean (Phaseolus vulgaris L.) symbiosis to high soil temperature

Summary Experiments were done to test whether N fixation is more sensitive to high soil temperatures in common bean than in cowpea or soybean. Greenhouse experiments compared nodulation, nitrogenase activity, growth and nitrogen accumulation of several host/strain combinations of common bean with the other grain legumes and with N-fertilization, at various root temperatures. Field experiments compared relative N-accumulation (in symbiotic relative to N-fertilized plants) of common bean with cowpea under different soil thermal regimes. N-fertilized beans were unaffected by the higher temperatures, but nitrogen accumulation by symbiotic beans was always more sensitive to high root temperatures (33°C, 33/28°C, 34/28°C compared with 28°C) than were cowpea and soybean symbiosis. Healthy bean nodules that had developed at low temperatures functioned normally in acetylene reduction tests done at 35°C. High temperatures caused little or no suppression of nodule number. However, bean nodules produced at high temperatures were small and had low specific activity. ForP. vulgaris some tolerance to high temperature was observed among rhizobium strains (e.g., CIAT 899 was tolerant) but not among host cultivars. Heat tolerance ofP. acutifolius andP. lunatus symbioses was similar to that of cowpea and soybean. In the field, high surface soil temperatures did not reduce N accumulation in symbiotic beans more than in cowpea, probably because of compensatory nodulation in the deeper and cooler parts of the soil.     

Effects of boron and calcium nutrition on the establishment of the Rhizobium leguminosarum–pea (Pisum sativum) symbiosis and nodule development under salt stress

The effects of modifying boron (B) and calcium (Ca²⁺) concentrations on the establishment and development of rhizobial symbiosis in Pisum sativum plants grown under salt stress were investigated. Salinity almost completely inhibited the nodulation of pea plants by Rhizobium leguminosarum bv. viciae 3841. This effect was prevented by addition of Ca²⁺ during plant growth. The capacity of root exudates derived from salt‐treated plants to induce Rhizobium nod genes was not significantly decreased. However, bacterial adsorption to roots was highly inhibited in plants grown with 75 mM NaCl. Moreover, R. leguminosarum 3841 did not grow in minimal media containing such salt concentration. High Ca²⁺ levels enhanced both rhizobial growth and adsorption to roots, and increased nodule number in the presence of high salt. Nevertheless, the nodules developed were not functional unless the B concentration was also increased. Because B has a strong effect on infection and cell invasion, these processes were investigated by fluorescence microscopy in pea nodules harbouring a R. leguminosarum strain that expresses green fluorescent protein. Salt‐stressed plants had empty nodules and only those treated with high B and high Ca²⁺ developed infection threads and exhibited enhanced cell and tissue invasion by Rhizobium. Overall, the results indicate that Ca²⁺ promotes nodulation and B nodule development leading to an increase of salt tolerance of nodulated legumes.     

The ethylene-inhibitor aminoethoxyvinylglycine restores normal nodulation by Rhizobium leguminosarum biovar. viciae on Vicia sativa subsp. nigra by suppressing the ‘Thick and short roots’ phenotype

Nodulation of Vicia sativa subsp. nigra L. by Rhizobium bacteria is coupled to the development of thick and short roots (Tsr). This root phenotype as well as root-hair induction (Hai) and root-hair deformation (Had) are caused by a factor(s) produced by the bacteria in response to plant flavonoids. When very low inoculum concentrations (0.5–5 bacteria·ml^-1) were used, V. sativa plants did not develop the Tsr phenotype and became nodulated earlier than plants with Tsr roots. Furthermore, the nodules of these plants were located on the primary root in contrast to nodules on Tsr roots, which were all located at sites of lateral-root emergence. The average numbers of nodules per plant were not significantly different for these two types of nodulation. Root-growth inhibition and Hai, but not Had, could be mimicked by ethephon, and inhibited by aminoethoxyvinylglycine (AVG). Addition of AVG to co-cultures of Vicia sativa and the standard inoculum concentration of 5·10⁵ bacteria·ml^-1 suppressed the development of the Tsr phenotype and restored nodulation to the pattern that was observed with very low concentrations of bacteria (0.5–5 bacteria·ml^-1). The delay in nodulation on Tsr roots appeared to be caused by the fact that nodule meristems did not develop on the primary root, but only on the emerging laterals. The relationship between Tsr, Hai, Had, and nodulation is discussed.Abbreviations AVG aminoethoxyvinylglycine - cfu colonyforming units - Had root-hair deformation - Hai root-hair induction - NB naringenin-bacteria filtrate - Tsr Thick and short roots     

Rhizobium leguminosarum Biovar viciae 1-Aminocyclopropane-1-Carboxylate Deaminase Promotes Nodulation of Pea Plants

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.     

Role of cellulose fibrils and exopolysaccharides of Rhizobium leguminosarum in attachment to and infection of Vicia sativa root hairs

Infection and subsequent nodulation of legume host plants by the root nodule symbiote Rhizobium leguminosarum usually require attachment of the bacteria to root-hair tips. Bacterial cellulose fibrils have been shown to be involved in this attachment process but appeared not to be essential for successful nodulation. Detailed analysis of Vicia sativa root-hair infection by wild-type Rhizobium leguminosarum RBL5523 and its cellulose fibril-deficient celE mutant showed that wild-type bacteria infected elongated growing root hairs, whereas cellulose-deficient bacteria infected young emerging root hairs. Exopolysaccharide-deficient strains that retained the ability to produce cellulose fibrils could also infect elongated root hairs but infection thread colonization was defective. Cellulose-mediated agglutination of these bacteria in the root-hair curl appeared to prevent entry into the induced infection thread. Infection experiments with V sativa roots and an extracellular polysaccharide (EPS)- and cellulose-deficient double mutant showed that cellulose-mediated agglutination of the EPS-deficient bacteria in the infection thread was now abolished and that infection thread colonization was partially restored. Interestingly, in this case, infection threads were initiated in root hairs that originated from the cortical cell layers of the root and not in epidermal root hairs. Apparently, surface polysaccharides of R. leguminosarum, such as cellulose fibrils, are determining factors for infection of different developmental stages of root hairs.     

Involvement of cytokinin in the stimulation of nodulation by low concentrations of ammonium in Pisum sativum

Nodulation in pea (Pisum sativum L.) grown in hydroponic and sand culture systems is stimulated by low concentrations (<1.0 mM) of ammonium, but the physiological mechanisms underlying this stimulation are unknown. The current study involves a series of experiments, which investigate if the ammonium‐induced stimulation of nodulation involves changes in endogenous hormone (auxin and cytokinin) levels. P. sativum L. cv. Express was grown in growth pouches for 1 week with mineral N (0.5 and 2.0 mM NH₄⁺ or NO₃^–) or for 3 weeks exposed to exogenous indole‐3‐acetic acid (IAA) or 6‐benzylaminopurine (BAP) at a range of concentrations (10^‐9?10^‐5 M). Ammonium enhanced nodulation on the basis of both early whole plant (nodules plant^?1) and specific nodulation (nodules g^?1 root DW), especially in 0.5 mM treatment in which nodulation was approximately 4‐fold of the mineral‐N‐free control 1 week after inoculation. Correspondingly, the roots treated with ammonium contained much higher levels of t‐zeatin (Z) and lower t‐zeatin riboside (ZR) than that the control or nitrate‐treated plants. There was no significant difference in IAA levels between the control and ammonium treatments. Exogenous application of BAP for 3 weeks at concentrations of 10^‐7?10^‐5 M strongly inhibited nodulation. However, 10^?9 M BAP, but not IAA, significantly enhanced nodulation. These data support the theory that a relatively high ratio of cytokinin:auxin in roots is favourable for nodule initiation, but that an excessively high level of cytokinin inhibits nodulation. Based on these results we propose that stimulation of nodulation by low concentrations of ammonium may be mediated through increasing Z level in roots, which alters the balance of cytokinin and auxin, which in turn induces cortical cell divisions leading to nodule initiation.     

Nitrogen uptake and recovery from urea and green manure in lowland rice measured by15N and non-isotope techniques

In the recent past considerable attention is paid to minimize dependence on purchased inputs such as inorganic nitrogen fertilizer. Green manure in the form of flood-tolerant, stem-nodulatingSesbania rostrata andAeschynomene afraspera is an alternative N source for rice, which may also increase N use efficiency. Therefore research was conducted to determine the fate of N applied to lowland rice (Oryza sativa L.) in the form ofSesbania rostrata andAeschynomene afraspera green manure and urea in two field experiments using¹⁵N labeled materials.¹⁵N in the soil and rice plant was determined, and¹⁵N balances established. Apparent N recoveries were determined by non-tracer method. ¹⁵N recoveries averaged 90 and 65% of N applied for green manure and urea treatments, respectively. High partial pressures of NH₃ in the floodwater, and high pH probably resulted from urea application and favoured losses of N from the urea treatment. Results show that green manure N can supply a substantial proportion of the N requirements of lowland rice. Nitrogen released fromSesbania rostrata andAeschynomene afraspera green manure was in synchrony with the demand of the rice plant. The effect of combined application of green manure and urea on N losses from urea fertilizer were also investigated. Green manure reduced the N losses from¹⁵N labeled urea possibly due to a reduction in pH of the floodwater. Positive added N interactions (ANIs) were observed. At harvest, an average of 45 and 25% of N applied remained in the soil for green manure and urea, respectively.Contribution from IRRI, Los Baños, Philippines and Justus-Liebig-University, Giessen, GermanyContribution from IRRI, Los Baños, Philippines and Justus-Liebig-University, Giessen, Germany     

Influence ofRhizobium leguminosarum biovartrifolii host specific nodulation genes on the ontogeny of clover nodulation

Summary The infection of white clover seedlings byRhizobium strains with different host range properties was assessed using various microscopic techniques. Several wild-type andRhizobium leguminosarum biovarvicias hybrid strains containing definedR. l. bv.trifolii host range genes were used. The morphological changes in the root tissue of uninoculated and rhizobia inoculated white clovers were identified and compared. In particular, changes were observed in the induction of inner cortical cell division, alterations to nodule development and lateral root formation. The responses of the infected roots and the types of structures formed support the hypothesis that lateral roots and nodules may be physiologically homologous structures. To establish a normal pattern of nodulation on white clover roots, both sets of known host specific nodulation genes (operonsnod FERL andnod MNX) ofR. l. bv.trifolii were required. However, some nodule development occurred when only thenod FERL genes were present in the hybrid strain.     

Nodulation ofMedicago sativa in solution culture

Summary Calcium and hydrogen ions interacted on nodulation. Increasing acidity from pH 5.6 to pH 4.8 increased the calcium concentration required to nodulate 50% of the plants, from 0.1 mM to 6 mM. Calcium concentration below 0.2 mM or pH below 4.8 inhibited nodulation at all tested levels of the other variable. Root extension and root-hair production were insufficiently affected by calcium or pH to explain reductions in nodule numbers. Initiation of infection, the most acid-sensitive stage of the nodulation process, was also the most calcium-demanding stage at pH 5.2. Once infections were initiated, infection threads still developed and nodules still grew despite transfer of the plants to solutions too low in calcium to have permitted infection to begin. Pretreatments at 0.5 mM and 8 mM calcium at pH 5.2 before inoculation had no significantly different effects on nodulation. Observations on root-hair distribution suggest that developing nodules can suppress further infection by suppressing the emergence of root hairs on newly developing roots.     

Effects of aluminium on nodulation of two Stylosanthes species grown in nutrient solution

Summary Effects of three solution aluminium concentrations (0, 25 and 100 M) on nodulation ofStylosanthes hamata andStylosanthes scabra inoculated with Rhizobium CB 756 were studied using nutrient solution culture. Aluminium strongly affected nodulation by delaying nodule appearance and reducing the number and dry weight of nodules in both species. The effects of aluminium toxicity on nodulation were more pronounced inStylosanthes scabra than inStylosanthes hamata. These effects of aluminium on nodulation occurred before any significant effect of aluminium on top growth, root growth or root elongation. A plant transfer experiment suggested that aluminium interfered with root infection and/or nodule initiation in both species. The detrimental effect of aluminium on nodulation appeared to be associated with a reduction in lateral root density, thus decreasing the potential number of sites for root infection and nodule formation.     

Strigolactones promote nodulation in pea

Strigolactones are recently defined plant hormones with roles in mycorrhizal symbiosis and shoot and root architecture. Their potential role in controlling nodulation, the related symbiosis between legumes and Rhizobium bacteria, was explored using the strigolactone-deficient rms1 mutant in pea (Pisum sativum L.). This work indicates that endogenous strigolactones are positive regulators of nodulation in pea, required for optimal nodule number but not for nodule formation per se. rms1 mutant root exudates and root tissue are almost completely deficient in strigolactones, and rms1 mutant plants have approximately 40% fewer nodules than wild-type plants. Treatment with the synthetic strigolactone GR24 elevated nodule number in wild-type pea plants and also elevated nodule number in rms1 mutant plants to a level similar to that seen in untreated wild-type plants. Grafting studies revealed that nodule number and strigolactone levels in root tissue of rms1 roots were unaffected by grafting to wild-type scions indicating that strigolactones in the root, but not shoot-derived factors, regulate nodule number and provide the first direct evidence that the shoot does not make a major contribution to root strigolactone levels.     

Relation between soybean nodulation byBradyrhizobium japonicum and intensity of infestation with the spider miteTetranychus cucurbitacearum (Saved) under different sowing dates and increasing amounts of nitrogenous fertilizer

Inoculation of soybeans withBradyrhizobium japonicum and also the time of sowing of soybeans were studied under N-fertilization with urea at 0, 96 and 192 kg N/ha. Intensity of infestation with the spider miteTetranychus cucurbitacearum (Saved) was followed over six weeks. Inoculation greatly enhanced nodule numbers and weight, dry weight and N content of 65-day-old plant, seed yield, seed N content, 100-seed weight and efficiency of use of N from urea. Counts of the spider mite were increased by inoculation and N fertilization but decreased sharply under late plantation. Fertilizer N proportionally decreased nodulation, N₂-fixation and productivity of chemical fertilizer. A late sowing date gave the least seed production but the maximum percentage increase in seed yield if inoculated. This indicated a lower efficiency of chemical fertilizer N utilization and the necessity for inoculation.     

A comparison of the effect of combined nitrogen on nodulation in non-legumes and legumes

Summary 1. The effect of the presence of combined (ammonium) nitrogen in the rooting medium on nodule development has been investigated inAlnus glutinosa andMyrica gale, the plants being grown by a water culture technique with provision for the control of the level of combined nitrogen and of pH. For purposes of comparison a similar experiment with a legume (Ulex europaeus) has been included.2. In some instances the differential treatment with combined nitrogen was commenced and inoculation effected at an early stage in plant development, while in others the plants were grown on to a larger size before these treatments were applied.3. In the two non-legumes the presence of combined nitrogen led to an increase in the weight of nodulus formed per plant, at least at the lower levels of nitrogen. Relative to the enhanced growth of the plant as a whole, nodule development was continuously depressed as the level of combined nitrogen was increased.4. InUlex when the nitrogen status of the plants at the time of inoculation was similar to that in the corresponding non-legume experiment, the above responses were also shown by the legume. It is concluded thatAlnus andMyrica resemble legumes closely in their responses to combined nitrogen.5. The data show that inAlnus the effect of a given level of combined nitrogen on nodulation depends on the nitrogen status of the plant at the time of inoculation, the adverse effect tending to be stronger if the plant is initially relatively high in nitrogen. This suggests that as in legumes the effect of the combined nitrogen is exerted internally in the plant tissues.