Genetic specificity of nitrogen nutrition in leguminous plants

Summary Mineral nitrogen did not increase grain yield and seed protein levels ofVicia faba L. andLupinus luteus L. in field trials and pot experiments. Fixed N₂ was substituted by mineral nitrogen in these cases because of inhibition of N₂ fixation by mineral nitrogen. Contrary to these results mineral nitrogen increased grain yields and seed protein amounts ofLupinus albus L.,Pisum sativum L., andGlycine max. (L.) Merr. The nitrogen effect was caused at an early stage by saving energy due to inhibition of N₂ fixation (measurement of gas exchange by means of IRGA). In case of the N application after flowering grain, yields and seed protein levels increased because the mineral N was an additional nitrogen source for plants. At this stage the plants had ceased fixing atmospheric nitrogen. The high sink activity of growing fruits induced a lack of assimilates in nodules (determined by means of¹⁴CO₂ application). The N effect was therefore the consequence of the lower assimilate pool for supplying root nodules in these plants in comparison withVicia faba L. andLupinus luteus L. Hence it follows that response to mineral nitrogen can be a criterion for discovering more effective Rhizobium-host combinations.     

Lupinus species differ in their requirements for iron

The effect of iron supply on the growth and nodulation ofLupinus angustifolius L. (Gungurru),Lupinus luteus L. (R-1171) andLupinus pilosus Murr. (P20957) was studied in acid solutions. Plants of the three species were grown together in the same solution and inoculated withBradyrhizobium (Lupinus) WU 425. Plants were then grown with or without applied NH₄NO₃. The lupin species differed greatly in their sensitivity to low iron concentrations in solution withL. pilosus being most tolerant andL. luteus most sensitive.L. pilosus had the highest iron concentration in tissues and had a higher ratio of iron concentration in the youngest fully expanded leaf blades (YEB) to that in roots than the other two species.L. luteus had higher iron concentrations in roots but lower iron concentration in YEB and shoots than didL. angustifolius. The requirements of internal iron for the maximal chlorophyll synthesis in YEB were 65 μg g^-1 forL. angustifolius andL. luteus, and 52 μg g^-1 forL. pilosus. In contrast to effects on growth, the three species had similar external iron requirements for nodule formation in roots and for maximal nitrogen concentrations in shoots. The results indicate that iron tolerant lupin species require lower internal and external iron supply and have a greater ability than sensitive species to translocate iron from roots to shoots.     

Carboxylate release of wheat, canola and 11 grain legume species as affected by phosphorus status

The capacity of plant roots to increase their carboxylate exudation at a low plant phosphorus (P) status is an adaptation to acquire sufficient P at low soil P availability. Our objective was to compare crop species in their adaptive response to a low-P availability, in order to gain knowledge to be used for improving crop P-acquisition efficiency from soils that are low in P or that have a high capacity to retain P. In the present screening study we compared 13 crop species, grown in sand at either 3 or 300 μM of P, and measured root mass ratio, cluster-root development, rhizosphere pH and carboxylate composition of root exudates. Root mass ratio decreased with increasing P supply for Triticum aestivum L., Brassica napus L., Cicer arietinum L. and Lens culinaris Medik., and increased only for Pisum sativum L., while the Lupinus species and Vicia faba L. were not responsive. Lupinus species that had the potential to produce root clusters either increased or decreased biomass allocation to clusters at 300 μM of P compared with allocation at 3 μM of P. All Lupinus species acidified their rhizosphere more than other species did, with average pH decreasing from 6.7 (control) to 4.3 for Lupinus pilosus L. and 5.9 for Lupinus atlanticus L.; B. napus maintained the most alkaline rhizosphere, averaging 7.4 at 300 μM of P. Rhizosphere carboxylate concentrations were lowest for T. aestivum, B. napus, V. faba, and L. culinaris than for the other species. Exuded carboxylates were mainly citrate and malate for all species, with the exception of L. culinaris and C. arietinum, which produced mainly citrate and malonate. Considerable variation in the concentration of exuded carboxylates and protons was found, even with a genus. Cluster-root forming species did not invariably have the highest concentrations of rhizosphere carboxylates. Lupinus species varied both in P-uptake and in the sensitivity of their cluster-root development to external P supply. Given the carbon cost of cluster roots, a greater plasticity in their formation and exudation (i.e. reduced investment in cluster roots and exudation at higher soil P, a negative feedback response) is a desirable trait for agricultural species that may have variable access to readily available P.     

Amount of nitrogen fixed by forage,pasture and grain legumes in Cyprus,estimated by the A-value and a modified difference method

The increasing need for protein at low cost has created a need to evaluate the biological nitrogen fixing potential of legumes in Cyprus. In field studies which were conducted over the growing years of 1982–3 and 1983–4, legumes which are traditionally grown in the country were evaluated for dry matter and nitrogen yield and biological nitrogen fixation (BNF). The legumes studied were medic (Medicago truncatula Gearth), ochrus vetch (Lathyrus ochrus L.), bitter vetch (Vicia ervilia L.) and faba bean (Vicia faba L. var major) in the first year and in addition chickpea (Cicer arietinum L.), woollypod vetch (Vicia dasycarpa Ten.) and tickbean (Vicia faba L. var minor) in the second year. Using the A-value method with barley and oats as reference crops, nitrogen (N) fixed by the various legumes in the first year was 30–50% and from 55–67% of total N yield for the two reference crops, respectively. In the second year the estimates of N fixed ranged from 70 to 80% with similar results obtained for the two reference crops barley and ryegrass. However, in the second year chickpea, which had limited nodulation, fixed only 40% of its N yield. Estimates of nitrogen from the atmosphere (Ndfa) obtained by the difference method (DM) were 10 to 14% lower than those from the A-value method. These results were obtained after correcting for the amount of N derived from the applied fertilizer. The two methods were highly correlated (r=0.98) for estimates of amount of BNF. The rates of N₂ fixation of uninoculated legumes which are nodulated by the indigenous populations of Rhizobium in Cyprus are comparable to those of legumes inoculated with selected strains of Rhizobium in other countries. An exception was the amount of N fixed by chickpea. The appearance of the first nodules at late stages of growth may be the reason for the low BNF of this crop.     

Pesticidal effect on soybean-rhizobia symbiosis

Summary Relative compatibility of selected pesticides at two levels of application (recommended rate and 5× or 10 ×) with soybean-rhizobia symbiosis was tested in pot culture experiments using a prepared peat inoculant.PCNB, carboxin and carboxin+captan at recommended level were innocuous to growth, nodulation, N₂-fixation and total N content of shoot. Carboxin and carboxin+captan but not PCNB at 10 times recommended level proved detrimental to nodulation and N₂-fixation. Carbaryl and malathion at recommended level had no adverse effect but at 10 times recommended level severely reduced N₂-fixation but not other parameters. Acephate, diazinon and toxaphene at both levels reduced N₂-fixation and total N content but not growth and nodulation. All five herbicides used at recommended and 5 times recommended level adversely affected nodulation and N₂-fixation. Glyphosate proved least toxic to all parameters. 2,4-DB at recommended level was less harmful to nodulation and N₂-fixation than trifluralin, alachlor and metribuzin.     

Nodulation and growth ofPhaseolus vulgaris in solution culture

Summary Conditions and techniques for achieving good nodulation ofPhaseolus vulgaris L. in continuously aerated solution were developed from greenhouse experiments.If nodules had been established, their growth and activity and the growth of the plant were at least as good in solution culture as in gravel culture. Nodule formation was observed within 10 days of inoculation in small volumes of solution culture (1 liter). In large volumes (19 liters), similarly prompt nodulation occurred only if the plants were inoculated before or immediately after the seedlings were transferred to the solution from gravel or vermiculite; and the nodules were restricted to the roots that had been present at the time of transfer. Delayed inoculation, 2 days after transfer to large volume solutions, led to sparse nodulation observed only after 3 weeks. Delay or inhibition of nodulation in large volumes of solution could not be explained by failute of bacteria to colonize roots or by sparsity of root hairs.Nodule initiation in solution culture was severely inhibited at pH below 5.4. An additional problem in growing N₂-dependent bean in solution culture was the buildup of Cl^– to toxic levels in the plant in nitrate-free media, even at solution concentrations as low as 0.4 mM Cl^–. Daily addition of 0.5 to 1.0 mg N per plant delayed nodule growth and activity slightly, but increased plant growth and alleviated the severe N-deficiency that otherwise developed before the onset of N₂-fixation.     

Root-hair infection and nodulation of four grain legumes as affected by the form and the application time of nitrogen fertilizer

The effects of application of combined nitrogen fertilizer (ammonium nitrate or urea) on root-hair infection and nodulation of four grain legumes were studied. Young roots of each legume were inoculated with their compatible rhizobia. The application of the two forms of combined N either at the early stages of plant growth and/or at the time of nodule formation depressed root-hair curling, infection and nodulation. Infection of hairs on the primary roots was more sensitive to the N fertilizer than hair infection of secondary roots in bothVicia faba andPisum sativum. The nodule number and total fresh mass of the four legumes were drastically affected by fertilizer application. The combined N added both at early and at later stages significantly reduced the nodulation ofV. faba, Phaseolus vulgaris andVigna sinensis. The inhibitory effect of urea on nodulation ofP. sativum was only observed when the fertilizer was applied at the late stages of plant growth. It is concluded that, although the nodulation of the four legumes was suppressed by combined N, the initial events ofRhizobium-legume symbiosis (infection of roots and nodule initiation) are more sensitive to combined N than the stages after nodule formation.     

Effect of substrate nitrogen on the performance ofin vitro propagatedAlnus glutinosa clones inoculated with Sp+ and Sp− Frankia strains

The addition of combined nitrogen to substrate at an appropriate rate can stimulate N₂-fixation thus inreasing the efficiency of the Alnus-Frankia symbiosis. To examine how nitrogen additions can effect the peformance of different pairs of symbionts, growth and time course of N₂-fixation were studied in plants supplied with NH₄NO₃. Two cloned ofAlnus glutinosa (L.) Gaertn., propagatedin vitro, were inoculated with two strains ofFrankia (AVP3d and ACN14a) and grown in a greenhouse. Calcined montmorillonite (Totfaice^R) was used as growth substrate. Six N treatments were made up of varied amounts of NH₄NO₃ supplied in one single addition shortly before inoculation. Weekly measurements of shoot height and repeated measurements of nitrogenase activity (acetylene reduction) performed on intact root systems were used to monitor the development of the symbioses. Nitrogen treatments containing from 0.10 to 0.68 mg N g⁻¹ dry substrate stimulated N₂-fixation as well as growth. The relative performance of the two clones was different according to N treatment; one clone showed a greater benefit from the nitrogen input. Our results support the recommendation that selection of symbionts according to performance should be carried out with an input of combined nitrogen. This can provide optimum conditions for the development of each pair of symbionts.     

Ecological roles of arbuscular mycorrhizal fungi in two wild legume plants

Two wild legume plants,Glycine soja andCassia mimosoides var.nomame, and a cultivated plant, soybean (Glycine max), were employed for a study of triple symbiosis with an inoculum ofScutellispora heterogama harvested from natural soils and an inoculum of their own rhizobial cells. The dry weight, colonization of arbuscular mycorrhizal fungus, nodule formation and N₂-fixation activity were estimated as the parameters of triple symbiosis. The two wild legume plants showed greater growth with colonization of arbuscular mycorrhizae than with nodulation, whereas the cultivated legume showed more nodulation than colonization of arbuscular mycorrhizae. Moreover,S. heterogama appeared to stimulate the triple symbiosis for the wild legume plants. The results suggested that spores ofS. heterogama are important in disturbed soils in Korea.     

Nitrogen fixation associated with non-legumes in agriculture

Summary This review examines the nitrogen cycle in upland agricultural situations where nonlegume N₂-fixation is likely to be important for crop growth. Evidence for associative fixation is adduced from accumulation of N in the top 15 cm soil under grasses, from N balances for crop production obtained from both pot and field experiments, in tropical and temperate environments, measurements of nitrogen (C₂H₂ reduction) activity, uptake of¹⁵N₂ by plants and¹⁵N isotope dilution. Factors influencing the activity such as the provision of carbon substrate by the plant and the efficiency of its utilisation by the bacteria, plant cultivar, soil moisture and N levels, and inoculation with N₂-fixing bacteria are discussed. Crop responses to inoculation withAzospirillum are detailed. The breakdown of crop residues, particularly straw, can support large levels of N₂-fixation. Cyanobacteria as crusts on the soil surface also fix nitrogen actively in many environments. Fixation by the nodulated, non-legume treesCasuarina andParasponia has beneficial effects in some cropping systems in Asia. I conclude that nonlegume N₂-fixation makes a significant contribution to the production of some major cereal crops in both temperate and tropical environments.     

Nodulation and growth response ofAllocasuarina andCasuarina species to phosphorus fertilization

To examine how soil phosphorus status affects nitrogen fixation by the Casuarinaceae —Frankia symbiosis,Casuarina equisetifolia and two species ofAllocasuarina (A. torulosa andA. littoralis) inoculated or fertilized with KNO₃ were grown in pots in an acid soil at 4 soil phosphate levels. InoculatedC. equisetifolia nodulated well by 12 weeks after planting and the numbers and weight of nodules increased markedly with phosphorus addition. Growth ofC. equisetifolia dependent on symbiotically fixed nitrogen was more sensitive to low levels of phosphorus (30 mg kg^–1 soil) than was growth of seedings supplied with combined nitrogen; at higher levels of phosphorus, the growth response curves were similar for both nitrogen fertilized and inoculated plants. The interaction between phosphorus and nitrogen treatments (inoculated and nitrogen fertilized) demonstrated that there was a greater requirement of phosphorus for symbiotic nitrogen fixation than for plant growth when soil phosphorus was low.WithAllocasuarina species, large plant to plant variation in nodulation occurred both within pots and between replicates. This result suggests genetic variation in nodulation withinAllocasuarina species. Nodulation ofAllocasuarina species did not start until 16 weeks after planting and no growth response due toFrankia inoculation was obtained at the time of harvest. Addition of nitrogen starter is suggested to boost plant growth before the establishment of the symbiosis. Growth ofAllocasuarina species fertilized with nitrogen responded to increasing levels of phosphorus up to 90 mg P/kg soil after which it declined by 69% forA. littoralis. The decrease in shoot weight ofA. littoralis, A. torulosa, C. equisetifolia andC. cunninghamiana at high phosphorus was confirmed in a sand culture experiment, and may be atributable to phosphorus toxicity.     

The effects of low root-zone temperature stress on two soybean (Glycine max) genotypes when combined with Bradyrhizobium strains of varying geographic origin

In areas with short growing seasons, poor early vegetative growth of soybean (Glycine max [L.] Merr.) is often attributed to the restrictive effect of cool soil conditions on nodulation and N₂-fixation by this subtropical grain legume. However, there are few studies regarding potential genetic variability of soybean and Bradyrhizobium japonicum genotypes for nodulation at cool root-zone temperatures (RZT). Experiments were conducted to (1) test for a threshold temperature for low RZT inhibition of soybean nodulation and (2) ascertain whether this threshold temperature response depends mainly on the micro- or macrosymbiont. In experiment 1 soybean seedlings (Glycine max [L.] Merr. cv. Maple Arrow) were inoculated with 1 ml of a log phase culture of B. japonicum strain 532C, H8 or H15 (the latter two strains were isolated from cold soils of Hokkaido, northern Japan) and maintained at either 16, 17.5, 19 or 25°C RZT. In experiment 2 seedlings of cv. Maple Arrow and a cold-tolerant Evans isoline were combined with strain 532C and two Hokkaido strains (H5, H30) at both 19 and 25°C RZT. Results indicated that N₂-fixation at 44 days after inoculation was substantially reduced (30–40%) by RZT as high as 19°C, due to development of less nodule mass and to a delay in the onset of N₂-fixation and a small decrease in the number of nodules formed. However, the number of nodules formed was sharply reduced and the time required for the first appearance of nodules was significantly delayed below an RZT of 17.5°C. Differences between cultivars for nodulation and N accumulation were apparent at 25°C, but were abolished by growth at 19°C, indicating that, in spite of differences in growth potential between the cultivars under optimum RZT, both cultivars were equally limited by low RZT. Differences between B. japonicum strains were consistent across temperatures and were largely attributable to higher rates of specific nodule activity recorded for strain 532C, which seemed well adapted to low RZT. These results suggest that the host plant mediates the sensitivity of N₂-fixation under low RZT and that inoculation with B. japonicum strains from cold environments is unlikely to enhance soybean N₂-fixation under cool soil conditions.     

Effects of salt stress on nitrogenase activity and growth of four legumes

The effects of salt stress on nitrogenase (N2-ase) activity, growth and nitrogen content ofVicia faba (L.),Medicago sativa (L.) Merrill,Glycine max andVigna sinensis (L.) were investigated. Four levels of salinity were applied and salt treatments were imposed on inoculated and N-fertilized plants.M. sativa tolerated mild levels of salinity but higher salt concentrations depressed N₂-ase activity of this species. The other three legumes were considerably affected by salt treatments, and N₂-ase activity was significantly reduced by salinity. Vicia faba, carrying elongate nodules, could restore a partial N₂-ase activity upon recovery from salt stress whereasG. max andV. sinesis, both with spherical nodules, could not regain significant activity when salinity was removed. Salt stress retarded growth of both inoculated and N-fertilized plants. The nitrogen content of both treatments was also affected by salinity and the effect was more severe for inoculated than N-fertilized plants.     

The effects of salinity on growth,nodulation and nitrogen fixation ofCasuarina equisetifolia

The growth, nodulation and nitrogen fixation ofCasuarina equisetifolia were compared at six levels (0–500mM NaCl) of salinity in sand culture. Dry weight of nodules, shoots and roots and N content of shoots increased at intermediate levels of salinity (50–100 mM) but decreased at 500 mM NaCl. Nodulation occurred at all NaCl levels, but at 500mM NaCl level, the nodule dry weight declined by 50% from the control. Increasing NaCl concentration of up to 200mM had little effect on the N₂-fixation rate, but at 500mM NaCl level the rate decreased to 40% of the control value.     

Effect ofLupinus seed diffusates onBradyrhizobium sp. growth and nodulation of lupine

Seeds of three species of lupine (Lupinus termis, L. triticale andL. albus) were tested to determine if the seed contains diffusable substances toxic to bradyrhizobia.L. albus seeds were less toxic to bradyrhizobia, followed byL. triticale. Six strains ofBradyrhizobium were evaluated for their resistance to the toxic substances in lupine seeds. Zones of growth inhibition were determined on yeast-mannitol-agar medium surrounding surface-sterilized seed. The effect of surface sterilization of seeds by different chemical treatments on seed toxicity was assessed. Seeds soaked in water for 1 h before placing on agar surface significantly decreased the inhibition zone. Also, the effect of soaking seeds in water for 4 h before planting and inoculation on nodulation, nitrogen fixation and plant growth were investigated. Addition of seed diffusate to soaked seeds significantly decreased nodulation and plant growth. Autoclaving the seed diffusate had no effect on the toxicity of the seed diffusate. Addition of the absorbent polyvinylpolypyrrolidone (PVPP) to seed diffusates significantly decreased the inhibitory effect of seed diffusate on nodulation and plant growth. Seed diffusate substances were water-soluble, heat-stable and partially bound to PVPP.     

Lutzomyia longipalpis:pH in the Gut, Digestive Glycosidases, and Some Speculations uponLeishmaniaDevelopment

Gontijo, N. F., Almeida-Silva, S., Costa, F. F., Mares-Guia, M. L., Williams, P., and Melo, M. N. 1998.Lutzomyia longipalpis:pH in the gut, digestive glycosidases, and some speculations uponLeishmaniadevelopment.Experimental Parasitology90, 212–219. Screening for digestive glycosidases in different parts of the gut and associated organs ofLutzomyia longipalpisis reported. Searches for the enzymes were made in blood-fed and non-blood-fed females and the enzymes were characterized as soluble or membrane-bound molecules. A total of four different activities were detected, corresponding to the following specificities: an α-glucosidase, anN-acetyl-β-d-glucosaminidase, anN-acetyl-β-d-galactosaminidase, and an α-l-fucosidase. Their possible role and importance forLeishmaniadevelopment are discussed and the α-glucosidase enzyme was partially characterized. The pH inside the gut of non-blood-fed phlebotomines was measured with pH indicator dyes. The pH ranges obtained for crop, midgut, and hindgut were, respectively, higher than pH 6, pH 6, and lower than pH 6. A hypothesis concerning these data andLeishmaniadevelopment is proposed.     

Untraditional N2-fixing bacteria as biofertilizers for wheat and barley

The screening of 27 isolates grown on nitrogen-free medium for nitrogen-fixing ability resulted in the isolation of five organisms belonging toBacillaceae, Enterobacteriaceae andPseudomonadaceae. Estimates of N₂-fixation efficiencies of these isolates indicated that they may be responsible for low rates of N₂-fixation in soil. The possible association of these isolates as well as ofAzotobacter andAzospirillum with wheat and barley was investigated in a greenhouse experiment. The highest values of nitrogenase activity on plant root were recorded in treatments inoculated with composite inocula of the isolated N₂-fixers, particularly whenAzotobacter and/orAzospirillum were added in combination. Inoculation with single inoculum of each of the N₂-fixing isolates had no significant influence on plant growth, except withPseudomonas andBacillus for wheat and barley, respectively. Highly significant increases in growth of both plants were recorded in all cases of multistrain inoculation.     

pH above 6.0 reduces nodulation in Lupinus species

Lupinus angustifolius and Lupinus pilosus differ substantially in root growth in response to high solution pH with L. angustifolius showing much greater sensitivity to pH above 6. This study examined the effect of pH above 6 on nodulation of these two species in buffered solution. Shoot weight and root weight and length in L. pilosus was not significantly affected by pH, whereas the growth of shoots and roots of L. angustifolius was markedly impaired by increasing pH. Total root length, the number of lateral roots, and the length of individual lateral roots were greatly decreased, resulting in decreased uptake of iron and phosphorus. In addition, L. angustifolius had a higher internal requirement for iron than L. pilosus. A solution pH above 6 decreased the number of nodule initials and nodules similarly in both species but decreased nodule mass much more in L. angustifolius. The effect of high pH on nodule formation occurred prior to that on host shoot growth. High pH also decreased nitrogen concentration and content in both species but to a greater extent for L. angustifolius. The results suggested that pH above 6 has a specific effect in the impairment of nodulation in lupins.     

Performance of chickpea,lentil and lupin nodulated with indigenous or inoculated rhizobia micropartners under nitrogen,boron, cobalt and molybdenum fertilization schedules

Chickpea (Cicer arietinum), lentil (Lens esculenta) and lupin (Lupinus albus) responded to inoculation with their respective symbiotants:Rhizobium loti, Rhizobium leguminosarum biovarviceae andBradyrhizobium sp. (Lupinus) alone or with (NH₄)₂SO₄ at 30 ppm N. Soil application of Na₂MoO₄.2H₂O at 3 ppm Mo, CoCl₂.6H₂O at 2 ppm Co²⁺ or Na₂B₄O₇.10H₂O at 1 ppm B with 0 and 30 ppm N increased nodule weight and plant dry weight and N-content 60 days after sowing and seed yield, seed size and the N and P contents of seed. Nodule numbers slightly decreased with application of such chemicals. Mo enhanced performance of the three plants more than Co and B. Yield, total N and P-contents of lupin were comparable with those of chickpea or lentil and lupin had the highest levels of both N₂-fixation and N absorption from the fertilizer. IndigenousR. leguminosarum biovarviceae was more established in the soil compared with the other two, chickpea and lupin, micropartners.     

l-Ascorbic Acid Biosynthesis in Ochromonas danica

This paper expands upon previous reports of ¹⁵N elevation in nodules (compared to other tissues) of N₂-fixing plants. N₂-Fixing nodules of Glycine max (soybeans), Vigna unguiculata (cowpea), Phaseolus vulgaris (common bean), Phaseolus coccineus (scarlet runner bean), Prosopis glandulosa (mesquite), and Olneya tesota (desert ironwood) were enriched in ¹⁵N. Nodules of Vicia faba (fava beans), Arachis hypogaea (peanut), Trifolium pratense (red clover), Pisum sativum (pea), Lathyrus sativus (grass pea), Medicago sativa (alfalfa), and Lupinus mutabilis (South American lupine) were not; nor were the nodules of nine species of N₂-fixing nonlegumes. The nitrogen of ineffective nodules of soybeans and cowpeas was not enriched in ¹⁵N. Thus, ¹⁵N elevation in nodules of these plants depends on active N₂-fixation. Results obtained so far on the generality of ¹⁵N enrichment in N₂-fixing nodules suggest that only the nodules of plants which actively fix N₂ and which transport allantoin or allantoic acid exhibit ¹⁵N enrichment.