Dinitrogen-fixation by three neotropical agroforestry tree species under semi-controlled field conditions

Cultivating dinitrogen-fixing legume trees with crops in agroforestry is a relatively common N management practice in the Neotropics. The objective of this study was to assess the N₂ fixation potential of three important Neotropical agroforestry tree species, Erythrina poeppigiana, Erythrina fusca, and Inga edulis, under semi-controlled field conditions. The study was conducted in the humid tropical climate of the Caribbean coastal plain of Costa Rica. In 2002, seedlings of I. edulis and Vochysia guatemalensis were planted in one-meter-deep open-ended plastic cylinders buried in soil within hedgerows of the same species. Overall tree spacing was 1 × 4 m to simulate a typical alley-cropping design. The ¹⁵N was applied as (NH₄)₂SO₄ at 10% ¹⁵N atom excess 15 days after planting at the rate of 20 kg [N] ha⁻¹. In 2003, seedlings of E. poeppigiana, E. fusca, and V. guatemalensis were planted in the same field using the existing cylinders. The ¹⁵N application was repeated at the rate of 20 kg [N] ha⁻¹ 15 days after planting and 10 kg [N] ha⁻¹ was added three months after planting. Trees were harvested 9 months after planting in both years. The ¹⁵N content of leaves, branches, stems, and roots was determined by mass spectrometry. The percentage of atmospheric N fixed out of total N (%N_f) was calculated based on ¹⁵N atom excess in leaves or total biomass. The difference between the two calculation methods was insignificant for all species. Sixty percent of I. edulis trees fixed N₂; %N_f was 57% for the N₂-fixing trees. Biomass production and N yield were similar in N₂-fixing and non-N₂-fixing I. edulis. No obvious cause was found for why not all I. edulis trees fixed N₂. All E. poeppigiana and E. fusca trees fixed N₂; %N_f was ca. 59% and 64%, respectively. These data were extrapolated to typical agroforestry systems using published data on N recycling by the studied species. Inga edulis may recycle ca. 100 kg ha⁻¹ a⁻¹ of N fixed from atmosphere to soil if only 60% of trees fix N₂, E. poeppigiana 60–160 kg ha⁻¹ a⁻¹, and E. fusca ca. 80 kg ha⁻¹ a⁻¹.     

Assessment of genetic variability for N2 fixation between and within provenances of Leucaena leucocephala and Acacia albida estimated by 15N labelling techniques

Nitrogen fixed in 13 provenances of Acacia albida and 11 isolines of Leucaena leucocephala inoculated with effective Rhizobium strains was measured by ¹⁵N techniques and the total N difference method. In the test soil, on the average, L. leucocephala derived about 65% of its total N from atmospheric N₂ fixation compared to about 20% by A. albida. Significant differences in the percentage of N derived from atmospheric N₂ (% Ndfa) occurred, between provenances or isolines within species. The % Ndfa ranged from 37 to 74% within L. leucocephala and from 6 to 37 within A. albida; (equivalent to 20–50 mg N plant^–1 and 4–37 mg N plant^–1 for the two species over three months, respectively) and was correlated with the nodule mass (r=0.91). The time course of N₂ fixation of three selected provenances (low, intermediate and good fixers) was followed at 12 weekly intervals over a 36 week period. The % Ndfa of all provenances and isolines increased with time; and except for one of the L. leucocephala provenances, % Ndfa was similar within species at the 36 weeks harvest. There was a significant correlation between % Ndfa and the amount of N₂ fixed (r=0.96). Significant interactions occurred between provenances and N treatments and often growth of uninoculated but N fertilized plants was less variable than for inoculated unfertilized plants.     

N recovery from legume prunings and priming effects are governed by the residue quality

Nitrogen recovery from 15N-labelled prunings of Gliricidia sepium, Peltophorum dasyrrachis, Calliandra calothyrsus and Leucaena leucocephala, each of two different chemical qualities, was followed over three cropping cycles in a growth room. Half of the pots of each treatment received a further addition of unlabelled pruning material, from the same species as that previously applied, before the second and third crop cycle. The cumulative maize total N accumulation revealed the largest benefit from N rich, low lignin and polyphenols Gliricidia prunings followed by Leucaena, Calliandra and Peltophorum. Cumulative N recovery measured using 15N over the three crop cycles ranged from 9% from Calliandra prunings to 44% from Gliricidia prunings. The vast majority of this N was recovered during the first crop cycle which agreed well with estimates using the N difference method. Recoveries in the second and third crops ranged from 0.4–5% (15N method) and 6–14% (N difference method) of the N initially applied. The protein binding capacity of polyphenols was the best predictor of N recovery at both initial and later crop cycles. Treatments which led to a large N recovery initially, continued to provide greater N benefits in subsequent cycles although with increasing harvest time this trend decreased. Thus, there was no compensation in initial N release from low quality prunings at later harvests and the agronomic implications of this are discussed. Addition of unlabelled Gliricidia prunings before the second and third cycle led to a positive apparent priming effect on previously applied 15N labelled prunings. By contrast, repeated additions of Peltophorum residues, rich in lignin and active polyphenols, resulted in a reduced recovery of initially applied pruning-15N. However, the maximum positive or negative effects on recovery of pruning N amounted to less than 2% recovery of the initial amount of N added over 14 weeks. Thus the scope for regulation of N release from tree prunings during these later stages of decomposition appears to be limited.     

Decomposition of leaf and root tissue of three perennial grass species grown at two levels of atmospheric CO2 and N supply

Leaf and root tissue of Lolium perenne L., Agrostis capillaris L. and Festuca ovina L. grown under ambient (350 μl l^-1 CO₂) and elevated (700 μl l^-1) CO₂ in a continuously ¹⁴C-labelled atmosphere and at two soil N levels, were incubated at 14°C for 222 days. Decomposition of leaf and root tissue grown in the low N treatment was not affected by elevated [CO₂], whereas decomposition in the high N treatment was significantly reduced by 7% after 222 days. Despite the increased C/N ratio (g g^-1) of tissue cultivated at elevated [CO₂] when compared with the corresponding ambient tissue, there was no significant correlation between initial C/N ratio and ¹⁴C respired. This finding suggests that the CO₂-induced changes in decomposition rates do not occur via CO₂-induced changes in C/N ratios of plant materials. We combined the decomposition data with data on ¹⁴C uptake and allocation for the same plants, and give evidence that elevated [CO₂] has the potential to increase soil C stores in grassland via increasing C uptake and shifting C allocation towards the roots, with an inherent slower decomposition rate than the leaves. An overall increase of 15% in ¹⁴C remaining after 222 days was estimated for the combined tissues, i.e., the whole plants; the leaves made a much smaller contribution to the C remaining (+6%) than the roots (+26%). This shows the importance of clarifying the contribution of roots and leaves with respect to the question whether grassland soils act as a sink or source for atmospheric CO₂. This revised version was published online in June 2006 with corrections to the Cover Date.     

Growth characteristics and total N content of a Leucaena/Sorghum agroforestry system

The growth characteristics and total N content of the woody legume Leucaena leucocephala and the companion crop Sorghum bicolor grown in the greenhouse with N-limiting (NH₄ ⁺ concentration = 447 mM) nutrient medium in sole and mixed cropping were examined. Plant height, dry weight, and total N content increased significantly in sorghum grown intercropped with nodulated Leucaena over the control, sole sorghum. Evidence is presented to indicate that in mixed culture, sorghum gains an average of 0.03 mg N day^–1 plant^–1 relative to sole-cropped sorghum in N-limiting sand culture. The gain in N content of intercropped sorghum, however, represented less than 1% of the N budget of N-fixing Leucaena and was inadequate to sustain normal physiological development of sorghum.     

Specific 15N, NH correlations for residues in15 N, 13C and fractionally deuterated proteins that immediately follow methyl-containing amino acids

A triple-resonance pulse scheme is described which records¹⁵N, NH correlations of residues that immediately follow amethyl-containing amino acid. The experiment makes use of a¹⁵N, ¹³C and fractionally deuterated proteinsample and selects for CH₂D methyl types. The experiment isthus useful in the early stages of the sequential assignment process as wellas for the confirmation of backbone ¹⁵N, NH chemical shiftassignments at later stages of data analysis. A simple modification of thesequence also allows the measurement of methyl side-chain dynamics. This isparticularly useful for studying side-chain dynamic properties in partiallyunfolded and unfolded proteins where the resolution of aliphatic carbon andproton chemical shifts is limited compared to that of amide nitrogens.     

Below-ground nitrogen transfer between different grassland species: Direct quantification by 15N leaf feeding compared with indirect dilution of soil 15N

Nitrogen (N) transfer from one species to another is important for the N cycling in low-input grassland. In the present work, estimates obtained by an indirect ¹⁵N dilution technique were compared with estimates obtained by a direct ¹⁵N leaf feeding technique over two complete growing seasons in red clover-ryegrass and white clover-ryegrass mixtures under field conditions. The direct technique confirmed that N transfer between clovers and ryegrass is a bi-directional process. The transfer of N from both clovers to ryegrass occurred within 25 days upon the first labelling event. A very high N transfer occurred from white clover to the associated ryegrass, 4.5 and 7.5 g m⁻² in the 1st and 2nd production year, respectively. The corresponding values for transfer from red clover to the associated ryegrass were 1.7 and 3.6 g m⁻². Quantified relatively to the total above-ground N content of white clover- ryegrass and red clover-ryegrass mixtures, the N transfer exceeded 50% and 10%, respectively, in three out of seven harvests. The N transfer from ¹⁵N labelled grass to associated clovers constituted a relatively constant proportion of approx. 8% of the above-ground N content of the mixtures. Estimates based on the soil ¹⁵N dilution technique generally underestimated the net N transfer by more than 50% compared to the direct ¹⁵N labelling technique. Furthermore, the indirect ¹⁵N dilution technique estimated only marginal differences between red and white clover in the quantities of N transferred, whereas the direct ¹⁵N labelling technique showed the N transfer from white clover to the associated ryegrass to be significantly higher than that involving red clover. It is concluded that N transfer is a much more dynamic and quantitatively important process in grassland than previously recognised. This revised version was published online in June 2006 with corrections to the Cover Date.     

2D and 3D TROSY-enhanced NOESY of 15N labeled proteins

Recently, several TROSY-based experiments have been designed for backbone chemical shift assignment and measurement of the NOEs of ²H, ¹³C and ¹⁵N labeled proteins. Here, we present TROSY-enhanced NOESY experiments, namely the 2D S3E-NOESY-S3E, 3D TROSY-NOESY-S3E and S3E-NOESY-TROSY experiments. These experiments use the spin-state selective excitation method (S3E), and have the TROSY effect in all the indirectly and directly detected dimensions, and so provide optimal resolution for amide protons. The first two experiments provide an additional useful feature in that the diagonal peaks of the amide proton region are cancelled or greatly reduced, allowing clear identification of NOE cross peaks that are close to diagonal peaks.     

Application of 15N and xylem ureide methods for assessing N2 fixation of three shrub legumes periodically pruned for forage

The apparently diminished capacity for N₂ fixation by the shrub legume Calliandra calothyrsus (Calliandra) relative to other woody perennial legumes was investigated in a field experiment in northern Queensland, Australia. In this trial, (i) the proportion of plant nitrogen (N) derived from symbiotic N₂ fixation (%Pfix) and the amounts of N₂ fixed were compared in Calliandra, Gliricidia sepium (Gliricidia) and Codariocalyx gyroides (Codariocalyx), (ii) variations in N₂ fixation due to season or tree age were determined, (iii) estimates of Pfix derived with the ¹⁵N natural abundance technique were compared with values obtained from ¹⁵N enrichment or xylem sap ureide procedures to determine whether the previous conclusions about Calliandra's ability to fix N had resulted from specific problems with the natural abundance methodology used in the earlier studies.Inoculated seedlings of each of the three shrub legume species were planted in dense stands (1.5 m rows, 0.5 m between trees) in two randomised blocks. The northern block was used solely for natural abundance measurements, while ¹⁵N-enriched KNO₃ (10 atom % ¹⁵N excess) was applied four times over a 52 week period to plots in the southern block. The non-nodulating tree legume Senna spectabilis (formally Cassia spectabilis) was used as a non-N₂-fixing reference for the ¹⁵N-based procedures, with Guinea grass (Panicum maximum) included as an additional non-fixing check. Growth by the trees above 75 cm was first cut and removed after 22 weeks and regrowth was subsequently pruned periodically for another 95 weeks. Sampling for dry matter production, N yield and estimates of Pfix were restricted to the central four of the 32 plants which constituted each replicate plot. Information generated during the 117 week study indicated that estimates of Pfix by ¹⁵N natural abundance were closely similar to values derived with ¹⁵N-enrichment or sap ureides. The data indicated that Calliandra had a reduced reliance upon N₂ fixation relative to Gliricidia and Codariocalyx for the first 65 weeks after establishment. This appeared to be due to more prolifc root growth by Calliandra than either of the other N₂-fixing species and an ability to extract a greater proportion of its N requirements from soil mineral N. However, after week 65 and for the remainder of the experiment, estimates of Pfix for Calliandra were similar to the other shrub legumes. Over 117 weeks, prunings from Calliandra and Gliricidia had removed 52–58 t dry matter ha^-1, and between 1471 and 1678 kg N ha^-1, of which 1026–1063 kg N ha^-1 was estimated to have been derived from N₂ fixation. At the time of final harvest, 65–73% of the fixed N was present in shoot regrowth of the N₂ fixing shrubs, 9–18% in the roots, 15% in the trunk, and 2–6% in fallen leaves.     

Variation in the export of 13C and 15N from soybean leaf: the effects of nitrogen application and sink removal

Translocation of carbon and nitrogen within a single source-sink unit, comprising a trifoliated leaf, the axillary pod and the subtending internode, and from this unit to the rest of the plant was examined in soybean (Glycine max L. cv. Akishirome) plant by feeding ¹³CO₂ and ¹⁵NO₃. The plants were grown at two levels of nitrogen in the basal medium, i.e. low-N (2 g N m^–2) and high-N (35 g N m^–2) and a treatment of depodding was imposed by removing all the pods from the plant, except the pod of the source sink unit, 13 days after flowering. The plants at high-N accumulated more biomass in its organs compared to low-N and pod removal increased the weight of the vegetative organs. When the terminal leaflet of the source-sink unit was fed with ¹³CO₂, almost all of the radioactive materials were retained inside the source-sink unit and translocation to rest of the plants was insignificant under any of the treatments imposed. Out of the¹³C exported by the terminal leaflet, less than half went into the axillary pod, as the lateral leaflets claimed equal share and very little material was deposited in the petiole. Pod removal decreased ¹³C export at high-N , but not at low-N. Similar to ¹³C, the source-sink unit retained all the ¹⁵N fed to the terminal leaflet at high-N. At low-N, the major part of ¹⁵N partitioning occurred in favour of the rest of the plant outside the source-sink unit, but removal of the competitve sinks from the rest of the plants nullified any partitioning outside the unit. Unlike the situation in ¹³C, no partitioning of ¹⁵N occurred in favour of the lateral leaflets from the terminal leaflet inside the unit. It is concluded that sink demand influences partitioning of both C and N and the translocation of carbon is different from that of nitrogen within a source-sink unit. The translocation of the N is more adjustive to a demand from other sink units compared to the C.     

A comparison of direct and indirect 15N isotope techniques for estimating crop N uptake from organic residues

Experiments were carried out to compare the direct approach for estimating crop N uptake from ¹⁵N labelled organic inputs, to two indirect approaches, ¹⁵N isotope dilution and A value. In the first experiment soils received 25, 50, 75, or 100 mg N kg soil⁻¹ in the form of Casuarina equisitifolia residues in addition to ammonium sulphate fertiliser, to give a total of 100 mg N kg soil⁻¹ added. This was a cross labelling design, thus two matching sets of treatments, were set up, identical in all but the position of the ¹⁵N label. Maize (Zea mays L.) plants were grown in the soils amended with residues for 11 weeks and N derived from residues (Ndfr) estimated using the A-value or the direct approach. The A-value approach appeared to significantly overestimate %Ndfr compared to the direct method. In the second experiment contrasting residues were added to soil, fababean (Vicia faba L. var. minor), alfalfa (Medicago sativa L.), soyabean fixing, (Glycine max (L.) Merrill), soyabean non-fixing, barley (Hordeum vulgare L.) and maize. This was also cross-labelling design, labelled and unlabelled residues were used. Maize plants were grown in these soils for 11 weeks and %Ndfr in the maize plants estimated using ¹⁵ N isotope dilution and the direct approach. The ¹⁵ N isotope dilution approach also overestimated %Ndfr compared to the direct method in this experiment. Pool substitution appeared to be responsible for the discrepancy between the direct and indirect techniques. It was concluded that ¹⁵N isotope dilution and A-value approaches as used in these experiments (i.e where residues and ¹⁵N label are added simultaneously) were not appropriate techniques for estimating N derived from organic residues in soils. This revised version was published online in June 2006 with corrections to the Cover Date.     

Manipulation of quality and mineralization of tropical legume tree prunings by varying nitrogen supply

The effect of N supply on the quality of Calliandra calothyrsus and Gliricidia sepium prunings was studied in a glasshouse over a 7-month growing period. Increasing the concentration of N supplied from 0.625 to 10.0 mM NO₃-N resulted in increased N concentration but decreased polyphenol concentration, protein-binding capacity and C:N ratio of prunings from both species. Lignin concentration was not consistently altered by the N treatment. Mineralization of N from the prunings was measured over a 14-week period under controlled leaching and non-leaching conditions. The results indicated a strong interaction between legume species and concentration of N supply in their influence on N mineralization of the prunings applied to the soil. Differences in the %N mineralized were dictated by the quality of the prunings. The (lignin + polyphenol):N ratio was the pruning quality factor which could be used most consistently and accurately to predict N mineralization of the legume prunings incubated under leaching conditions, and the relationship was best described by a linear regression. Under non-leaching conditions, however, the protein-binding capacity appeared to be the most important parameter in determining the patterns of N release from the prunings studied. The relationship between the N mineralization rate constant and the protein-binding capacity was best described by a negative exponential function, y=0.078 exp(–0.0083x). The present study also indicated that the release of N from legume prunings containing a relatively high amount of polyphenol could be enhanced by governing the N availability conditions under which the plant is grown, for example whether or not it is actively fixing nitrogen. Estimates of pruning N mineralization after 14 weeks with the difference method averaged 6% (leaching conditions) and 22% (nonleaching conditions) more than with the ¹⁵N method for all legume prunings studied. The recovery of pruning by maize (4–38%) was well correlated with the % pruning N mineralized suggesting that incubation data closely reflect the pruning N value for a given catch crop under non-leaching conditions.     

Determination of root biomasses of three species grown in a mixture using stable isotopes of carbon and nitrogen

A method is evaluated that employs variation in stable C and N isotopes from fractionations in C and N acquisition and growth to predict root biomasses of three plant species in mixtures. Celtis laevigata Willd. (C₃), Prosopis glandulosa Torr. (C₃, legume) and Schizachyrium scoparium (Michx.) Nash (C₄), or Gossypium hirsutum L. (C₃), Glycine max (L.) Merr. (C₃ legume), and Sorghum bicolor (L.) Moench (C₄) were grown together in separate, three-species combinations. Surface roots (0–10 cm depth) of each species from each of the two combinations were mixed in various proportions, and the relative abundances of ¹⁵N and ¹⁴N and ¹³C and ¹²C in prepared mixtures, surface roots of single species, and roots extracted from the 80-cm soil profile in which each species combination was grown were analyzed by mass spectrometry. An algebraic determination which employed the δ ¹³C, % ¹⁵N, and C and N concentrations of root subsamples of individual species accounted for more than 95% of the variance in biomass of each species in prepared mixtures with G. max, G. hirsutum, and S. bicolor. A similar analysis demonstrated species-specific differences in rooting patterns. Root biomasses of the C₄ monocots in each combination, S. scoparium and S. bicolor, were concentrated in the upper 20 cm of soil, while those of G. hirsutum and the woody P. glandulosa were largest in lower soil strata. Analyses of stable C and N isotopes can effectively be used to distinguish roots of species which differ in ratios of ¹⁵N to ¹⁴N and ¹³C to ¹²C and thus to study belowground competition between or rooting patterns of associated species with different C and N isotope signatures. The method evaluated can be extended to quantify aboveground and belowground biomasses of component species in mixtures with isotopes of other elements or element concentrations that differ consistently among plants of interest.     

The use of a double isotope (15N and 34S) labelling technique to assess the suitability of various reference crops for estimating nitrogen fixation in Gliricidia sepium and Leucaena leucocephala

In this study, an approach involving a double isotope (¹⁵N and ³⁴S) labelling technique was used to examine which of five reference crops (Eucalyptus camaldulensis, Cassia siamea, Cassia spectabilis, Lolium perenne and Eucalyptus grandis) would be suitable for measuring N fixed by Gliricidia sepium and Leucaena leucocephala. The rationale is that the ratio of fertilizer-derived S to soil-derived S in a suitable reference crop is similar to that measured in the nitrogen fixing tree (NFT) since the N ratios in the two crop types cannot be measured directly. E. camaldulensis and E. grandis were found to be suitable reference crops because they absorbed fertilizer and soil S in the same ratio as G. sepium and L. leucocephala.     

Compared cycling in a soil-plant system of pea and barley residue nitrogen

Field experiments were carried out on a temperate soil to determine the decline rate, the stabilization in soil organic matter and the plant uptake of N from ¹⁵N-labelled crop residues. The fate of N from field pea (Pisum sativum L.) and spring barley (Hordeum vulgare L.) residues was followed in unplanted and planted plots and related to their chemical composition. In the top 10 cm of unplanted plots, inorganic N was immobilized after barley residue incorporation, whereas the inorganic N pool was increased during the initial 30 days after incorporation (DAI) of pea residues. Initial net mineralization of N was highly correlated to the concentrations of soluble C and N and the lignin: N ratio of residues. The contribution of residue-derived N to the inorganic N pool was at its maximum 30 DAI (10–55%) and declined to on average 5% after 3 years of decomposition.Residual organic labelled N in the top 10 cm soil declined rapidly during the initial 86 DAI for all residue types. Leaching of soluble organic materials may have contributed to this decline. At 216 DAI 72, 59 and 45% of the barley, mature pea and green pea residue N, respectively, were present in organic N-forms in the topsoil. During the 1–3 year period, residual organic labelled N from different residues declined at similar rates, mean decay constant: 0.18 yr^-1. After 3 years, 45% of the barley and on average 32% of the pea residue N were present as soil organic N. The proportion of residue N remaining in the soil after 3 years of decomposition was most strongly correlated with the total and soluble N concentrations in the residue. The ratio (% inorganic N derived from residues): (% organic N derived from residues) was used as a measure of the rate residue N stabilization. From initial values of 3–7 the ratios declined to on average 1.9 and 1.6 after 2 and 3 yrs, respectively, indicating that a major part of the residue N was stabilized after 2 years of decomposition. Even though the largest proportion of residue N stabilized after 3 years was found for barley, the largest amount of residue N stabilized was found with incorporation of pea residues, since much more N was incorporated with these residues.In planted plots and after one year of decomposition, 7% of the pea and 5% of the barley residue N were recovered in perennial ryegrass (Lolium perenne L.) shoots. After 2 years the cumulative recovery of residue N in ryegrass shoots and roots was 14% for pea and 15% for barley residue N. The total uptake of non-labelled soil N after 2 years of growth was similar in the two residue treatments, but the amount of soil N taken up in each growth period varied between the treatments, apparently because the soil N immobilized during initial decomposition of residues was remineralized later in the barley than in the pea residue treatment. Balances were established for the amounts of barley and mature pea residue N remaining in the 0–10 cm soil layer and taken up in ryegrass after 2 years of decomposition. About 24% of the barley and 35% of the pea residue N were unaccounted for. Since these apparent losses are comparable to almost twice the amounts of pea and barley residue N taken up by the perennial ryegrass crop, there seems to be a potential for improved crop residue management in order to conserve nutrients in the soil-plant system.     

The nitrogen mineralization rate of legume residues in soil as influenced by their polyphenol,lignin, and nitrogen contents

A 12-week greenhouse experiment was conducted to determine the effect of the polyphenol, lignin and N contents of six legumes on their N mineralization rate in soil and to compare estimates of legume-N release by the difference and ¹⁵N-recovery methods. Mature tops of alfalfa (Medicago sativa L.), round leaf cassia (Cassia rotundifolia Pers., var. Wynn), leucaena (Leucaena leucocephala Lam., deWit), Fitzroy stylo (Stylosanthes scabra Vog., var Fitzroy), snail medic (Medicago scutellata L.), and vigna (Vigna trilobata L., var verde) were incorporated in soil at the rate of 100 mg legume N kg^-1 soil. The medic and vigna were labeled with ¹⁵N. Sorghum-sudan hybrid (Sorghum bicolor, L. Moench) was used as the test crop. A non-amended treatment was used as a control. Net N mineralization after 12 weeks ranged from 11% of added N with cassia to 47% of added N for alfalfa. With the two legumes that contained less than 20 g kg^-1 of N, stylo and cassia, there was net N immobilization for the first 6 weeks of the experiment. The legume (lignin + polyphenol):N ratio was significantly correlated with N mineralization at all sampling dates at the 0.05 level and at the 0.01 level at 6 weeks (r²=0.866). Legume N, lignin, or polyphenol concentrations or the lignin:N ratio were not significantly correlated with N mineralization at any time. The polyphenol:N ratio was only significantly correlated with N mineralization after 9 weeks (r²=0.692). The (lignin + polyphenol):N ratio appears to be a good predictor of N mineralization rates of incorporated legumes, but the method for analyzing plant polyphenol needs to be standardized. Estimates of legume-N mineralization by the difference and ¹⁵N recovery methods were significantly different at all sampling dates for both ¹⁵N-labeled legumes. After 12 weeks, estimates of legume-N mineralization averaged 20% more with the difference method than with the ¹⁵N recovery method. This finding suggests that estimates of legume N available to subsequent crops should not be based solely on results from ¹⁵N recovery experiments.     

Hyphal N transport by a vesicular-arbuscular mycorrhizal fungus associated with cucumber grown at three nitrogen levels

Cucumis sativus L. cv. Aminex (F1 hybrid) was grown alone or in symbiosis with Glomus intraradices Schenck and Smith in containers with two hyphal compartments (HC_A and HC_B) on either side of a root compartment (RC) separated by fine nylon mesh. Plants received a total of either 100, 200 or 400 mg N which were applied gradually to the RC during the experiment. ¹⁵N was supplied to HC_A 42 d after plating, at 50 mg ¹⁵NH₄ ⁺-N kg^–1 soil. Lateral movement of the applied ¹⁵N towards the roots was minimized by using a nitrification inhibitor and a hyphal buffer compartment.Non-mycorrhizal controls contained only traces of ¹⁵N after a 27 d labelling period irrespective of the amount of N supplied to the RC. In contrast, 49, 48 and 27% of the applied ¹⁵N was recovered in mycorrhizal plants supplied with 100, 200 and 400 mg N, respectively. The plant dry weight was increased by mycorrhizal colonization at all three levels of N supply, but this effect was strongest in plants of low N status. The results indicated that this increase was due partly to the improved inflow of N via the external hyphae. Root colonization by G. intraradices was unaffected by the amount of N supplied to the RC, while hyphal length increased in HC_A compared to HC_B. Although a considerable ¹⁵N content was detected in mycorrhizal roots adjacent to HC_B, only insignificant amounts of ¹⁵N were found in the external hyphae in HC_B. The external hyphae depleted the soil of inorganic N in both HC_A and HC_B, while the concentration of soil mineral N was still high in non-mycorrhizal containers at harvest. An exception was plants supplied with 400 mg N, where some inorganic N was present at 5 cm distance from the RC in HC_A. The possibility of a regulation mechanism for hyphal transport of N is discussed.     

Simulation of C and N mineralisation during crop residue decomposition: A simple dynamic model based on the C:N ratio of the residues

C and N mineralisation kinetics obtained in laboratory incubations during decomposition of crop residues under non-limiting nitrogen conditions were simulated using a simple dynamic model. This model includes three compartments: the residues, microbial biomass and humified organic matter. Seven parameters are used to describe the C and N fluxes. The decomposed C is either mineralised as CO₂ or assimilated by the soil microflora, microbial decay producing both C humification and secondary C mineralisation. The N dynamics are governed by the C rates and the C:N ratio of the compartments which remain constant in the absence of nitrogen limitation. The model was parameterised using apparent C and N mineralisation kinetics obtained for 27 different residues (organs of oilseed rape plants) that exhibited very wide variations in chemical composition and nitrogen content. Except for the C:N ratio of the residues and the soil organic matter, the other five parameters of the model were obtained by non-linear fitting and by minimising the differences between observed and simulated values of CO₂ and mineral N. Three parameters, namely the decomposition rate constant of the residues, the biomass C:N ratio and humification rate, were strongly correlated with the residues C:N ratio. Hyperbolic relationships were established between these parameters and the residues C:N ratio. In contrast, the other two parameters, i.e. the decay rate of the microbial biomass and the assimilation yield of residue-C by the microbial biomass, were not correlated to the residues C:N ratio and were, therefore, fixed in the model. The model thus parameterised against the residue C:N ratio as a unique criterion, was then evaluated on a set of 48 residues. An independent validation was obtained by taking into account 21 residues which had not been used for the parameterisation. The kinetics of apparent C and N mineralisation were reasonably well simulated by the model. The model tended to over-estimate carbon mineralisation which could limit its use for C predictions, but the kinetics of N immobilisation or mineralisation due to decomposition of residues in soil were well predicted. The model indicated that the C:N ratio of decomposers increased with the residue C:N ratio. Higher humification was predicted for substrates with lower C:N ratios. This simple dynamic model effectively predicts N evolution during crop residue decomposition in soil.     

Symbiotic N2-fixation by the cover crop Pueraria phaseoloides as influenced by litter mineralization

The perennial legume Pueraria phaseoloides is widely used as a cover crop in rubber and oil palm plantations. However, very little knowledge exists on the effect of litter mineralization from P. phaseoloides on its symbiotic N₂-fixation. The contribution from symbiotic N₂-fixation (Ndfa) and litter N (Ndfl) to total plant N in P. phaseoloides was determined in a pot experiment using a ¹⁵N cross-labeling technique. For determination of N₂-fixation the non-fixing plant Axonopus compressus was used as a reference. The experiment was carried out in a growth chamber during 9 weeks with a sandy soil and 4 rates of ground litter (C/N=16,2.8% N). P. phaseoloides plants supplied with the highest amount of litter produced 26% more dry matter and fixed 23% more N than plants grown in soil with no litter application, but the percentage of Ndfa decreased slightly, but significantly, from 87 to 84%. The litter N uptake was directly proportional to the rate of application and constituted 10% of total plant N at the highest application rate. Additionally, a positive correlation was found between litter N uptake and the amount of fixed N₂. The total recovery of litter N in plants averaged 26% at harvest (shoot + root) and was not affected by the quantity added. A parallel incubation experiment also showed that, as an average of all litter levels, 26% of the litter N was present in the inorganic N pool. The amounts of fertilizer and soil N taken up by plants decreased with litter application, probably due to microbial immobilization and denitrification. It is concluded that, within the litter levels studied, litter mineralization will result in a higher amount of N₂-fixed by P. phaseoloides.     

Effects of N-deficiency and timing of N supply on the recovery and distribution of labeled 15N in contrasting maize hybrids

Little information exists on the pattern of nitrogen (N) uptake, remobilization and N use efficiency (NUE) in Leafy and stay-green (SG) maize (Zea mays L.) genotypes. A pot experiment was conducted under controlled nutrition and growing conditions to determine the response of Leafy and SG maize genotypes to different levels of N-deficiency and timing of N supply. Three contrasting maize hybrids, Pioneer 3905 (a conventional hybrid with moderate SG characteristics), Pioneer 39F06 Bt (with a high score of SG trait) and Maizex LF850-RR (with a Leafy trait) were grown in 6 L plastic pots. Five different N treatments [no supply of N until V8 (N₁), no supply of N after V8 (N₂), no supply of N after silking (N₃), no supply of N beyond 3 weeks after silking (N₄), and continuous N supply from emergence to physiological maturity (N₅; standard check)] were imposed through modified Hoagland solution applied manually. Labeled ¹⁵N of 5% ¹⁵N₂–NH₄NO₃fertilizer was applied at 3 g per pot at the start of each schedule N treatment. Total amounts of N applied in different treatments were 3.13, 1.32, 1.90, 2.63 and 3.40 g, respectively in N₁, N₂, N₃, N₄ and N₅. Dry matter, N concentration, ¹⁵N (atom% enrichment) and NUE were determined in roots, stalk, leaves and grains at crop maturity. The three contrasting hybrids did not differ in grain yield, total N acquisition, partitioning of ¹⁵N and NUE. Restriction of N supply until V8, and from V8 to physiological maturity significantly reduced grain yield and N-uptake in all hybrids. Irrespective of the level of N-deficiency in plant and timing when the labeled fertilizer was applied, the amount of ¹⁵N recovered in the matured plant was the same in all N treatments. It has been evident that maize continued to take up N beyond 3 weeks after silking and the later N was applied during the development, the higher proportion of it was partitioned to grains. Of the total ¹⁵N uptake, 78% was partitioned to kernels in the N₄ treatment compared to only 61% in the control. Our data showed no evidence of differential N uptake, remobilization and NUE in the SG or Leafy hybrids tested, but the timing of N application and level of N-deficiency in plant significantly influenced N uptake, remobilization and N-dynamics in maize.