Effect of nitrogen supply on nitrogen and carbohydrate constituent accumulation in rhizomes and storage roots of Curcuma alismatifolia Gagnep.

Abstract

Curcuma (Curcuma alismatifolia cv. Gagnep.), a tropical flowering plant known as “Siam tulip”, were cultivated in a pot with vermiculite and supplied with different levels of nitrogen (N). Rhizomes with storage roots were harvested at 215 days after planting. Results indicated that a high level of N supply increased flower numbers and promoted continuous new rhizome formation, but storage root growth was depressed. The N supply to the plants increased the N concentrations both in the rhizomes and in the storage roots. The predominant nitrogenous compounds related to total N increase were proteins in the rhizomes. The N of the insoluble fraction of 80% ethanol or the N of the soluble fraction of 10% trichloroacetic acid was the predominant fraction of N that accumulated in the storage roots. A lack of N supply increased the starch concentration both in the rhizomes and in the storage roots. These results suggested that a high level of N supply to the curcuma plant increased new rhizome formation because of increased flower numbers, but depressed new storage root formation because of reduced starch accumulation.     

Immobilization of 15N in forest litter studied by 15N CPMAS NMR spectroscopy

Solutions labelled with ¹⁵N were applied as (¹⁵NH₄)₂SO₄ or K¹⁵NO₃ to isolated microplots in the floor of mountain beech forest (Nothofagus solandri var. cliffortioides) and incubated for 135 days under field conditions of moisture and temperature. Solid state ¹⁵N CPMAS NMR spectra of the forest litter layer showed that more than 80% of the total signal intensity was attributable to the secondary amide-peptide peak. The degree of ¹⁵N enrichment or form of N did not alter the relative intensity of signals attributable to ¹⁵N in peptides, nucleic acids and aliphatic amine groups (amino sugars and free NH₂ on amino acids). Combinations of ¹³C and ¹⁵N-NMR spectra, edited by a process that exploited differences in proton spin properties between distinct categories of organic matter, indicated incorporation of ¹⁵N in humified organic matter rather than partly degraded plant material. This application demonstrated that solid state ¹⁵N CPMAS NMR has potential for use in studies of N immobilization under field conditions and with materials containing little N and small ¹⁵N enrichment.     

Influence of pool substitution on the interpretation of fertilizer experiments with 15N

The uptake of labelled and unlabelled N by wheat was measured in pot and field experiments with ¹⁵N-labelled fertilizer. Soils from two sites on the same series were used in the pot experiment; one had been bare-fallowed for 22 years and contained 1.6% organic C, the other had been under grass for many years and contained 3.8% organic C. Fertilizer N increased the uptake of unlabelled soil N in both soils, i.e. there was a positive ‘added nitrogen interaction’ (ANI). There was no ANI in the field experiment. A simulation model is used to show how positive ANIs can arise as a result of ‘pool substitution’—labelled inorganic fertilizer N standing proxy for unlabelled inorganic soil N that would otherwise have been immobilized. In the low-organic fallow soil, pool substitution accounted for the whole of the observed ANI and fertilizer N did not enhance either gross or net mineralization of soil N. Pool substitution also operated in the high organic grassland soil, but here net mineralization of soil N increased with increasing additions of fertilizer, giving rise to a ‘real’ ANI in addition to the larger ‘apparent’ ANI caused by pool substitution. This increase in net mineralization is probably caused by a decrease in immobilization of N as fertilizer N additions increase, not by an increase in gross mineralization of soil N. For pool substitution to operate, fertilizer N and soil inorganic N must occupy the same pool. This occurred in the pot experiment but not in the field experiment, where fertilizer and soil inorganic N remained separate and there was no ANI. When pool substitution occurs, fertilizer use efficiency is predictably lower as measured by the isotopic method than as measured by the conventional non-isotopic procedure.     

Real-time imaging of nitrogen fixation in an intact soybean plant with nodules using 13N-labeled nitrogen gas

Real-time images of nitrogen fixation in an intact nodule of hydroponically cultured soybean ( Glycine max [L] Merr.) were obtained. In the present study, we developed a rapid method to produce and purify ¹³N-labeled radioactive nitrogen gas (half life: 9.97 min). ¹³N was produced from a ¹⁶O (p, α) ¹³N nuclear reaction. The target chamber was filled with CO₂ and irradiated for 10 min with protons at an energy of 18.3 MeV and an electric current of 5 μA, which was delivered from a cyclotron. All CO₂ in the collected gas was absorbed and removed with powdered soda-lime in a syringe and replaced with helium gas. The resulting gas was injected into gas chromatography and separated and a 35 mL fraction, including the peak of [¹³N]-nitrogen gas, was collected by monitoring the chromatogram. The obtained gas was mixed with 10 mL of O₂ and 5 mL of N₂ and used in the tracer experiment. The tracer gas was fed into the underground part of intact nodulated soybean plants and serial images of the distribution of ¹³N were obtained non-invasively using a positron-emitting tracer imaging system (PETIS). The rates of nitrogen fixation of the six test plants were estimated to be 0.17 ± 0.10 μmol N₂ h⁻¹ from the PETIS image data. The decreasing rates of assimilated nitrogen were also estimated to be 0.012 ± 0.011 μmol N₂ h⁻¹. In conclusion, we successfully observed nitrogen fixation in soybean plants with nodules non-invasively and quantitatively using [¹³N]N₂ and PETIS.     

Uptake and translocation of nitrogen in patumma (Curcuma alismatifolia) by leaves or root

An adequate supply of nitrogen (N) is important for patumma growth and flower quality. This study aimed to compare the uptake and translocation of N by foliar and root application. Fertilization with ¹⁵ nitrate (NO₃)-N via roots or leaves was carried out at four stages, at the 1st to 4th fully expanded leaf (FEL) stages, and the plants were sampled at each successive stage. The uptake and translocation of ¹⁵N from foliar or root applications showed relatively similar patterns at all stages. Although the N fertilizer utilization rate by roots was higher than that via leaves, the foliar application stimulated reproductive growth by earlier flowering. The N supplied at the 1st FEL and the 2nd FEL was utilized mainly in leaves, whereas supplying N at the 3rd and 4th FEL promoted flower quality. Fertilizer application method and stage of application influence the utilization rate and translocation of N to the sink organs.     

Denitrification losses of nitrogen fertilizer applied to winter wheat following ley and arable rotations as estimated by acetylene inhibition and 15N balance

We studied the fate of 222 kg N ha^?1 applied in spring as K¹⁵NO₃ to winter wheat test crops which followed either continuous arable cropping (Arable) or a rotation in which a 3-year grass/clover ley preceded the wheat (Ley). Denitrification losses (measured by an acetylene-inhibition method) of over 1 kg N ha^?1 d^?1 were measured for short periods following heavy rain in mid-May. However the generally dry and cool weather resulted in accumulated losses by denitrification between fertilizer application and anthesis equivalent to only 5.3% and 3.6% (±2%) of the applied N for the arable and ley treatments respectively. The smaller loss from the ley was despite this treatment containing more inorganic N and available carbon. ¹⁵N balance indicated that, at anthesis, 1.5% and 11.5% (± 7%) of the labelled N was lost from the arable and ley treatments respectively. Given the precision of the 15N and the acetylene-inhibition methods, the results are not significantly different. However, the larger difference between methods for losses from the ley treatment may be an underestimate because ¹⁵N balance does not measure losses of unlabelled N. These were probably very small on the arable treatment but could have increased total N loss by 25% to c. 32 kg ha^?1 on the ley treatment compared with the 8 kg ha-1 measured as denitrified. Such a large difference is unlikely to be an error but was probably due to ammonia volatilization from this crop which was severely infected by mildew. The results were thus a poor test of the acetylene-inhibition method, but revealed another loss process which could be significant in some situations.     

Nitrogen in particle size fractions of soils incubated for five years with 15N-ammonium and 14C-hemicellulose

Four soils with a range of clay and silt contents were incubated for 5 a with ¹⁵N-labelled (NH₄)SO₄ and ¹⁴C-labelled hemicellulose and then fractionated according to particle size by ultrasonic dispersion and sedimentation. The distribution of labelled and native N between clay, silt and sand fractions was determined and elated to previous results on the C distributions. Between 29% and 48% of the added N was found in organic form. The ¹⁵N atom percentage excess decreased in the order: clay > whole soil > silt > sand. For both clay and silt, the enrichment factor for labelled and native N decreased with increasing fraction weight. Clay enrichment was higher for labelled than for native N, the converse being true for silt. The distribution of whole soil labelled organic N was: clay 77–91%, silt 4–11%, and sand <0.5%. Corresponding values for native N were 69–74%, 16–22%, and 1–2%, respectively. All soils had higher proportions of labelled than of native N in the clay, the converse was true for the silt. The C/N ratio of the native silt organic matter was higher and that of clay organic matter lower than whole soil C/N ratios. Differences between the C/N ratio distributions of native and labelled organic matter were small. The relative distribution of labelled N and C was very similar confirming that the turnover of C and N in soil organic matter is closely interrelated.     

Measuring gross nitrogen mineralization, and nitrification by 15 N isotopic pool dilution in intact soil cores

The isotope dilution method for measuring gross rates of N mineralization, immobilization, and nitrification was applied to intact soil cores so that the effects of soil mixing were avoided. Soil cores were injected with solutions of either ¹⁵NH₄⁺ or ¹⁵NO₄^?; gross mineralization rates were calculated from the decline in “N enrichment of the NH: pool during a 24-h incubation; gross nitrification rates were calculated from the decline in ¹⁵N enrichment of the NO^?₃ pool; gross rates of NH₄⁺ and NO₃^? consumption were calculated from disappearance of the ¹⁵N label. The assumptions required for application of this method to intact cores are evaluated. Sensitivity analysis revealed that homogeneous mixing of added “N with ambient pools was not a necessary assumption but that bias in distribution of added label, coincident with a non-random distribution of microbial processes, would cause significant errors in rate estimates. Rate estimates were also sensitive to errors in initial ¹⁵N and ¹⁴N pool size estimates, In a silt loam soil from a grassland site, abiotic processes consumed over 30% of the added ¹⁵NH₄⁺ within minutes of adding the label to sterilized soil. Extracting a subset of soil cores at the beginning of an incubation is recommended for obtaining initial pool size estimates. Gross immobilization is probably stimulated by addition of inorganic ¹⁵N substrate and, therefore, is overestimated by the isotope dilution method. As an alternative method, a non-linear equation is given for calculating the gross immobilization rate from the appearance of ¹⁵N in chloroform-labile microbial biomass; but incomplete extraction of biomass N may result in low estimates. Details of the isotope dilution methodology (injection rates, concentrations, experimental artefacts, etc.) are described and discussed. When care is taken to understand the underlying assumptions and sources of error, the isotope dilution method provides a powerful tool for measuring gross rates of microbial transformations of soil nitrogen in intact soil cores.     

Field measurements of symbiotic nitrogen fixation in an established pasture using acetylene reduction and a 15N method

The C₂H₂ reduction (A.R.) assay was investigated for quantitative measurement of symbiotic N₂ fixation in established legume-based pastures under field conditions.It was found that the rate of C₂H₄ production was relatively constant for approx 6 h. For an accurate estimation of the N₂ fixing activity, many field samples are required to overcome the errors due to the inherent spatial distribution of white clover (Trifolium repens L.) within the pasture. Furthermore, it is necessary to physically integrate the day-to-day and diurnal variations in the N₂ fixing activity of the legume to obtain a reliable estimate of the rate of C₂H₄ production and hence symbiotic N₂ fixation.The A.R. assay and an ¹⁵N technique were compared for measuring symbiotic N₂ fixation in established pastures. A 3 h incubation in the A.R. assay gave the best estimate of symbiotic N₂ fixation relative to the ¹⁵N technique. The 1 h incubation over-estimated and the 6 h incubation under-estimated the rate of symbiotic N₂ fixation.     

A new method for evaluating symplastic cadmium absorption in the roots of Solanum melongena using enriched isotopes 113Cd and 114Cd

Radioisotope techniques are well known as methods for evaluating symplastic ion absorption in roots. In the present study, a new method for evaluating symplastic cadmium (Cd) absorption in plant roots was developed using the enriched isotopes ¹¹³Cd and ¹¹⁴Cd. Seedlings of Solanum melongena were exposed to an enriched isotope solution of ¹¹³Cd at 25°C for 30 min. The roots were excised from each seedling and were then immersed in a cold buffer solution without Cd at 2°C for 120 min to suppress the metabolic activity of the roots. Finally, the roots were treated with a cold buffer solution containing enriched stable isotope ¹¹⁴Cd at 2°C for 120 min, whereby the apoplastically bound ¹¹³Cd was desorbed. We tested the validity of our method for evaluating symplastic Cd in roots compared with the conventional method based on differences in the amount of Cd absorbed at 2°C and 25°C using unlabeled Cd. There was no difference in the symplastic Cd content of the roots between the two methods. These results indicate that it is possible to evaluate the symplastic Cd content in roots using the enriched isotopes ¹¹³Cd and ¹¹⁴Cd.     

Decomposition of 15N-labelled catch-crop residues in soil: evaluation of N mineralization and plant-N uptake potentials under controlled conditions

The decomposition of ¹⁵N-labelled catch-crop materials (rape, radish and rye), obtained from field experiments, was studied in a chalky Champagne soil during a 60-week incubation at 28°C. Mineralized N was assumed to come from either labile or recalcitrant fractions of plant residues. The labile fraction represented about one-third of the catch-crop N; its mineralization rate constant varied from 0.06 to 0.12 d^?1. The decomposition rate of the recalcitrant N fraction ranged from 0.03 × 10^?2 to 0.06 × 10^?2 d^?1. Catch-crop species and rate of incorporation had no effect on N residue mineralized at the end of incubation. The decomposition of labelled rye was monitored in the same soil during a 5-month pot experiment to determine the N availability to an Italian ryegrass crop and the effect of plants on the decomposition processes. The ¹⁵N-rye decomposed rapidly both in the presence or absence of Italian ryegrass, but the amounts of N mineralized were influenced by the presence of living roots: 42% of the ¹⁵N in labelled rye was present as inorganic N in the pots without plants after 5 months, compared with only 32% in the ryegrass crop. Comparison of microbial-biomass dynamics in both treatments suggested that there had been preferential utilization by soil micro-organisms of materials released from the living roots than the labelled plant residues.     

Recovery of 15N-labelled fertilizer in crops, drainage water and soil using monolith lysimeters in south-east Nigeria

Labelled urea was applied to monolith lysimeters in the 1st year of a 2-year experiment at Onne in south-east Nigeria. On eight lysimeters maize and rice were grown in each of the 2 years. Four lysimeters were similarly cropped in the 2nd year after being uncropped in the 1st year. Measurements were made over the 2-year period of labelled and unlabelled mineral nitrogen in the drainage water, and labelled and unlabelled nitrogen in the crops. At the end of the experiment, weeds and the soil were also analysed for labelled and unlabelled nitrogen.
The total recovery of ¹⁵N in crop, soil and leachate varied between 70 and 93%. It was lowest when applied to the second season rice crop, which recovered only 15%, and highest when it was leached in the 1st year or was taken up by the maize crop. The highest crop uptake was 31%. The results indicate that, depending on the treatment, between 10 and 30% of the ¹⁵N was immobilized in the soil, lysimeters cropped in the 1st year lost between 22 and 29% of the ¹⁵N in drainage water, and between 7 and 30% was lost by denitrification. The accuracy of these figures is discussed.     

不同基因型糯玉米氮素吸收利用效率的研究 II.氮素积累动态的基因型差异

为了解糯玉米氮素积累进程的变化规律,阐明不同生育时期氮素积累的基因型差异及其对产量形成的作用,分析了31个糯玉米品种在同一施氮水平下四叶期、拔节期、大口期(12叶期)、开花(吐丝)期、鲜穗采收期和成熟期的植株氮素积累量。结果表明,植株氮素含量随生育进程逐渐下降,植株氮素积累量随生育进程的增加呈不对称的S型曲线变化,可用Richards方程拟合。不同品种各生育时期的氮素含量和积累量均存在显著差异。鲜穗高产品种主要在大口至开花阶段增加了吸氮量;而鲜子粒及成熟子粒高产品种主要在大口至开花,其次在开花至鲜穗采收阶段增加了吸氮量。通径分析表明,氮素积累过程主要影响氮素积累总量的高低,而对氮素利用效率影响较小。氮素积累过程S型曲线的Richards方程特征参数品种间差异显著。最大积累速率大、活跃积累期长、快增期的积累速率大和持续时间长对提高品种的氮素吸收总量有利。属于高产、氮素吸收量大、氮素利用效率高的基因型有6个品种,其大口至开花及开花至鲜穗采收阶段的吸氮量平均值分别为1.136和0.554 g/plant,比其它品种分别高24.3%和37.8%;最大积累速率和快增期的积累速率分别为0.068和0.059 g/(d.plant),比其它品种分别高15.8%和15.9%。活跃积累期和快增期的持续时间平均值分别为63.4和29.5 d,比其它品种分别延长了1.9和0.9d。     

Dual inoculation of peanut with Glomus sp. and Bradyrhizobium sp. enhanced the symbiotic nitrogen fixation as assessed by 15N-Technique

Abstract

The response of peanut (Arachis hypogaea L.) to inoculation with vesicular-arbuscular mycorrhizal (VAM) fungi (Glomus etunicatum) and Bradyrhizobiurn sp. was studied in pots by the acetylene reduction activity (ARA) and ‘A-value’ methods. The soil used was a Light-coloured Andosol and the treatments consisted of the inoculation of VAM fungi only, inoculation of Bradyrhizobium only, dual inoculation of VAM fungi and Bradyrhizobium and control, under non-sterilized and sterilized soil conditions.

In the non-sterilized soil the ARA and nitrogen fixation determined by the ‘A-value’ method increased significantly only by dual inoculation of VAM fungi and Bradyrhizobium at 100 days after planting (DAP), but no significant difference was observed at 70 DAP. In the case of dual inoculation, 75% of the nitrogen of the plant was derived from fixation whereas the plants inoculated only with Bradyrhizobium derived 68% of their nitrogen from fixation and the control plants, 64%. Amount of P in plant increased significantly only by dual inoculation with VAM fungi and Bradyrhizobium.

In the sterilized soil a highly significant increase in the ARA was observed of the dual inoculation at all the sampling times. Nitrogen fixation determined by the A-value technique and N and P contents in plant also increased significantly by dual inoculation. Results obtained by the A-value method showed that plants with dual inoculation derived 68% of their nitrogen from fixation while the plants inoculated only with Bradyrhizobium, 38%.

From our this study we conclude that nitrogen fixation as well as N and P contents in peanut increased significantly only by dual inoculation with VAM fungi and Bradyrhizobium.