Content and Partial Characterization of Unknown N in Humic Acids

INTRODUCTION Nitrogen is a key component of soil organic matter. Only when we have succeeded in characterizing the major part of organic N-containing compounds will we be able to understand fully the transformation reactions in the soil and to use soil-N more efficiently. However, only about 1/4-1/2 of the total N in humic acid (HA), one of the major constituents of soil organic matter, can be accounted for as amino acids and amino sugars, and most of the remainder has still to be accounted for.     

N Cycle, N Flow Trends in Japan, and Strategies for Reducing N2O Emission and NO3^- Pollution

To feed an increasing population, large amounts of chemical nitrogen fertilizer have been used to produce much of our food, feed and fiber thereby increasing nitrogen levels in soils, natural waters, crop residues, livestock wastes,and municipal and agricultural wastes, with national and international concern about its potential adverse effects on environmental quality and public health. To understand these phenomena and problems, first the nitrogen cycle and the environment are described. Then recent trends for nitrogen cycling through the food and feed system, N2O emissions from fertilized upland and paddy soils, and NO3^- pollution in ground water in Japan are reported. Finally, mitigation strategies in Japan for reducing N2O emission and NO3^- pollution are proposed, including nitrification inhibitors, controlled release fertilizers, utilization of plant species that could suppress nitrification, utilizing the toposequence, government policy, and appropriate agricultural practices. Of all the technologies presented, use of nitrification inhibitors and controlled release fertilizers are deemed the most important with further development of these aspects of technologies being expected. These practices, if employed worldwide, could help reduce the load, or environmental deterioration, on the Earth‘s biosphere.     

Control of ammonia volatilization with N‐(N‐butyl) thiophosphoric triamide in loamy sands

Abstract

In a laboratory study, ammonia (NH₃) was trapped from 10 g soil units treated with 10 mg urea‐N, 10 mg urea‐N plus 50 ug N‐(n‐butyl) thiophosphoric triamide (NBPT), or 10 mg urea‐N plus 50 ug phenyl‐phosphorodiamidate (PPD). The soil was a Dothan loamy sand with pH levels adjusted to 6.0, 6.5, and 6.9 prior to N application. After 12 days, NBPT reduced NH₃ volatilization 95 to 97%, while PPD reduced it 19 to 30%. Although NH₃ loss was positively related to initial soil pH, there was no interaction between pH and urease inhibitor. In a field study, NH₃ was trapped in semi‐closed chambers from 134 kg N/ha surface applied to corn (Zea mays L.) 6 weeks after planting. Nine days after N application, NH₃ losses were 20.5, 1.5, 1.5, and 0.2 kg N/ha from urea, urea plus 0.25% NBPT, urea plus 0.50% NBPT, and ammonium nitrate, respectively. Covariance analysis showed that percent organic matter was negatively related to NHL losses. The soil properties, initial pH, CEC, and percent sand, did not vary enough to affect NH₃ volatilization. In conclusion, in both the laboratory and the field, NBPT exhibited strong control of NH₃ volatilization, and could thereby prevent significant loss of surface‐applied urea‐N to crops.     

Extractability of microbial N as influenced by C : N ratio in the flush after drying or fumigation

 Extractability of microbial N was estimated using in situ labelling of the microbial population with ¹⁵N. Four arable soils (one grey forest soil and three chernozems with different long-term fertilization) were amended with (NH₄)₂SO₄ (unlabelled or labelled with ¹⁵N) and d-glucose with a C : N ratio of 10 : 1 or 20 : 1 for the grey forest soil and 50 : 1 for the chernozems. d-glucose and labelled N with a C : N ratio of 20 : 1 did not cause microbial immobilization of unlabelled N. The use of substrates with a C : N ratio of 50 : 1 led to a pronounced priming action on soil N and decreased the extractability of immobilized ¹⁵N. Values of the extractable biomass N fraction (k _EN ) assessed for the fumigation-extraction and rehydration procedures were similar and varied in inverse proportion to the C : N ratio of the flush. The k _EN factor was calculated using values of the C : N ratio in flushes and the fixed C : N ratio of structural cell components, with the assumption that the C : N ratio of the extractable cytoplasmic cell fraction is variable. The ratio between the extractable and non-extractable biomass N fraction (k _EC ) and the C : N ratio of non-extractable cell components were assessed as equation parameters optimized for the measured k _EN and C : N ratio of flush data. Received: 31 October 1997     

Mercury Transport in a Low-Arctic River in Kobbefjord, West Greenland (64° N)

Mercury (Hg) transport was studied in a river in Kobbefjord, near Nuuk in West Greenland, during the 2009 and 2010 summer periods. The river drains an area of 32?km², and the Kobbefjord area is considered representative to low-Arctic West Greenland. The river water origins from both precipitation and melting of small glaciers and annual water discharges for 2009 and 2010 were estimated to be 29 and 26 million?m³, respectively. Mean Hg concentrations (±SD) were 0.46?±?0.17 and 0.26?±?0.17?ng?L^?1 for 2009 and 2010. The annual Hg transport was estimated to 14 and 6.4?g, corresponding to a transport rate of 0.45 and 0.20?g Hg km^?2?year^?1 from the river basin. The highest Hg concentrations (up to 1.0?ng?L^?1) and discharges were measured in spring 2009 along with melting of extensive amounts of snow deposited during the 2008?C2009 winter period. In contrast, the following 2009?C2010 winter period was relatively dry with less snowfall. This indicates that a major fraction of the Hg in this area is likely to come from Hg deposited along with winter precipitation (as wet deposition) released upon snowmelt. Also, the results show that while Hg concentrations were low in Kobbefjord River compared to other sub-Arctic/Arctic rivers, the annual Hg transport rates from the basin area were within the range reported for other sub-Arctic/Arctic areas.     

Differing N status and N retention processes of soils under old-growth lowland forest in Eastern Amazonia,Caxiuanã, Brazil

Indirect evidence of the nitrogen (N) status of tropical forests strongly suggests that in heavily weathered soils under old-growth lowland tropical forests nitrogen is in relative excess. However, within the lowland forests of the Amazon basin, there is substantial evidence that soil texture influences soil NH₄⁺ and NO₃^? concentrations and hence possibly N availability and retention in the soil. Here, we evaluate the soil N status of two heavily weathered soils which contrast in texture (sandy versus clay Oxisol). Using ¹⁵N pool dilution, we quantified gross rates of soil N cycling and retention. We also measured the δ¹⁵N signatures from the litter layer down to 50-cm depth mineral soil and calculated the overall ¹⁵N enrichment factor (ε) for each soil type. The clay soil showed high gross N mineralization and nitrification rates and a high overall ¹⁵N enrichment factor, signifying high N losses. The sandy soil had low gross rates of N cycling and ¹⁵N enrichment factor, manifesting a conservative soil N cycling. Faster turnover rates of NH₄⁺ compared to NO₃^? indicated that NH₄⁺ cycles faster through microorganisms than NO₃^?, possibly contributing to better retention of NH₄⁺ than NO₃^?. However this was opposite to abiotic retention processes, which showed higher conversion of NO₃^? to the organic N pool than NH₄⁺. Our combined results suggest that clay Oxisol in Amazonian forest have higher N availability than sandy Oxisol, which will have important consequences for changes in soil N cycling and losses when projected increase in anthropogenic N deposition will occur.     

Does the nitrate fraction account for differences between dumas‐N and Kjeldahl‐N values in vegetable leaves?

With recent advances in nitrogen (N) analyzers, the Dumas method may replace the traditional Kjeldahl method for the routine diagnosis of N in plants. Because of its nature, the Dumas method truly determines total N. The Kjeldahl method only converts protein N and some nitrate (NO₃‐N) into ammonium. Therefore, the N‐NO₃ ^‐ fraction may explain the difference observed between Kjeldahl‐N (Kn) and Dumas‐N (Dn) values. This study was conducted to (1) determine the Kn:Dn ratio for vegetable crops and (2) evaluate the effect of the size of the nitrate fraction on the Kn:Dn ratio. Over the 0.9–7.0% N range, Dn was a good predictor of Kn in vegetable samples. The Kn may be estimated from Dn as Kn=0.68 Dn (n=134 obs., R²=0.71, p<0.01). For all vegetable crops combined, the mean Kn:Dn ratio was 0.75. This ratio suggests that approximately 25% of N in the samples was recovered by the Dumas method but not by Kjeldahl digestion. This percentage is much higher than the actual N‐NO₃ foliar content. These results suggest that when N‐NO₃ is not known (as in most routine samples), Kn may be estimated from Dn as Kn=0.75 Dn. These results also suggest that under a wide range of NO₃‐N, NO₃‐N alone does not account for the difference between Kjeldahl‐N and Dumas‐N.     

Changes in δ15N in a soil–plant system under different biochar feedstocks and application rates

The application of biochar in soils has been hypothesised to improve soil quality whilst enhancing carbon (C) sequestration. However, its effect on nitrogen (N) dynamics in the soil–plant system is still not fully understood. In the present work, N isotope composition (δ¹⁵N) was used to facilitate the understanding of the processes involved in the N cycling when biochar is applied. We evaluated, through a wheat pot trial, the effect of different application rates of two types of biochar produced from jarrah and pine woodchips on the wheat biomass at harvest and on the soil and plant C and N contents and δ¹⁵N. In addition, the potential benefit of using nutrient-saturated biochar for the soil–plant system was also investigated. Whilst biochar produced from different feedstocks had similar effects on soil and plant nutrient contents, they induced differences in wheat grain biomass and plant δ¹⁵N. The effect of the biochar application rate was more pronounced, and at rates higher than 29 t ha^?1, the application of biochar decreased grain biomass by up to 39 % and potentially increased N losses. Isotopic analyses indicated that this acceleration of N dynamics had probably occurred before the stage of wheat grain formation. The application of nutrient-enriched biochar resulted in an improved wheat grain production, most likely due to the enhanced nutrient availability, and in reduced N cycling rates in the plant–soil system, which could offset the competition between biochar and plants for nutrients and could decrease adverse environmental impacts due to N losses.     

Fate of 15N after combined application of rabbit manure and inorganic N fertilizers in a rice–wheat rotation system

The present study was carried out on pot experiments with rice (Oryza sativa L. cv. Wuyujing 7) and winter wheat (Triticum aestivum L. cv. Yangmai 6) rotation in a sandy and a clayey soil fertilized with ¹⁵N-labeled ammonium sulfate (AS) and ¹⁵N-labeled rabbit feces so as to study the mechanisms of reduction of fertilizer N loss by organic fertilizers. The treatments included: (1) control without any N fertilizer application; (2) fertilization with ¹⁵N-labeled AS (IF); (3) fertilization with labeled rabbit feces (OF); (4) fertilization with either 40% ¹⁵N-labeled rabbit feces and 60% unlabeled AS (IOF1) or (5) 40% unlabeled rabbit feces and 60% ¹⁵N-labeled AS (IOF2). In the rice season, the IOF treatments compared to the IF treatment decreased the percentage of lost fertilizer N from the sandy and clayey soils, whereas it increased the percentage of fertilizer N, present as mineral N and microbial biomass N (MBN). During the second season, when soils were cropped to winter wheat, the IOF treatments in comparison with the IF or OF treatment increased mineral N and MBN contents of soils sampled at tillering, jointing, and heading stages, and such increases were derived from the organic N fertilizer in the sandy soil and from the inorganic N fertilizer in the clayey soil. The increased MBN in the IOF treatments was derived from inorganic fertilizers applied both soils. Therefore, in the IOF treatment, during the rice season, the organic N increased the immobilization of inorganic N in MBN, while the inorganic N fertilizer applied to both soils stimulated the uptake of organic N and the organic N fertilizer increased the uptake of inorganic N by winter wheat; the inorganic N increased the recovery of organic N in the plant-soil system after harvesting the winter wheat.     

Determination of (Nitrate + Nitrite )‐N in alkaline permanganate solutions

Abstract

The quantitative reduction of nitrate in an acid medium with reduced Fe was applied to the alkaline permanganate solution used to absorb NO and NO₂ evolved from soils during denitrification reactions. The method involves addition of H₂SO₄ to acidify the solution and ensure oxidation of nitrite to nitrate, and treatment with reduced Fe at 100°C to reduce nitrate to ammonium. The solution is made alkaline with NaOH and ammonium determined by standard distillation procedures. It is simple and precise, and applicable to nitrogen isotope ratio analysis of NO and NO₂ evolved from soils.     

Soil organic N - An under-rated player for C sequestration in soils?

The availability of Soil Organic Nitrogen (SON) determines soil fertility and biomass production to a great extent. SON also affects the amounts and turnover rates of the soil organic carbon (SOC) pools. Although there is increasing awareness of the impact of the nitrogen (N) cycle on the carbon (C) cycle, the extent of this interaction and the implications for soil organic matter (SOM) dynamics are still under debate. Therefore, present knowledge about the inter-relationships of the soil cycles of C and N as well as current ideas about SON stabilization are summarized in this paper in order to develop an advanced concept of the role of N on C sequestration. Modeling global C-cycling, it was already recognized that SON and SOC are closely coupled via biomass production and degradation. However, the narrow C/N ratio of mature soil organic matter (SOM) shows further that the impact of SON on the refractory SOM is beyond that of determining the size of the active cycling entities. It affects the quantity of the slow cycling pool and as a major contributor it also determines its chemical composition. Although the chemical nature of SON is still not very well understood, both improved classical wet chemical analyses and modern spectroscopic techniques provide increasing evidence that almost the entire organic N in fire-unaffected soils is bound in peptide-like compounds and to a lesser extent in amino sugars. This clearly points to the conclusion, that such compounds have greater importance for SOM formation than previously assumed. Based on published papers, I suggest that peptides even have a key function in the C-sequestration process. Although the mechanisms involved in their medium and long-term stabilization are far from understood, the immobilization of these biomolecules seems to determine the chemistry and functionality of the slow cycling SOM fraction and even the potential of a soil to act as a C sink. Pyrogenic organic N, which derives mostly from incomplete combustion of plant and litter peptides is another under-rated player in soil organic matter preservation. In fire-prone regions, its formation represents a major N stabilization mechanism, leading to the accumulation of heterocyclic aromatic N, the stability of which is still not elaborated. The concept of peptide-like compounds as a key in SOM-sequestration implies that for an improved evaluation of the potential of soils as C-sinks our research focus as to be directed to a better understanding of their chemistry and of the mechanisms which are responsible for their resistance against biochemical degradation in soils.     

Kinetics of Nitrification in Selected Iowa Soils Treated with Stay‐N 2000

Abstract

The effectiveness of Stay‐N 2000 or reformulated nitrapyrin [2‐chloro‐6‐(tricholoromethyl) pyridine] was investigated in two Iowa soils representative of Clarion and Okoboji soils that differed in organic carbon, pH, and texture. A nonlinear regression was used to estimate kinetic parameters. The maximum nitrification rate (K _max) and the duration of lag period (t′) were derived from the equation to characterize the nitrification process in both soils. Stay‐N 2000 appeared to be a better inhibitor than nitrapyrin to extend t′ and as effective as nitrapyrin in reducing K _max. Stay‐N 2000 reduced K _max an appreciable amount in the Okoboji soil at the rate of 12 µg a.i. g^?1 soil or three times the recommended rate. Nitrification rates were affected by the rates of nitrogen (N) applied to both soils; the higher the N rates, the higher K_max, and the more the nitrate (NO₃ ^?)‐N accumulation.     

15N2–DNA–stable isotope probing of diazotrophic methanotrophs in soil

Nucleic acid stable isotope probing (SIP) is a powerful tool that can identify and characterize the microorganisms that mediate specific soil processes and explore the flow of C and N through functional groups in the soil food web. While ¹³C–SIP has been used successfully in a range of applications, methodological constraints have limited the applicability of ¹⁵N-labelled compounds in nucleic acid SIP. However, ¹⁵N–DNA–SIP can now be achieved and this method when used with ¹⁵N₂ provides a powerful new tool for characterizing free-living diazotrophs in natural ecosystems. A diverse array of non-cultivated diazotrophs have been observed in soil and yet the characteristics of these organisms and their environmental significance remain almost completely unknown. ¹⁵N₂–DNA–SIP can identify those diazotrophs that are active in situ while providing access to gene sequences and genome fragments that can yield insights on their evolutionary history and functional capacities. Further insights on the ecology of free-living diazotrophs in soil can be provided by performing ¹⁵N₂–DNA–SIP on microcosms in which the response of the diazotrophic community is determined in relation to experimental manipulation. We describe the use of ¹⁵N₂–DNA–SIP to explore linkages between different C sources and N-fixation by specific diazotroph populations in soil. Methane addition to soil was observed to stimulate N-fixation and the organisms that were found to be responsible for this activity were Type II methanotrophs most closely related to the genus Methylocystis. This report provides insights on the use of nucleic acid SIP to identify and characterize microorganisms that mediate specific soil processes and represents the first time that a specific group of methanotrophs has been shown to mediate N-fixation while in the soil environment.     

Evaluation of N‐butyl phosphorothioic triamide for retardation of urea hydrolysis in soil

Abstract

Work reported showed that N‐butyl phosphorothioic triamide (NBPT) is considerably more effective than phenylphosphorodiamidate (PPD) as a soil urease inhibitor and merits consideration as a fertilizer amendment for retarding hydrolysis of urea fertilizer in soil. Studies to determine the factors influencing the effectiveness of NBPT for retardation of urea hydrolysis in soil showed that the inhibitory effect of NBPT on hydrolysis of urea by soil urease increased markedly with the amount of NBPT added and decreased markedly with time and with increase in temperature from 10 to 40°C. They also showed that the ability of NBPT to retard urea hydrolysis in 13 surface soils selected to obtain a wide range in properties was significantly correlated with organic C content (r = ‐0.70**), total N content (r = ‐0.76**), cation‐exchange capacity (r = ‐0.67* ), sand content (r = 0.61*), clay content (r = ‐0.63*), and surface area (r = ‐0.66*), but was not significantly correlated with pH, silt content, urease activity, or CaCO₃ equivalent. Multiple‐regression analyses indicated that the effectiveness of NBPT for retardation of urea hydrolysis in soil tends to increase with decrease in soil organic‐matter content.     

Potential of phenylphosphorodiamidate and N‐(N‐butyl) thiophosphoric triamide for inhibiting urea hydrolysis in simulated oxidized and reduced soils

Abstract

A laboratory study was conducted to assess the effectiveness of phenylphosphorodiamidate (PPD) and N‐(n‐butyl) thiophosphoric triamide (NBT) in retarding urea hydrolysis in four flooded rice soils under simulated oxidized and reduced conditions. Urea (400 μg N g^‐1soil) with PPD or NBT (2.0% w/w) was added to preincubated soils and analyzed for urea content 1, 3, 5, 7 and 15 days after N application. N‐(n‐butyl) thiophosphoric triamide was more effective in delaying urea hydrolysis under oxidizing conditions and at 5 days 57% of the added urea remained in the oxidized soils compared to only 4% under reduced soil conditions. In three soils, PPD was observed to be effective under reducing soil constraints. At 5 days 56 and 31% of the added urea was unhydrolyzed under reducing and oxidizing soil conditions, respectively, with the addition of PPD. For two soils 48% of the added urea remained at the 15 day sampling for the urea + NBT treatment     

Effects of chitin degradation products N-acetylglucosamine and N,Nʹ-diacetylchitobiose on chitinase activity and bacterial community structure in an incubated upland soil

ABSTRACT

Chitin, which is the polymer of N-acetylglucosamine (GlcNAc) linked by β1,4 glycoside bonds, has been reported as a soil amendment to mitigate plant soil diseases, increasing the population of chitin-degrading bacteria, and chitinolytic enzymatic activity in the soil. In some chitin-degrading bacteria, whose chitinolytic systems have been intensively studied, the chitin degradation product N,N?-diacetylchitobiose {(GlcNAc)₂} induces expression of genes for chitinases whereas GlcNAc does not. To evaluate the effects of these mono- and disaccharides on the population and activity of chitinolytic bacteria in soil, we investigated the chitinolytic enzyme activity and bacterial community structure in an incubated upland soil supplemented with GlcNAc or (GlcNAc)₂. The added GlcNAc and (GlcNAc)₂ (2 mg g^?1) were consumed within 1 d when incubated at 25°C. Chitinase activity was induced by (GlcNAc)₂ and chitin after 1-d and 7-d incubation, respectively, but not by GlcNAc. N-acetylglucosaminidase (GlcNAcase) activity was induced by GlcNAc but was lower than those by (GlcNAc)₂ and chitin. Amplicon sequencing analysis targeting 16S rRNA genes demonstrated that both GlcNAc and (GlcNAc)₂ significantly increased the rate of the order Bacillales, but the compositions of Bacillales differed from each other: the family Planococcaceae significantly increased in either GlcNAc- or (GlcNAc)₂-added soil, but the genera Bacillus and Paenibacillus were increased mainly by GlcNAc and (GlcNAc)₂, respectively. The family Streptomycetaceae of the order Actinomycetales was significantly increased by (GlcNAc)₂ and chitin, but GlcNAc did not. Thus, GlcNAc and (GlcNAc)₂, which were promptly consumed in the incubated soil, indicated partly similar but distinctive effects on chitinolytic enzyme activity and bacterial communities. Both aminosugars increased GlcNAcase activity and the population size of Planococcaceae. GlcNAc increased Bacillus. Chitinase activity and the populations of Paenibacillus and Streptomycetaceae, a number of strains of which are known as potent chitin-degraders, were increased by (GlcNAc)₂, but not by GlcNAc.     

The relationship between applied nitrogen and the concentration of nitrate‐N in cotton petioles

Abstract

Cotton petioles from irrigated plants grown under varying nitrogen regimes were analyzed for nitrate‐N (NO₃‐N). The most recent, fully matured petioles were selected. The concentration of NO₃‐N in the petioles was related to applied N rates and the yields of lint obtained. The concentration of NO₃‐N for any given N application declined as the season progressed. The concentration of petiole nitrate increased at all sampling dates as the rate of applied N increased. The relationship between applied N and NO₃‐N concentrations was most predictable when samples were collected two weeks after the initiation of squaring. The influence of applied N on the concentration of petiole nitrate was also greatest at this stage. The diagnosis of either N deficiency or excess N would be feasible by petiole analysis when the effects of stage of growth could be separated from the effects of soil N.     

N, P and K Fertilizer Response of Rape in Haimen Ⅰ: Fertilizing Responses

2007-2009年度在海门市进行了油菜3414肥料效应试验.结果表明:海门市油菜产量与氮、磷、钾肥用量之间呈三元二次回归效应关系,2007-2008年度和2008-2009年度试验点综合肥料效应方程分别为:y2007-2008=98.220+ 4.876N -0.23 1N2+ 13.399P - 1.748P2+ 6.346K - 0.621K2+ 0.377NP+ 0.126NK+ 0.090PK和y2008-2009=110.352+ 7.636N - 0.311N2+ 7.178P -0.929P2+ 3.923K - 0.550K2+ 0.152NP+ 0.175NK+ 0.173PK.平均每千克养分增产油菜籽分别为:N 5.36kg,P2O57.78kg,K2O4.14kg; N+P2O53.74kg,N+K2O2.83kg,P2O5+K2O2.18kg,N+P2O5+K2O3.85kg.     

Symbiotic N nutrition, C assimilation, and plant water use efficiency in Bambara groundnut (Vigna subterranea L. Verdc) grown in farmers’ fields in South Africa, measured using 15N and 13C natural abundance

Bambara groundnut (Vigna subterranea L. Verdc) is the second most important indigenous food legume in Africa. The aim of this study was to evaluate plant growth, N₂ fixation, N contribution, C accumulation, and plant water relations of Bambara groundnut grown in 26 farmers’ fields in Mpumalanga Province of South Africa. The data revealed marked (p?≤?0.05) differences in plant dry matter (DM) yield, N concentration and content, δ¹⁵N, the proportion of N derived from symbiotic fixation (%Ndfa), and actual amounts of N-fixed between and among the 26 farms surveyed. Bambara groundnut plants obtained 33–98 % (mean?=?72 %) of their N nutrition from symbiotic fixation and contributed 4–200 kg N-fixed ha^?1 (mean?=?102 kg N-fixed ha^?1). Plant density correlated positively with %N (r?=?0.31***), δ¹⁵N (r?=?0.126***), and amount of N-fixed (r?=?0.15*), indicating that the high %Ndfa values obtained for Bambara groundnut in this study and the low symbiotic N yield associated with some farms were due to low plant density rather than poor symbiotic functioning. Bambara groundnut obtained more N from soil (e.g., 173 kg N ha^?1) than from symbiosis (e.g., 135 kg N-fixed ha^?1) in some fields, implying that the N₂-fixing efficacy of the microsymbionts nodulating Bambara groundnut was low at some locations in South Africa. The data from this study showed δ¹³C values ranging from ?28.01 to ?26.20?‰, which indicates differences in plant water use efficiency on the different fields studied. Furthermore, the positive correlations between δ¹³C and N-fixed (r?=?0.15*) and between δ¹³C and N content (r?=?0.14*) suggest a functional relationship between water use efficiency and N₂ fixation, just as the positively significant correlations between δ¹⁵N and DM yield (r?=?0.24***), N-fixed and DM weight (r?=?0.76**), and N content and DM yield (r?=?0.99*), as well as N-fixed and C content (r?=?0.76**) also indicate a functional relationship between N₂ fixation and photosynthesis. In the same way, the positive correlation between δ¹³C and DM weight (r?=?0.14*), or δ¹³C and C content (r?=?0.15*), also implies a functional link between water use efficiency and plant growth. Thus, an increase in water use efficiency in Bambara groundnut, whenever it occurs, seems to functionally enhance plant growth, symbiotic N₂ fixation, and photosynthetic activity, just as N₂ fixation in nodules also seems to stimulate leaf photosynthesis.