The addition of magnesium sulfate and borax to urea reduced soil NH3 emissions but increased N2O emissions from soil with grass.

Nitrogen (N) use efficiency can be increased by the addition of substances to urea. Magnesium sulfate (MgSO4) and boron were considered as plant nutrients, while zeolite was used as soil conditioner. The addition of these substances may affect soil NH3 and N2O emissions, by increasing N use efficiency. We conducted an 30 days incubation experiment with ryegrass using fertilizer treatments (12 g N m-2) as follows: urea (U); urea + MgSO4 (UM); urea + MgSO4 + borax (UMB); zeolite + urea + MgSO4 (Z-UM); and zeolite + urea + MgSO4 + borax (Z-UMB). We measured NH3 and N2O emissions and the aboveground N uptake of ryegrass. Cumulative NH3 emissions of UM, UMB, Z-UM and Z-UMB were 10%, 53%, 21% and 58% lower than U, respectively, while their N2O emissions were 32%, 133%, 43% and 72% higher than U, respectively. Aboveground N uptake of UM, UMB, Z-UM and Z-UMB were 9%, 6%, 12% and 13% higher than U, respectively. Overall, we suggest that the addition of MgSO4 and borax were effective in reducing NH3 emissions and potentially increase plant N uptake. However, the risk of higher denitrification and N2O emissions also needs to be considered. This study reveals the considerable effect of MgSO4 and borax in soil N cycles. Future research should evaluate how the application of urea + MgSO4 + borax effects gaseous emissions and crop yield of dicotyledons and in drier soil conditions.

Optimization of Potassium Supply under Osmotic Stress Mitigates Oxidative Damage in Barley.

Potassium (K) is the most abundant cation in plants, playing an important role in osmoregulation. Little is known about the effect of genotypic variation in the tolerance to osmotic stress under different K treatments in barley. In this study, we measured the interactive effects of osmotic stress and K supply on growth and stress responses of two barley cultivars (Hordeum vulgare L.) and monitored reactive oxygen species (ROS) along with enzymatic antioxidant activity and their respective gene expression level. The selected cultivars (cv. Milford and cv. Sahin-91Sahin-91) were exposed to osmotic stress (-0.7 MPa) induced by polyethylene glycol 6000 (PEG) under low (0.04 mM) and adequate (0.8 mM) K levels in the nutrient solution. Leaf samples were collected and analyzed for levels of K, ROS, kinetic activity of antioxidants enzymes and expression levels of respective genes during the stress period. The results showed that optimal K supply under osmotic stress significantly decreases ROS production and adjusts antioxidant activity, leading to the reduction of oxidative stress in the studied plants. The cultivar Milford had a lower ROS level and a better tolerance to stress compared to the cultivar Sahin-91. We conclude that optimized K supply is of great importance in mitigating ROS-related damage induced by osmotic stress, specifically in drought-sensitive barley cultivars.

Assessment of soil fertility and potato crop nutrient status in central and eastern highlands of Kenya.

Inherent low soil fertility remains a hindrance to potato production in Kenya and continues to pose a threat to food security. A study was conducted in Nyandarua and Meru counties to assess the soil fertility status in smallholder potato farms. Soil and plant tissue samples were collected and analysed for selected nutrients (pH, OC, N, P, K, S, Ca, Mg, Zn, B and Cu) from 198 farms. Critical nutrient levels were used to assess the sufficiency levels of nutrients for potato growth. Soils in the sampled farms were weakly to strongly acidic (pH-CaCl2 3.9-6.6) and had low to high soil organic matter content (1.5-97.5 g Kg-1). The percent of farms in Meru and Nyandarua with nutrient contents below critical levels were 66% and 20% for N, 46% and 85% for P, 67% and 31% for S, 9% and 51% for Cu, and 87% and 80% for B, respectively. Low tissue nutrient concentrations were observed for N, P, K, and S irrespective of the sites. Soil pH correlated strongly with majority of the analyzed soil and tissue nutrients. These results affirm the need to design integrative soil fertility management strategies to correct the impoverished soil fertility status in the study area.

Effects of resource addition on recovery of production and plant functional composition in degraded semiarid grasslands.

Degradation of semiarid ecosystems from overgrazing threatens a variety of ecosystem services. Rainfall and nitrogen commonly co-limit production in semiarid grassland ecosystems; however, few studies have reported how interactive effects of precipitation and nitrogen addition influence the recovery of grasslands degraded by overgrazing. We conducted a 6-year experiment manipulating precipitation (natural precipitation and simulated wet year precipitation) and nitrogen (0, 25 and 50 kg N ha-1) addition at two sites with different histories of livestock grazing (moderately and heavily grazed) in Inner Mongolian steppe. Our results suggest that recovery of plant community composition and recovery of production can be decoupled. Perennial grasses provide long-term stability of high-quality forage production in this system. Supplemental water combined with exclosures led, in the heavily grazed site, to the strongest recovery of perennial grasses, although widespread irrigation of rangeland is not a feasible management strategy in many semiarid and arid regions. N fertilization combined with exclosures, but without water addition, increased dominance of unpalatable annual species, which in turn retarded growth of perennial species and increased inter-annual variation in primary production at both sites. Alleviation of grazing pressure alone allowed recovery of desired perennial species via successional processes in the heavily grazed site. Our experiments suggest that recovery of primary production and desirable community composition are not necessarily correlated. The use of N fertilization for the management of overgrazed grassland needs careful and systematic evaluation, as it has potential to impede, rather than aid, recovery.

Quantitative limitations to photosynthesis in K deficient sunflower and their implications on water-use efficiency.

Potassium (K) is crucial for crop growth and is strongly related to stress tolerance and water-use efficiency (WUE). A major physiological effect of K deficiency is the inhibition of net CO2 assimilation (AN) during photosynthesis. Whether this reduction originates from limitations either to photochemical energy conversion or biochemical CO2 fixation or from a limitation to CO2 diffusion through stomata and the leaf mesophyll is debated. In this study, limitations to photosynthetic carbon gain of sunflower (Helianthus annuus L.) under K deficiency and PEG- induced water deficit were quantified and their implications on plant- and leaf-scale WUE (WUEP, WUEL) were evaluated. Results show that neither maximum quantum use efficiency (Fv/Fm) nor in-vivo RubisCo activity were directly affected by K deficiency and that the observed impairment of AN was primarily due to decreased CO2 mesophyll conductance (gm). K deficiency additionally impaired leaf area development which, together with reduced AN, resulted in inhibition of plant growth and a reduction of WUEP. Contrastingly, WUEL was not affected by K supply which indicated no inhibition of stomatal control. PEG-stress further impeded AN by stomatal closure and resulted in enhanced WUEL and high oxidative stress. It can be concluded from this study that reduction of gm is a major response of leaves to K deficiency, possibly due to changes in leaf anatomy, which negatively affects AN and contributes to the typical symptoms like oxidative stress, growth inhibition and reduced WUEP.

Long-term non-invasive and continuous measurements of legume nodule activity.

Symbiotic nitrogen fixation is a process of considerable economic, ecological and scientific interest. The central enzyme nitrogenase reduces H(+) alongside N2 , and the evolving H2 allows a continuous and non-invasive in vivo measurement of nitrogenase activity. The objective of this study was to show that an elaborated set-up providing such measurements for periods as long as several weeks will produce specific insight into the nodule activity's dependence on environmental conditions and genotype features. A system was developed that allows the air-proof separation of a root/nodule and a shoot compartment. H2 evolution in the root/nodule compartment can be monitored continuously. Nutrient solution composition, temperature, CO2 concentration and humidity around the shoots can concomitantly be maintained and manipulated. Medicago truncatula plants showed vigorous growth in the system when relying on nitrogen fixation. The set-up was able to provide specific insights into nitrogen fixation. For example, nodule activity depended on the temperature in their surroundings, but not on temperature or light around shoots. Increased temperature around the nodules was able to induce higher nodule activity in darkness versus light around shoots for a period of as long as 8 h. Conditions that affected the N demand of the shoots (ammonium application, Mg or P depletion, super numeric nodules) induced consistent and complex daily rhythms in nodule activity. It was shown that long-term continuous measurements of nodule activity could be useful for revealing special features in mutants and could be of importance when synchronizing nodule harvests for complex analysis of their metabolic status.

Short-Term Molecular Acclimation Processes of Legume Nodules to Increased External Oxygen Concentration.

Nitrogenase is an oxygen labile enzyme. Microaerobic conditions within the infected zone of nodules are maintained primarily by an oxygen diffusion barrier (ODB) located in the nodule cortex. Flexibility of the ODB is important for the acclimation processes of nodules in response to changes in external oxygen concentration. The hypothesis of the present study was that there are additional molecular mechanisms involved. Nodule activity of Medicago truncatula plants were continuously monitored during a change from 21 to 25 or 30% oxygen around root nodules by measuring nodule H2 evolution. Within about 2 min of the increase in oxygen concentration, a steep decline in nitrogenase activity occurred. A quick recovery commenced about 8 min later. A qPCR-based analysis of the expression of genes for nitrogenase components showed a tendency toward upregulation during the recovery. The recovery resulted in a new constant activity after about 30 min, corresponding to approximately 90% of the pre-treatment level. An RNAseq-based comparative transcriptome profiling of nodules at that point in time revealed that genes for nodule-specific cysteine-rich (NCR) peptides, defensins, leghaemoglobin and chalcone and stilbene synthase were significantly upregulated when considered as a gene family. A gene for a nicotianamine synthase-like protein (Medtr1g084050) showed a strong increase in count number. The gene appears to be of importance for nodule functioning, as evidenced by its consistently high expression in nodules and a strong reaction to various environmental cues that influence nodule activity. A Tnt1-mutant that carries an insert in the coding sequence (cds) of that gene showed reduced nitrogen fixation and less efficient acclimation to an increased external oxygen concentration. It was concluded that sudden increases in oxygen concentration around nodules destroy nitrogenase, which is quickly counteracted by an increased neoformation of the enzyme. This reaction might be induced by increased formation of NCR peptides and necessitates an efficient iron supply to the bacteroid, which is probably mediated by nicotianamine. The paper is dedicated to the 85th birthday of Prof. Dr. Günther Schilling, University of Halle/Wittenberg, Germany, https://de.wikipedia.org/wiki/Günther_Schilling.

RNA-seq transcriptome profiling reveals that Medicago truncatula nodules acclimate N2 fixation before emerging P deficiency reaches the nodules.

Legume nodules are plant tissues with an exceptionally high concentration of phosphorus (P), which, when there is scarcity of P, is preferentially maintained there rather than being allocated to other plant organs. The hypothesis of this study was that nodules are affected before the P concentration in the organ declines during whole-plant P depletion. Nitrogen (N2) fixation and P concentration in various organs were monitored during a whole-plant P-depletion process in Medicago truncatula. Nodule gene expression was profiled through RNA-seq at day 5 of P depletion. Until that point in time P concentration in leaves reached a lower threshold but was maintained in nodules. N2-fixation activity per plant diverged from that of fully nourished plants beginning at day 5 of the P-depletion process, primarily because fewer nodules were being formed, while the activity of the existing nodules was maintained for as long as two weeks into P depletion. RNA-seq revealed nodule acclimation on a molecular level with a total of 1140 differentially expressed genes. Numerous genes for P remobilization from organic structures were increasingly expressed. Various genes involved in nodule malate formation were upregulated, while genes involved in fermentation were downregulated. The fact that nodule formation was strongly repressed with the onset of P deficiency is reflected in the differential expression of various genes involved in nodulation. It is concluded that plants follow a strategy to maintain N2 fixation and viable leaf tissue as long as possible during whole-plant P depletion to maintain their ability to react to emerging new P sources (e.g. through active P acquisition by roots).

The activity of nodules of the supernodulating mutant Mtsunn is not limited by photosynthesis under optimal growth conditions.

Legumes match the nodule number to the N demand of the plant. When a mutation in the regulatory mechanism deprives the plant of that ability, an excessive number of nodules are formed. These mutants show low productivity in the fields, mainly due to the high carbon burden caused through the necessity to supply numerous nodules. The objective of this study was to clarify whether through optimal conditions for growth and CO2 assimilation a higher nodule activity of a supernodulating mutant of Medicago truncatula (M. truncatula) can be induced. Several experimental approaches reveal that under the conditions of our experiments, the nitrogen fixation of the supernodulating mutant, designated as sunn (super numeric nodules), was not limited by photosynthesis. Higher specific nitrogen fixation activity could not be induced through short- or long-term increases in CO2 assimilation around shoots. Furthermore, a whole plant P depletion induced a decline in nitrogen fixation, however this decline did not occur significantly earlier in sunn plants, nor was it more intense compared to the wild-type. However, a distinctly different pattern of nitrogen fixation during the day/night cycles of the experiment indicates that the control of N2 fixing activity of the large number of nodules is an additional problem for the productivity of supernodulating mutants.

Soil C and N availability determine the priming effect: microbial N mining and stoichiometric decomposition theories.

The increasing input of anthropogenically derived nitrogen (N) to ecosystems raises a crucial question: how does available N modify the decomposer community and thus affects the mineralization of soil organic matter (SOM). Moreover, N input modifies the priming effect (PE), that is, the effect of fresh organics on the microbial decomposition of SOM. We studied the interactive effects of C and N on SOM mineralization (by natural (13) C labelling adding C4 -sucrose or C4 -maize straw to C3 -soil) in relation to microbial growth kinetics and to the activities of five hydrolytic enzymes. This encompasses the groups of parameters governing two mechanisms of priming effects - microbial N mining and stoichiometric decomposition theories. In sole C treatments, positive PE was accompanied by a decrease in specific microbial growth rates, confirming a greater contribution of K-strategists to the decomposition of native SOM. Sucrose addition with N significantly accelerated mineralization of native SOM, whereas mineral N added with plant residues accelerated decomposition of plant residues. This supports the microbial mining theory in terms of N limitation. Sucrose addition with N was accompanied by accelerated microbial growth, increased activities of ß-glucosidase and cellobiohydrolase, and decreased activities of xylanase and leucine amino peptidase. This indicated an increased contribution of r-strategists to the PE and to decomposition of cellulose but the decreased hemicellulolytic and proteolytic activities. Thus, the acceleration of the C cycle was primed by exogenous organic C and was controlled by N. This confirms the stoichiometric decomposition theory. Both K- and r-strategists were beneficial for priming effects, with an increasing contribution of K-selected species under N limitation. Thus, the priming phenomenon described in 'microbial N mining' theory can be ascribed to K-strategists. In contrast, 'stoichiometric decomposition' theory, that is, accelerated OM mineralization due to balanced microbial growth, is explained by domination of r-strategists.

An RNA sequencing transcriptome analysis reveals novel insights into molecular aspects of the nitrate impact on the nodule activity of Medicago truncatula.

The mechanism through which nitrate reduces the activity of legume nodules is controversial. The objective of the study was to follow Medicago truncatula nodule activity after nitrate provision continuously and to identify molecular mechanisms, which down-regulate the activity of the nodules. Nodule H2 evolution started to decline after about 4 h of nitrate application. At that point in time, a strong shift in nodule gene expression (RNA sequencing) had occurred (1,120 differentially expressed genes). The most pronounced effect was the down-regulation of 127 genes for nodule-specific cysteine-rich peptides. Various other nodulins were also strongly down-regulated, in particular all the genes for leghemoglobins. In addition, shifts in the expression of genes involved in cellular iron allocation and mitochondrial ATP synthesis were observed. Furthermore, the expression of numerous genes for the formation of proteins and glycoproteins with no obvious function in nodules (e.g. germins, patatin, and thaumatin) was strongly increased. This occurred in conjunction with an up-regulation of genes for proteinase inhibitors, in particular those containing the Kunitz domain. The additionally formed proteins might possibly be involved in reducing nodule oxygen permeability. Between 4 and 28 h of nitrate exposure, a further reduction in nodule activity occurred, and the number of differentially expressed genes almost tripled. In particular, there was a differential expression of genes connected with emerging senescence. It is concluded that nitrate exerts rapid and manifold effects on nitrogenase activity. A certain degree of nitrate tolerance might be achieved when the down-regulatory effect on late nodulins can be alleviated.

Soil denitrification potential and its influence on N2O reduction and N2O isotopomer ratios.

RATIONALE: N2O isotopomer ratios may provide a useful tool for studying N2O source processes in soils and may also help estimating N2O reduction to N2. However, remaining uncertainties about different processes and their characteristic isotope effects still hamper its application. We conducted two laboratory incubation experiments (i) to compare the denitrification potential and N2O/(N2O+N2) product ratio of denitrification of various soil types from Northern Germany, and (ii) to investigate the effect of N2O reduction on the intramolecular (15)N distribution of emitted N2O. METHODS: Three contrasting soils (clay, loamy, and sandy soil) were amended with nitrate solution and incubated under N2 -free He atmosphere in a fully automated incubation system over 9 or 28 days in two experiments. N2O, N2, and CO2 release was quantified by online gas chromatography. In addition, the N2O isotopomer ratios were determined by isotope-ratio mass spectrometry (IRMS) and the net enrichment factors of the (15)N site preference (SP) of the N2O-to-N2 reduction step (η(SP)) were estimated using a Rayleigh model. RESULTS: The total denitrification rate was highest in clay soil and lowest in sandy soil. Surprisingly, the N2O/(N2O+N2) product ratio in clay and loam soil was identical; however, it was significantly lower in sandy soil. The IRMS measurements revealed highest N2O SP values in clay soil and lowest SP values in sandy soil. The η(SP) values of N2O reduction were between -8.2 and -6.1‰, and a significant relationship between δ(18)O and SP values was found. CONCLUSIONS: Both experiments showed that the N2O/(N2O+N2) product ratio of denitrification is not solely controlled by the available carbon content of the soil or by the denitrification rate. Differences in N2O SP values could not be explained by variations in N2O reduction between soils, but rather originate from other processes involved in denitrification. The linear δ(18)O vs SP relationship may be indicative for N2O reduction; however, it deviates significantly from the findings of previous studies.

Novel laser spectroscopic technique for continuous analysis of N2O isotopomers--application and intercomparison with isotope ratio mass spectrometry.

RATIONALE: Nitrous oxide (N(2)O), a highly climate-relevant trace gas, is mainly derived from microbial denitrification and nitrification processes in soils. Apportioning N(2)O to these source processes is a challenging task, but better understanding of the processes is required to improve mitigation strategies. The N(2)O site-specific (15)N signatures from denitrification and nitrification have been shown to be clearly different, making this signature a potential tool for N(2)O source identification. We have applied for the first time quantum cascade laser absorption spectroscopy (QCLAS) for the continuous analysis of the intramolecular (15)N distribution of soil-derived N(2)O and compared this with state-of-the-art isotope ratio mass spectrometry (IRMS). METHODS: Soil was amended with nitrate and sucrose and incubated in a laboratory setup. The N(2)O release was quantified by FTIR spectroscopy, while the N(2)O intramolecular (15)N distribution was continuously analyzed by online QCLAS at 1 Hz resolution. The QCLAS results on time-integrating flask samples were compared with those from the IRMS analysis. RESULTS: The analytical precision (2σ) of QCLAS was around 0.3‰ for the δ(15)N(bulk) and the (15)N site preference (SP) for 1-min average values. Comparing the two techniques on flask samples, excellent agreement (R(2)= 0.99; offset of 1.2‰) was observed for the δ(15)N(bulk) values while for the SP values the correlation was less good (R(2 )= 0.76; offset of 0.9‰), presumably due to the lower precision of the IRMS SP measurements. CONCLUSIONS: These findings validate QCLAS as a viable alternative technique with even higher precision than state-of-the-art IRMS. Thus, laser spectroscopy has the potential to contribute significantly to a better understanding of N turnover in soils, which is crucial for advancing strategies to mitigate emissions of this efficient greenhouse gas.

Contribution of nitrification and denitrification to nitrous oxide emissions from soils after application of biogas waste and other fertilizers.

The attribution of nitrous oxide (N(2)O) emission to organic and inorganic N fertilizers requires understanding of how these inputs affect the two biological processes, i.e. denitrification and nitrification. Contradictory findings have been reported when the effects of organic and inorganic fertilizers on nitrous oxide emission were compared. Here we aimed to contribute to the understanding of such variation using (15)N-labelling techniques. We determined the processes producing N(2)O, and tested the effects of soil moisture, N rates, and the availability of organic matter. In a pot experiment, we compared soil treated with biogas waste (BGW) and mineral ammonium sulphate (Min-N) applied at four rates under two soil moisture regimes. We also tested biogas waste, conventional cattle slurry and mineral N fertilizer in a grassland field experiment. During the first 37 days after application we observed N(2)O emissions of 5.6 kg N(2)O-N ha(-1) from soils supplied with biogas waste at a rate of 360 kg N ha(-1). Fluxes were ca. 5-fold higher at 85% than at 65% water holding capacity (WHC). The effects of fertilizer types and N rates on N(2)O emission were significant only when the soil moisture was high. Organic fertilizer treated soils showed much higher N(2)O emissions than those receiving mineral fertilizer in both, pot and field experiment. Over all the treatments the percentage of the applied N emitted as N(2)O was 2.56% in BGW but only 0.68% in Min-N. In the pot experiment isotope labelling indicated that 65-95% of the N(2)O was derived from denitrification for all fertilizer types. However, the ratio of denitrification/nitrification derived N(2)O was lower at 65% than at 85% WHC. We speculate that the application of organic matter in conjunction with ammonium nitrogen first leads to a decrease in denitrification-derived N(2)O emission compared with soil receiving mineral fertilizer. However, at later stages when denitrification becomes C-limited, higher N(2)O emissions are induced when the soil moisture is high.

Diffusion technique for 15N and inorganic N analysis of low-N aqueous solutions and Kjeldahl digests.

Diffusion of ammonia is a common sample preparation method for the stable isotope analysis of inorganic nitrogen in aqueous solution. Classical diffusion methods usually require 6-12 days of diffusion and often focus on (15)N/(14)N analysis only. More recent studies have discussed whether complete N recovery was necessary for the precise analysis of stable N isotope ratios. In this paper we present a newly revised diffusion technique that allows correct and simultaneous determination of total N and (15)N at% from aqueous solutions and Kjeldahl digests, with N concentrations down to sub-0.5-mg N L(-1) levels, and it is tested under different conditions of (15)N isotope labelling. With the modification described, the diffusion time was reduced to 72 h, while the ratios of measured and expected (15)N at% were greater than 99% and the simultaneous recovery of total N was >95%. Analysis of soil microbial biomass N and its (15)N/(14)N ratio is one of the most important applications of this diffusion technique. An experiment with soil extracts spiked with (15)N-labelled yeast showed that predigestion was necessary to prevent serious N loss during Kjeldahl digestion of aqueous samples (i.e. soil extracts). The whole method of soil microbial biomass N preparation for (15)N/(14)N analysis included chloroform fumigation, predigestion, Kjeldahl digestion and diffusion. An experiment with soil spiked with (15)N-labelled yeast was carried out to evaluate the method. Results showed a highly significant correlation of recovered and added N, with the same recovery rate (0.21) of both total N and (15)N. A k(N) value of 0.25 was obtained based on the data. In conclusion, the diffusion method works for soil extracts and microbial biomass N determination and hence could be useful in many types of soil/water studies.