Three polyphenol oxidases from red clover (Trifolium pratense) differ in enzymatic activities and activation properties

Polyphenol oxidases (PPOs) oxidize o-diphenols to o-quinones, which cause browning reactions in many wounded fruits, vegetables, and plants including the forage crop red clover (Trifolium pratense L.). Production of o-quinones in red clover inhibits postharvest proteolysis during the ensiling process. The cDNAs encoding three red clover PPOs were expressed individually in alfalfa (Medicago sativa L.), which lacks detectable endogenous foliar PPO activity and o-diphenols. Several physical and biochemical characteristics of the red clover PPOs in alfalfa extracts were determined. In transgenic alfalfa extracts, red clover PPOs exist in a latent state and are activated (10-40-fold increase in activity) by long incubations (>2 days) at ambient temperature or short incubations (<10 min) at > or =65 degrees C. PPO1 appears to be more stable at high temperatures than PPO2 or PPO3. During incubation at ambient temperature, the molecular masses of the PPO enzymes were reduced by approximately 20 kDa. The apparent pH optima of latent PPO1, PPO2, and PPO3 are 5.5, 6.9, and 5.1, respectively, and latent PPO1 is slightly activated (~5-fold) by low pH. Activation of the PPOs shifts the pH optima to approximately 7, and the activated PPOs retain substantial levels of activity as the pH increases above their optima. The latent and activated PPOs were surveyed for ability to oxidize various o-diphenols, and activation of the PPOs had little effect on substrate specificity. Activation increases the V max but not the affinity of the PPO enzymes for caffeic acid. Results indicate red clover PPOs undergo structural and kinetic changes during activation and provide new insights to their effects in postharvest physiology.     

Seasonal variation of red clover (Trifolium pratense L., Fabaceae) isoflavones and estrogenic activity

Red clover (Trifolium pratense L., Fabaceae) dietary supplements are currently used to treat menopausal symptoms because of their high content of the mildly estrogenic isoflavones daidzein, genistein, formononetin, and biochanin A. These compounds are estrogenic in vitro and in vivo, but little information exists on the best time to harvest red clover fields to maximize content of the isoflavones and thus make an optimal product. Samples of cultivated red clover above-ground parts and flower heads were collected in parallel over one growing season in northeastern Illinois. Generally, autohydrolytic extracts of above-ground parts contained more isoflavones and had more estrogenic activity in Ishikawa endometrial cells as compared with extracts of flower heads. Daidzein and genistein contents peaked around June to July, while formononetin and biochanin A contents peaked in early September. Flower head and total above-ground parts extracts exhibited differential estrogenic activity in an Ishikawa (endometrial) cell-based alkaline phosphatase induction assay, whereas nondifferential activity was observed for most extracts tested in an MCF-7 (breast) cell proliferation assay when tested at the same final concentrations. Ishikawa assay results could be mapped onto the extracts' content of individual isoflavones, but MCF-7 results did not show such a pattern. These results suggest that significant metabolism of isoflavones may occur in MCF-7 cells but not in Ishikawa cells; therefore, caution is advised in the choice of bioassay used for the biological standardization of botanical dietary supplements.     

Latent and active polyphenol oxidase (PPO) in red clover (Trifolium pratense) and use of a low PPO mutant to study the role of PPO in proteolysis reduction

Polyphenol oxidase (PPO) activity in leaf extracts of wild type (WT) red clover and a mutant line expressing greatly reduced levels of PPO (LP red clover) has been characterized. Both latent and active forms of PPO were present, with the latent being the predominant form. PPO enzyme and substrate (phaselic acid) levels fluctuated over a growing season and were not correlated. Protease activation of latent PPO was demonstrated; however, the rate was too low to have an immediate effect following extraction. A novel, more rapid PPO activation mechanism by the enzyme's own substrate was identified. Rates of protein breakdown and amino acid release were significantly higher in LP red clover extracts compared with WT extracts, with 20 versus 6% breakdown of total protein and 1.9 versus 0.4 mg/g FW of free amino acids released over 24 h, respectively. Inclusion of ascorbic acid increased the extent of protein breakdown. Free phenol content decreased during a 24 h incubation of WT red clover extracts, whereas protein-bound phenol increased and high molecular weight protein species were formed. Inhibition of proteolysis occurred during wilting and ensilage of WT compared with LP forage (1.9 vs 5 and 17 vs 21 g/kg of DM free amino acids for 24 h wilted forage and 90 day silage, respectively). This study shows that whereas constitutive red clover PPO occurs predominantly in the latent form, this fraction can contribute to reducing protein breakdown in crude extracts and during ensilage.     

Development of nitrification hot-spots around degrading red clover (Trifolium pratense) leaves in soil

 Litterbags with clover leaves in soil were buried in the field to study the influence of degrading clover leaves on the development of elevated nitrification activity (hot-spots) in space and time. Potential NH₃ oxidation activity indicating the population size of ammonia-oxidizing bacteria was measured in soil samples taken in a zone a few millimetres thick around the leaves and in bulk soil. On 3 sampling days during leaf degradation, the potential NH₄ ⁺ oxidation was significantly higher in leaf-associated soil than in bulk soil, the largest difference (factor of 2) occurring 24 days after burial. At all sampling occasions, NH₄ ⁺ oxidation rates followed a normal distribution, except for a log-normal distribution in the leaf-associated soil sampled 24 days after burial when some very active samples (hot-spots) appeared. In a similar laboratory experiment we examined the effects of soil water on the development of nitrification hot-spots. We observed that the development of hot-spots did not take place in soil incubated at 60% of water-holding capacity (WHC), whereas they developed faster when the soil was incubated at approximately 100% WHC. It was concluded that soil water in combination with easily degradable organic N were essential parameters for the development of nitrification hot-spots in this soil. Received: 18 April 2000     

Potassium uptake of rye-grass (Lolium perenne) and red clover (Trifolium pratense) as related to root parameters

Summary Rye-grass (Lolium perenne) is known to be a strong competitor to red clover (Trifolium pratense) for soil K⁺ under conditions of low K availability in the soil. The objective of this study was to clarify whether this competitive behaviour of the two species can be explained by root morphology. Total K⁺ uptake ofL. perenne andT. pratense was studied under field conditions in relation to root fresh weight, root density, root cation exchange capacity, root surface and root length. The soil was an Alfisol, Udalf. All root parameters, when calculated per unit soil surface (M²), were higher inL. perenne than inT. pratense. In addition,L. perenne had longer root hairs and a denser root hair system thanT. pratense. The greatest difference in root morphology between species was root length, withL. perenne roots averaging 4–6 times longer than those ofT. pratense.Significant correlations were found between the total K⁺ uptake and all root parameters examined, with highest correlationsforroot fresh weight (r,0.92***T. pratense; 0.94***L. perenne) and root length (r, 0.91***T. pratense;r, 0.93***L. perenne). Potassium uptake per unit root fresh weight, root surface and root length were all significantly higher forT. pratense than for L. perenne. Differences in the rate of K⁺ uptake between species were particularly high when expressed per unit root length. Because of its greater root length and surface area,L. perenne can take up more soil K⁺ thanT. pratense, particularly where there is a low K^– supply in the soil. Under such conditionsL. perenne will be a particularly strong competitor toT. pratense.     

Comparison of the in vitro estrogenic activities of compounds from hops (Humulus lupulus) and red clover (Trifolium pratense)

Because the prevailing form of hormone replacement therapy is associated with the development of cancer in breast and endometrial tissues, alternatives are needed for the management of menopausal symptoms. Formulations of Trifolium pratense L. (red clover) are being used to alleviate menopause-associated hot flashes but have shown mixed results in clinical trials. The strobiles of Humulus lupulusL. (hops) have been reported to contain the prenylflavanone, 8-prenylnaringenin (8-PN), as the most estrogenic constituent, and this was confirmed using an estrogen receptor ligand screening assay utilizing ultrafiltration mass spectrometry. Extracts of hops and red clover and their individual constituents including 8-PN, 6-prenylnaringenin (6-PN), isoxanthohumol (IX), and xanthohumol (XN) from hops and daidzein, formononetin, biochanin A, and genistein from red clover were compared using a variety of in vitro estrogenic assays. The IC50 values for the estrogen receptor alpha and beta binding assays were 15 and 27 microg/mL, respectively, for hops and 18.0 and 2.0 microg/mL, respectively, for the red clover extract. Both of the extracts, genistein, and 8-PN activated the estrogen response element (ERE) in Ishikawa cells while the extracts, biochanin A, genistein, and 8-PN, significantly induced ERE-luciferase expression in MCF-7 cells. Hop and red clover extracts as well as 8-PN up-regulated progesterone receptor (PR) mRNA in the Ishikawa cell line. In the MCF-7 cell line, PR mRNA was significantly up-regulated by the extracts, biochanin A, genistein, 8-PN, and IX. The two extracts had EC50 values of 1.1 and 1.9 microg/mL, respectively, in the alkaline phosphatase induction assay. On the basis of these data, hops and red clover could be attractive for the development as herbal dietary supplements to alleviate menopause-associated symptoms.     

Measurement of pH at the root surface of red clover (Trifolium pratense) grown in soils differing in proton buffer capacity

Summary Measurements of pH were made at the root surface of Trifolium pratense, using Sb electrodes. Nodulated plants were grown in rhizotrones on a sandy soil free of carbonate and on a clay soil rich in carbonate. In the sandy soil, pH at the surface of root laterals was about 1 unit lower than in the bulk soil. The lowest pH values were found at the root tips. In the calcareous soil, pH measured at the root surface did not differ from pH in the bulk soil. This soil had a much higher H⁺ buffer capacity than the sandy soils. It seems likely that H⁺ ions excreted from the roots grown in the calcareous soil were directly neutralized by soil carbonate.     

Inhibition of extrahepatic human cytochromes P450 1A1 and 1B1 by metabolism of isoflavones found in Trifolium pratense (red clover)

Biochanin A and formononetin are the predominant isoflavones in red clover. In a previous study (J. Agric. Food Chem. 2002, 50, 4783-4790), it was demonstrated that human liver microsomes converted biochanin A and formononetin to genistein and daidzein. This paper now shows CYP1B1-catalyzed O-demethylation of biochanin A and formononetin to produce genistein and daidzein, respectively, which inhibit CYP1B1. Recombinant human CYP1A1 or CYP1B1 was incubated with biochanin A or formononetin. CYP1A1 catalyzed isoflavone 4'-O-demethylation and hydroxylations with similar efficiency, whereas CYP1B1 favored 4'-O-demethylation over hydroxylations. Three of the biochanin A metabolites (5,7,3'-trihydroxy-4'-methoxyisoflavone, 5,7,8-trihydroxy-4'-methoxyisoflavone, and 5,6,7-trihydroxy-4'-methoxyisoflavone) were characterized by 1H NMR spectroscopy and mass spectrometry. Daidzein (Ki = 3.7 microM) exhibited competitive inhibition of CYP1B1 7-ethoxyresorufin O-deethylase activity, and genistein (Ki = 1.9 microM) exhibited mixed inhibition. Biochanin A and/or formononetin may exert anticarcinogenic effects directly by acting as competitive substrates for CYP1B1 or indirectly through their metabolites daidzein and genistein, which inhibit CYP1B1.     

Repellency of insecticides and the effect of thiacloprid on bumble bee colony development in red clover (Trifolium pratense L.) seed crops

The study intended to compare repellency of three insecticides on bumble bees and honey bees in Norwegian red clover (Trifolium pratense L.) seed crops, and to examine effects of thiacloprid on bumble bee colony development in the field. The repellency study was carried out in a large-scale field trial in SE Norway in 2013. On average for observations during the first week after spraying, 17 and 40% less honey bees (P?=?.03) and 26 and 20% less bumble bees (P?=?.36) were observed on plots sprayed with the pyrethroids lambda-cyhalothrin and alpha-cypermethrin, respectively, than on unsprayed control plots. No pollinator repellency was found on plots sprayed with the neonicotinoid thiacloprid. Compared with unsprayed control the seed yield increases were 22% on plots sprayed with thiacloprid vs. 12–13% on plots sprayed with pyrethroids (P?=?.10). Follow-up studies in 2014–2016 focused on the effect of thiacloprid on bumble bee colony development in commercially reared nests of Bombus terrestris placed into red clover seed crops at the start of flowering. Unsprayed control crops were compared with crops sprayed either at the bud stage or when 18–44% of flower heads were in full bloom. Chemical analyses of adult bumble bees showed that thiacloprid was taken up in bees when crops were sprayed during flowering, but not detected when crops were sprayed at the bud stage. The bumble bees in late-sprayed crops also developed weaker colonies than in unsprayed crops. Dead bees with a high internal concentration of thiacloprid were found in one crop sprayed during the night at 35% flowering. This shows that thiacloprid is not bee-safe if sprayed after anthesis and that spraying has to be conducted at the bud stage to reduce its contamination of nectar and pollen.     

Effect of sowing methods and sowing rate in organic seed production of timothy (Phleum pratense L.), meadow fescue (Festuca pratensis Huds.) and red clover (Trifolium pratense L.)

Different sowing methods and sowing rates were evaluated in organic seed production of timothy (two trials), meadow fescue (two trials) and red clover (one trial) in Southeast Norway, during 2010–2013. The plan included: (1) broadcast sowing of grass/clover, cover crop sown at 12 cm row distance; (2) sowing of cover and seed crop in crossed rows, both at 12 cm row distance; and (3) sowing of cover crop and seed crop in every other row. The three sowing rates were 5, 10 and 15 kg ha–1 in timothy and meadow fescue and 3, 6 and 9 kg ha–1 in red clover. On average for sowing rates and all trials with timothy, meadow fescue and red clover, first year’s seed yields were 5–6%, 20–25% and 19–25% higher on plots sown with cover crop and seed crop in every other row than on plots where seed crop had been broadcast or sown perpendicularly to the cover crop. The different sowing methods had no effect on weed coverage or weed contamination in the cleaned seed. Increasing sowing rate usually had a negative influence on seed yield, while weed coverage/contamination was not significantly affected. It is concluded that organic seed crops should be established with cover crop and seed crop in every other row at a low sowing rate. However, in an organic production system, even this favorable method will not always be sufficient to meet the requirement for seed crop purity.     

Kinetics of inorganic and organic P release from red clover (Trifolium pratense L.) and ryegrass (Lolium multiflorum L.) upon frost or drying

An incubation study with fresh, frozen, and dried clover and rye-grass shoots gathered in autumn was performed in the laboratory (43 days). The aim was to study inorganic and organic phosphorus (P) release from plant material during decomposition without soil. Plant materials (2?cm size) were mixed with small glass beads and placed in large syringes for consecutive water extractions on 7 occasions. Leachates were analysed for inorganic and total P. At the first leaching event, 8% of total crop P was released from fresh, 24% from frozen and 27% from dried plant material. During decomposition, both inorganic and organic P was released following first order kinetics, with grass releasing mostly inorganic P and clover mostly organic P. After 43 days, 42%–50% of total crop P from fresh, 49%–51% from frozen and 57%–69% from dried material was released, with significant differences between treatments. Using the results in calculations on field scale showed that P released from overwintering crops under cold climate conditions can amount to several kg P ha^?1. Thus, senescence of overwintering aboveground biomass can be a significant source for P leaching from soils.     

Interaction Effects of Nitrogen and Phosphorus on Nodulation in Red Clover (Trifolium pratense L.)

Red clover (Trifolium pratense L.) is one of the most important plants in forage production, especially in northern areas. Fertilisation practices are focused on high yield and forage quality but effects of nutrients on nodulation and N₂ fixation are poorly understood. The aim of this work was to study how nitrogen (N) and phosphorus (P) separately as well as in combination affected nodulation. Red clover plants were grown in pots with gravel in a greenhouse for 11 weeks. To resemble field conditions the root temperature was kept lower than the shoot temperature. Plants were given five different combinations of N and P concentrations during growth. The result showed that at high N concentrations P had a counteracting effect on the N inhibition. The N₂-fixation parameters, nodule number, nodule dry matter and specific nitrogenase activity, were six times higher in plants grown with high N and high P than in plants with high N and low P. When the N₂-fixation parameters and the dry matter of roots and shoots were related to total plant dry matter, there was a stronger effect of P on nodulation parameters than on roots and shoots. This indicates that P has a direct effect on the N₂-fixation parameters, rather than an indirect effect via increased plant growth. These results demonstrate the importance to studying the effects of more than one nutrient at a time.     

几种控释氮肥的饲料玉米肥效及其生理效应研究

通过田间试验研究了几种控释氮肥对饲料玉米分蘖、叶片叶绿素含量、光合速率及氮肥利用率的影响,并探讨了这些生理指标与饲料玉米生物量之间的关系。结果看出,控释氮肥处理,玉米分蘖数在分蘖前期比等养分尿素略低,分蘖后期明显高于尿素处理;叶片叶绿素含量、叶片光合速率与等养分尿素相比,都有明显提高。相关分析表明,各处理饲料玉米后期分蘖数、叶片叶绿素含量、叶片光合速率与其生物量达到显著相关,相关系数(r)分别为0.9677、0.8967、0.8979。控释氮肥可明显降低饲料玉米硝酸盐含量,比施同等养分尿素,其硝酸盐降低幅度为73.37～709.41mg/kg;与等重量尿素相比,其硝酸盐降低幅度为469.26～1150.00mg/kg,控释氮肥的降硝效果明显。控释氮肥处理比尿素处理明显提高氮肥利用率,且保肥效果好。     

Microbial growth and nitrogen immobilization in the root zone of barley (Hordeum vulgare L.), Italian ryegrass (Lolium multiflorum Lam.), and white clover (Trifolium repens L.)

Summary The effect of roots on microbial growth and N immobilization was investigated in a pot experiment with barley, Italian ryegrass, and white clover. We used a silty subsoil with a low soil organic matter content (0.16%C and 0.012%N), which allowed us to measure N immobilization as an increase in total soil organic N (planted versus unplanted). At sampling, the soil was easily removed from intact roots by gentle washing, with a negligible loss of root material. Plant growth and extra mineral N (in planted soil only) gave increased total counts (fluorescence microscopy) and viable counts (plate dilution) of bacteria, a higher proportion of larger cells, and increased viable counts as a percentage of total counts. Under monocots, 12–17% of the added fertilizer N was recovered as soil organic N. Although this N immobilization was attributed to microbial assimilation, less than 1/4 was actually recovered as microbial biomass N, as measured with the chloroform fumigation/N-extraction method or calculated from total bacterial counts. The white clover accumulated substantial amounts of N due to N₂ fixation. However, microbial N immobilization represented only 3% of the total N accumulation, showing that the microorganisms obtained a smaller share of biologically fixed N₂ than of the N applied as fertilizer. Extra additions of mineral N (monocots) enhanced microbial N assimilation, partly due to increased plant growth. The results also strongly indicated, however, that the microbial growth under monocots was N-limited in the latter part of the experiment and that fertilizer N had a direct effect on microbial growth. In the early phase of plant growth, N immobilization ranged from 33 to 58 mg N g^-1 root C. This level of immobilization required a release of organic C into the soil representing a minimum of 60–100% of that found in intact roots.     

Yield and Quality of Mixed Seed Crops of Timothy ( Phleum pratense ) and Clovers ( Trifolium spp.) in an Organic Cropping System

Seed production of timothy ( Phleum pratense L.) in mixed crops with alsike ( Trifolium hybridum L.), white ( T. repens L.) or red ( T. pratense L.) clover was evaluated in an organic cash crop system. No fertilizer was applied except for household compost in the sowing year. While the first year crops of the alsike clover/timothy and red clover/timothy leys were harvested for clover seed, the first year crop of the white clover/timothy ley and all second year crops were harvested for timothy seed. The botanical composition of the alsike clover/timothy crop averaged 89/11 in the first ley year, and 8/92 in the second year. The corresponding yields averaged 443 kg ha -1 clover seed and 849 kg ha -1 timothy seed, respectively. Second year seed yields of timothy from mixtures with red and white clover were, in turn, 7% higher and 44% lower than from the corresponding mixture with alsike clover. First year seed yields of alsike clover from the alsike clover/timothy combination, and first year yields of timothy from the timothy/white clover combination were mostly rejected because of insufficient purity. In contrast, first year seed yields of red clover from the red clover/ timothy combination, and second year yields of timothy from all combinations, never failed to met the international requirement of maximum 1% contamination of one particular species in certified seed.     

Surface runoff phosphorus (P) loss in relation to phosphatase activity and soil P fractions in Florida sandy soils under citrus production

Phosphorus losses by surface runoff from agricultural lands have been of public concern due to increasing P contamination to surface waters. Five representative commercial citrus groves (C1-C5) located in South Florida were studied to evaluate the relationships between P fractions in soils, surface runoff P, and soil phosphatase activity. A modified Hedley P sequential fractionation procedure was employed to fractionate soil P. Soil P consisted of mainly organically- and Ca/Mg-bound P fractions. The organically-bound P (biological P, sum of organic P in the water, NaHCO₃ and NaOH extracts) was dominant in the acidic sandy soils from the C2 and C3 sites (18% and 24% of total soil P), whereas the Ca/Mg-bound P (HCl-extractable P) accounted for 45-60% of soil total P in the neutral and alkaline soils (C1, C4 and C5 soils). Plant-available P (sum of water and NaHCO₃ extractable P fractions) ranged from 27 to 61 mg P kg⁻¹ and decreased in the order of C3>C4>C1>C2>C5. The mean total P concentrations (TP) in surface runoff water samples ranged from 0.51 to 2.64 mg L⁻¹. Total P, total dissolved P (TDP), and PO₄³⁻-P in surface runoff were significantly correlated with soil biological P and plant-available P forms (p<0.01), suggesting that surface runoff P was directly derived from soil available P pools, including H₂O- and NaHCO₃- extractable inorganic P, water-soluble organic P, and NaHCO₃- and NaOH-extractable organic P fractions, which are readily mineralized by soil microorganisms and/or enzyme mediated processes. Soil neutral (55-190 mg phenol kg⁻¹ 3 h⁻¹) and natural (measured at soil pH) phosphatase activities (77-295 mg phenol kg⁻¹ 3 h⁻¹) were related to TP, TDP, and PO₄³⁻-P in surface runoff, and plant-available P and biological P forms in soils. These results indicate that there is a potential relationship between soil P availability and phosphatase activities, relating to P loss by surface runoff. Therefore, the neutral and natural phosphatase activities, especially the natural phosphatase activity, may serve as an index of surface runoff P loss potential and soil P availability.     

Continuous defoliation of perennial ryegrass (Lolium perenne) and white clover (Trifolium repens) and associated changes in the composition and activity of the microbial population of an upland grassland soil

A microcosm study was conducted to investigate the effect of continuons plant defoliation on the composition and activity of microbial populations in the rhizosphere of perennial ryegrass (Lolium perenne) and white clover (Trifolium repens). Continuons defoliation of ryegrass and clover resulted in sigmficant (P <0.01) increases in soil microbial biomass, although whilst increases were measured from day 2 in soil sown with clover significant increases were only seen from day 21 in soil sown with ryegrass. These increases were paralleled, from day 10 onwards, by increases in the numbers of culturable bacteria. Numbers ofPsendomonas spp. also increased in the later stages of the study. No influence on culturable fungal populations was detected. Whilst shifts in the composition of the microbial populations were measured in response to defoliation there was little effect on microbial activity. No changes in either dehydrogenase activity or microbial respiration in the rhizosphere of ryegrass or clover were measured in response to defoliation, but both dehydrogenase activity and microbial respiration were greater in ryegrass than clover when values over the whole study were combined. Continuous defoliation resulted in significant (P <0.001) reductions in the root dry weight of ryegrass and clover, of the order 19% and 16%, respectively.     

Diurnal and seasonal patterns of nitrogenase activity of red alder in comparison with white clover in silvopastoral agroforestry systems

 Simultaneous measurements were made to assess the diurnal and seasonal patterns of nitrogenase activity of red alder (Alnus rubra Bong.) and white clover (Trifolium repens L.) growing together in a silvopastoral agroforestry system using the acetylene reduction assay. Diurnal measurements were made in the summer and autumn at 3-h intervals whereas seasonal nitrogenase activity was assessed based on observations made at midday in July, September and January to represent the summer, autumn and winter seasons, respectively. No obvious diurnal patterns of nitrogenase activity were found in either red alder or white clover in summer and no significant variations in nitrogenase activity were observed between day and night. However, in autumn, pronounced diurnal patterns were observed in both species. Significantly higher rates of nitrogenase activity per unit dry weigh (dwt) of nodules were detected at 1500 hours in red alder, whereas, in white clover, significantly higher rates were obtained at 2100 hours. There was no significant correlation between diurnal nitrogenase activity and air temperature, photosynthetically active radiation and soil temperature at 10 cm depth in either red alder or white clover. Seasonal rates of nitrogenase activity showed significantly higher activity in summer, which subsequently decreased in autumn, to reach very low levels in the winter. The rates of nitrogenase activity of white clover were consistently higher than those of red alder both diurnally and seasonally. In the three seasons sampled, the average nitrogenase activity for white clover was 66.42 μmol C₂H₄ g dwt^–1 h^–1, which was 3.5 times higher than the 18.67 μmol C₂H₄ g dwt^–1 h^–1 obtained for red alder. Received: 11 November 1997     

Decomposition and distribution of N labelled mustard litter (Sinapis alba) in physical soil fractions of a cropland with high- and low-yield field areas

High-yield (HY) areas of an agricultural cropland were characterized by different positions on a slope and lower silt and clay contents, compared to low-yield (LY) areas, and this was associated with differences in water regime and C and N turnover. To understand differences in N flows of HY and LY areas, a combination of ¹⁵N tracer techniques and physical fractionation procedures was applied. Within 570 d after application of ¹⁵N labelled mustard litter to an agricultural cropland, the distribution of ¹⁵N was measured in particulate organic matter (POM) fractions and in fine mineral fractions (fine silt- and clay-sized fractions). After 570 d, only 2.5% of the initial ¹⁵N amount was found in POM fractions, with higher amounts in POM occluded in aggregates than in free POM. After this period, stabilization of the initial ¹⁵N in fine silt- and clay-sized fractions amounts to 10% in HY, but 20% in LY soils. 70% to 85% of the added ¹⁵N were lost. Initial decomposition of labelled material was faster in HY than in LY areas during the first year, but the remaining ¹⁵N amounts in POM fractions of the different areas were similar after 570 d. ¹⁵N amounts and concentrations in mineral-associated fractions increased within 160 d after application. From 160 to 570 d, HY and LY areas showed different ¹⁵N dynamics, resulting in a decline of ¹⁵N amounts in HY, but constant ¹⁵N amounts in LY soils. The results indicate faster decomposition processes in HY than in LY areas, due to different soil conditions, such as soil texture and water regime. The higher silt and clay contents of LY areas seem to promote N stabilization in fine mineral fractions. As a whole, N flows were higher in HY compared to LY areas, thus supporting higher yields and accelerated organic matter degradation due to higher N supply.     

Quantification of airborne N‐input in long‐term field experiments and its validation through measurements using 15N isotope dilution

The N‐deposition in Germany is commonly calculated as values of about 20—30 kg/ha·yr. This range is based on the measurements of the nitrate and ammonium nitrogen bulk deposition, which does not include the gaseous N‐deposition and the direct N‐uptake by plants. The calculation of airbone N‐deposition from N‐balances of the Static Fertilization Experiment Bad Lauchstädt came to 50—58 kg/ha·yr. This is consistent with results from other European long‐term experiments. Using the newly developed ¹⁵N‐based ITNI‐system, the total airborne N‐deposition can be determined. For Bad Lauchstädt analogous to results of former measuring periods an annual N‐deposition of 65 kg/ha·yr was measured in 1998, a figure greater than the balanced values. The balanced and measured values show, that airborne N‐deposition is often underestimated and amounts to at least 50 kg/ha·yr, which is a significant burden on natural ecosystems. By taking this extra N‐input into account in calculations for fertilizer recommendations in agriculture a decrease of N‐losses can be achieved which, in turn can also induce a decrease in airborne N‐deposition.