Below-ground carbon distribution in barley (Hordeum vulgare L.) with and without nitrogen fertilization

The distribution of net assimilated C in barley (Hordeum vulgare L.) grown at two N-levels was determined in a growth chamber. The N-fertilization involved 0 and 3.61 mol N g^-1 dry soil. After growth for seven weeks in an atmosphere with continuously ¹⁴C-labelled CO₂, ¹⁴C was determined in shoots, roots, rhizosphere respiration and soil. At the low N-level, 32% of the net assimilated ¹⁴C was translocated below ground, whereas at the high N-level 27% was translocated below ground. The release of C from roots (root respiration, microbial respiration originating from decomposition of ¹⁴C-labelled root material and ¹⁴C remaining in soil) was greater with no N-supply (19% of net assimilated ¹⁴C) than in the treatment with N-supply (15%). Thus, the effect of N-supply on both translocation of assimilated ¹⁴C below ground and the release of ¹⁴C from growing roots was relatively small.     

Influence of light intensity on the distribution of carbon and consequent effects on mineralization of soil nitrogen in a barley (Hordeum vulgare L.)-soil system

A pot experiment was conducted in a ¹⁴C-labelled atmosphere to study the influence of living plants on organic-N mineralization. The soil organic matter had been labelled, by means of a 200-days incubation, with ¹⁵N. The influence of the carbon input from the roots on the formation of microbial biomass was evaluated by using two different light intensities (I). Mineralization of ¹⁵N-labelled soil N was examined by following its fate in both the soil biomass and the plants. Less dry matter accumulated in shoots and roots at the lower light intensity. Furthermore, in all the plant-soil compartments examined, with the exception of rhizosphere respiration, the proportion of net assimilated ¹⁴C was lower in the low-I treatment than in the high-I treatment. The lower rates of ¹⁴C and ¹⁵N incorporation into the soil biomass were associated with less root-derived ¹⁴C. During the chamber period (¹⁴CO₂-atmosphere), mineralized amounts of ¹⁵N (measured as plant uptake of ¹⁵N) were small and represented about 6.8 to 7.8% of the initial amount of organic ¹⁵N in the soil. Amounts of unlabelled N found in the plants, as a percentage of total soil N, were 2.5 to 3.3%. The low availability of labelled N to microorganisms was the result of its stabilization during the 210 days of soil incubation. Differences in carbon supply resulted in different rates of N mineralization which is consistent with the hypothesis that roots induce N mineralization. N mineralization was higher in the high-I treatment. On the other hand, the rate of mineralization of unlabelled stable soil N was lower than labelled soil ¹⁵N which was stabilized. The amounts of ¹⁵N mineralized in planted soil during the chamber period (43 days) which were comparable with those mineralized in unplanted soil incubated for 210 days, also suggested that living plants increased the turnover rate of soil organic matter.     

Gaseous nitrogen losses from field plots grown with pea (Pisum sativum L.) or spring barley (Hordeum vulgare L.) estimated by 15N mass balance and acetylene inhibition techniques

In a mass balance of ¹⁵N-labelled nitrate added to soil grown with pea or barley, denitrification estimates using the acetylene-inhibition technique were compared with unaccounted for ¹⁵N. During the growth season of 1989, which was drier than average, N losses due to denitrification estimated by the acetylene-inhibition technique were negligible. A substantial amount of fertilizer N was unaccounted for by the ¹⁵N mass balance, especially in the pea plots. The loss took place during the period of grain-filling in which no leaching occurred, and was accompanied by a decrease in ¹⁵N content of the plants. Volatilization of ammonia from the aerial parts of the plants is a possible explanation of the observed loss. An estimation of denitrification relying only on the ¹⁵N mass balance would have resulted in an overestimation of denitrification.     

Root contribution to nitrate reduction in barley seedlings (Hordeum vulgare L.)

Summary The leaf and root nitrate reductase activities were measured in 7 day-old barley seedlings by anoxic nitrite accumulation in darkness, during 48h after the transfer from a N-starved medium to a 1.5 mM K¹⁵NO₃ medium. Thisin situ nitrate reduction was compared with the¹⁵N incorporation in the reduced N fraction of the whole seedlings.The nitrate reduction integrated fromin situ measurements was lower than the reduced¹⁵N accumulation. The rootin situ nitrate reductase activity seemed to account for only the third of the real root nitrate reduction, which may have been responsible for the overall underestimation. This discrepancy was partly explained by the ability of the root to reduce nitrite in an anoxic environment.These results suggest that, after correction of thein situ estimation of the nitrate reduction. the roots contribute to about 50% of the total assimilation.     

Carbon and nitrogen metabolism in barley (Hordeum vulgare L.) mutants lacking ferredoxin-dependent glutamate synthase

Five mutant lines of barley (Hordeum vulgare L.), which are only able to grow at elevated levels of CO₂, contain less than 5% of the wild-type activity of ferredoxin-dependent glutamate synthase (EC 1.4.7.1). Two of these lines (RPr 82/1 and RPr 82/9) have been studied in detail. Leaves and roots of both lines contain normal activities of NADH-dependent glutamate synthase (EC 1.4.1.14) and the other enzymes of ammonia assimilation. Under conditions that minimise photorespiration, both mutants fix CO₂ at normal rates; on transfer to air, the rates drop rapidly to 15% of the wild-type. Incorporation of ¹⁴CO₂ into sugar phosphates and glycollate is increased under such conditions, whilst incorporation of radioactivity into serine, glycine, glycerate and sucrose is decreased; continuous exposure to air leads to an accumulation of ¹⁴C in malate. The concentrations of malate, glutamine, asparagine and ammonia are all high in air, whilst aspartate, alanine, glutamate, glycine and serine are low, by comparison with the wild-type parent line (cv. Maris Mink), under the same conditions. The metabolism of [¹⁴C]glutamate and [¹⁴C]glutamine by leaves of the mutants indicates a very much reduced ability to convert glutamine to glutamate. Genetic analysis has shown that the mutation in RPr 82/9 segregates as a single recessive nuclear gene.Abbreviations GDH glutamate dehydrogenase (EC 1.4.1.2) - GS glutamine synthetase (EC 6.3.1.2) - RuBP ribulose 1,5-bisphosphate     

Distribution of15N in the soil-plant system during a four-year field lysimeter study with barley (Hordeum distichum L.) and perennial meadow fescue (Festuca pratensis Huds.)

An annual cereal, barley, and a perennial grass ley, meadow fescue, were grown in field lysimeters in Sweden and fertilized with 12 and 20g Ca(NO₃)₂-N m⁻² yr⁻¹, respectively. Isotope-labeled (¹⁵N) fertilizer was added during year 1 of the study, whereafter similar amounts of unlabeled N were added during years 2 and 3. The grass ley lysimeters were ploughed after the growing season of year 3 and sown with barley during year 4. The barley harvest in year 1 removed 59% of the added fertilizer N, while the fertilizer N export by two meadow fescue harvests in year 1 was 65%. The labeled N export decreased rapidly after year 1, especially in the barley, but increased slightly after ploughing of the grass ley. The microbial biomass, measured with the chloroform fumigation method, incorporated a maximum of 1.4–1.7% of the labeled N during the first seven weeks after application. Later on, the incorporation stabilized at less than 1% in both cropping systems. The susceptibility of the residual labeled N to mineralization was evaluated three years after application by means of long-term laboratory incubations. The curves of cumulative mineralized N were described by a two-component first-order regression model that differentiated between an available and a more recalcitrant fraction of potentially mineralizable N. There was no difference in the amounts of potentially mineralizable N between the cropping systems. The labeled N comprised 5 and 2% of the amounts of potentially mineralizable N in the available and more recalcitrant fraction, respectively. The mineralization rate constants for the labeled N were almost twice as high as for the total potentially mineralizable N. The available fraction of the total potentially mineralizable N was 12%, while twice that proportion of the labeled N was available. It was concluded that the short-term ley did not differ from the annual crop with respect to the early disposition of the fertilizer N and the behaviour of the residual organic N.     

Availability of the residual nitrogen from a single application of 15N-labelled fertilizer to subsequent crops in a long-term continuous barley experiment

In an earlier paper we presented data from an experiment in which nitrogen-15-labelled fertilizer was applied in spring to barley on the Rothamsted long-term Spring Barley Experiment, at rates of 48, 96 or 144 kg N ha^–1. A substantial proportion (between 28 and 39%) of this ¹⁵N remained in the soil (0–70 cm) and stubble at harvest, mostly in organic form. The present paper follows the fate of this `residual' <sup>15</sup>N over the following 2 years. Small amounts of `residual' ¹⁵N were recovered in the following two spring barley crops; 8% in the first and 3% in the second. The overall loss of `residual' <sup>15</sup>N (i.e. `residual' ¹⁵N not recovered in crops and soil to a depth of 70 cm) over the 2 years was 23%. This is equivalent to just 8% of the total ¹⁵N originally applied. There was surprisingly little difference in the behaviour of the `residual' ¹⁵N in soils containing very different quantities of soil organic matter.     

Effect of anther orientation on microspore-callus production in barley (Hordeum vulgare L.)

Barley anthers from cold pretreated spikes produced no or few calluses when plated with both loculi in contact with the medium (flat). When anthers were plated with only one loculus in contact with the medium (up), a high proportion of the anthers produced calluses. The top loculus of the up anthers was most productive. Flat anthers, when compared with up anthers, were not only slower to produce multicellular pollen grains (MCPs) and microcalluses, but also produced fewer of them and ceased production earlier. The MCPs and microcalluses in flat anthers grew more slowly and few developed beyond the 30 cell stage. These results establish the importance of anther orientation for barley anther culture.     

Optimizing somatic embryogenesis and particle bombardment of barley (Hordeum vulgare L.) immature embryos

Summary A highly regenerable target tissue and a high-frequency DNA delivery system are required for the routine production of transgenic barley. This project separately optimized tissue culture and particle bombardment parameters. Immature zygotic embryos (0.7 to 1.2 mm) were excised and culture on B5L solid medium. Klages and H930-36 cultivars regenerated significantly more green plants than Sabarlis and Bruce. The regeneration pathway shifted from organogenesis to somatic embryogenesis when maltose was used as the medium carbohydrate source instead of sucrose. More somatic embryos were induced on 5 mg/liter 2,4-dichlorophenoxyacetic acid than 2 mg/liter. Gene delivery was optimized using anthocyanin regulatory genes as a transient marker. A 3-mm rupture disc-to-macrocarrier gap distance, a 1-day prebombardment embryo culture period, and a maltose carbohydrate source were each significantly better than other treatments. Double bombardments per plate, a 6-mm macrocarrier fly distance, and 650-psi rupture discs each had the highest number of transiently expressing cells in individual experiments, although the results were not statistically significant compared to the other treatments. Using the optimized parameters, over 200 cells routinely expressed anthocyanin in a bombarded immature embryo. In tissue culture experiments, 350 to 400 green plants regenerated per 100 immature embryos. The improvement of green plant regeneration and gene delivery forms a strong basis to develop a practical barley transformation system.     

Si amelioration of Al toxicity in barley (Hordeum vulgare L.) growing in two Andosols

The influence of silicon on aluminium toxicity in barley (Hordeum vulgare L. cv. Shunrai) was studied in two Andosols. Silicon sources were a solution of sodium metasilicate with pH adjusted to 5.0, silica gel, and an industrial waste, porous hydrated calcium silicate. The waste is produced in large amounts in the manufacturing processes of autoclaved light concrete, and has been used as a silicon source for rice plants. The addition of the waste increased the concentration of Si in the soil solution, soil pH and amelioration of aluminium toxicity was observed. The addition of silica gel and sodium metasilicate solution to both soils increased significantly (p<0.05) the Si concentration of the soil solutions, but no amelioration of aluminium toxicity was observed. An amelioration of aluminium toxicity by the waste porous hydrated calcium silicate was probably due to the increase in soil pH rather than to the increase of silicon concentration in the soil solution.     

Fate of 15N-labelled fertilizer applied to spring barley grown on soils of contrasting nutrient status

An experiment with ¹⁵N-labelled fertilizer was superimposed on the Rothamsted Hoosfield Spring Barley Experiment, started in 1852. Labelled ¹⁵NH₄ ¹⁵NO₃ was applied in spring at (nominal) rates of 0, 48, 96 and 144 kg N ha^-1. The labelled fertilizer was applied to microplots located within four treatments of the original experiment: that receiving farmyard manure (FYM) annually, that receiving inorganic nutrients (PK) annually and to two that were deficient in nutrients: applications were made in two successive years, but to different areas within these original treatments. Maximum yields in 1986 (7.1 t grain ha^-1) were a little greater than in 1987. In 1987, microplots on the FYM and PK treatments gave similar yields, provided enough fertilizer N was applied, but in 1986 yields on the PK treatment were always less than those on the FYM treatment, no matter how much fertilizer N was applied. In plots with adequate crop nutrients, about 51% of the labelled N was present in above-ground crop and weed at harvest, about 30% remained in the top 70 cm of soil (mostly in the 0–23 cm layer) and about 19% was unaccounted for, all irrespective of the rate of N application and of the quantity of inorganic N in the soil at the time of application. Less than 4% of the added fertilizer N was present in inorganic form in the soil at harvest, confirming results from comparable experiments with autumn-sown cereals in south-east England. Thus, in this experiment there is no evidence that a spring-sown cereal is more likely to leave unused fertilizer in the soil than an autumn-sown one. With trace applications (ca. 2 kg N ha^-1) more labelled N was retained in the soil and less was in the above-ground crop. Where P and K were deficient, yields were depressed, a smaller proportion of the labelled fertilizer N was present in the above-ground crop at harvest and more remained in the soil.Although the percentage uptake of labelled N was similar across the range of fertilizer N applications, the uptake of total N fell off at the higher N rates, particularly on the FYM treatment. This was reflected in the appearance of a negative Added Nitrogen Interaction (ANI) at the highest rate of application. Fertilizer N blocked the uptake of soil N, particularly from below 23 cm, once the capacity of the crop to take up N was exceeded. Denitrification and leaching were almost certainly insufficient to account for the 19% loss of spring-added N across the whole range of N applications and other loss processes must also have contributed.     

The effects of DNA hypomethylating drugs on androgenesis in barley (Hordeum vulgare L.)

Summary The effects of DNA hypomethylating drugs (azacytidine and ethionine) on induction of microspore-derived calluses and embryos were studied in barley (Hordeum vulgare L.) ev. Igri. The results were as follows: (1) Yield of calluses and embryos pretreated with the different concentrations of azacytidine for 3 d was several-fold higher than that of the control. The highest yield of calluses and embryos in all treatments appeared at a concentration of 3 mg l⁻¹, which reached 11.03 per anther. It was 110-fold higher than the control. (2) There was a significant difference in yield of calluses and embryos between the different days of pretreatment. The highest yield was obtained at a 3-d pretreatment. If the period of pretreatment was shorter or longer than 3 d, yield of calluses and embryos was reduced sharply, and was similar to that of the control. (3) The data obtained with ethionine pretreatment were very similar to those obtained with azacytidine. (4) Tests on the different methods of pretreatment showed that yield of calluses and embryos pretreated with distilled H₂O, mannitol, azacytidine, and ethionine was much higher than other pretreatments and the control, and reached 6.53–11.39 per anther. The yield of calluses and embryos pretreated with DNA hypomethylating drugs was higher than with mannitol. However, pretreatment with hypomethylation drugs supplemented with induction medium was not effective.     

Variability in nitrogen fixation of common bean (Phaseolus vulgaris L.) intercropped with maize

Thirty one selected bean lines were evaluated in the field for ability to support N₂ fixation when intercropped with maize which received 0, 30 and 60 kg N ha^–1 as ammonium sulphate. The amount of fixed N₂ was estimated using the natural variation of ¹⁵N and wheat as the standard non-fixing crop. Nitrogen as low as 15 kg N ha^–1 at sowing suppressed nodule weight and activity (acetylene reduction activity) but not nodule number, suggesting that the main effect of mineral N was on nodule development and function. ¹⁵N data revealed a high potential of the bean genotypes to fix N₂, with the most promising ones averaging between 50–60% of seed N coming from fixation. Bean lines CNF-480, Puebla-152, Mexico-309, Negro Argel, CNF-178, Venezuela-350 and WBR22-3, WBR22-50 and WBR22-55 were ranked as good fixers.     

Retrotransposon BARE-1 is a major, dispersed component of the barley (Hordeum vulgare L.) genome

The barley BARE-1 is a transcribed, copia-like retroelement with well-conserved functional domains, an active promoter, and a copy number of at least 3 × 10⁴. We examined its chromosomal localization by in situ hybridization. The long terminal repeat (LTR) probe displayed a uniform hybridization pattern over the whole of all chromosomes, excepting paracentromeric regions, telomeres, and nucleolar organizer (NOR) regions. The integrase probe showed a similar pattern. The 5-untranslated leader (UTL) probe, expected to be the most rapidly evolving component, labeled chromosomes in a dispersed and non-uniform manner, concentrated in the distal regions, possibly indicating a targe site preference.     

Labeled nitrogen fertilizer research with urea in the semi-arid tropics

Summary Field studies with bordered microplots were conducted on an Alfisol in the semiarid tropics of India to determine (1) the fate of¹⁵N-labeled urea applied to dryland sorghum in two successive rainy seasons and (2) the effect of method of application on N fertilizer efficiency. Recoveries of¹⁵N-labeled fertilizers by above-ground plant parts ranged from 46.7% to 63.6% in 1981 when the rainfall was above the average and from 54.4% to 66.9% in 1980 when the rainfall was near the average. Small (0.014 g) pellets of urea applied twice as postemergent applications in separate 5 cm deep bands were more effective than single preemergent applications either surface applied or incorporated. Both banding and the split applications contributed to overall fertilizer efficiency. Large (1.0 g) pellets of urea (supergranules) placed at a depth of 5 cm were also superior to the incorporated, small-pellet treatment in 1981. The¹⁵N-balance data for the soil (0–90 cm in depth)-plant system in 1981 showed that the unaccounted-for fertilizer N ranged from 5.1% to 20.6%. An important finding was that high grain yields, in excess of 6,000 kg/ha, with N fertilizer losses of less than 10% could be obtained through fertilizer management during a very wet season. The data from the Alfisol experiments were compared with data from similar Vertisol experiments; N fertilizer losses resulting from incorporated and surface applications were greater for Vertisols than for Alfisols in the wetter year.     

Turnover of residual 15N-labelled fertilizer N in soil following harvest of oilseed rape (t Brassica napus L.)

We studied the fate of ¹⁵N-labelled fertilizer nitrogen in a sandy loam soil after harvest of winter oilseed rape (Brassica napus L. cv. Ceres) given 100 or 200 kg N ha^-1 in spring, with or without irrigation. Our main objective was to quantify the temporal variations of the soil mineral N, the extractable soil organic N and soil microbial biomass N, and fertilizer derived N in these pools during autumn and winter. Nitrogen use efficiency of the oilseed rape crop varied from 47% of applied N in the 100N, irrigated treatment to 34% in the 200N, non-irrigated treatment. However, only in the latter treatment did we find significantly higher fertilizer derived soil mineral N than in the three other treatments which all had low soil mineral N contents at the first sampling after harvest (8 days after stubble tillage). Between 31% and 42% of the applied N could not be accounted for in the harvested plants or 0-15 cm soil layer at this first sampling. Over the following autumn and winter none of the remaining fertilizer derived soil N was lost from the 0–5 cm depth, but from the 5–15 cm depth a marked proportion of N derived from fertilizer was lost, probably by leaching. Negligible amounts of fertilizer derived extractable soil organic and mineral N (<1 kg N ha^-1, 0-15 cm) were found in all treatments after the first sampling.Soil microbial biomass N was not significantly affected by treatments and showed only small temporal variability (±11% of the mean 76 kg N ha^-1, 0- 15 cm depth). Surprisingly, the average amount of soil microbial biomass N derived from fertilizer was significantly affected by the treatments, with the extremes being 5.5 and 3.1 kg N ha^-1 in the 200N, non-irrigated and 100N, irrigated treatments, respectively. Also, the estimated exponential decay rate of microbial biomass N derived from fertilizer, differed greatly (2 fold) between these two treatments, indicating highly different microbial turnover rates in spite of the similar total microbial biomass N values. In studies utilising ¹⁵N labelling to estimate turnover rates of different soil organic matter pools this finding is of great importance, because it may question the assumption that turnover rates are not affected by the insertion of the label.     

The fate of residual 15N-labelled fertilizer in arable soils: its availability to subsequent crops and retention in soil

An earlier paper (Macdonald et al., 1997; J. Agric. Sci. (Cambridge) 129, 125) presented data from a series of field experiments in which ¹⁵N-labelled fertilizers were applied in spring to winter wheat, winter oilseed rape, potatoes, sugar beet and spring beans grown on four different soils in SE England. Part of this N was retained in the soil and some remained in crop residues on the soil surface when the crop was harvested. In all cases the majority of this labelled N remained in organic form. In the present paper we describe experiments designed to follow the fate of this `residual' <sup>15</sup>N over the next 2 years (termed the first and second residual years) and measure its value to subsequent cereal crops. Averaging over all of the initial crops and soils, 6.3% of this `residual' ¹⁵N was taken up during the first residual year when the following crop was winter wheat and significantly less (5.5%) if it was spring barley. In the second year after the original application, a further 2.1% was recovered, this time by winter barley. Labelled N remaining after potatoes and sugar beet was more available to the first residual crop than that remaining after oilseed rape or winter wheat. By the second residual year, this difference had almost disappeared. The availability to subsequent crops of the labelled N remaining in or on the soil at harvest of the application year decreased in the order: silty clay loam>sandy loam>chalky loam>heavy clay. In most cases, only a small proportion of the residual fertilizer N available for plant uptake was recovered by the subsequent crop, indicating poor synchrony between the mineralization of ¹⁵N-labelled organic residues and crop N uptake. Averaging over all soils and crops, 22% of the labelled N applied as fertilizer was lost (i.e., unaccounted for in harvested crop and soil to a depth of 100 cm) by harvest in the year of application, rising to 34% at harvest of the first residual year and to 35% in the second residual year. In the first residual year, losses of labelled N were much greater after spring beans than after any of the other crops.     

Resin-core and buried-bag estimates of nitrogen transformations in Costa Rican lowland rainforests

We compared the resin-core and buried-bag incubation methods for estimating nitrogen (N) transformation rates using the ¹⁵N pool dilution technique in alluvial soils of an early successional forest (ESF) and an old-growth forest (OGF) at the La Selva Biological Station in Costa Rica. Soil cores (38×100-mm) from both forests were incubated in situ for 7 days. The two methods gave generally similar estimates of net N mineralization rates for the two forests. Estimates of ammonium production by the resin-core method were higher than those by the buried-bag method in ESF, but did not differ significantly in OGF (p<0.05). Estimates of nitrate production by the two methods did not differ significantly. Nitrate averaged 74% and 81% of the total inorganic N production in ESF and OGF, respectively. Net N mineralization in ESF (6.6 mmol m^-2d^-1) did not differ significantly from that in OGF (5.0 mmol m^-2d^-1). Fluxes of ammonium and nitrate were high for both forests, but the OGF tended to have higher gross mineralization and nitrification rates than ESF. Approximately 60% of the gross nitrate production and less than 30% of the ammonium were immobilized by microorganisms.