Nitrogen mineralisation and plant nitrogen acquisition in a nitrogen-limited calcareous grassland

Environmental and Experimental Botany 40 (1998) 209 – 219

Nitrogen mineralisation and plant nitrogen acquisition in a nitrogen-limited calcareous grassland Murray Unkovich a,*, Nicola Jamieson b, Ross Monaghan 1,b, Declan Barraclough b a

Department of Botany and Centre for Legumes in Mediterranean Agriculture, Uni6ersity of Western Australia, Nedlands, WA 6907, Australia b Department of Soil Science, Uni6ersity of Reading, P.O. Box 233, Reading RG6 2DW, UK Received 23 March 1998; received in revised form 5 June 1998; accepted 8 June 1998

Abstract A field study measured the rate of soil mineral N supply and its effects on plant biomass and N accumulation in a 13-year-old, naturally regenerating, calcareous grassland. Gross rates of N mineralisation (2 mg g − 1 day − 1, i.e. 0.69 kg ha − 1 day − 1), assessed using 15N pool dilution, were at the lower end of the range previously reported for grasslands. Weekly additions of liquid N fertiliser ([NH4]2SO4, NH4NO3 or KNO3) and, to a lesser extent the addition of water, increased plant growth substantially, demonstrating that the primary constraint to plant growth was low N availability. In plants that had received NO3− , the activity of the inducible enzyme nitrate reductase in shoots initially increased in proportion to the amount of NO3− supplied. However, as above-ground herbage accumulated, nitrate reductase activity declined to similar low levels in all treatments, despite the continuance of the constant NO3− additions. The decline in NR specific activity reflected declining tissue NO3− concentrations, although total plant NRA may have remained constant during the period of study. The study has shown that plant growth is limited by low N mineralisation rates and indeed the soil is a sink for much added N. Low water availability provides an additional constraint on N mineralisation in this calcareous grassland soil. Any disturbances in the N cycle which increase the availability of mineral N will result in a substantial increase in plant growth within this ecosystem. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Ammonium; Nitrate; Nitrate reductase; Nitrification; Nitrogen-15; Chalk grassland; Holcus lanatus; Pastinaca sati6a; Poa tri6ialis

1. Introduction

* Corresponding author. Tel.: +61 89 3802205; Fax: +61 89 3801001; E-mail: [email protected] 1 Present address: Invermay Agricultural Centre, Private Bag 50034, Mosgiel, New Zealand.

Changes in the availability of soil mineral N may alter the relative abundance or proportional biomass of plant species, depending on their varied abilities to respond to such changes within the ecosystem (for example, Lee and Stewart, 1978; Pate, 1981). There are few reliable estimates

S0098-8472/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0098-8472(98)00038-0

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Table 1 Forms and amounts of N added to plots and additions of Treatment

15

N

Weekly N additionsa NH4-N

15 N additions for mineralisation/nitrification estimates (14 June)a

NO3-N

g m−2

mg kg−1 soil

g m−2

mg kg−1 soil

Water (NH4)2SO4 NH4NO3

0 1.15 0.575

0 28.27 14.14

0 0 0.575

0 0 14.14

KNO3 Complete

0 0.238

0 5.86

1.15 0.912

28.27 22.43

a

1.15 g N m−2 applied Monday of each week with 2 mm water for 6 weeks, with on sixth Monday for mineralisation/nitrification estimates.

of N mineralisation in the literature on which to predict the ability of the soil in a given ecosystem to supply NH4+ and NO3− for plant growth, and this is principally due to a lack of suitable, easily accessible, techniques. Estimates of net soil N mineralisation (for example, Raison et al., 1987; Gill et al., 1995) afford limited insight into the several processes that provide mineral N for plant and soil biota. Where estimates of net N mineralisation from field incubation studies are low, it cannot be inferred that the actual rate of mineralisation is low, since mineralised N can be simultaneously consumed by microbial immobilisation, nitrate leaching or denitrification (Jansson and Persson, 1982). A proper assessment of the total capacity of a given soil to supply mineral N from soil organic matter should therefore measure the gross rate of NH4+ supply. A conceptual framework for estimating gross rates of N mineralisation, using 15N pool dilution (Kirkham and Bartolomew, 1954), has been available for some time and this is now being used to study soil N mineralisation in situ (for example, Davidson et al., 1991; Yevdokimov and Blagodatsky, 1993; Smith et al., 1994; Monaghan and Barraclough, 1995; Murphy et al., 1997). Such an approach is being used to assess potential changes in soil mineral N supply in response to artificial changes in climate (increased temperature and/or changes in rainfall) in a calcareous grassland in southern England (TIGER, 1994). Shifts in plant species

10A%15N (NH4)2SO4 10A%15N (NH4)2SO4 10.1A%15N 15NH14 4 NO3 or 11.5A%15N 14NH15 4 NO3 10.31A%15N KNO3 – 15

N applied to 0.125 m2 subplots at same N rate

composition in the grassland following the same changes in climate are also being studied at the site. However, it was not anticipated that changes in soil mineral N availability at the site, in amount or form, would affect plant growth rates and plant nitrogen uptake, a hypothesis that this study then was designed to test.

2. Materials and methods

2.1. Experimental site and treatments The study site, a 1 ha area of ungrazed naturally regenerating calcareous grassland at Wytham (Oxon, UK National Grid Reference 462 083) has been described in detail previously (Gibson et al., 1987). The soil is a Sherbourne series over Jurassic limestone with a maximum depth of 10 cm, pH of 7–8 (H2O), total soil N of 0.52% and 7 mg kg − 1 available P. Average annual rainfall at the site is 601 mm, average temperature is 10.1°C, and average annual soil moisture deficit is 140 mm. Within the study site, the vegetation on 30×1 m2 plots was lightly trimmed to approximately 20 mm with a strimmer on 29 April 1994 and the following treatments (listed in Table 1) were imposed from 9 May with fivefold replication. Nitrogen was added weekly for 6 weeks at a rate of 1.15 g N m − 2 week − 1, with the N applied as (NH4)2SO4, NH4NO3, KNO3 or in a ‘complete’ nutrient fertiliser (Phostrogen, Phostrogen, Cor-

M. Unko6ich et al. / En6ironmental and Experimental Botany 40 (1998) 209–219

wen, Wales) containing NO3− N and NH4+ − N in a 4:1 ratio, P, K, Ca, Mn, Fe and S. Nitrogen additions were made as solutions with 2 mm water per application. A further set of five plots received only the 2 mm weekly watering, and a final (control) set were left unamended after initial cutting.

2.2. Estimation of gross mineralisation and nitrification rates Gross nitrogen mineralisation and nitrification rates were estimated using the 15N pool dilution method detailed by Barraclough (1991). This method relies on the addition of a small amount of 15N-labelled NH4+ to the soil and measurement of the subsequent dilution of this 15NH4+ by unlabelled NH4+ arising from mineralisation. Nitrification rates can be calculated similarly following 15N-labelled NO3− additions. 15N-labelled N fertiliser solutions as detailed in Table 1 were applied to 0.125 m2 subplots on 13 June 1994 with 2 mm water, followed immediately with a further 2 mm water to wash the N solutions off of the foliage and into the soil. On the following 2 days, 4 and 3 mm of water were similarly applied to reduce the effects of a substantial soil water deficit during the final 10 days of the experiment when rainfall had been nil and evapotranspiration totalled 34 mm, conditions atypical of the preceding period of the study. On the 14 June (t0) and 17 June (t3), two replicate 75×50 mm2 deep soil cores were taken from each subplot that had received 15N. Core samples were immediately transported to the laboratory and 75 g fresh weight soil shaken with 300 ml 1 M KCl for 1 hour after which the sample was filtered through glass fibre filter paper and the extract stored at 3°C. Extracts were analysed for NH4+ and NO3− by flow injection colorimetry (Tecator 5010 analyser). N isotope analysis was conducted using a VG Micromass 622 mass spectrometer linked to a Europa Scientific Roboprep combustion analyser, following separate diffusion of the NH4+ and NO3− (Brooks et al., 1989). Gross N mineralisation was calculated by solving the following equation for m, the mineralisation rate, (Barraclough, 1991):

211

A*/(1+ ut /A0)m/u A*= t 0 where A *t is the excess 15N abundance of the NH4+ pool at time t, A *0 is the excess 15N abundance of the NH4+ pool at time 0, u is the rate in the change of the NH4+ pool size between times 0 and t, A0 is the size of the NH4+ pool at time 0, and m is the rate of gross N mineralisation. Nitrification rates were calculated similarly following 15N-labelled NO3− addition (Barraclough and Puri, 1995).

2.3. Biomass and nitrogen accumulation measurement Above-ground plant material was harvested to approximately 10 mm on 16 June (38 days growth), dried in an oven (80°C) for at least 48 h and weighed, with results expressed on a per unit area basis. Tissue N contents and 15N were measured on subsamples of finely ground plant material by combustion in a Roboprep automatic Nitrogen Analyser (Europa Scientific, Crewe). Tissue NO3− concentrations were measured in water extracts of dry shoots (10:1) by colorimetry according to the method of Best (1977).

2.4. Assay of nitrate reductase acti6ity Relative capacities of the shoots of the principal plant species to utilise the soil NO3− available in the different plots were assessed on four occasions using an in vivo assay of nitrate reductase (Stewart et al., 1986), the substrate-inducible enzyme responsible for the first step in the assimilation of NO3− in plant tissues. Species selected for assay were the grasses Holcus lanatus L. and Poa tri6ialis L., and the biennial herb Pastinaca sati6a L., since these were the only species present in all of the plots in sufficient number to allow repeated sampling. Assays were conducted on 11 May and 2, 9 and 16 June, each assay 48 h after the previous N addition. The assays involved a 1 h dark incubation at 30°C of 0.1 g chopped fresh plant shoot tissue sample, vacuum infused with 5 ml of phosphate buffer (pH 7.5) containing 1.5% propanol and 100 mM KNO3. The NO2− produced by the enzyme was measured by spec-

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212

trophotometer at 540 nm after reaction of an aliquot of the incubated sample solution with sulpahanilic acid and naphthyl-ethylenediamine dihydrochloride. Results are expressed as pkat g − 1 (pmol s − 1 [g tissue fresh wt − 1]).

2.5. Assessment of N2 fixation by legumes using 15 N natural abundance Soils are often naturally enriched in the stable isotope 15N when compared with atmospheric N2 (for example, Ledgard et al., 1984), making it possible to estimate the proportional dependence of a legume on atmospheric and soil N by comparing the 15N content of the legume in question with that of an adjacent ‘reference’ non-legume relying solely on soil N. To assess the importance of N2 fixation to the N nutrition of the predominant legumes at the Wytham study site samples were taken for 15N natural abundance measurement. The natural abundance of 15N is normally expressed as parts per thousand (‰) deviation from atmospheric N2 (0.3663 at.% 15N (Junk and Svec, 1958)), which by definition is given a delta (d) 15N value of 0‰. Conversion of at.% 15N values to the delta notation utilises the following formula; d 15N(‰)=

at.%

×

15

N sample − at.% at.% 15N air

15

N air

1000 1

The following formula can then be used to calculate the percentage of plant nitrogen derived from the atmosphere (%Ndfa); %Ndfa =

d 15N reference plant − d 15N legume d 15N reference plant −B ×

100 1

where the factor B refers to the d 15N value of the effectively nodulated legume grown in media totally lacking combined N (Bergersen et al., 1986) and compensates for isotope fractionation within the plant after N2 fixation. Successful application of the technique requires a difference in N isotope composition of at least

10 times the analytical precision of d 15N measurement between plant available soil N (as assessed by non-N2 fixing ‘reference’ plants) and atmospheric N2 (Unkovich et al., 1994). In the present study, shoots of the most common legumes at the site, Trifolium repens L. and Vicia sati6a L., and adjacent (non-legumes) grasses, were taken on 13 June outside the N treatment plots at five disparate locations as determined by the co-occurrence of the two legume species within the 1 ha site. Samples were oven dried at 80°C for at least 48 h and analysed for d 15N as detailed in Unkovich et al. (1993). 3. Results

3.1. Mineralisation and nitrification rates In view of the weekly addition of some 28 mg of mineral N kg − 1 of soil, the concentrations of NH4+ and NO3− recorded in the soil were low, averaging only 6.5 (NH4+ ) and 1.0 (NO3− ) mg N kg − 1 soil 24 h after the final 15N addition, and falling to 3.4 (NH4+ ) and 1.5 (NO3− ) mg N kg − 1 4 days after addition. The 15N-labelled NH4+ and NO3− disappeared rapidly from the soil mineral N pool with only 2–6% of the 15N added recovered as NH4+ plus NO3− 24 hours after addition, and B 2% 4 days after addition. Changes in the 15N enrichments of the soil NH4+ (2.25–1.13 A%15N) and NO3− (2.66–1.28 A%15N) pools were sufficient to calculate gross N mineralisation and nitrification and these data are presented in Table 2 on a mg N kg − 1 dry soil day − 1 and kg N ha − 1 day − 1 (to 50 mm) basis. Overall, the rates of N mineralisation were low, averaging 2 mg N kg − 1 day − 1. Differences in the calculated mineralisation rates between the NH4NO3 (3.6 mg N kg − 1 day − 1), water and NH4+ fed plots (both 1.2 mg N kg − 1 day − 1) were not significant by analysis of variance (ANOVA) (P =0.075). The average mineralisation rate of 0.66 kg N ha − 1 day − 1 indicates that over the 38 day period of study, 25 kg N ha − 1 would have been mineralised in the top 5 cm of soil. The average nitrification rate (0.67 mg N kg − 1 day − 1) was one-third of N mineralisation (Table 2). The difference between the calculated nitrification rates in the NH4NO3 (0.82 mg N

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213

Table 2 Calculated gross N mineralisation and nitrification rates (kg N ha−1 day−1, to 50 mm depth) Treatment Mineralisation Water NH4 NH4NO3 Nitrification NH4NO3 NO3

mg N kg soil−1 day−1

S.D.

kg N ha−1 day−1

S.D.

1.2 1.2 3.6

1.02 1.36 4.05

0.40 0.40 1.18

0.38 0.49 1.35

0.82 0.53

0.44 0.48

0.27 0.17

0.17 0.13

Differences between treatments are not significant (P\0.05).

kg − 1 day − 1) and NO3− fed plots (0.53 mg N kg − 1 day − 1) was not significant by ANOVA (P \0.05). Total uptake by plants of the fertiliser NH4+ or NO3− averaged 27% of the 15N added 96 h after application (Table 3) and was not significantly different between N addition treatments (P\ 0.05). However, in the plots that had previously received only water, significantly more of the 15N was incorporated into plant roots (P B 0.01) than in all of the other plots that received 15N.

3.2. Shoot biomass and nitrogen accumulation Herbage growth totalled 0.57 t ha − 1 over the 38 day study period in the control plots, increasing by 75% to 1 t ha − 1 in response to the addition of water, and 300% (on average, 1.7 t ha − 1) where water and N were added (Fig. 1). Total shoot biomass production and N uptake were significantly increased (P B0.05) by N fertiliser addition regardless of N form. The inclusion of P, K, Ca, Mn, Fe and S in the ‘complete’ treatment did not lead to further increase in shoot biomass accumulation. In plots receiving N, the increases in shoot N accumulation, over and above that observed in the water only treated plants, represent 20–25% of the amount of N applied as fertiliser. In plots receiving NH4+ or NH4+ and NO3− , shoot N concentrations (Table 4) were significantly higher (P B0.05) than control, water or NO3− fed plants (average, 1.39%). Shoot total N uptake (Fig. 2) in the watered plots did not increase as much total biomass accumulation (46

versus 75% increase), resulting in a lowering of shoot N concentration in the herbage of plots fed water only (1.2%) compared to control plots (1.47%), although the reduction was not significant (Table 4).

3.3. Nitrate reductase acti6ity Averaged over the period of study (11 May–16 June), nitrate reductase activity (NRA) (Table 5) was below 500 pkat g − 1 for the predominant plant species, although there was a positive and proportionate response to increased nitrate supply. However, this averaged data belies a significant downward trend in NRA with time for all nitrate applications despite the constant weekly N additions. For example, in plots receiving the greatest NO3− addition, NRA of grass shoots (Fig. 3) declined from 1123 pkat g per weight fraction 2 days after the first NO3− addition to 73 pkat g − 1 2 days after the final NO3− addition, by which time NRA was not significantly different between any treatments (range, 37–73 pkat g per weight fraction). Analysis of plant shoots for free NO3− at harvest revealed no more than a trace of NO3− (B 1 mg g − 1 tissue dry weight, data not shown) in all of the shoots.

3.4. Natural abundance of legumes

15

N in grasses and

The mean shoot d 15N values (Table 6) were −0.6‰ for grasses, and for the two most common legumes, T. repens and V. sati6a, were 0.5‰ and 0.8‰, respectively. It was not possible to

M. Unko6ich et al. / En6ironmental and Experimental Botany 40 (1998) 209–219

214 Table 3 Recovery of added

15

N (percentage of

15

N added) in plant shoot and root material (to 50 mm) 96 h after Roots

15

N application

Plot

Shoots

Total

treatment

% Label recovered

S.D.

% Label recovered

S.D.

% Label recovered

Water NH4 NH4NO3 NO3

10.2 12.8 9.9 10.2

4.2 5.3 3.3 4.2

23.3a 15.2b 14.7b 11.7b

7.9 7.3 5.0 3.4

32.0 26.4 25.1 21.1

Values followed by the same letter within a column are not significantly different by ANOVA (PB0.05).

calculate the dependence on N2 fixation of these two legumes using the 15N natural abundance technique due to the low, indeed slightly negative, d 15N value of the ‘reference’ grass shoots.

4. Discussion Rates of gross N mineralisation were estimated to be 1.2–3.6 mg N g − 1 day − 1 (0.07 g N m − 2

day − 1 or 0.69 kg N ha − 1 day − 1, to 50 mm depth) and at the lower end of the range previously reported in the literature. In a grassland in Berkshire, Geens et al. (1991) recorded gross N mineralisation rates of 0.5–2.6 kg N ha − 1 day − 1, with the higher rates being associated with instances where NH4+ fertiliser additions had occurred some weeks prior to the mineralisation estimates. At another drought-prone pasture in southern England, Monaghan and Barraclough (1997) recorded gross N mineralisation rates of 0.11–1.20 mg N g − 1 day − 1, while in the USA, rates of mineralisation in grassland have been found to be 1.37 mg g day − 1 in spring (Davidson et al., 1991) and 19 mg g day − 1 6 weeks after soil rewetting in autumn (Schimel, 1996). In the present study, the total amount of N taken up by plants receiving water but no N was about double that estimated to have been mineralised over the 38 day period of the experiment, the discrepancy due to the mineralisation sampling being underTable 4 Shoot tissue N concentrations (%) at harvest on 16 June, 38 days after commencement of N additions

Fig. 1. Shoot biomass accumulations (t ha − 1) in a calcareous grassland following weekly addition of water, water+N (as (NH4)2SO4, NH4NO3 or KNO3) or water + complete nutrient fertiliser (complete). Values atop bars are S.E. of mean and letters indicate significant differences between treatments by analysis of variance (PB 0.05).

Treatment

%N

S.D.

Nil Water Water+NH4 Water +NH4NO3 Water+NO3 Water+nutrients

1.47a 1.23a 1.76b 1.68b 1.46a 1.90c

0.15 0.19 0.29 0.26 0.16 0.10

Values followed by the same letter are not significantly different by ANOVA (PB0.001).

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Fig. 2. Shoot N accumulations (kg ha − 1) in a calcareous grassland following weekly addition of water, water+ N (as (NH4)2SO4, NH4NO3 or KNO3) or water + complete nutrient fertiliser (complete). All N additions at a rate of 1.15 g N m − 2 week − 1. Values atop bars are S.E. of mean and letters indicate significant differences between treatments by analysis of variance (P B 0.05).

taken to only 5 cm in depth and the plant root zone extending beyond this. While the estimated total gross N mineralisation was slightly higher in plots receiving NH4NO3 than the total amount of N accumulated in shoots, the variance in these estimates were substantial, reflecting the inherent variability in soil 15N and mineral N pools (see Monaghan, 1995). The estimates of nitrification at Wytham of 0.5–0.8 mg g − 1 day − 1 are also at the lower end of the range reported elsewhere for grasslands (0.89– 7 mg g − 1 day − 1 (Davidson et al., 1991; Schimel, 1996)) and woodlands (0.96 – 2.6 mg g − 1 day − 1 (Barraclough and Puri, 1995)). Low soil moisture availability during the study at Wytham would reduce nitrification more so than mineralisation, since it is well documented (Haynes, 1986) that the organisms responsible for nitrification are much more sensitive to environmental variables than the range of heterotrophs engaged in ammonification. The higher rates of nitrification in the NH4NO3 fed plots may have resulted from increased substrate (NH4+ ) availability. Although

215

Havill et al. (1977) used increased nitrate reductase activity in grass shoots as evidence for increased nitrification rates in calcareous grassland following NH4+ addition, there does not appear to have been any increase in NRA of plant shoots following NH4+ additions in the present study (see Table 5), where soil NO3− concentration remained below 2 mg g − 1 despite frequent fertiliser N application. Although the plants in the grassland at Wytham are shown to be N limited, only 27% of the fertiliser N supplied was recovered as aboveground herbage. The bulk of the added N appears to have been immobilised into root biomass or soil organic matter. In the present study, analysis of root N was extremely difficult since the roots formed a very dense mat, making it difficult to confidently separate soil from root. However, from the analyses that were done (data not shown), it was estimated that 14% of the added N could have been immobilised in roots. Leaching of the N or denitrification losses would be minimal at the site given the low soil water status. Immobilisation of N in grassland soils has been reported previously by Bristow et al. (1987), who recovered 37% of added NH4+ in the microbial biomass just 28 h after application. Similarly, Jackson et al. (1989) found that, 24 h after NH4NO3 application to a grassland, microbes contained five times more of the applied NH4+ than the grass, while the ratio for the added NO3− was about 2:1. It would thus appear that microorganisms may be greater sinks for NH4+ than plants in unfertilised grassland ecosystems. That plant growth was limited by the rate of mineralisation of soil organic N is further evidenced by the decline in herbage shoot N concentrations in plots supplied with water but not N. This poor capacity to mineralise N from the large bulk of organic matter in the soil results from a high ratio of soil carbon to inorganic N (Bosatta and Agren, 1995) and, perhaps to a lesser extent, to the C:N ratio of the organic material in the soil (Warren and Whitehead, 1988; Monaghan and Barraclough, 1997), and to the low availability of water at Wytham at this time of the year. The increase in herbage growth with watering indicates that water availability also limits plant

216

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Table 5 Averaged nitrate reductase activity in the principal grass species (predominantly Holcus lanatus and Agrostris stolonifera) and in Pastinaca sati6a Treatment (g NO3-N m−2 week−1)

Nil –

Water –

NH4 –

NH4NO3 0.58

Complete 0.92

NO3 1.15

Grasses Pastinaca sati6a

128 185

79 215

144 144

200 314

376 378

462 438

Values are meaned (pkat g per weight fraction) for the four sampling dates (11 May, 2, 9 and 16 June).

growth, although the overriding constraint appeared to be N. A similar finding was made on calcareous grassland in southern England by Lloyd and Pigott (1967) who concluded that ‘‘under natural conditions an inadequate water supply causes a nitrogen deficiency in the soil’’. During the summer, in the shallow soil at Wytham, the limited water available is rapidly transpired by plants to the detriment of microbial activity and N mineralisation (see Puri and Ashman, 1998). Other studies in Britain have found calcareous grasslands to be responsive to P application as well as N (for example, Morecroft et al., 1994) or to both N and K (for example, Smith et al., 1971). The available P status of the soil at Wytham is low (7 mg kg − 1) but, based on the lack of increase in DM yield following application, was apparently not limiting native grass growth. The lack of a response to additional nutrients in terms of herbage growth may be due to an overriding water, or chronic N, limitation. Indeed, this was reported by Lloyd and Pigott (1967) who found chalk grassland to be responsive to P only after water and N constraints were removed. It is therefore concluded that the amounts of N and water added in the present study were suboptimal for plant growth and, consequently, responses to other nutrients were unlikely. Given the low N status of this soil, it is difficult to understand why the eight legume species recorded (Gibson et al., 1987) at the site made up such a minor proportion of the sward biomass. Vetches and clovers examined at the site appeared to be adequately nodulated, though the effectiveness of these nodules in N2 fixation has not been assessed. Under conditions of low mineral N availability, as seen at Wytham, it might be ex-

pected that N2 fixing legumes would have a competitive advantage. Low P availability and low water availability over the summer months may curtail growth of nodulated clover more than grasses (Whitehead, 1995), especially since the symbiotic unit is much more sensitive to environmental stresses than non-nodulated plants. Unfortunately, the d 15N values for available mineral N, as assessed by the grasses at this site (−0.6‰), were not sufficiently different from atmospheric N2 to estimate the dependence of these legumes on N2 fixation (Unkovich et al., 1994). These low d 15N values for non-legumes at the site may constitute a legacy of fertiliser N inputs from when the land was arable and a relatively ‘tight’ contemporary N cycle (Hogberg, 1990; Nommik et al., 1994; Hogberg, 1997). The proportionate increases in activity of the inducible nitrate reductase enzyme in herbage following application of NO3− were not surprising given the low N status of the soil. However, the decline in NRA with time following constant N additions was not expected. Havill et al. (1977) observed a decline in NRA with time in a calcareous grassland associated with a concomitant decline in soil water status. In their study, NO3− accumulated in plant tissues as water availability declined and so it was suggested that low water availability impaired the activity of nitrate reductase. While soil water content also declined over the period of our study at Wytham, NO3− did not accumulate in plant tissues and so there is little evidence for a decline in NRA due directly to low water availability. Only a very small proportion of the decline in NRA activity in the grassland at Wytham was attributed to a reduction in growth rate (see nil treatment in Fig. 3), and hence de-

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217

Fig. 3. Nitrate reductase activity (NRA) (pkat g − 1) of grass shoots following weekly application of N as NH4NO3 (0.575 g NO3-N m − 2 week − 1), KNO3 (1.15 g NO3-N m − 2 week − 1) or in a complete nutrient fertiliser (0.92 g NO3-N m − 2 week − 1).

mand for N. Since NO3− was supplied at a constant weekly rate, declining tissue NO3− concentrations resulted from the incoming NO3− being distributed to an ever increasing shoot biomass. Consequently, while total shoot NRA might have remained the same the specific activity of the enzyme, as measured in this study, declined inversely to biomass accumulation. This study has shown that the soil under this grassland has a limited capacity to supply mineral N when compared to plant demand, and indeed the soil is a sink for much fertiliser N. Rates of N mineralisation are restricted, in part, by a low water availability during the spring and summer when evapotranspiration exceeds rainfall. Nitrate

is sparingly available in the soil and, in the absence of N fertiliser additions, there appears to be little assimilation of NO3− in above-ground herbage. Supplementary water and N result in a substantial increase in plant growth, regardless of the form of N added, and thus any changes to N mineralisation rates will be immediately reflected in plant growth. Further studies are needed to ascertain why N2 fixing legumes play such a minor role in the N dynamics of the system given their potential advantage in being independent of soil N mineralisation.

Table 6 15 N natural abundance (d 15N, ‰) of shoots of the principal legume components and mixed grass species at Wytham

Deborah Court, Jan Barron and Andrew Wilson are thanked for their assistance in the laboratory and field. MJU is indebted to The British Council for the award of a Postgraduate Bursary, to the A.W. Howard Memorial Trust for a travel grant and to The Centre for Legumes in Mediterranean Agriculture for continuing support.

Grasses Trifolium repens Vicia sati6a

d 15N (‰)

S.D. (9)

−0.60 0.53 0.80

0.59 1.10 0.43

Acknowledgements

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