Directory UMM :Data Elmu:jurnal:A:Applied Soil Ecology:Vol13.Issue3.Dec1999:

Mineralization and immobilization of nitrogen in heath

soil under intact

Calluna

, after heather beetle

infestation and nitrogen fertilization

H.L. Kristensen

a,b,*

, G.W. McCarty

b

a_{Department of Terrestrial Ecology, National Environmental Research Institute, Vejlsùvej 25, P.O. Box 314, DK-8600 Silkeborg, Denmark} b_{Environmental Chemistry Laboratory, USDA-ARS, Bldg. 007, BARC-West, Beltsville, MD 20705, USA}

Received 5 November 1998; received in revised form 25 April 1999; accepted 8 May 1999

Abstract

The maintenance of low availability of mineral N in heath soils is thought to be a key factor for the stability of heathland ecosystems. We investigated the turnover of NH4‡and NO3ÿin the organic surface layer of soils from a Danish heathland using15_{N isotope techniques in laboratory incubations. The soils were sampled under intact and dead}

Callunavegetation. The deadCallunavegetation had been fertilized at rates of 0, 15 or 35 kg N haÿ1per year and the death of vegetation had been caused by a naturally occurring heather beetle infestation. In the soil under intactCalluna, the NH4‡pool was very low with no net mineralization, while a substantial mineralization-immobilization turnover of NH4‡was found with a large capacity for short term net NH4‡immobilization (36mg N gÿ1during 1 h; 135mg N gÿ1during 24 h). The metabolic inhibitor mercury chloride completely inhibited assimilation of NH4‡indicating the process was biological. The immobilization of NH4‡had no short or long-term (38 days) effect on soil respiration while NH4‡immobilization stimulated net mineralization of soil N during long-term incubation. The soils sampled under dead and dead/fertilized Calluna had large pools and high net mineralization rates of NH4‡with a decrease of gross NH4‡immobilization relative to the soil under intactCalluna. Neither net nor gross nitri®cation activity could be detected in any of the soils. The results indicate that the effects of an increased atmospheric N deposition to the heathland may be delayed because of the tight cycling of NH4‡and the storage capacity for N in the soil and vegetation. The ecosystem may, however, be susceptible to disruption of the tight NH4‡cycling because of the limited capacity of the ecosystem to remove excess mineral N from the soil. This may increase the risk of conversion of the heath into grassland.#1999 Elsevier Science B.V. All rights reserved.

Keywords: Calluna vulgaris; Gross ammoni®cation; Immobilization;Lochmaea suturalis; Nitri®cation

1. Introduction

Lowland heath ecosystems once covered extensive areas of Western Europe, but this coverage has been

greatly reduced during the last two centuries as a result of land use change. During the last two decades much work has been done to increase our understanding of heathland ecology and to enable the continued pre-servation of the remnant heathlands. There has been growing evidence that the heath areas are threatened by increased atmospheric deposition of anthropogenic *Corresponding author. Tel.: +45-8920-1764; fax: +45-8920-1414

E-mail adress:hlk@dmu.dk (H.L. Kristensen)

N that changes heaths into grasslands and by natural succession of vegetation leading to the development of forest (Gimingham et al., 1979; Aerts and Heil, 1993). The dry lowland heaths are dominated byCalluna vulgaris (L.) Hull. and the litter of this species is known to be of low palatability for decomposers, which is thought to be the cause of organic matter accumulation in the mor layer (O horizon) (Read, 1991). Field and laboratory studies have found little or no apparent soil N mineralization, resulting in mineral N pools being maintained at very low content (Adams, 1986; Rangeley and Knowles, 1988; Kris-tensen and Henriksen, 1998). In addition, the heath mor has been found to immobilize substantial amounts of mineral N (Kristensen and Henriksen, 1998). The mor layer is, however, considered to be the main source for plant N uptake and it has been suggested that the apparent lack of mineral N in the soil is a key factor for the functioning of heathland ecosystems (Read, 1991). Studies have indicated theCallunaand other ericaceous species may be able to grow under these conditions because they live in symbiosis with ericoid mycorrhiza which have an ability to degrade recalcitrant organic N accumulated in the mor layer. The resulting simple N compounds can then be assimi-lated by the mycorrhiza and transferred to the host plant in exchange for photosynthetic products (Smith and Read, 1997). This may giveCallunaand related species an advantage in the competition with grasses and herbs which may have arbuscular mycorrhiza and, therefore, are thought to depend primarily on mineral N (Michelsen et al., 1996, 1998) or amino acid N for growth. The latter pathway has recently been indicated for the grassDeschampsia ¯exuosa(L.) Trin (NaÈsholm et al., 1998) which is indigenous to lowland heaths (Gimingham et al., 1979). Free living soil microor-ganisms may likewise use organic instead of mineral N during decomposition of organic matter (Jennings, 1995; Barraclough, 1997). With this background, it can be questioned to what degree mineralization± immobilization turnover of N is operating inCalluna mor which has no net mineralization activity. Net ammoni®cation and nitri®cation have been reported to occur in several studies of lowland heath soils and this occurrence has been related to changes in the species composition of the vegetation as well as to increased atmospheric N deposition to the ecosystem (Berendse, 1990; Troelstra et al., 1990; Van Vuuren et

al., 1992; Kristensen and Henriksen, 1998). The atmo-spheric N deposition has increased in the industria-lized countries during the last century as a consequence of emission of N compounds from agri-culture, industry and transportation. In Denmark the total atmospheric N deposition was, for example, on average 15 kg N haÿ1 per year in 1996 (Bak et al., 1999). The deposition is thought to increase the availability of mineral N in the heathland ecosystem, which may increase the ability of grasses to compete with theCallunavegetation. Studies have found that grasses like D. ¯exuosa are replacing the Calluna vegetation in parts of European lowland heath areas (Aerts and Heil, 1993; Marrs, 1993). This change can be related to the in¯uence of increased atmospheric N deposition on decomposition and mineralization in Calluna mor (French, 1988; Kristensen and Henrik-sen, 1998), but can also be due to increased N avail-ability in combination with damage to the Calluna vegetation (Prins et al., 1991). Such damage can result from naturally occurring epidemic attacks onCalluna by the heather beetle (Lochmaea suturalis Thoms.) which may increase in severity and frequency as a result of increased N input to heathland ecosystems (Brunsting and Heil, 1985). Little is known, however, about the in¯uence of beetle infestations on miner-alization±immobilization turnover in the mor under Callunaor the extent to which atmospheric N deposi-tion affects such processes.

The purpose of this study was to investigate the turnover of NH4‡and NO3ÿ(i.e., gross mineralization

and immobilization processes) inCallunamor which has no net mineralization activity (Kristensen and Henriksen, 1998). In addition, N and C mineralization was studied in the mor underCallunawhich had been killed due to infestation by heather beetles (Lochmaea suturalis) in combination with increased N availabil-ity, as these perturbations of the ecosystem are expected to be key factors in the conversion ofCalluna lowland heaths to grasslands.

2. Materials and methods

2.1. Study sites and sampling

in Western Jutland, Denmark, on a ¯uvio-glacial sand plain from the Weichselian period (10 000 BP). The soil was a typical Haplorthod; and the total wet and dry atmospheric N deposition amounted to an average of 18 kg N haÿ1per year (Hansen and Nielsen, 1998). A detailed site description is presented by Kristensen and Henriksen (1998). Three experimental plots (59.5 m2) were established on the heathland during the summer of 1993. They were fertilised six times a year in the periods September±November and March± May with NH4NO3in annual doses of 0, 15, and 35 kg

N haÿ1. The fertilizer was applied above the vegeta-tion as 15 l of soluvegeta-tion per ®eld plot with a 2.5 m spraying fan. The western part of the heathland study area containing the experimental plots was subjected to a natural epidemic attack by the heather beetle (Lochmaea suturalis) in July of 1994 which caused the Callunato turn reddish and defoliate. The attack did not spread to the eastern part of the heathland and this part was therefore left with an intact stand ofCalluna vegetation. The experimental plots were sampled together with an additional plot which was situated within a distance of 500 m from the other plots in the area that was not subjected to beetle infestations. The experimental plots are distinguished as shown in Table 1. At the time of soil sampling the I plot was dominated (>90% coverage) by intactCallunain the building phase (last cut in 1991). The D, D‡15, and D‡35 plots were covered by patches of dead and decomposing Calluna as well as live Empetrum nigrum ssp migrum L., D. ¯exuosa, mosses and lichens. The vegetation in the three plots with dead Callunais described in details by Riis-Nielsen (1997) who found no effect of N fertilization on the phaner-ogam vegetation in 1995. Soil samples were taken in November 1995 on a date 21 days after an N fertilizer application. The samples consisted of three replicate turfs (1522 cm; bulk density 0.3 g cmÿ3) of 2±4 cm thick organic mor layer (the O horizon). The turfs

were taken randomly within each plot, but always under intact or deadCallunavegetation. Any above-ground vegetation and fresh litter was removed and the soils were kept moist and cool until the turfs were sieved (4 mm mesh size), pooled, ad stored in plastic bags at 4₈C.

2.2. Nitrogen kinetics

The gross processes of N turnover presented in Fig. 1 were estimated by use of 15N labelling and equations based on the principles of isotope dilution (Hart et al., 1994):

mˆ …‰NH‡₄Š₀ÿ ‰NH‡₄Š_t†=t …log…APE0=APEt††=

…log…‰NH‡₄Š₀=‰NH‡₄Št†† (1)

iˆmÿ …‰NH‡₄Š_tÿ ‰NH‡₄Š₀†=t (2) where mˆgross N mineralization rate, iˆNH4‡

immobilization rate, tˆtime, APE0ˆatom% 15N

excess of the NH4‡ pool at time 0, APEtˆatom% 15

N excess of the NH4‡pool at timet, [NH4‡]0ˆtotal

NH4‡concentration at time 0, [NH4‡]tˆtotal NH4‡

concentration at timet. Gross nitri®cation and NO3ÿ

immobilization rates were estimated by replacing NH4‡ with NO3ÿ in the equations. The experiment

involved incubation of soil samples (15 g DW) in slurries of deionized water (soil : water ratio 1 : 8) in 250 ml Erlenmeyer ¯asks on an orbital shaker

Table 1

The four experimental plots that were sampled in November 1995

Plot Treatment

I IntactCallunavegetation

D DeadCallunadue to heather beetle infestation in July 1994

D‡15 DeadCallunadue to heather beetle infestation in July 1994, fertilized with 15 kg N haÿ1_{per year since Sept. 1993}

D‡35 DeadCallunadue to heather beetle infestation in July 1994, fertilized with 35 kg N haÿ1_{per year since Sept. 1993}

(180 rpm) at 22₈C. At the start of the experiment (time zero), 5% enriched15N solution was added to each of three replicate slurries of each soil. A solution of

15

NH4NO3 was added to the slurries for estimation

of gross ammoni®cation and NH4‡ immobilization

rates, while K15NO3 was added to the slurries for

estimation of gross nitri®cation and NO3

immobiliza-tion rates. The ®nal concentraimmobiliza-tion obtained was 200mg gÿ1DW soil equivalent to 25mg mlÿ1slurry of NH4‡-N or NO3-N, respectively. After intervals of

1, 24, and 48 h, each slurry was subsampled for 25 g of slurry and 3 M KCl was added to a ®nal concentration of 1 M and soil : solution ratio of 1 : 12.5. These subsamples were extracted for 1 h by shaking; cen-trifuged at 10 000 rpm for 10 min; and ®ltered through prerinsed Gelman Acordis glass ®lters. Finally, the subsamples were stored at 3₈C until analysis for amount and isotopic content of N. Additional slurries of each soil were incubated with KNO3and C2H2to

test for occurrence of denitri®cation by analysis of N2O in gas samples obtained from the sealed

incuba-tion ¯asks.

2.3. Inhibition of NH4‡immobilization and short term CO2production

To investigate the chemical/biological nature of the immobilization process in the soil sampled under intact Calluna vegetation, the rates of gross NH4‡

immobilization were measured in soil slurries treated with a metabolic inhibitor by use of isotope dilution techniques (Eqs. (1) and (2)). The experiment included a 48 h preincubation at 258C of six replicate samples of moist soil (10 g DW) in Erlenmeyer ¯asks (250 ml) with mercury chloride (20 mg HgCl2gÿ1

DW soil) added to two of the samples to inhibit biological activity (Wolf and Skipper, 1994). After preincubation, deionized water was added to all the samples to make soil slurries (soil : water ratio 1 : 8) which were then placed on an orbital shaker (180 rpm) at 228C. The N transformation assay was initiated by addition of 200mg15NH4‡-N gÿ

1

DW soil in the form of 15NH4NO3 (5%

15

N enriched) in solution to all slurries (time zero) except to two replicates which were incubated without any additions. Subsampling of slurry for analysis of amount and isotopic content of N was conducted after 0.25, 6 and 12 h by the same produce as in the N kinetic experiment. During

incu-bation the ¯asks were sealed and the headspace was subsampled with a gas syringe for CO2analysis.

2.4. Long-term N mineralization and CO2production

To study the long-term effects of increased N availability on net ammoni®cation, nitri®cation, and respiration, the soils were incubated with additions of NH4NO3 during a 38 days incubation period. Two

replicate soil samples (10 g DW) from each plot were slightly dried and then brought back to the original moisture content by drop wise addition and mixing of either deionized water or NH4NO3 solution. The

NH4NO3 addition was equivalent to 200mg NH4‡

-N gÿ1DW soil. The samples were incubated at 25₈C in 220 ml bottles which were sealed and subsampled with a gas syringe for CO2analysis. The bottles were

opened for aeration every 5±7 days during incubation. Samples of each soil were extracted at the start and the end of the experiment by shaking for 1 h in 1 M KCl (soil : solution ratio 1 : 5) followed by centrifugation, ®ltration, and storage of the ®ltrate for a maximum of two weeks at 38C until mineral N analysis. The calculations of the net ammoni®cation rate for the soil sampled under intact Calluna vegetation were performed using estimates of the initial NH4‡pool

as being equal to the sum of added NH4‡and

endo-genous NH4‡.

2.5. The microbial biomass and sample analysis

The microbial biomass was estimated by substrate induced respiration (SIR) as described by Anderson and Domsch (1978) which included incubation of two replicate samples (10 g DW) of each soil with addi-tions of 3000mg glucose gÿ1DW soil. The soils were analyzed for soil water content by drying at 1058C for 24 h and all results were calculated on a soil dry weight basis.The total amount of C and N in the soils was measured by dry combustion using a C and N analyzer (Leco CNS-2000). Soil pH was measured in 1 M KCl (soil : solution ratio 1 : 12.5). All soil KCl extracts were analyzed colorimetrically for NH4‡and

NO3 content by use of an automated ¯ow-injection

analyzer (Lachat Instruments). The 15_{N enriched}

extracts were prepared for isotopic analysis of NH4‡and NO3using the diffusion method of Brooks

an isotope mass spectrometer interfaced with an auto-mated N±C analyzer (Europa Scienti®c). The CO2and

N2O content of the gas samples were measured by gas

chromatography (Tremetrics) (McCarty and Blicher-Mathiesen, 1996).

2.6. Statistical analysis

The three replicate turfs taken from each ®eld treatment were combined to form one composite sample. This procedure eliminated information on the variation of properties for each treatment and this limits the ability to perform statistical analyses for detecting differences between the ®eld treatments (Hurlbert, 1984). However, the properties found for each composite soil represented averages for the ®eld plot. This allows us to use major differences in proper-ties found between the four soil samples as indications of the differences between the four treatments. Due to the limitations on statistical analysis, however, con-clusions about differences resulting from the ®eld treatments should be undertaken with some caution. All laboratory incubations were replicated and stan-dard errors were calculated for data obtained from each incubation. Differences between means of laboratory treatments were tested statistically within each soil by analysis of variance (SAS Institute, Cary, NC).

3. Results

3.1. Soil N pools and N kinetics

Some chemical and biological properties of the I, D, D‡15 and D‡35 soils are seen in Table 2. The NH4‡ pool was very low in the I soil while it was

180mg N gÿ1in the D soil with an even greater pool in the D‡15 and a maximum of 370mg N gÿ1in the D‡15 soil. No NO3ÿwas detected in the I and D

soils and only a small amount (9mg N gÿ1) was found in the D‡15 and D‡35 soils. The rates of net change in inorganic N pools during the N kinetic experiment are presented as a combination of net changes of the NH4‡ pool in the NH4NO3 slurries

and net changes in the NO3ÿpool in the KNO3slurries

(Fig. 2). A signi®cant part of the NH4‡that was added

to the slurries of I soil was immobilized at the ®rst sampling event of the experiment and the added plus the endogenous NH4‡pool was therefore used as the

time zero value of the NH4‡pool during calculation of

rates in this soil. The I soil showed a large net NH4‡

immobilization of almost 3mg N gÿ1hÿ1, while the D, D‡15 and D‡35 soils showed net ammoni®ca-tion rates in the range of 0.4±0.9mg N gÿ1hÿ1. In addition, NO3ÿwas subject to net immobilization in

the I soil at a rate of 0.4mg N gÿ1hÿ1when applied as KNO3(Fig. 2) and at a lower rate of 0.1mg N gÿ1hÿ1 when applied as NH4NO3 (results not shown)

Table 2

Some chemical and biological properties and N pools of the O horizon of the four heath soils (termed according to Table 1)

Soil water % H2O

pH(KCl) C/N C % N % Microbial

biomass (mg C gÿ1)

NH4‡

pool (mg N gÿ1)

NO3ÿ

pool (mg N gÿ1)

I 150 2.90 26.6 36.3 1.37 5.21 1 0.9

D 185 3.07 26.3 35.5 1.35 3.96 180 1.1

D‡15 160 3.22 23.0 30.6 1.33 3.88 300 8.6

D‡35 200 3.21 24.4 35.8 1.47 4.86 370 8.9

Fig. 2. The net rates of production and immobilization of NH4‡

and NO3ÿin the four heath soils (termed according to Table 1)

during 48 h of incubation. Changes in the NH4‡and NO3ÿpools

were measured after addition of NH4NO3and KNO3, respectively.

(p< 0.01). In either case, the rate of net NO3ÿ

immo-bilization was much lower than that of net NH4‡

immobilization (p< 0.001). Net nitri®cation was not detectable in any of the soils (Fig. 2).

The gross ammoni®cation rate in the I soil during the N kinetic experiment (Table 3) was 1.4mg N gÿ1hÿ1which was in the range of the gross ammo-ni®cation rates in the soils under deadCalluna vege-tation (1.2±1.7mg N gÿ1hÿ1). The gross NH4‡

immobilization rate was 4.3mg N gÿ1hÿ1 in the I soil, which was much higher than the rates in the D, D‡15 and D‡35 soils (0.6±1.0mg N gÿ1hÿ1). None of the soils showed any gross nitri®cation, and NO3ÿimmobilization was seen in the I soil only.

In the I soil, the NH4‡process rates were found not to

be constant during the 48 h experimental period of the N kinetic experiment. This is illustrated in Fig. 3 by showing the remaining NH4‡pool in the slurry at each

time of subsampling as well as the calculated gross process rates between sampling events. The NH4‡

concentration in the slurry decreased very quickly to an amount around 160mg N gÿ1 within the ®rst hour of incubation and then decreased to 66mg N gÿ1 during the 1±24 h interval. Thereafter, the pool remained almost constant until the 48 h subsampling leaving a pool of 60mg NH4‡-N gÿ1in the slurry at

the end of the experiment. The gross ammoni®cation rate decreased from 3.4 to 0.8mg N gÿ1hÿ1during the experiment while the gross NH4‡immobilization rate

decreased from 39 to 5.0 and then to 1.1mg N gÿ1hÿ1 over the 0±1, 1±24 and 24±48 h interval, respectively. No denitri®cation was detected from any of the soils during the incubations with C2H2.

3.2. Inhibition of NH4‡immobilization and CO2 production

The gross NH4‡immobilization rates in the slurries

of I soil that were treated with HgCl2are presented in

Fig. 4. The rate in the soil with NH4NO3additions was

Table 3

Gross rates of N turnover in the four heath soils (termed according to Table 1) as measured by isotope dilution techniques during 48 h incubation. Numbers in brackets are standard errors for means obtained during incubations of a composite sample of each soil

Ammonification NH4‡immobilization Nitrification NO3ÿimmobilization

mg N gÿ1hÿ1

I 1.39 (0.04) 4.31 (0.02) 0 0.37 (0.05)

D 1.19 (0.05) 0.80 (0.10) 0 0

D‡15 1.43 (0.04) 0.56 (0.15) 0 0

D‡35 1.67 (0.06) 1.02 (0.20) 0 0

Fig. 3. Net changes (leftyaxis) in the NH4‡pool ÐÐÐ; and

gross rates (right y-axis) of ammonification & and NH4‡

immobilization & in NH4NO3 treated soil which was obtained

from under intactCallunavegetation. Error bars indicate standard errors.

Fig. 4. Effect of a metabolic inhibitor on the rate of gross NH4‡

immobilization in NH4NO3treated soil which was obtained from

under intactCallunavegetation. The treatments were: no inhibitors (Control); or HgCl2 to inhibit biological acitivity. Error bars

7.0mg N gÿ1hÿ1while the rate was close to zero in the slurries which had been treated with HgCl2 and

NH4NO3No CO2was produced in the HgCl2treated

slurries which indicated that this treatment effectively stopped soil microbial activity (results not shown). Comparison of the net change in the NH4‡pool in the

slurries without additions and the slurries with NH4NO3 additions showed that NH4‡ was

immobi-lized at a rate of 5.3mg N gÿ1hÿ1in the latter, while the slurries without additions showed no net change in the NH4‡ pool of 1.8mg N gÿ1 during the 12 h experimental period (results not shown). The respira-tion rate measured in the two treatments did not, however, differ either during the ®rst (p< 0.58) or the last half (p< 0.54) of the experimental period (Fig. 5).

3.3. Long-term N mineralization and CO2production

The net ammoni®cation rates in the four soils during the 38-day incubation period were generally higher in the samples with N additions as compared to the soils without additions (Fig. 6). The difference was signi®cant (p< 0.01) except for the D‡35 soil (p< 0.59). The largest increase in the net ammoni®-cation rate when N was added was seen in the I soil where the rate increased more than threefold to 0.3mg N gÿ1hÿ1. In the soils without N additions, the rates increased in the order, I, D, D‡15 and D‡35 while the order was I, D‡15, D and D‡35 with N additions. No net nitri®cation or NO3ÿimmobilization

was observed in any soil during the experiment

(results not shown). The respiration rate was unchanged with N addition in the I soil (p< 0.20), while it decreased in the D, D‡15 and D‡35 soils in the samples where N was added when compared to the samples where no N was added (Fig. 7), however, the difference was only statistically signi®cant in the D‡15 soil while it was close to signi®cant in the D‡35 soil and the D soil.

4. Discussion

4.1. Turnover and net immobilization of NH4‡in the Calluna mor

The mor under intactCallunavegetation showed a substantial capacity for turnover of NH4‡ (Fig. 3)

with gross ammoni®cation rates (0.8±3.4mg N gÿ1hÿ1) comparable to those found by Tietema (1998) in acid organic forest soils (i.e., 0.4±0.8mg N gÿ1hÿ1) with similar contents of organic matter and microbial biomass. Thus, the results obtained under intact Callunagive no support to the theory that the microbial activity in heath mor is inhibited by the release of toxic compounds from theCalluna vegeta-tion (Jalal and Read, 1983). This conclusion is in accordance with the work of Rangeley and Knowles (1988) who found that microbial activity as measured by CO2production in limed Scottish heath mor was

comparable to that of agricultural soil. The present study found, however, an unusual ability for rapid net immobilization of added NH4‡(Fig. 2). In the heath

Fig. 5. The CO2 production after no addition or addition of

NH4NO3to soil obtained from under intact Callunavegetation.

Error bars indicate standard errors.

Fig. 6. The net rates of ammonification after no addition or addition of NH4NO3to the four heath soils (termed according to

soil, the NH4‡ pool was maintained at a very low

content (Table 2) and the rate of gross NH4‡

immo-bilization was much higher than that of gross ammo-ni®cation after addition of NH4‡ (Fig. 3). This

indicates that the addition of NH4‡ stimulated the

immobilization process. Moreover, this suggests that the mineralized NH4‡in general may be

re-immobi-lized in this soil immediately after being released to the soil NH4‡pool, and this keeps the pool as well as

the net ammoni®cation rate very low which was also found in ®eld studies with soil from the same site (Kristensen and Henriksen, 1998). The cause of this may be the chemical environment created in the soil by theCallunavegetation. For example,Callunalitter is known to have a high content of soluble phenolic compounds which enhance the formation of recalci-trant humic complexes through condensation and microbially mediated immobilization of organic N (Kuiters, 1990; Read, 1991).

The ability of heathland mor to rapidly immobilize N could have consequences for the fate of N that is

deposited on the soil from the atmosphere. The con-ditions for immobilization of inorganic N in short term slurry experiments differ greatly from ®eld conditions as regards availability of substrate, temperature, soil disturbance, etcetera. The results obtained in the pre-sent study can, however, indicate the potential capa-city of the heath mor under intact Calluna to immobilize N and was for the 48 h experiment found to be equivalent to 10±15 kg NH4‡-N haÿ1and 1±2 kg

NO3ÿ-N haÿ1. Such a capacity for immobilization is

substantial when considering that the total annual N deposition has been estimated to be 18 kg N haÿ1per year for the heathland under study with 65% being deposited as NHxand the remainder as NOy(Hansen

and Nielsen, 1998). The mor may in this way act as a short term sink for N from atmospheric deposition, which may prevent that inorganic N from being leached to deeper soil layers. This was con®rmed by studies from the same heathland where intact cores of the mor layer taken under intactCallunashowed net immobilization of both NH4‡ and NO3ÿ from

rain-Fig. 7. The cumulative CO2production after no addition or addition of NH4NO3to the four heath soils (termed according to Table 1) during

water during a 35-day laboratory experiment (Kris-tensen and Henriksen, 1998). Likewise, studies of percolating soil water collected under intactCalluna from this heathland found no NO3ÿand only traces

of NH4‡to be leached below the mor layer (Nielsen

et al., 1999). The high capacity for net immobilization of the mor layer may also maintain low availability of inorganic N for plant uptake. This probably strength-ens the competitive ability of the ericaceous vegeta-tion at the expense of the herbaceous vegetavegeta-tion since the symbiosis with ericoid mycorrhiza may give the plant access to recalcitrant organic N (Smith and Read, 1997).

4.2. Turnover of N in mor under dead Calluna vegetation

The tight cycling of N in the mineralization±immo-bilization system in the mor under intact Calluna was greatly in¯uenced by the heather beetle attack on the Calluna vegetation. The availability of inor-ganic N increased from a very low to a high level as shown by both large pools of NH4‡and occurrence

of net ammoni®cation (Table 2, Fig. 2). This was indicated to be a result of a decrease in the gross immobilization rate while the gross ammoni®ca-tion rates in the three soils sampled under dead Calluna vegetation were about the same as the rate in the soil under intact vegetation (Table 3). The increase of the NH4‡ pool under dead as compared

to intact Calluna was, when calculated on an areal basis, in the range of the amount of NHxwhich had

been deposited from the atmosphere since the occur-rence of the heather beetle infestation (both around 16 kg N haÿ1). These changes in availability of inor-ganic N were probably due to a combined effect of the defoliation and death of theCallunavegetation with the heather beetle infestation which resulted in the cessation of uptake of N by vegetation while litter production from the dead plants, faeces and dead beetles was increased. The decrease in gross immo-bilization after the heather beetle attack could, how-ever, also indicate that the capacity for NH4‡

immobilization under intactCalluna was caused by a direct in¯uence from the intactCallunavegetation, possibly in the form of soluble phenolic compounds that may be released from leaves and roots (Kuiters, 1990).

4.3. Turnover of NO3ÿ

Despite the high availability of NH4‡as substrate

for autotrophic nitri®cation in the three soils under dead Calluna vegetation (Table 2), no net or gross nitri®cation could be detected in any of these soils nor in the soil under intact Calluna vegetation (Fig. 2, Table 3). This is one of the few studies that we know of which has documented a total lack of gross nitri-®cation activity in soil. The small amount of NO3ÿ

found in the fertilized plots (Table 2) probably origi-nated from the NH4NO3 fertilizer added 21 days

before the sampling of soil for this study. The lack of gross nitri®cation can possibly be explained by the very low pH in the soils (pHKClaround 3 equivalent to

pH_H2O around 4 (Kristensen and Henriksen, 1998)). However, other studies of for example Dutch heath soils have found net nitri®cation to occur under similar acidic conditions (Troelstra et al., 1990). Furthermore, substantial gross nitri®cation and NO3ÿ

immobiliza-tion was found in undisturbed forest soils at pH below 4 even though net nitri®cation could not be detected (Stark and Hart, 1997). It was surprising that there was a lack of nitri®cation in the soil after the death of the Callunavegetation as perturbation of the vegetation in general is thought to stimulate nitri®cation and this may lead to N removal from the ecosystem through NO3ÿleaching and denitri®cation. The vegetation is

in this way thought to exert biological control over N losses from some natural ecosystems (Tamm, 1991) but there was no indication that liveCalluna vegeta-tion exerted any direct control over nitri®cavegeta-tion. The lack of nitri®cation and the subsequent limited capa-city for mineral N removal from the heath ecosystem can help explain the accumulation of NH4‡in the soil

after perturbation of the vegetation (Table 2) as NH4‡

is adsorbed in the soil and not readily leached with percolating water. This accumulation of NH4‡could

be expected to enhance the destabilizing effects of the beetle attack on the ecosystem by increasing the availability of inorganic N for competing plant species.

Nitrate was found to be immobilized in the soil under intact Calluna vegetation but the process was found to be inhibited when NH4‡was also available.

This indicates that the immobilization of NO3ÿwas

known to be inhibited by microbial assimilation of NH4‡(McCarty and Bremner, 1992).

4.4. The NH4‡immobilization process

The use of HgCl2 as an inhibitor of biological

activity showed that the NH4‡ immobilization was

a fully biological process (Fig. 4). The rate of gross NH4‡ immobilization during the ®rst hour of the

incubation was found to be extraordinarily high at 39mg N gÿ1hÿ1(Fig. 3). This rate may tentatively be compared with the maximum gross NH4‡

immobili-zation rate of 23mg N gÿ1hÿ1found by Schimel and Firestone (1989) in acid coniferous forest ¯oor mate-rial. They found that 19% of the immobilization was due to abiotic processes for gross immobilization rates in the range of 4±9mg N gÿ1hÿ1. However, in the present study all of the NH4‡was immobilized by the

microbial population, and the immobilized15N could therefore be expected to constitute a signi®cant part of the biologically active N pool in the soil. An assump-tion made in Eqs. (1) and (2) is that no immobilized

15

N will be re-mineralized during the experiment (Hart et al., 1994). With the immobilization of a large quantity of 15N into the biologically active pool in the soil, it is likely that this assumption was violated to some degree. This may have contributed to decreases in gross ammoni®cation and NH4‡

immobilization rates with the successive periods of sampling (Fig. 3).

The large microbial assimilation of added NH4‡in

the soil under intactCallunasuggests that the micro-bial community was limited by availability of N. But we found that CO2production was not stimulated by

addition of NH4NO3to soil slurries (Fig. 5) and this

contrasts with the expected result if the N assimilation had stimulated the activity or the growth of the heterotrophic microorganisms. It is possible, however, that the assimilated NH4‡ was a type of `luxury'

uptake as described by Fog (1988), where N is assimilated and stored for later use. For example, fungi have been found to accumulate amino acids and it has been suggested that protein inclusions and other fungal cell structures may act as storage for N in insoluble form (Jennings, 1995). Moreover, Kerley and Read (1997) have hypothesized that N may be incorporated into fungal cell walls through mela-nisation to enable fungi to persist in the heath soil and

this may further increase the resistance of the immo-bilized N to decomposition.

4.5. Long-term effects of N on mineralization

The addition of N during the long-term incubation experiment was found to increase the net ammoni®ca-tion rate in the soil sampled from under intactCalluna vegetation while the respiration was largely unaf-fected. The latter trend was also observed in the short-term experiment (Figs. 6 and 7). It is possible that the increase in N availability induced the decomposition of N-rich organic compounds with a subsequent release of excess N with no in¯uence on C mineralization. Studies of acid organic forest soils have also found either no in¯uence or a negative effect of N addition on decomposition and respiration, but the mechanisms behind these ®ndings are not well understood (Fog, 1988; Martikainen, 1996). Our results provide evidence, however, that a single addi-tion of NH4‡of the size of the annual atmospheric

N deposition will induce the release of N from the large pool of organic N bound in the heath mor. This suggests that N additions will be detrimental to the stability of the ecosystem as they may in¯uence the competition between Calluna and grasses by increasing nutrient availability. But net ammoni®-cation was also observed in the soil under intact Callunawithout N addition (Fig. 6), which otherwise has been found to be very low in ®eld and laboratory incubations at temperatures closer to those measured under ®eld conditions (Kristensen and Henriksen, 1998). Moreover, the amount of N added in our experiment was well above the amount of N deposited in a single rain event, and the in¯uence of an intact Calluna vegetation was missing during the incuba-tion. This indicates that the results of the experiment may not be directly transferable to the heathland ecosystem.

Net ammoni®cation was also increased with N addition during the long-term incubation of the soils sampled from under dead Calluna (Fig. 6), but, respiration was found to decrease with the addition of N (Fig. 7). This may for example have been caused by a salt effect on the microbial population (Marti-kainen, 1996) because of the high concentration of NH4‡after addition of this compound to the already

of the NH4‡pool under deadCallunawhen fertilized

with annual doses of 15 and 35 kg N haÿ1could, when calculated on an areal basis (11 and 17 kg N haÿ1, respectively), be accounted for by the amount of fertilizer NH4‡applied since the heather beetle attack

(10 and 23 kg N haÿ1, respectively). In addition, the size of the net ammoni®cation rates during the short-and long-term incubation experiments of the three soils sampled under deadCalluna were in the same range and no clear relationship to N fertilization rate was evident (Figs. 2 and 6). These results indicate that an increase of N input to the ecosystem will not in¯uence mineralization of the mor after a heather beetle attack. This was expected due to the already large accumulation of NH4‡ in the soil with the

heather beetle attack and the limited capacity of the ecosystem to remove mineral N in the absence of nitri®cation.

5. Conclusion

TheCalluna mor was found to have a substantial capacity for mineralization±immobilization turnover of NH4‡ which was comparable to values reported

in the literature for acid forest soils. The lack of net mineralization and the high capacity for immo-bilization of mineral N in the heath soil may point to a unique in¯uence of the Calluna vegetation on microbial cycling of N within the ecosystem. The ability to rapidly immobilize large amounts of N could play a major role in the competition between Calluna and grass species because ericaceous vegetation would be favored by the maintenance of low N availability. An increased N input to the heathland ecosystem may increase the frequency of heather beetle infestations, which in turn was found to change the balance of N cycling processes in the ecosystem, resulting in substantial increases in net ammoni®cation in the soil. These destabi-lizing in¯uences are likely enhanced by the inability of the ecosystem to remove excess N because of the complete lack of nitri®cation in the soil. Therefore, large accumulations of NH4‡ occur in

soil under Calluna vegetation damaged by heather beetle attack. Together, these in¯uences may increase the ability of grasses to gain dominance on the heathland.

Acknowledgements

This work was supported by a fellowship from the National Environmental Research Institute of Den-mark, the Danish Research Academy, and Aalborg University. We thank the Hjerl Foundation for permis-sion to use the ®eld site and Knud Erik Nielsen for the establishment and maintenance of the ®eld plots.

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