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Comparison of a wet and dry 15N isotopic dilution technique as a short-term nitrification assay
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Soil Biol. Biochem. Vol. 30, No. 5, pp. 661±663, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0038-0717(97)00214-9 0038-0717/98 $19.00 + 0.00
SHORT COMMUNICATION COMPARISON OF A WET AND DRY 15 N ISOTOPIC DILUTION TECHNIQUE AS A SHORT-TERM NITRIFICATION ASSAY T. W. WILLISON*, J. C. BAKER, D. V. MURPHY and K. W. T. GOULDING Soil Science Department, IACR-Rothamsted, Harpenden, Herts., AL5 2JQ, U.K. (Accepted 18 August 1997)
The nitrifying activity of soil is generally measured as the amount of nitrate produced during an incubation of known duration. The assay may involve the use of sieved soil or intact cores and generally incubations are for a duration of up to 8 weeks (Robertson, 1982; Lensi et al., 1986). In some cases the incubations are conducted at a controlled temperature in the laboratory, or alternatively soil samples are buried in the ®eld in an attempt to mimic in situ nitri®cation rates. The principal criticism of these methods is that they do not give a true index of nitri®er activity at a particular time because they are conducted over relatively long periods. In addition, the number of interacting factors makes any mechanistic interpretation virtually impossible. Nitri®cation assays where much shorter incubations have been used generally involve adding ammonium (NH+ 4 )-containing solution with or without nitri®cation inhibitors such as chlorate or acetylene (Belser and Mays, 1980; Lensi et al., 1986) and subsequently determining the oxidation and nitrate (NOÿ products nitrite (NOÿ 2) 3 ). Nitri®cation assays which use a soil slurry system are likely to generate anaerobic conditions, with the consequent increase in denitri®cation. More recently it has been demonstrated that adding an NH+ 4 source has a ``priming'' eect which gives an overestimate of the endogenous rate of nitrate production (Fisk and Schmidt, 1996). The principal advantage of 15 N isotopic dilution for characterising microbial processes is that the product and not a substrate is added to the experimental medium (soil or water), eliminating the possibility of a priming eect. A pre-requisite for measuring gross nitri®cation using isotopic dilution is the uniform labelling of the soil NOÿ 3 pool with 15 N. In laboratory experiments this has typically *Author for correspondence. @bbsrc.ac.uk.
E-mail:
toby.Willison 661
involved the application of 15 N labelled salt solutions at near optimal soil water contents (Ambus et al., 1992; Hart et al., 1994; Barraclough and Puri, 1995). The addition of solution to soil has two main disadvantages: if the soil is already moist sites of anaerobiosis may be created, and if the soil is dry, rewetting may result in a mineralization ¯ush. An alternative to adding solution is the use of a dry method of 15 N labelling. Stark and Firestone (1995) have used 15 N labelled nitric oxide gas to label the nitrate pool but the expense and toxicity problems associated with this methodology make its widespread use impractical. Alternatively, an inert powder could be used as the carrier of 15 N. We describe such a technique and compare the results with a more conventional wet method in soils from three dierent land uses. Soils were taken from three sites at Rothamsted Experimental Station, Hertfordshire: section 3, plot 5 (zero N fertilizer) of the Broadbalk continuous wheat experiment; unfertilized areas of the Park Grass continuous hay experiment; and Knott Wood, a mixed deciduous wood. These three sites are <1.0 km apart, the soils are silt to silty clay loams with a clay content between 21 and 28% in the topsoil (0±23 cm) and overlying clay-with-¯ints; they are chromic luvisols classi®ed as Batcombe series (Avery and Catt, 1995). Other soils were used to compare soil texture and were collected from two pastures at least 40 y old. Grasslands were located on a sandy colluvial brown earth (Stackyard series) at Woburn Experimental Farm, Woburn, and a silty clay loam (Bromyard series) at ADAS Rosemaund, Worcester. Wet 15 N isotopic dilution incubations to measure gross nitri®cation were conducted as laboratory incubations similar to those described by Barraclough and Puri (1995) and Watkins and Barraclough (1996). Subsamples of fresh soil (35.0 g) were weighed into glass jars (5 cm dia) so that the soil
662
T. W. Willison et al.
depth was approximately 2 cm. Thereafter 3 ml of 15 N labelled solution was added to the soil surface as K15NO3 (at 10.3 at.%) at a rate equivalent to ÿ1 5.0 mg NOÿ dry soil. This volume was 3 ±N g required to provide a relatively uniform distribution of solution throughout the soil sample to minimise errors associated with non-uniform distribution of 15 N. For the dry incubations, 15 N-labelled powder (3 g) was applied to the surface of the soil and the jars were lightly tapped to distribute the powder through the soil. The powder was prepared by dissolving K15NO3 (at 10.3 at.%) in a slurry of silica ¯our and deionised water which, after mixing for 1 h, was dried in an oven at 1058C for 24 h and milled to a ®ne talc using a disk mill (Tema, model T100). The resulting 15 N labelled powder contained the same amount of K15NO3 in 3 g as was present in 3 ml of solution (i.e. 5.0 mg N gÿ1 dry soil). In addition 3 ml of a solution containing (NH4)2SO4 was added as an additional treatment to the three soils from Rothamsted (Broadbalk, Park Grass and Knott Wood) at a rate equivalent to 5.0 mg N gÿ1 dry soil. This enabled comparison of net nitri®cation rates determined using a traditional short-term nitri®cation assay. ÿ The 15 N-to-14 N ratio of the NH+ 4 or NO3 soil extracts (3 replicates taken 1,2,3,4 and 8 d after addition of N and shaken for 1 h with 60 ml of 2.0 M KCl) were determined on an Europa mass spectrometer linked to a combustion analyzer (Europa Scienti®c Roboprep) after diusion onto acidi®ed glass wool disks (Brooks et al., 1989). Gross rates of nitri®cation were calculated using the formula of Barraclough (1991). Rates were calculated between 2 and 8 d, no remineralization of 15 NOÿ 3 was detected during this period. The incubation period for measurement of gross nitri®cation started 2 d after 15 N addition. This delay was necessary to allow the 15 NOÿ 3 in the powder to dissolve and reach an equilibrium with the soil NOÿ 3 ±N pool. For the same soils the amount and enrichment of the nitrate pool was not signi®-
Fig. 1. Comparison of net nitri®cation rates (mg N kgÿ1 dÿ1) showing the net change in the nitrate pool calculated during a 6 d incubation. The ®gure compares the rates calculated by 15 N isotopic dilution following addition of either (1) labelled powder, (2) solution or (3) by using a conventional nitri®cation assay with addition of ammonium sulphate (AS). Rates are means plus and minus standard errors (n = 3).
cantly dierent 2 d after application when the label was added in solution or powder form (data not shown). With the exception of the Broadbalk and Park Grass soils the gross nitri®cation rates were signi®cantly lower in the soils where 15 N was applied as powder (range: 90±580 mg N kgÿ1 dÿ1) compared to solution (range: 170±1200 mg N kgÿ1 dÿ1). This re¯ects the stimulation of mineralization following wetting (Table 1); there is a greater change in moisture content following solution addition (range: +27 to +70%) than powder (range: ÿ11 to ÿ16%). In the case of Broadbalk soil the lack of dierence in the gross nitri®cation rates following powder or solution application was attributed to the lack of NH+ 4 ±N as a substrate for ammonium oxidizers. When an NH+ 4 ±N source was applied to the three soils from Rothamsted the net rate of nitri®cation increased greatly in the Broadbalk soil and, to a lesser extent, in Knott Wood soil but there was no change in the Park Grass soil (Fig. 1). The lack of increase in Park Grass is because ammonium oxidizers were inhibited by low pH (4.5) of this soil (Johnston et al., 1986).
Table 1. Gross nitri®cation rates (ngross, mg N kgÿ1 dÿ1) and the change in gravimetric soil water content (w/w) after the application of 15 N in solution or powder form. Values in parentheses are standard errors (n = 3). Plus and minus values are percentage changes in moisture content following application of 15 N in solution or powder form Soil
Initial water content
15
N applied as solution
ngross Broadbalk
13.25
Knott Wood
24.56
Park Grass
41.80
Woburn
18.45
Rosemaund
19.00
0.17 (0.04) 1.20 (0.11) 0.17 (0.02) 0.70 (0.08) 1.00 (0.29)
water content 22.71 +70 35.00 +42 53.28 +27 28.35 +50 28.28 +50
15
N applied as powder
ngross 0.09 (0.03) 0.47 (0.03) 0.15 (0.02) 0.58 (0.03) 0.41 (0.10)
water content 12.03 ÿ13 22.16 ÿ11 37.80 ÿ11 15.45 ÿ16 16.27 ÿ15
15N Isotopic dilution technique
The rates of gross nitri®cation reported by the methods we describe in this paper are generally in the range reported by other workers. Rates of 5.3 mg N kgÿ1 dÿ1 (Gleyic cambisol), 2.0 mg N kgÿ1 dÿ1 (Gleyic podzoluvisol) and 1.7 mg N kgÿ1 dÿ1 (Rendzina) for cultivated soils using isotopic dilution and a 0±6 d incubation period were reported by Recous and Mary (1990). Davidson et al. (1992) measured gross nitri®cation rates using 15 N pool dilution with cores incubated in situ in open grass prairie (590±810 mg N kgÿ1 dÿ1) and grass under oak (Quercus wislizenii A.) in spring and winter (1.38±3.47 mg N kgÿ1 dÿ1). The dry method of 15 N application that we have described oers an alternative method for quantifying gross rates of nitri®cation where solution addition is not desired. The bene®ts of applying the 15 N isotopic dilution technique as a short-term nitri®cation assay are clear: a short incubation period, reduced alteration of soil moisture content and no risk of introducing a priming eect due to substrate addition. AcknowledgementsÐWe would like to thank James Wake®eld for assistance in 15 N analysis and Wendy Wilmer for colorimetric determination of ammonium and nitrate. Anne Bhogal kindly supplied the Rosemaund soil. This work was funded by the U.K. Ministry of Agriculture, Fisheries and Food. IACR-Rothamsted receives grant-aided support from the Biotechnology and Biological Sciences Research Council. REFERENCES
Ambus P., Mosier A. and Christensen S. (1992) Nitrogen turnover rates in a riparian fen determined by 15 N dilution. Biology and Fertility of Soils 14, 230±236. Avery B. W. and Catt J. A. (1995) The Soil at Rothamsted. Lawes Agricultural Trust Ltd., Harpenden. Barraclough D. (1991) The use of mean pool abundance's to interpret nitrogen-15 tracer experiments. I. Plant and Soil 131, 89±96.
663
Barraclough D. and Puri G. (1995) The use of 15 N pool dilution and enrichment to separate the heterotrophic and autotrophic pathways of nitri®cation. Soil Biology & Biochemistry 27, 17±22. Belser L. W. and Mays W. L. (1980) Speci®c inhibition of nitrate oxidation by chlorate and its use in assessing nitri®cation in soils and sediments. Applied and Environmental Microbiology 39, 505±510. Brooks P. D., Stark J. M., McInteer B. B. and Preston T. (1989) Diusion method to prepare soil extracts for automated nitrogen-15 analysis. Soil Science Society of America Journal 53, 1701±1711. Davidson E. A., Hart S. C. and Firestone M. K. (1992) Internal cycling of nitrate in soils of a mature coniferous forest. Ecology 73, 1148±1156. Fisk M. C. and Schmidt S. K. (1996) Microbial responses to nitrogen additions in alpine tundra soil. Soil Biology & Biochemistry 28, 751±755. Hart S. C., Stark J. M., Davidson E. A. and Firestone, M. K. (1994) Nitrogen mineralization, immobilization and nitri®cation. In: Methods of Soil Analysis, Part 2. Microbiological and Biochemical Properties, eds. R. W. Weaver, S. Angle, P. Bottomley, D. Bezdicek, S. Smith, A. Tabatabai and A. Wollum, pp. 985±1018. Soil Science Society of America, Madison. Johnston A. E., Goulding K. W. T. and Poulton P. (1986) Soil acidi®cation during more than 100 years under permanent grassland and woodland at Rothamsted. Soil Use and Management 2, 3±10. Lensi R., Mazurier S., GourbieÁre F. and Josserand A. (1986) Rapid determination of the nitri®cation potential of an acid forest soil and assessment of its variability. Soil Biology & Biochemistry 18, 239±240. Recous S. and Mary B. (1990) Partitioning of fertilizer N between soil micro¯ora and winter wheat crop. In Proceedings of the 1st Congress European Society of Agronomy, ed. A Scaife Colmar, p. 45. European Society of Agronomy. Robertson G. P. (1982) Nitri®cation in forested ecosystems. Philosophical Transactions of the Royal Society of London B 296, 445±457. Stark J. M. and Firestone M. K. (1995) Isotopic labelling of soil nitrate pools using nitrogen-15-nitric oxide gas. Soil Science Society of America Journal 59, 844±847. Watkins N. and Barraclough D. (1996) Gross rates of N mineralization associated with the decomposition of plant residues. Soil Biology & Biochemistry 28, 169± 175.
Soil Biol. Biochem. Vol. 30, No. 5, pp. 661±663, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0038-0717(97)00214-9 0038-0717/98 $19.00 + 0.00
SHORT COMMUNICATION COMPARISON OF A WET AND DRY 15 N ISOTOPIC DILUTION TECHNIQUE AS A SHORT-TERM NITRIFICATION ASSAY T. W. WILLISON*, J. C. BAKER, D. V. MURPHY and K. W. T. GOULDING Soil Science Department, IACR-Rothamsted, Harpenden, Herts., AL5 2JQ, U.K. (Accepted 18 August 1997)
The nitrifying activity of soil is generally measured as the amount of nitrate produced during an incubation of known duration. The assay may involve the use of sieved soil or intact cores and generally incubations are for a duration of up to 8 weeks (Robertson, 1982; Lensi et al., 1986). In some cases the incubations are conducted at a controlled temperature in the laboratory, or alternatively soil samples are buried in the ®eld in an attempt to mimic in situ nitri®cation rates. The principal criticism of these methods is that they do not give a true index of nitri®er activity at a particular time because they are conducted over relatively long periods. In addition, the number of interacting factors makes any mechanistic interpretation virtually impossible. Nitri®cation assays where much shorter incubations have been used generally involve adding ammonium (NH+ 4 )-containing solution with or without nitri®cation inhibitors such as chlorate or acetylene (Belser and Mays, 1980; Lensi et al., 1986) and subsequently determining the oxidation and nitrate (NOÿ products nitrite (NOÿ 2) 3 ). Nitri®cation assays which use a soil slurry system are likely to generate anaerobic conditions, with the consequent increase in denitri®cation. More recently it has been demonstrated that adding an NH+ 4 source has a ``priming'' eect which gives an overestimate of the endogenous rate of nitrate production (Fisk and Schmidt, 1996). The principal advantage of 15 N isotopic dilution for characterising microbial processes is that the product and not a substrate is added to the experimental medium (soil or water), eliminating the possibility of a priming eect. A pre-requisite for measuring gross nitri®cation using isotopic dilution is the uniform labelling of the soil NOÿ 3 pool with 15 N. In laboratory experiments this has typically *Author for correspondence. @bbsrc.ac.uk.
E-mail:
toby.Willison 661
involved the application of 15 N labelled salt solutions at near optimal soil water contents (Ambus et al., 1992; Hart et al., 1994; Barraclough and Puri, 1995). The addition of solution to soil has two main disadvantages: if the soil is already moist sites of anaerobiosis may be created, and if the soil is dry, rewetting may result in a mineralization ¯ush. An alternative to adding solution is the use of a dry method of 15 N labelling. Stark and Firestone (1995) have used 15 N labelled nitric oxide gas to label the nitrate pool but the expense and toxicity problems associated with this methodology make its widespread use impractical. Alternatively, an inert powder could be used as the carrier of 15 N. We describe such a technique and compare the results with a more conventional wet method in soils from three dierent land uses. Soils were taken from three sites at Rothamsted Experimental Station, Hertfordshire: section 3, plot 5 (zero N fertilizer) of the Broadbalk continuous wheat experiment; unfertilized areas of the Park Grass continuous hay experiment; and Knott Wood, a mixed deciduous wood. These three sites are <1.0 km apart, the soils are silt to silty clay loams with a clay content between 21 and 28% in the topsoil (0±23 cm) and overlying clay-with-¯ints; they are chromic luvisols classi®ed as Batcombe series (Avery and Catt, 1995). Other soils were used to compare soil texture and were collected from two pastures at least 40 y old. Grasslands were located on a sandy colluvial brown earth (Stackyard series) at Woburn Experimental Farm, Woburn, and a silty clay loam (Bromyard series) at ADAS Rosemaund, Worcester. Wet 15 N isotopic dilution incubations to measure gross nitri®cation were conducted as laboratory incubations similar to those described by Barraclough and Puri (1995) and Watkins and Barraclough (1996). Subsamples of fresh soil (35.0 g) were weighed into glass jars (5 cm dia) so that the soil
662
T. W. Willison et al.
depth was approximately 2 cm. Thereafter 3 ml of 15 N labelled solution was added to the soil surface as K15NO3 (at 10.3 at.%) at a rate equivalent to ÿ1 5.0 mg NOÿ dry soil. This volume was 3 ±N g required to provide a relatively uniform distribution of solution throughout the soil sample to minimise errors associated with non-uniform distribution of 15 N. For the dry incubations, 15 N-labelled powder (3 g) was applied to the surface of the soil and the jars were lightly tapped to distribute the powder through the soil. The powder was prepared by dissolving K15NO3 (at 10.3 at.%) in a slurry of silica ¯our and deionised water which, after mixing for 1 h, was dried in an oven at 1058C for 24 h and milled to a ®ne talc using a disk mill (Tema, model T100). The resulting 15 N labelled powder contained the same amount of K15NO3 in 3 g as was present in 3 ml of solution (i.e. 5.0 mg N gÿ1 dry soil). In addition 3 ml of a solution containing (NH4)2SO4 was added as an additional treatment to the three soils from Rothamsted (Broadbalk, Park Grass and Knott Wood) at a rate equivalent to 5.0 mg N gÿ1 dry soil. This enabled comparison of net nitri®cation rates determined using a traditional short-term nitri®cation assay. ÿ The 15 N-to-14 N ratio of the NH+ 4 or NO3 soil extracts (3 replicates taken 1,2,3,4 and 8 d after addition of N and shaken for 1 h with 60 ml of 2.0 M KCl) were determined on an Europa mass spectrometer linked to a combustion analyzer (Europa Scienti®c Roboprep) after diusion onto acidi®ed glass wool disks (Brooks et al., 1989). Gross rates of nitri®cation were calculated using the formula of Barraclough (1991). Rates were calculated between 2 and 8 d, no remineralization of 15 NOÿ 3 was detected during this period. The incubation period for measurement of gross nitri®cation started 2 d after 15 N addition. This delay was necessary to allow the 15 NOÿ 3 in the powder to dissolve and reach an equilibrium with the soil NOÿ 3 ±N pool. For the same soils the amount and enrichment of the nitrate pool was not signi®-
Fig. 1. Comparison of net nitri®cation rates (mg N kgÿ1 dÿ1) showing the net change in the nitrate pool calculated during a 6 d incubation. The ®gure compares the rates calculated by 15 N isotopic dilution following addition of either (1) labelled powder, (2) solution or (3) by using a conventional nitri®cation assay with addition of ammonium sulphate (AS). Rates are means plus and minus standard errors (n = 3).
cantly dierent 2 d after application when the label was added in solution or powder form (data not shown). With the exception of the Broadbalk and Park Grass soils the gross nitri®cation rates were signi®cantly lower in the soils where 15 N was applied as powder (range: 90±580 mg N kgÿ1 dÿ1) compared to solution (range: 170±1200 mg N kgÿ1 dÿ1). This re¯ects the stimulation of mineralization following wetting (Table 1); there is a greater change in moisture content following solution addition (range: +27 to +70%) than powder (range: ÿ11 to ÿ16%). In the case of Broadbalk soil the lack of dierence in the gross nitri®cation rates following powder or solution application was attributed to the lack of NH+ 4 ±N as a substrate for ammonium oxidizers. When an NH+ 4 ±N source was applied to the three soils from Rothamsted the net rate of nitri®cation increased greatly in the Broadbalk soil and, to a lesser extent, in Knott Wood soil but there was no change in the Park Grass soil (Fig. 1). The lack of increase in Park Grass is because ammonium oxidizers were inhibited by low pH (4.5) of this soil (Johnston et al., 1986).
Table 1. Gross nitri®cation rates (ngross, mg N kgÿ1 dÿ1) and the change in gravimetric soil water content (w/w) after the application of 15 N in solution or powder form. Values in parentheses are standard errors (n = 3). Plus and minus values are percentage changes in moisture content following application of 15 N in solution or powder form Soil
Initial water content
15
N applied as solution
ngross Broadbalk
13.25
Knott Wood
24.56
Park Grass
41.80
Woburn
18.45
Rosemaund
19.00
0.17 (0.04) 1.20 (0.11) 0.17 (0.02) 0.70 (0.08) 1.00 (0.29)
water content 22.71 +70 35.00 +42 53.28 +27 28.35 +50 28.28 +50
15
N applied as powder
ngross 0.09 (0.03) 0.47 (0.03) 0.15 (0.02) 0.58 (0.03) 0.41 (0.10)
water content 12.03 ÿ13 22.16 ÿ11 37.80 ÿ11 15.45 ÿ16 16.27 ÿ15
15N Isotopic dilution technique
The rates of gross nitri®cation reported by the methods we describe in this paper are generally in the range reported by other workers. Rates of 5.3 mg N kgÿ1 dÿ1 (Gleyic cambisol), 2.0 mg N kgÿ1 dÿ1 (Gleyic podzoluvisol) and 1.7 mg N kgÿ1 dÿ1 (Rendzina) for cultivated soils using isotopic dilution and a 0±6 d incubation period were reported by Recous and Mary (1990). Davidson et al. (1992) measured gross nitri®cation rates using 15 N pool dilution with cores incubated in situ in open grass prairie (590±810 mg N kgÿ1 dÿ1) and grass under oak (Quercus wislizenii A.) in spring and winter (1.38±3.47 mg N kgÿ1 dÿ1). The dry method of 15 N application that we have described oers an alternative method for quantifying gross rates of nitri®cation where solution addition is not desired. The bene®ts of applying the 15 N isotopic dilution technique as a short-term nitri®cation assay are clear: a short incubation period, reduced alteration of soil moisture content and no risk of introducing a priming eect due to substrate addition. AcknowledgementsÐWe would like to thank James Wake®eld for assistance in 15 N analysis and Wendy Wilmer for colorimetric determination of ammonium and nitrate. Anne Bhogal kindly supplied the Rosemaund soil. This work was funded by the U.K. Ministry of Agriculture, Fisheries and Food. IACR-Rothamsted receives grant-aided support from the Biotechnology and Biological Sciences Research Council. REFERENCES
Ambus P., Mosier A. and Christensen S. (1992) Nitrogen turnover rates in a riparian fen determined by 15 N dilution. Biology and Fertility of Soils 14, 230±236. Avery B. W. and Catt J. A. (1995) The Soil at Rothamsted. Lawes Agricultural Trust Ltd., Harpenden. Barraclough D. (1991) The use of mean pool abundance's to interpret nitrogen-15 tracer experiments. I. Plant and Soil 131, 89±96.
663
Barraclough D. and Puri G. (1995) The use of 15 N pool dilution and enrichment to separate the heterotrophic and autotrophic pathways of nitri®cation. Soil Biology & Biochemistry 27, 17±22. Belser L. W. and Mays W. L. (1980) Speci®c inhibition of nitrate oxidation by chlorate and its use in assessing nitri®cation in soils and sediments. Applied and Environmental Microbiology 39, 505±510. Brooks P. D., Stark J. M., McInteer B. B. and Preston T. (1989) Diusion method to prepare soil extracts for automated nitrogen-15 analysis. Soil Science Society of America Journal 53, 1701±1711. Davidson E. A., Hart S. C. and Firestone M. K. (1992) Internal cycling of nitrate in soils of a mature coniferous forest. Ecology 73, 1148±1156. Fisk M. C. and Schmidt S. K. (1996) Microbial responses to nitrogen additions in alpine tundra soil. Soil Biology & Biochemistry 28, 751±755. Hart S. C., Stark J. M., Davidson E. A. and Firestone, M. K. (1994) Nitrogen mineralization, immobilization and nitri®cation. In: Methods of Soil Analysis, Part 2. Microbiological and Biochemical Properties, eds. R. W. Weaver, S. Angle, P. Bottomley, D. Bezdicek, S. Smith, A. Tabatabai and A. Wollum, pp. 985±1018. Soil Science Society of America, Madison. Johnston A. E., Goulding K. W. T. and Poulton P. (1986) Soil acidi®cation during more than 100 years under permanent grassland and woodland at Rothamsted. Soil Use and Management 2, 3±10. Lensi R., Mazurier S., GourbieÁre F. and Josserand A. (1986) Rapid determination of the nitri®cation potential of an acid forest soil and assessment of its variability. Soil Biology & Biochemistry 18, 239±240. Recous S. and Mary B. (1990) Partitioning of fertilizer N between soil micro¯ora and winter wheat crop. In Proceedings of the 1st Congress European Society of Agronomy, ed. A Scaife Colmar, p. 45. European Society of Agronomy. Robertson G. P. (1982) Nitri®cation in forested ecosystems. Philosophical Transactions of the Royal Society of London B 296, 445±457. Stark J. M. and Firestone M. K. (1995) Isotopic labelling of soil nitrate pools using nitrogen-15-nitric oxide gas. Soil Science Society of America Journal 59, 844±847. Watkins N. and Barraclough D. (1996) Gross rates of N mineralization associated with the decomposition of plant residues. Soil Biology & Biochemistry 28, 169± 175.