soil indicators of c and n transformations under pure and mixed grass–clover swards

European Journal of Agronomy 9 (1998) 157–172

Soil indicators of C and N transformations under pureand mixed grass–clover swards

G. Alvarez a,*, R. Chaussod b, P. Loiseau c, R. Delpy ca ENITA Clermont-Ferrand, Departement Agriculture et Espaces, Marmilhat, 63 370 Lempdes, France

b INRA, Laboratoire de Microbiologie des Sols, 7 Rue Sully, 21034 Dijon, Cedex, Francec INRA, Unite d’Agronomie, Domaine de Crouel 234 avenue du Brezet, 63039 Clermont-Ferrand, Cedex 2, France

Accepted 30 June 1998

Abstract

The changes occurring in the soil organic status under grassland swards after 3 years were examined as a functionof the initial soil nitrogen supplying capacity (SNSC: 135 or 225 kg N ha−1 year−1) and of the soil cover: bare soil,pure white clover, pure rye-grass or a mixture of both species. The amounts of C and N of the coarse particle sizeOM, the microbial biomass-C, the C and N in water extracts (microbial metabolites) and the C and N mineralizationin 28-day incubation tests were used as indicators of the transformation of the organic C and N inputs to the soilunder these contrasting managements. The part of the variation of these indicators explained by the initial soil Nsupplying capacity decreased from the metabolite-C to the coarse plant residues-C to the microbial biomass-C (MB-C)soil compartments. The part of the variation explained by plant cover increased in the same order. For each variable,the effect of soil fertility was maintained under pure grass and was reduced or suppressed under pure legume. Thesoil organic status under the mixed stand was similar to that of the pure clover and intermediate between that of thepure clover and the pure grass stands. The amount of N mineralization was similar with similar N amounts in theplant residues, whereas amounts of C in the plant residues and MB-C were in the order; pure clover<mixedstand<pure grass. Higher soil respiration and microbial specific respiration in incubation tests were seen in the puregrass stand. Soil organic status was examined as a function of the amounts of C and N and the C:N ratio of theorganic inputs and from their impact on the microbial activity in situ. The ratio of C mineralization to N mineralizationin laboratory incubations is proposed as an indicator of the dry matter and N harvests, of the degree of accumulationof the non-harvested C and N in the plant residues, and of the risk of nitrate leaching. The optimum value of thisindicator, about 14, was obtained under the mixed sward. © 1998 Elsevier Science B.V. All rights reserved.

Keywords: Microbial biomass; Soil water-extracts; Particle-size fraction; Organic matter; Respiration; Mineralization;Grassland; White clover; Rye-grass; C:N ratio; N leaching; Diagnosis

* Corresponding author. Tel: +33 473 98 1381; fax: +33 473 98 1380.

Abbreviations: DM, dry matter; MIX, mixture of Lolium perenne and Trifolium repens; OM, organic matter; RGp, pure rye-grass;SNSC, Soil nitrogen supplying capacity; WCp, pure white clover

1161-0301/98/$ – see front matter © 1998 Elsevier Science B.V. All rights reserved.PII S1161-0301 ( 98 ) 00034-3

158 G. Alvarez et al. / European Journal of Agronomy 9 (1998) 157–172

1. Introduction favours the accumulation of recently synthesized‘metabolites’ (Grace et al., 1993).

The concept of quality of substrate has beenStudies on rye-grass/white clover mixtures havemainly concerned above-ground C and N fluxes introduced in several studies on soil organic matter

dynamics. Grass and legumes supply the soil withsuch as dry matter (DM ) and N harvested, nitro-gen fixation, nitrogen transfer to the grass and the residues of a very different N concentration and

lignin content. Bosatta and Agren (1994) formal-competition between plant species (Soussana andArregui, 1995). However, the way in which ized the dynamics of C decomposition through a

series of parameters that all depend on the qualitygrass/clover mixtures depend on, and modify, thesoil organic matter status and the C and N cycles of the substrate. The parameters were the microbial

growth rate, the ratio of microbial production toremains poorly understood.In grazed grass–clover swards, the amount and assimilation, the transformation in quality of the

substrate and the mortality kinetics of the micro-quality of organic soil inputs from non-harvestedparts of the sward and from animal returns depend bial biomass. Incorporation of legumes into rota-

tions increases soil microbial biomass (Angerson the grass/clover ratio. In legume-associated andin legume-intercropped systems, the level of et al., 1993a; Kirchner et al., 1993; Harris et al.,

1994), soil enzymatic activity (Bolton et al., 1985;organic returns influences the accumulation ofcarbon (Williams and Haynes, 1990) and nitrogen Kirchner et al., 1993), soil respiration (Fraser

et al., 1988) and soil N availability (Francisin the soil compartments, and therefore, the subse-quent soil N supplying capacity (SNSC) and the et al., 1994).

Conversely, the amounts and the quality of planttransfer of the symbiotically fixed nitrogen to thecompanion grass or the succeeding crop (Stern, residues under grass/clover swards, receiving no

mineral N fertilizer, may be controlled by soil1993; Francis et al., 1994). Soil microbial biomassand microbial activities are strongly affected by factors such as the soil N supplying capacity. Soil

factors can also interact with soil inputs on thethe amount and quality of these organic inputs(Bosatta and Agren, 1994; Fraser et al., 1994). It microbial biomass and activity. Consequently,

both soil and vegetation quality may both controlis not clear how these parameters depend on soilcharacteristics and on organic inputs in order to the organic status of the soil and a number of

biological indicators of the C and N fluxes in thebe useful as indicators of the C and N cycles insuch ecosystems. soil ecosystem. Suitable parameters of soil OM

status may contain information on the amountsSeveral authors have shown a positive relationbetween microbial biomass-C and amount of and quality of the soil OM and of the recent

organic inputs.organic substrates derived from plant roots. Undera perennial rye-grass/white clover pasture, the evo- Soil OM consists of fractions with different

turnover times. For example, in the 3-year experi-lution of microbial biomass-C during the growingseason reflects the short-term pattern of root pro- ment of Jensen (1994), the labelled pea residue-N

was recovered in two main soil compartmentsduction (Bristow and Jarvis, 1991). Under annualcrops, the positive correlation between microbial differing in their turnover time, the microbial bio-

mass and the more recalcitrant residual organicbiomass-C and crop yields (Insam et al., 1991) onthe time-scale of a year can be attributed to an N. The young labile OM fractions involved in the

soil nitrogen supplying capacity represent only aincrease of carbon allocation to plant roots(Russell, 1977). Similarly, the higher microbial small part of the total N and characterize the

recent impact of the sward on soil organic matter.biomass under perennial grassland than underannual crops (Patra et al., 1990; Robertson et al., Labile OM must be examined using complemen-

tary methods (Biederbeck et al., 1994) and two1993; Fraser et al., 1994) and under no-till systemscompared with ploughed systems is related to a major pathways involved in short-term soil organic

matter accumulation considered (Fig. 1). In themore abundant root phytomass (Lynch andPanting, 1980). An absence of soil cultivation also detritical pathway, especially important in grass-

159G. Alvarez et al. / European Journal of Agronomy 9 (1998) 157–172

with mixed swards, in order to compare the impactof the long-term acquired fertility and that of themore recent sward activity. Measuring dry matteryields, harvested N, urine and faeces inputs, symbi-otic nitrogen fixation and nitrate leaching duringthe 3 years made it possible to estimate soil C andN inputs and N outputs. The final status of thedifferent organic matter compartments was studiedon the basis of the two major pathways describedabove and related to the estimated soil C and Ninputs and N outputs.

2. Materials and methods

2.1. Experimental site and treatments

The long-term field experiment used for thisstudy consisted of a set of 50 lysimeters (3 m2surface area), located at Theix (Puy de Dome,France), 890 m above sea level, on a slightly acid

Fig. 1. Fate of organic inputs. The detritical and biological path- sandy soil (15% clay, 3.9% OM, C:N 9, pH 6).ways involved in soil organic matter transformation and

The average annual rainfall and temperature overaccumulation.the experiment were 746 mm and 7.6°C, respec-tively. Three forage cropping systems (annuallands, the most resistant part of the organic inputscrops, rotation including 4 years of temporary ley,is accumulated as plant residues in the lighter andpermanent grassland) at four N fertilization levelscoarser fractions of the soil organic matter. Thesewere run during 20 years from 1968, and resultedfree plant residues, measured by particle size frac-in different soil N balances. In 1988, the soils were

tionation (Christensen, 1992), become physically tilled and kept bare for 3 years until 1991. Theor biologically split into finer particle-size frac- nitrate leaching was measured under bare soilstions. No-till systems, such as grasslands, are char- during three successive drainage periods from 1988acterized by an accumulation of these coarse plant to 1991 (Loiseau et al., 1994; 1995). During theresidues (Angers et al., 1993b; Alvarez et al., drainage period and before this1995). In the biological pathway, the most degrad- experiment, soil fertility ranged from 94 toable part of the plant residues and animal returns 227 kg N ha−1 year−1 according to the previousis metabolized by the microbial biomass, which treatments.allows the accumulation of secondary metabolic In August 1991, four managements were appliedproducts (Pool II or ‘humads’, Molina et al., 1983; to these soils. Pure rye-grass (RGp), pure whiteassociated microbial products, Grace et al., 1993), clover ( WCp) or a mixture of both species (MIX )which represent about 30% of the soil OM were sown in 15-cm spaced lines, except in three(Nicolardot et al., 1994). lysimeters, which were kept bare as controls. The

Our objective was to assess the 3-year effect of swards were managed in a simulated rotationalthe grass/legume ratio and of the initial soil N grazing. The swards were cut four or five timessupplying capacity on the subsequent status and during each growing season. The harvested mate-activities of soil organic matter compartments. In rial was hand-sorted by species, measured for DMlysimeters, two soils differing in their initial soil yield and grass/clover ratio and analysed for its Nnitrogen supplying capacities, were taken as bare content. the DM and N harvests at each cut were

used to calculate the amounts of faeces and urineor as sown with pure grass, with pure clover or


that sheep would have returned to the soil under and sieved to 2 mm, for measuring carbon andnitrogen in the coarser particle-size fractions andgrazing. Natural sheep faeces and reconstitutedin the total soil.urine were applied to simulate animal returns. No

mineral nitrogen was supplied to the swards, and2.3. Microbial biomass and metabolites, andsoil nitrogen inputs in the grazed ecosystem camemicrobial activitiesexclusively from symbiotic nitrogen fixation in the

legume. P and K fertilizers were applied in non-Carbon in the microbial biomass was measuredlimiting quantities. Dry matter and N harvests

in three replicates by the fumigation-extractionwere measured from 1992 to 1995. During thismethod (Chaussod et al., 1988), on the bulked soilperiod, the water drainage and the N-NO3 concen-of each treatment. After removing the supernatanttration of the drainage water were measured everythat contained the extractable microbial biomass,2 weeks to calculate N-NO3 leaching.the residual soil was used to determine themetabolite-C by autoclaving the pellet in water for2.2. Soil sampling16 h at 120°C (Lemaitre et al., 1995a). By 14Clabelling, the same authors have shown the micro-Two levels of initial soil nitrogen supplyingbial origin of this fraction, composed of aminocapacity (SNSC) were used for the soil measure-acids and neutral sugars (Lemaitre et al., 1995b).ments 135 (SNSC-) and 225 (SNSC+)

Soil respiration and net N mineralization werekg N ha−1 year−1 (Table 1). Eight 80-cm-deepmeasured in three replicates during short-termlysimeters were chosen, corresponding to threelaboratory incubations (28 days at 28°C) of thesward types and one bare soil at each SNSC. Thefresh soil maintained at 85% of field capacity. Thesoils were collected in late April 1995 from the topCO2 accumulated in NaOH traps was measured

20 cm of the soil profile, i.e. in the fourth year after 7, 14, and 28 days of incubation and analysedafter sowing, using a mixture of 15 cores taken at colorimetrically in continuous flow (Chaussodrandom with an earth drill (45 mm in diameter). et al., 1986). The accumulated mineralA subsample of each composite sample was dried N (NH+4 , NO−2 , NO−3 ) was extracted by 1 M KClovernight at 100°C for total C and N analysis. and measured by colorimetry in a continuous flowThe remainder of the fresh soil cores were sieved analysis (Technicon).to 5 mm, homogenized and stored for 3 days at4°C prior to measuring the microbial biomass-C, 2.4. C and N in the coarse particle-size fractionsthe metabolites and the biological activities. InDecember 1995, new soil samples were collected Light and coarse particle-size fractions of OM

were fractionated according to a method adaptedin triplicate from the upper 20-cm layer, air-dried

Table 1N fluxes during the cultivation period

SNSC (kg N ha−1) Leaching (kg N ha−1) DM harvest (t ha−1) N harvest (kg N ha−1)SNSC Poor Rich Poor Rich Poor Rich Poor Rich

Bare soils 144 230 344 484 0.0 0.0 0 0Pure grass 138 224 9 7 6.7 17.7 122 394Pure clover 133 224 142 176 20.3 24.8 809 996Mixture 132 222 33 20 30.4 36.2 898 972Associated grass 19.0 26.7 471 612Associated clover 11.4 9.5 427 360

SNSC: initial soil N supplying capacity measured by N leaching during the last drainage period 1990–1991 before sowing. Poor andrich correspond to 135 and 225 kg N ha−1 year−1, respectively. Harvests: cumulative DM or N harvests during the years 1992, 1993and 1994. Leaching: total NO3-N leaching between 1992 and 1995.


from Williams (1983). Air-dried soil samples 3.2. Microbial biomass(600–900 g) were wet-sieved with a continuous

At sowing in 1991, the initial microbialwater flow through a series of three brass sievesbiomass-C (MB-C) in the bare soils was 221 andwith 1-, 0.2- and 0.05-mm mesh sizes. The free359 mg C kg−1 in the poor (SNSC-) and richmacro-OM fraction, recovered in the 1-mm and(SNSC+) soils, respectively. This decreased to0.2-mm sieves, was separated from the sand by166 mg C kg−1 (−25%) and to 184 mg C kg−1densimetry in water. The aggregated organo-min-(−49%) in 1995 (Table 2). The positive effect oferal material recovered on the last sievethe initial soil-nitrogen supplying capacity on the(50–200 mm) was washed until the water was clear.MB-C decreased with time from +62% in 1991 toThe two macro-OM fractions of plant residues+11% in 1995. In 1995, the SNSC accounted forwere, respectively, named Fr1000 and Fr200 andonly 4% of the total variability of the microbialthe aggregated organo-mineral fraction, Fr50.biomass, as opposed to 90% for the type of cover.Each particle-size fraction was dried at 40°C forCompared with bare soil of the same SNSC, the2 days, weighed, ground and analysed for organicpresence of plants increased the MB-C from 102C and total N content using an automated elemen-to 175%. Statistical analysis (ANOVA) showed atal analyzer (Fisons 1500).significant interaction between the SNSC and thesoil cover (P<0.0001). The effect of the initialSNSC on the MB-C remained high in RGp(+34%), positive under MIX (+10%) and disap-

3. Results peared under WCp (Table 2).

3.1. N fluxes during the cultivation period3.3. Metabolite-C

In the absence of mineral N fertilizer, the yieldswere low in the pure rye-grass (RGp) and higher In the bare soils, the SNSC effect was higher on

the metabolite-C (+42%) than on the MB-Cin the pure white clover ( WCp) swards (Table 1).The dry-matter harvest (DM) in the mixture (+11%). In 1995, SNSC still explained 58% of the

total variation of the metabolite compartment. In(MIX ) exceeded the sum of the harvests of thepure swards by 13% at the low initial soil N the presence of the swards, SNSC increased the

metabolites by 34% ( WCp) to 65% (MIX ). Soilsupplying capacity (SNSC-) and was 15% lower atthe high initial soil N supplying capacity cover accounted for 36% of the total variation. As

observed for the MB-C, the metabolite-C increased(SNSC+). The N removed in MIX harvests wasalways lower than the sum of N harvests in pure in the presence of a stand, but this increase, relative

to bare soil of the same SNSC, was only from 33stands (−4% at SNSC-, −30% at SNSC+). Theeffect of the initial soil N supplying capacity was to 61%. The metabolite compartment was slightly

larger under WCp than under RGp for both levelsmaximum for RGp, increasing the DM harvest by164% and the N harvest by 223%. The presence of SNSC (Table 2).

Statistical analysis (ANOVA) showed a signifi-of clover reduced or negated the effect of the soil-nitrogen supplying capacity. The SNSC effect fell cant interaction between sward type and initial

SNSC. Under MIX, the SNSC increased theto 22% (DM) and 23% (N ) in WCp, and to 19%(DM) and 8% (N) in MIX (Table 1). N leaching metabolites 1.8 and 1.7 times more than under

WCp and RGp, respectively. In the poor soils, thewas maximum under bare soil, moderate in WCpand negligible in RGp. Adding the legume to grass metabolites were slightly higher under WCp than

under RGp and MIX. In the rich soils, the highest(i.e. MIX ) significantly, but poorly, increased Nleaching compared with RGp (Table 1, statistical amounts of metabolite-C were observed under

MIX.analysis not shown).


Table 2Soil organic status and mineralization in incubation tests: effect of soil cover and initial soil N supplying capacity

(A)

Bare soil Pure rye-grass Pure clover Mixture

TreatmentsPoor Rich Mean Poor Rich Mean Poor Rich Mean Poor Rich Mean LSD

VariablesMB-C (ppm) 166a 184b 175a∞ 378c 507f 443c∞ 384c 373c 378b∞ 416d 458e 437c∞ 17Metabolites 905a 1286cd 1096a∞ 1226b 1710e 1468b∞ 1310d 1755f 1532c∞ 1253bc 2072g 1662d∞ 50

(ppm)C mineralization 169a 214b 191a∞ 627e 838f 732d∞ 509c 530c 519b∞ 579d 615e 597c∞ 44

(ppm)N mineralization 17a 19a 18a∞ 26b 46c 36b∞ 47c 53d 50d∞ 45c 45c 45c∞ 7

(ppm)Accumulated C in:Residues: Fr1000 42a 47a 44a∞ 355b 629d 492b∞ 434bc 560cd 497b∞ 476c 501c 488b∞ 137

(ppm)Residues: Fr200 112a 148a 130a∞ 530a 701a 616b∞ 837ab 1557b 1197c∞ 798ab 1010ab 904b∞c∞ 834

(ppm)Aggregates: Fr50 2604a 2204a 2404a∞ 3060a 2246a 2653a∞ 3958a 3825a 3891a∞ 2908a 4982a 3945a∞ 2801

(ppm)Fr200/Fr1000 3.80c 2.51bc 3.16b∞ 1.54ab 1.12a 1.33a∞ 1.93ab 2.76bc 2.34a∞b∞ 1.67ab 2.06ab 1.87a∞ 1.35C:N of:Metabolites 9.3a 10.0ab 9.6a∞ 9.8ab 9.7ab 9.7a∞ 10.4b 9.9ab 10.1a∞ 10.0ab 9.7ab 9.8a∞ 0.8Residues: Fr1000 21.7b 16.2a 18.9b∞ 26.1c 29.1c 27.6c∞ 14.8a 14.9a 14.8a∞ 17.6ab 17.2ab 17.4a∞b∞ 5.5Residues: Fr200 18.3bc 15.6ab 16.9b∞ 19.4c 22.8d 21.1c∞ 13.5a 14.0a 13.7a∞ 16.5abc 16.5abc 16.5b∞ 3.3Aggregates: Fr50 11.0a 10.8a 10.9a∞ 11.6a 11.7a 11.7a∞ 10.7a 11.1a 10.9a∞ 12.4a 11.5a 11.9a∞ 2.7

(B)

Biological pathway C accumulation in the C:Ndetritic pathway

Effect on MB-C Met-C C min N min Fr1000 Fr200 Fr50 Fr200/- Metabolites Fr1000 Fr200 Fr50Fr1000

ofSNSC 4**** 58**** 3.5**** 6**** 6*** NS NS NS NS NS NS NSSoil cover 90**** 36**** 92**** 78**** 83**** 57*** NS 44* NS 79**** 74**** NSSNSC cover 5**** 5.8**** 3.5**** 8**** 6** NS NS NS 28** 8** 13*** NS

(A) Means and LSD; means within a column followed by the same letter are not significantly different at the 95% confidence level;see text for the number of replicates; xppm=mgx kg−1drysoil.(B) ANOVA: significance level (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001), and percentage of the total variation explainedby the factors, Met-C, Cmin and Nmin. These are abbreviations of metabolite-C, C mineralization and N mineralization, respectively.

3.4. C and N mineralization whereas soil cover had a large effect (92% of thevariance). Compared with bare soil of the sameSNSC, the presence of plants increased soil respira-Soil respiration during laboratory incubations

exhibited trends similar to those observed for the tion by 148% to 292%. At both SNSC levels, soilrespiration increased significantly in the order baremicrobial biomass. The initial SNSC had a limited

impact on respiration (3% of the variance), soil<WCp<RGp (Table 2). Statistical analysis


(ANOVA) showed a significant interaction coarse fractions. C or N accumulated in bothFr200 or Fr1000 was much greater under thebetween the initial SNSC and soil cover. Soil

respiration in bare soils was 26% higher in the rich stands than under the bare soils. Under the stands,it was significantly greater under WCp than underthan in the poor soil (Table 2). The positive effect

of the SNSC on soil respiration was high under RGp and similar in WCp and in MIX. Soil fertilityinteracted with soil cover only in the case of theRGp (34%), low under MIX (6%) and absent

under WCp. The increase in soil respiration due coarse macro-OM fraction (Fr1000). The SNSCstimulated carbon accumulation under RGp butto the SNSC was 4.7 and 5.9 higher under RGp

than under bare soil and MIX, respectively. not in the presence of clover (Table 2).The C:N ratio of the Fr50 fraction was neverNet N mineralization during laboratory incub-

ations was 51–182% higher under the planted soils affected by the treatments. It decreased signifi-cantly with particle size under RGp, but not inthan under the bare soils. Responses to SNSC

differed significantly between soil cover (Table 2). the presence of clover (Table 2). The C:N ratio ofthe fractions Fr1000 and Fr200 decreased fromNo SNSC effect was observed in the bare soils. In

the planted soils, initial SNSC significantly RGp to WCp and was intermediate for MIX(Table 2). In the presence of clover, the SNSCincreased N mineralization by 76% under RGp,

11% under WCp, and showed no effect in MIX. showed no mean effect on the C:N ratio, whateverthe fraction (Table 2).The SNSC effect on net N mineralization was

particularly high under RGp, where the absoluteincrease was 13 and 3.7 times higher than in the 3.6. Microbial biomass, activity and the coarse

organic fractionbare soils and under WCp, respectively. In thepoor soils, the presence of clover increased the Nmineralization. In the rich soils, the highest value The MB-C was better correlated with the carbon

(Table 3 and Fig. 2a) than with the nitrogen levelwas measured under WCp, and no significantdifference was seen between the MIX and RGp in Fr1000 (Table 3 and Fig. 2b). At a similar level

of N residue, MB-C was higher under RGp, intreatments.keeping with the higher C:N ratio of the residues.The MB-C (mg C kg soil−1) increased with the C3.5. Organic residuescontent and the C:N ratio of the Fr1000(mg C kg soil−1) according to the multiple regres-Accumulation in Fr1000, Fr200 and Fr50 repre-

sented 1.5, 2.9 and 13.1% of the total soil C and sion:0.8, 1.8 and 12.1% of the total soil N, respectively.

MB−C=0.5CFr1000+7C:NFr1000 (r2=0.86, n=8).The amounts of C in the different particle-sizefractions were positively correlated: r2=0.49 The correlation between the C mineralization

and the amounts of C in the plant residues (Table 3(P<0.001) between the amounts of C in Fr1000and Fr200, and r2=0.24 (P<0.05) between the and Fig. 3a) was not as high as the correlation

between the N mineralization and the amounts ofamounts of C in Fr200 and Fr50. The same patternwas observed for the amounts of N: r2=0.63 N in the residues (Table 3 and Fig. 3b). At a

similarly available C level in the Fr1000, the(P<0.0001) between Fr1000 and Fr200, andr2=0.24 (P<0.05) between Fr200 and Fr50. in-vitro soil respiration was higher in the soils

cultivated with pure rye-grass.However, no correlation was observed between theamounts of C or N between Fr1000 and Fr50. The C mineralization was positively correlated

with the MB-C (Table 3). However, the slopeThe treatments explained 95% of the variabilityof the C accumulated in the Fr1000 and 57% in representing the specific microbial respiration

activity was higher in the case of RGp than in thethe Fr200, whereas no significant effect of thetreatments was observed on the aggregated OM presence of clover (Fig. 4a). Conversely, the net N

mineralization to MB-C ratio was higher underfraction Fr50 (Table 2). Soil cover was a majorfactor affecting the accumulation of carbon in the clover (Fig. 4b).


Table 3Correlation between the variables of the soil organic status and activities

Metabolite-C Metabolites-N C Fr1000 N Fr1000 Cmin Nmin

MB-C NS NS 0.95*** 0.75* 0.97*** 0.82*Metabolite-C — 0.99**** 0.71* NS NS NSMetabolite-N — — NS NS NS NSC Fr1000 — — — 0.87** 0.92** 0.92**N Fr1000 — — — — NS 0.97***Cmin — — — — — 0.71*

Correlation coefficients (r) calculated on the average values of eight treatments and significance level (*p<0.05; **p<0.01; ***p<0.001;****p<0.0001); Cmin and Nmin are the abbreviated forms of C and N mineralizations, respectively.

Fig. 2. Microbial Biomass-C as a function of the coarse OM fraction. (A) Soil C in the Fr1000 fraction. (B) Soil N in the Fr1000fraction. Open and solid symbols represent poor soils (135 kg N ha−1 year−1) and rich soils (225 kg N ha−1 year−1), respectively.The regression lines for each cover include the bare soils.

Fig. 3. Mineralization as a function of the coarse OM fraction. (A) Respiration (C mineralization) in incubation test vs. carbon inthe Fr1000 fraction. (B) N mineralization in incubation test vs. nitrogen in the Fr1000 fraction. Open symbols: poor soils(135 kg N ha−1 year−1); closed symbols: rich soils (225 kg N ha−1 year−1). The regression lines for each cover include the bare soils.

4. Discussion cultivation. The absence of soil inputs into thebare soil resulted in a decrease in the amounts ofeasily decomposable organic substrates and the4.1. Microbial biomassMB-C between 1991 and 1995. On the contrary,3 years of perennial swards increased the MB-C,Microbial biomass depends on the available

organic substrate, either initially accumulated or as already shown in the literature in comparisonsof arable soils and pastures (Edwards, 1983; Fraserintroduced into the soil by sward activity during


Fig. 4. Specific activity of the microbial biomass. (A) Respiration (C mineralization) in incubation test vs. microbial biomass-C(specific respiratory activity or qCO2). (B) N mineralization in incubation test vs. BM-C. Open and solid symbols represent poorsoils (135 kg N ha−1 year−1) and rich soils (225 kg N ha−1 year−1), respectively. The lines represent the mean specific activities undereach sward.

et al., 1994). Both sward type and the initial SNSC mixture in the grass harvest than in the cloverharvest. As a result, not only the amounts ofinfluenced the level of microbial biomass-C. The

preservation of the SNSC effect under RGp shows available carbon but also the quality of the sub-strate supplied by the plant species must be consid-that the pure grass sward maintained the initial

soil characteristics as favourable for the microbial ered when explaining the level of microbialbiomass. With similar quantities of carbon alloca-biomass. Soil N supplying capacity did not directly

determine soil MB-C but did do indirectly by tion to the soil, the quality of organic inputs fromthe clover limits the microbial biomass size com-stimulating grass growth, and presumably by allo-

cating organic substrates to the soil (Robertson pared with inputs from the grass.et al., 1993). Under WCp, the disappearance ofthe initial SNSC effect on the microbial biomass 4.2. Metabolitesshows that different soil N supplying capacitiesresulted in similar substrate availabilities for The residence time of the metabolite compart-microbial growth. This might be due to an ment is apparently long enough to reflect theincreased symbiotic fixation in the poor soil, result- previous soil N supplying capacity, yet shorting in similar clover growth. In the same way, in enough to depend on the recent activity of thethe mixture, the SNSC effect was reduced in com- swards. Thus, a double determination of the accu-parison with pure grass because the presence of mulation of the metabolites as affected by theclover improved grass nutrition in the poor soil initial SNSC and by recent ecosystem activity canand reduced the positive SNSC effect on the grass be represented by the double linear correlations:DM harvest (+40% in MIX vs. +164% in RGp;

Metabolite−C=1.8 MB−C+4.54 SNSCTable 1) and presumably also on the carbon alloca-tion from grass to soil. However, the SNSC effect (r2=0.84, n=8)was maintained by its correlation with higher grass

Metabolite−C=1.1 CFr1000+5.7 SNSCgrowth rate. Consequently, the mean activity ofthe mixed clover did not completely compensate (r2=0.80, n=8).for the initial differences in the soil SNSC, owingto competition with the companion grass. According to Lemaitre et al. (1995a,b), the

metabolite compartment results from the microbialAssuming that organic inputs from the swardare positively related to the DM harvests, the ratio activities in situ and represents a labile organic

pool, intermediate between the microbial biomassof the microbial biomass to the DM harvestsremained higher in the pure grass than under the and humic substances. Consequently, their C:N

ratio was nearly constant at 9.8, and was onlypure clover. The ratio was more constant in the


increased slightly by the percentage of clover C substrates remained available to the microbialbiomass in the rich than in the poor soil. However,(Table 2). The small amounts of metabolites after

incubation in the bare soils and their decrease N mineralization as well as amounts of metaboliteswere similar, via the faster depletion of the labileduring laboratory incubation (Grace et al., 1993;

Lemaitre et al., 1995a) show that they supply a fractions in the richer soil during the 3 years ofincubation in situ, and as shown by the higher Nlarge part of the organic substrates used by the

microbial biomass. As a result, the metabolites leaching (Table 3). In contrast, the positive effectof plant cover on C and N mineralization is duemay be involved in the soil-N supplying capacity.

Nevertheless, we found no relationship between to an increased availability of new organic sub-strates accumulated during the cultivation period.soil-N mineralization in short incubation tests and

the level of metabolites. Respiration of the grass- Under RGp, the soil N fertility strongly influencesC and N mineralization and confirms that grassland soils was mainly related to the amounts of

plant residues, whose residence time was much growth and the input of grass material to the soildepend on soil N fertility. In the presence of clover,shorter. The maintenance of the initial SNSC effect

after four years is in accordance with the long the reduced or zero effect of the initial SNSCconfirms that the carbon and nitrogen fixed by theresidence time of the metabolites. This shows that

the metabolites were not used as substrates in the legume and allocated to the sward leads to similarfinal substrate availabilities for the microbial bio-short-term incubation in the soil sampled under

the swards, but only in the bare soils. As shown mass. Consequently, as for the clover harvests, soilinputs from the clover were inversely correlatedby the correlation with the SNSC, the metabolites

consist essentially of a storage compartment of with the initial soil N supplying capacity.In the laboratory, the mineralization of decom-labile soil OM.

Both the amount and quality of the organic posable substrates is stimulated by favourable tem-perature, moisture and oxygenation conditionsinputs affect microbial turnover and activity

(Bosatta and Agren, 1994) and the accumulation generated by soil disturbance. The effect of soildisturbance has been confirmed in the field byof metabolites of microbial origin during the culti-

vation period (Molina et al., 1983; Nicolardot comparing no-till with plough–tillage systems(Angers et al., 1993b; Alvarez et al., 1995;et al., 1994; Lemaitre et al., 1995a,b). The organic

inputs were characterized by high C:N ratios in Franzluebbers et al., 1995). Therefore, respirationintensities in incubation tests do not necessarilygrass (26–29) and low C:N ratios in clover (15)

(Table 2). The larger amounts of metabolites reflect differences in soil respiration between treat-ments in the field. Biederbeck et al. (1994) andfound in the presence of clover than in grass

suggest that the high N content of the organic Alvarez et al. (1995) ascribed high laboratorymineralization to an accumulation of labile formsresidues from clover induces a faster turnover rate

and an increased activity of the microbial biomass. of organic matter but did not conclude that therewas any increase in soil respiration in the field. InThe positive interaction between clover and grass

effects found on the richer soil implies that the consequence, the larger respiration measured inthe laboratory in RGp implies a lower in-situaccumulation of metabolites was N-limited under

RGp and C-limited under WCp. The synergy mineralization of the carbon inputs. Conversely,the lower in-vitro respiration of the soils cultivatedbetween the organic substrates from grass and

legume results in an optimum C:N ratio of 17–18 in the presence of clover corresponds to a lowercarbon accumulation in situ. This latter effect mayfor the accumulation of the metabolites.be due to either fewer carbon inputs from theclover than from the grass or to a higher soil4.3. C and N mineralizationactivity in the field in the presence of clover.

Concerning N mineralization, the highestIn bare soil, the positive effect of SNSC on thesoil respiration during laboratory incubation amounts of mineralized N measured under WCp

are attributable to high organic-N contents and(+26%) shows that more previously accumulated


low C:N ratios of the organic inputs from the in mineral N. The effect of grass vs. clover playsa role here similar to the no-tillage vs. tillage effectlegume, and presumably to low soil N immobiliza-

tion. On the contrary, the high C:N ratio of the in cultivated fields by decreasing the degradationrate of the residues, and increasing their accumula-grass inputs in addition to high N immobilization

may explain the lower amounts of mineralized N. tion rate at a given soil availabitlity (Angerset al., 1993b).

4.4. Organic residues4.5. Microbial biomass, activity and the coarseorganic fractionCorrelations between the C or N in the succes-

sive particle size fractions reflect the continuum ofthe detritical pathway. Compartments of decreas- Similar net N mineralization was observed for

similar N levels in the Fr1000. However, at aing size represent cohorts of plant residues atdifferent stages of degradation. Under grass, this similar level of available C in the Fr1000, the

in-vitro soil respiration was higher under pure rye-continuum is characterized by a decrease in theC:N ratio with decreasing particle size (Hassink grass. This satisfies the N-requirements of the

microbial biomass in a N-poor substrate situationet al., 1993; Preston et al., 1994). The free coarseorganic residues represented only 4% of the total (Houot and Chaussod, 1995) and corresponds to

an increase in the specific respiration activity ofsoil C. The final accumulation occurred in theaggregated fraction Fr50 (13% of total soil C ). As the microbial biomass.

In incubation tests, the respiration of agricul-a result, plant residues changed from free OM tothe aggregated fractions, leading to a physical tural soils is positively correlated with the MB-C

(Vekemans et al., 1989). In our grassland soils,protection by minerals (Mcgill and Myers, 1987;Breland, 1994) associated with an increase in the the relation remains significant (Table 3), but the

microbial activity adapted itself to the contrastedresidence time of the compartment.This representation makes it possible to interpret qualities of the substrates.

A given mineralization was obtained at a similarthe effects of the sward. As the treatments affectedonly the first two stages of the residues without N content in Fr1000 under pure grass and in the

presence of clover ( Wcp and MIX ), but at aany effect on the aggregated fraction, the Fr200-to-Fr1000 ratio is a useful indicator of the activity of higher available C, soil respiration, MB-C and

microbial specific respiration under pure grass.the detritical pathway. Due to the absence oforganic inputs, bare soils showed the most Carbon substrate availability determines or

limits the level of the microbial biomassadvanced stage of residue evolution. This is inaccordance with the greater degree of decomposi- (Robertson et al., 1993), but the availability of

substrate N determined the ratio between net Ntion of the organic matter of the sand fraction incultivated soils than in native grassland or forest mineralization and MB-C (Fig. 5). Consequently,

the specific respiratory activity in the incubationsoils (Preston et al., 1994; Gregorich et al., 1995).Among grassland stands, the presence of clover test depended on the relative N availability, i.e. on

the C:N ratio of the substrate.increased the accumulation and evolution of resi-dues because increased inputs by symbiotic N The microbial biomass is not affected by the soil

disturbance that occurs in the incubation testfixation led to an increase in both the amountsand in the N concentration of the soil organic (Grace et al., 1993). In another respect, the micro-

bial specific respiration (qCO2) has been reportedmatter inputs. In RGp, the higher soil N availabil-ity resulted in a larger Fr1000, as does mineral to decrease with the fungal-to-bacterial biomass

ratio (Sakamoto and Oba, 1994). It is doubtfulfertilizer application on the light OM fraction incontinuous cropping systems (Janzen et al., 1992; that the fungal-to-bacterial ratio increased in the

presence of clover, as compared with the soil underBiederbeck et al., 1994; Gregorich, 1996). Thedegradation rate of Fr1000 was slowed both by the unfertilized grass. These statements imply that

the specific microbial activity measured in labora-the high C:N ratios and by the reduced availability


correlated with the C:N mineralization ratio(Fig. 6b). This identifies the coarse fraction Fr1000as the substrate assimilated by the microbial bio-mass. C and N mineralization were more highlycorrelated to the amounts of C and N, respectively,in the plant residues than to the amounts of C andN in the metabolites, as discussed previously.

4.6. Dry-matter production and accumulation offree soil organic matter

The amounts of plant residues did not give agood indication of the amounts of litter inputsfrom the sward. They result from a balance

Fig. 5. Relation between the N content of the Fr1000 and between residue inputs, persistence, and decompo-the N mineralized per microbial biomass unit under the sition as determined by the soil environment andswards. Open and solid symbols represent poor soils

the nature of the residues. The harvests measured(135 kg N ha−1 year−1) and rich soils (225 kg N ha−1 year−1),during the 4 years can be used as an indicator ofrespectively. The relation between the mean N mineralization

per unit of biomass-C and the amounts of N in the Fr1000 is the soil C inputs. The relation between the harvestssimilar whatever the sward. Another factor than N in the and the accumulation in the Fr1000 shows thatFr1000 is involved under the bare soils. the accumulation rate of the C inputs must have

been higher under RGp than in the presence ofclover (Fig. 7a), whereas the accumulation rate oftory conditions is not driven by the size or the

composition of the microbial biomass but depends N in Fr1000 would have depended first on the soilN inputs (Fig. 7b).primarily on the quality of the soil organic matter

accumulated in the field. Such an increase in The higher rates of C accumulation under thepure grass is attributable to the higher C:N ratiospecific respiration activity has been shown by

Houot and Chaussod (1995) in the unfertilized of the soil organic inputs, limiting net N availabil-ity through N immobilization (Jenkinson, 1981).treatments of a long-term field experiment.

The in-vitro C:N mineralization ratio increased The lack of available N for microbial needs limitsthe soil microbial activity, in agreement with thefrom WCp to MIX and RGp (Fig. 6a). The C:N

status of the coarse fraction >1 mm was positively results establishing a lower evolution rate of the

Fig. 6. Relation between C and N status and activities. (A) Relation between C mineralization and N mineralization in incubationtests. (B) Relation between the C:N ratios of the plant residues and the ratio C:N mineralization. Open and solid symbols representpoor soils (135 kg N ha−1 year−1) and rich soils (225 kg N ha−1 year−1), respectively. The lines represent the mean ratios undereach sward.


Fig. 7. Relation between the DM and N harvests, and the C and N contents of the plant residues. (A) Relation between the DMharvests (1992–1994) and the C accumulation in Fr1000. Open and solid symbols represent poor soils (135 kg N ha−1 year−1) andrich soils (225 kg N ha−1 year−1), respectively. The regression line includes all the treatments.

plant residues in the detritical pathway. This leads time. The residence time of the plant residuesdecreased from RGp to MIX and pure legume.to an in-situ accumulation of metabolizable C.

Other co-limiting factors such as soil oxygenation The bare soils were characterized by the poorestN contents at the more advanced stages of residueand soil aggregate structure (Cambardella and

Elliott, 1994) must be involved in that protection, evolution.These indicators, based on a set of diversifiedwhich is removed when the soil is disturbed in

laboratory incubations. Consequently, the same situations, allow an a-posteriori diagnosis of grass-land productivity and its risks for the environment.high C:N ratios of the substrates could be responsi-

ble both for lowering the specific microbial activity No simple indicator exists for a triple diagnosis ofdry matter harvest, N harvests and N leaching.under pure grass in the field and for its increase

in laboratory conditions. Tests of N mineralization or the MB-C alone giveno direct indication of the dry matter or N produc-tivity. One of the best indicators is given by the4.7. Soil indicatorsratio between C:N mineralization in laboratoryincubation tests. The DM an N harvests are maxi-These results help to assess some indicators of

soil OM dynamics and C and N cycle activities mized at the optimum of the C:N mineralizationratio near 14 (Fig. 8a). This corresponds to aunder perennial grassland ecosystems. Among the

biological variables, the microbial specific respira- minimum rate of accumulation of the litter inputsand to a small risk for drainage water (Fig. 8b).tory activity and the N mineralized per biomass

unit, as measured in the laboratory, provide the Values of the C:N mineralization ratio below 14do not induce a higher dry matter or nitrogenmost useful information on the limitation of soil

activity by N and C, respectively. Under pure harvests, but increase the N contents of the resi-dues and the nitrate leaching (Fig. 8b). Values ofunfertilized grass, a high specific respiration reflects

an adaptation to N-limited conditions. Under the C:N mineralization ratio above 14 correspondto low dry matter and N harvests and to a highWCp, a high N mineralization per biomass unit

arises from adaptation to C-limited conditions. accumulation rate of litter inputs, which limit therisk of nitrate leaching.Under MIX, a medium specific respiration and N

mineralization per biomass unit is due to C–Nco-limited microbial activities. In bare soils, verylow specific respiration activities are due to the use 5. Conclusionof more recalcitrant but N-rich substrates.

Among the variables of the detritical pathway, A comparison of the non-fertilized grass andclover swards on the basis of the effects of theboth the ratios of coarse to fine plant residues and

their C:N ratio give an indication of their residence initial soil N supplying capacity and the quality of


Fig. 8. Ratio C:N mineralization as indicator of the C and N fluxes in the grassland ecosystem. (A) DM harvest. (B) N harvest. (C)Ratio C Fr1000: DM harvest (D) N leaching. Open and solid symbols represent poor soils (135 kg N ha−1 year−1) and rich soils(225 kg N ha−1 year−1), respectively. The bar is centred on the mean, and its width corresponds to the standard error (±SE) onthe abscissa.

the vegetation made it possible to characterize the Gerard L’homme for making time available to usto write the paper.indications given by some soil compartmental and

biological parameters. Whereas the microbial bio-mass, the amounts of C and N and the C:N ratioof the coarse OM fraction reflected the recenteffects of the swards, the microbial metabolites Referencesextracted by autoclaving characterized the storageof labile organic matter in the longer term. The Alvarez, R., Diaz, R.A., Barbero, N., Santanatoglia, O.J.,

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