Claire Jouany
email: claire.jouany@inrae.fr

Christian Morel

Noura Ziadi

Gilles Bélanger

Sokrat Sinaj

Ciprian Stroia

Pablo Cruz

Jean-Pierre Theau

Michel Duru

Plant and soil tests to optimize phosphorus fertilization management of grasslands

Keywords: phosphorus, grassland, long-term field experiment, phosphorus nutrition index, soil plant available P, Olsen P

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Grasslands provide an essential source of cattle feed, and the development of more sustainable agricultural systems tends to increase their contribution to livestock production systems [START_REF] Carrère | Valoriser les services rendus par la prairie. Une voie pour assurer la durabilité des systèmes d'élevage herbagers ? In Les services écosystémiques dans les espaces agricoles[END_REF]. In a given environment, forage production is determined largely by the amounts of nutrients supplied by soil reserves and/or organic or mineral fertilizers applied by farmers. The aim of phosphorus (P) fertilization is to meet crop requirements, which are determined largely by the nitrogen (N) supply [START_REF] Bélanger | Effects of 25 years of N, P and K fertilization on yield, persistence and nutritive value of a timothy sward[END_REF][START_REF] Schellberg | Long-term effects of fertilizer on soil nutrient concentration, yield, forage quality and floristic composition of a hay meadow in the Eifel Mountains, Germany[END_REF][START_REF] Griffin | Yield response of long-term mixed grassland swards and nutrient cycling under different nutrient sources and management regimes[END_REF][START_REF] Valkama | Meta-analysis of grass ley response to phosphorus fertilisation in Finland[END_REF]. Environmental and economic concerns require developing more sustainable forage systems. Excess P threatens the integrity of terrestrial and aqueous ecosystems [START_REF] Sharpley | The impact of soil and fertilizer phosphorus on the environment[END_REF][START_REF] Janssens | Relationship between soil chemical factors and grassland diversity[END_REF][START_REF] Ceulemans | A trait-based analysis of the role of phosphorus vs. nitrogen enrichment in plant species loss across North-west European grasslands[END_REF], while readily available and high-quality reserves of phosphate rocks will be exhausted in the medium term (Cordel et al., 2011).

Developing more sustainable forage systems requires effective decision support tools for fertilization management. To this end, plant-and soil-based indicators with threshold values for assessing nutrient status and managing fertilizers have been developed. Nutrient concentration ratios in plant tissues were developed as assessment tools for alfalfa (Medicago sativa L.; [START_REF] Walworth | Preliminary DRIS norms for alphalpha in the southeastern United States and a comparison with midwestern norms[END_REF] and perennial ryegrass (Lolium perenne L.) [START_REF] Bailey | The diagnosis and recommendation integrated system (DRIS) for diagnosing the nutrient status of grassland swards: I. Model establishment[END_REF][START_REF] Bailey | The Diagnosis and Recommendation Integrated System (DRIS) for diagnosing the nutrient status of grassland swards: III Practical applications[END_REF]. These ratios are also used for natural ecosystems to determine whether biomass production in terrestrial plant communities is N-or P-limited or co-limited by both nutrients [START_REF] Güsewell | N:P ratios in terrestrial plants: variation and functional significance[END_REF]. Soil P tests inform on soil plant available P (SPAP) and provide response thresholds useful in decision support tools for fertilization management [START_REF] Schulte | Quantifying responses to phosphorus in Irish grasslands: Interactions of soil and fertiliser with yield and P concentration[END_REF][START_REF] Reinjeveld | Changes in the soil phosphorus status of agricultural land in the Netherlands during the 20th century[END_REF]. However, both plant and soil tests remain of limited general value. Ratios that indicate of N or P limitation vary according to the type of ecosystems. For instance, they are much lower in upland grasslands than in wetlands (Mamolos et al., 2005;[START_REF] Craine | Nutrient concentration ratios and co-limitation in South African grasslands[END_REF]. Similarly, there is no agreement on a universal soil P test likely to provide, for a given crop or grassland sward, a single threshold value regardless of the soil type [START_REF] Schulte | Quantifying responses to phosphorus in Irish grasslands: Interactions of soil and fertiliser with yield and P concentration[END_REF][START_REF] Jordan-Meille | An overview of fertilizer-P recommendations in Europe: soil testing, calibration and fertilizer recommendations[END_REF].

More recently, significant advances have been made with the development of innovative assessment tools. The approach of nutrition indices based on the nutrient concentration of shoot biomass allows grassland P nutrition status to be assessed during growth [START_REF] Duru | A nitrogen and phosphorus herbage nutrient index as a tool assessing the effect of N and P supply on the dry matter yield of permanent pastures[END_REF]. For P, this approach is more reliable than those based on a single critical concentration since it considers changes in nutrient concentration as a function of sward biomass accumulation and concentration of other nutrients [START_REF] Duru | A nitrogen and phosphorus herbage nutrient index as a tool assessing the effect of N and P supply on the dry matter yield of permanent pastures[END_REF]. The P nutrition index (PNI) is an effective tool for P fertilization management in grasslands since it assesses the sward P nutrition status well during growth and can verify and validate fertilization practices a posteriori [START_REF] Thélier-Huché | L'analyse d'herbe: un outil pour le pilotage de la fertilisation phosphatée et potassique des prairies naturelles et temporaires[END_REF]. The PNI is adequate for the interpreting of the effect of plant P nutrition status on plant growth in grasslands managed at different intensities (Liebisch et al., 2013); as well, PNI provides appropriate plant nutrients status evaluation at the interface between agricultural land and saline wetlands in protected saline habitats [START_REF] Luna | Soil composition and plant nutrients at the interface between crops and saline wetlands in arid environments in NE Spain[END_REF]. At an ecosystem level, the N nutrition index (NNI) and PNI provide appropriate evaluation of the functional response of species and communities to fertility gradients induced by practices [START_REF] Garnier | Assessing the effects of land-use change on plant traits, communities and ecosystem functioning in grasslands : A standardized methodology and lessons from an application to 11 European sites[END_REF][START_REF] Lavorel | A plant functional traits data base for the Alps[END_REF].

At the same time, [START_REF] Morel | Caractérisation de la phytodisponibilité du phosphore du sol par la modélisation du transfert des ions phosphates entre le sol et la solution[END_REF] developed a mechanistic model based on the assumption that the SPAP pool represents the sum of the amount of orthophosphate ions (oPion) in the soil solution (Qw) and the amount of soil P that can diffuse from the soil to the solution over time (P r ). This model assumes that (i) diffusion of oPion at the solid-to-solution interface of soils is quantitatively the dominant process in plant P nutrition and (ii) depletion of oPion concentration at the root surface, due to absorption, creates a gradient of oPion concentration between the root surface, the soil solution, and the soil solid phase. This gradient is the driving force behind the flux of diffusive oPion from the soil solid phase to the soil solution. A general model, based on a Freundlich kinetic equation, was developed to calculate Pr as a function of the oPion concentration in soil solution (CP) and time [START_REF] Morel | Caractérisation de la phytodisponibilité du phosphore du sol par la modélisation du transfert des ions phosphates entre le sol et la solution[END_REF]. This model correctly simulated the changes in SPAP in the 0-5 cm soil horizon oven seven years from a long-term grassland experiment with contrasting P fertilization regimes [START_REF] Stroia | Dynamics of diffusive soil phosphorus in two grassland experiments determined both in field and laboratory conditions[END_REF]. In the present study, we examined the abilities of CP [START_REF] Morel | Transfer of phosphate ion between soil and solution. Perspectives in soil testing[END_REF], Olsen P (Olsen et al., 1954), and the sum (Qw + Pr) to assess SPAP in grasslands. Both CP and Olsen P are used around the world for this purpose [START_REF] Jordan-Meille | An overview of fertilizer-P recommendations in Europe: soil testing, calibration and fertilizer recommendations[END_REF][START_REF] Ziadi | Assessment and modeling of soil available phosphorus in sustainable cropping systems[END_REF].

The objectives of this study were to: (i) evaluate the relationship between PNI and the yield response to P fertilization over a range of soil types and climate conditions; (ii) analyze relationships between PNI and three SPAP indicators in order to compare their abilities to assess the soil P status and (iii) evaluate PNI assets for P diagnosis in forage system. The study relied on five long-term experiments that measured the response of forage yield to P fertilization under contrasting environments representative of grassland ecosystems in Canada, Switzerland, France and Romania. The four sites offered the opportunity to explore large gradients in soil P fertility caused by cumulative effects of P fertilization over several years.

Materials and Methods

Overview of the five sites

Five long-term grassland experiments at five sites across Europe and North America were used (Table 1). At each site, a control treatment without P fertilization was compared to one or more P fertilization treatments. Sites differed primarily in the duration of the experiment, soil and climate characteristics, and species composition (Table 1; Table 2).

LÉVIS, CANADA (CA-LEV). A grassland experiment, sown with timothy (Phleum pratense L. cv. Champ), was established in 1998 at Lévis, Canada (Table 1). The experimental design was a split plot, with four application rates of P fertilizer as triple super phosphate [0 (P0), 15 (P15), 30 (P30), and 45 (P45) kg P ha -1 ] assigned to main plots, and four application rates of N fertilizer as calcium ammonium nitrate [0 (N0), 60 (N60), 120 (N120), and 180 (N180) kg N ha -1 ] assigned to subplots. Experimental treatments were replicated in four blocks. For this study, plots that received the four P application rates and 120 kg N ha -1 were selected within the experimental setup. From 1999-2006, fertilizers were applied each year before the start of growth in the first week of May. Potassium (K) as KCl was applied at 84 kg K ha -1 as the same time as P and N to ensure that K did not limit plant growth.

LES VERRIÈRES, SWITZERLAND (CH-LES). A permanent grassland experiment was

established in 1993 on a Cambisol at Les Verrières, Switzerland (Table 1), with a mixture of red fescue (Festuca rubra L.), common bent (Agrostis capillaris L.), and orchard grass (Dactylis glomerata L.) (Table 1). The experiment consisted of four application rates of P [0 (P0), 9 (P9), 17 (P17), and 26 (P26) kg P ha -1 ] in plots arranged in a randomized completeblock design with three replicates. P was applied each year in a single application as triple super phosphate in October. K was applied as KCl in a single application in October at different rates according to the P treatment: 0, 29, 58 and 116 kg K ha -1 for treatments P0, P9, P17, and P26, respectively. N was applied at a rate of 25 kg N ha -1 as ammonium nitrate in all treatments once a year after the first cut. ERCÉ (FR-ERC) AND GRAMOND (FR-GRA), FRANCE. An experiment was conducted at Ercé and Gramond, France, on permanent multi-species grasslands (Table 1). The experiment was established in 1999 at Ercé and in 1998 at Gramond. At both sites, the experiment consisted of two rates of P fertilizer [0 (P0) and 50 (P50) kg P ha -1 ] applied each year in February as triple super phosphate on plots arranged in a randomized complete-block design with four replicates. N was applied as ammonium nitrate at rates of 100 kg N ha -1 in February and 60 kg N ha -1 after the first cut. K as KCl was applied at 200 kg K ha -1 at the same time as P and N to ensure that K did not limit plant growth. DÂMBOVICIOARA, ROMANIA (RO-DÂM). An experiment was established in 1963 on a permanent grassland in the Southern Carpathians Mountains in the Rucar-Bran-Dragoslavele corridor, Romania (Table 1). The grassland had a mixture of F. rubra and A. capillaris. The experiment consisted of two rates of P fertilizer [0 (P0) and 33 (P33) kg P ha -1 ] applied in autumn each year as super phosphate on plots arranged in a randomized complete-block design with four replicates. N was applied as ammonium nitrate at 100 kg N ha -1 in early spring and 50 kg N ha -1 after the first cut. Potash salt was added at a rate of 108 kg K ha -1 once a year in autumn to ensure that K did not limit plant growth (Ciubotariu et al., 2002).

More details on the five long-term experiments can be found in Bélanger et al. (2008) and [START_REF] Bélanger | Phosphorus and nitrogen relationships during spring growth of an aging timothy sward[END_REF] for CA-LEV, in [START_REF] Jeangros | Besoins en phosphore et en potassium d'une prairie de fauche du Jura riche en fétuque rouge[END_REF] for CH-LES, in [START_REF] Stroia | Etude de fonctionnement de l'écosystème prairial en conditions de nutrition N et P sub-limitante ; application au diagnostic de nutrition[END_REF] for FR-ERC and FR-GRA, and in Ciubotariu et al. (2002) for RO-DÂM.

Yield and nutrition index determination

Dry matter (DM) yield and nutrient concentration were measured at the end of the first growth cycle (Table 2), a period of the year when growth is rarely limited by water. Forage production in this first growth cycle represented 40%-70% of the average total annual production, depending on the site and treatment. All plots were harvested again during the rest of the growing season, but no measurements were taken. DM yield was measured by cutting an area of at least 1m 2 to a height of 5-cm. A fresh sample of ca. 300-500 g was taken, dried at 55 °C for 48 h, and ground.

At CA-LEV, dried and ground (1 mm) forage samples (0.1 g) were wet-digested with 1.5 mL H2SO4-H2SeO3 and 2.0 mL H2O2 [START_REF] Isaac | Determination of total nitrogen in plant tissue, using a block digester[END_REF]. N and P concentrations were measured by colorimetry using an automated continuous-flow injection analyzer (QuickChem nitroprusside procedure for total N (method 13-107-06-2-E) and the vanadomolybdate reaction for total P (method 15-301-3).

At CH-LES, concentrations of P and N in plant tissues were determined in samples that had been oven-dried (55°C for 72 h) and ground in a Retsch rotor. Total N was determined after combustion using the Dumas method [START_REF] Masson | Determination of Major and Trace Elements in Plant Samples by Inductively Coupled Plasma-Mass Spectrometry[END_REF], while total P was determined by radial ICP-AES (Varian Vista RL Simultaneous) after incineration (480°C for 5 h) and solubilization in hydrofluoric acid [START_REF] Masson | Determination of Major and Trace Elements in Plant Samples by Inductively Coupled Plasma-Mass Spectrometry[END_REF].

At FR-ERC, FR-GRA, and RO-DÂM, concentrations of P and N in plant tissues were determined in dried and ground (0.5 mm) samples. Total P was determined after wet digestion in H2SO4-H2O2 with ceruleomolybdic blue colorimetry [START_REF] Murphy | A modified single solution method for the determination of phosphate in natural waters[END_REF]. Total N concentration was determined with a CN gas analyzer (LECO Corporation, St Joseph, Michigan, USA). When present in samples, legumes were sorted by hand and excluded before analysis, as recommended by [START_REF] Jouany | Diagnosing phosphorus status of natural grassland in the presence of white clover[END_REF].

The NNI, expressed as a percentage, was calculated as the sample N concentration (Nmeasured, mg g -1 DM ) divided by the critical N concentration (Ncritical, mg g -1 DM) which was estimated from the critical N-dilution curve [START_REF] Lemaire | N Uptake and Distribution in Plant Canopies[END_REF], as follows:

NNI = Nmeasured/Ncritical × 100 (1) Ncritical = 48 (shoot DM) -0.32 (2)
with Nmeasured and Ncritical expressed in mg g -1 DM and shoot DM in t ha -1 .

The PNI and KNI, expressed as a percentages, were calculated as the sample P (Pmeasured, mg g -1 DM) or K (Kmeasured, mg g -1 DM) concentration divided by the critical P (Pcritical, mg g -1 DM)

or K (Pcritical, mg g -1 DM) concentration. PNI = Pmeasured/Pcritical × 100;

(3) KNI = Kmeasured/Kcritical × 100;

(4) Pcritical and Kcritical were estimated from [START_REF] Duru | A nitrogen and phosphorus herbage nutrient index as a tool assessing the effect of N and P supply on the dry matter yield of permanent pastures[END_REF], as follows:

Pcritical = 1.50 + 0.065 × Nmeasured (5) Kcritical = 1.6 + 0.525 × Nmeasured (6)
with Pmeasured and Pcritical expressed in mg g -1 DM.

Grassland vegetation characterization

At the four permanent multi-species grassland sites (CH-LES, FR-ERC, FR-GRA, and RO-DÂM), species composition was measured at the beginning of the experiments using exhaustive sorting of handfuls of vegetation, as described by De [START_REF] De Vries | Methods used in botanical grassland research in the Netherlands and their application[END_REF]. At least four species contributed 80% of community biomass (Table 1). Grasslands species compositions did not significantly change between control and fertilized plots for the whole period tested.

Soil physical and chemical properties

Before the start of the experiment at each site, soil samples were taken air-dried, sieved (2 mm) and stored at ambient temperature before analysis (Table 1). Soils were analyzed using the Swiss national reference methods for CH-LES [START_REF] Fal | Méthodes de référence des stations fédérales de recherches agronomiques[END_REF] and the method recommended in the province of Quebec for CA-LEV, as reported by [START_REF] Bélanger | Phosphorus and nitrogen relationships during spring growth of an aging timothy sward[END_REF]. For FR-ERC, FR-GRA and RO-DÂM sites, the soil samples were analyzed by the Laboratoire d'Analyses des Sols of the National Research Institute for Agriculture, Food and Environment (INRAE, 62000 Arras, France) using French standards [START_REF] Afnor | Qualité des sols[END_REF], as reported by [START_REF] Stroia | Dynamics of diffusive soil phosphorus in two grassland experiments determined both in field and laboratory conditions[END_REF].

SPAP was determined from existing recommendations in each country. The Olsen procedure [START_REF] Olsen | Estimation of available phosphorus in soils by extraction with sodium bicarbonate[END_REF] was used at the FR-ERC, FR-GRA, and RO-DÂM sites. A ammonium acetate-EDTA mixture was used at the CH-LES site [START_REF] Demaria | Exchangeability of phosphate extracted by four chemical methods[END_REF], and Mehlich-3 extractable P was used at the CA-LEV site [START_REF] Tran | Mehlich-3 extractable elements[END_REF].

Soil plant available P

Soil samples were taken in each replicate plot of each treatment at one date (Table 2). The soil samples were air-dried, sieved (2 mm), and stored at ambient temperature before analysis.

Three methods were used to estimate SPAP. Two of them were laboratory tests based on soil / solution-extraction procedures. CP, which corresponds to the pool of immediately available P, was measured using 1g of soil in 10 mL of distilled water [START_REF] Morel | Transfer of phosphate ion between soil and solution. Perspectives in soil testing[END_REF]. This test was performed for soils from all sites, except RO-DÂM (Table 2). The Olsen P [START_REF] Olsen | Estimation of available phosphorus in soils by extraction with sodium bicarbonate[END_REF] represents the pool of readily extractable P. It was measured using a mass of 1 g of soil in 20 mL of 0.5M sodium bicarbonate solution (pH=8.5). This test was performed for soils from all sites, except CA-LEV and RO-DÂM (Table 2). Both tests are suitable for a wide range of soil pH and widely used for soil testing and fertilizer recommendations [START_REF] Jordan-Meille | An overview of fertilizer-P recommendations in Europe: soil testing, calibration and fertilizer recommendations[END_REF][START_REF] Ziadi | Assessment and modeling of soil available phosphorus in sustainable cropping systems[END_REF][START_REF] Zehetner | Correlation of extractable soil phosphorus (P) with plant P uptake: 14 extraction methods applied to 50 agricultural soils from Central Europe[END_REF][START_REF] Johnston | Phosphorus in Agriculture: A Review of Results from 175 Years of Research at Rothamsted, UK[END_REF].

We also tested the functional-and process-based approach previously developed for soils with annual crops [START_REF] Morel | Modeling of phosphorus dynamics in contrasting agroecosystems using long-term field experiments[END_REF][START_REF] Messiga | Soil test phosphorus and cumulative phosphorus budgets in fertilized grassland[END_REF] and perennial forages [START_REF] Stroia | Dynamics of diffusive soil phosphorus in two grassland experiments determined both in field and laboratory conditions[END_REF][START_REF] Messiga | Process-based mass-balance modeling of soil phosphorus availability in a grassland fertilized with N and P[END_REF], and for rivers sediments [START_REF] Némery | Phosphorus budget in the Marne Watershed (France): urban vs diffuse sources, dissolved vs particulate forms[END_REF]. The model assumes that (i) roots absorb oPion only from the soil solution and (ii) this absorption generates a gradient of oPion concentration between the soil solid phase and solution that drives the oPion diffusion [START_REF] Barbier | Sur la diffusivité des ions phosphates du sol[END_REF][START_REF] Barber | Soil nutrient bioavailability. A mechanistic approach[END_REF]. This method provides an experimental data-set within a few hours for parameterizing of the function that describes the total amount of oPion that can diffuse (Pr, mg P kg-1 soil) over time (t, minutes) and the soil-solution oPion concentration (CP, mg P L-1 solution). This equation, the Freundlich kinetic equation, is as follows:

Pr = v × CP w × t p with Pr < PrLIMIT (7)
where v is the value or Pr at time t = 1 min and C P = 1 mg P L -1 , w is the non-linear increase in P r as a function of C P , and p is the non-linear increase in P r as a function of time (t). Parameters v, w and p are specific to soil at each site. The value of P r LIMIT, which cannot be determined experimentally, is estimated to be lower than the soil inorganic P content.

The related experiments were performed for soils from all sites except RO-DÂM (Table 2), by combining studies of sorption-desorption in soil suspensions in which isotopic dilution kinetics reached a steady-state for a few hours, as described by [START_REF] Stroia | Dynamics of diffusive soil phosphorus in two grassland experiments determined both in field and laboratory conditions[END_REF], [START_REF] Messiga | Process-based mass-balance modeling of soil phosphorus availability in a grassland fertilized with N and P[END_REF], and [START_REF] Morel | Calibration of maize phosphorus status by plantavailable soil P assessed by common and process-based approaches. Is it soil-specific or not?[END_REF]this issue). In this approach, SPAP equaled Qw + P r . Qw was calculated by multiplying CP by the solution-to-soil ratio (solution volume /soil mass).

In order to better appreciate the amount of soil plant available P and compare the different sites-treatments we expressed both Olsen P and the amount of diffusible P (Qw + P r) as kg P ha - 1 by using soil dry matter (Table 1).

Data analysis

All statistics were performed with Statgraphics Centurion XV (version 15.2.06, StatPoint, Inc., Herndon, Virginia, USA). Analyse of variance was performed for each treatment and site for PNI, CP, (Qw + Pr60min) and Olsen P. Statistical significance (P<0.05) was tested using a Fisher's exact test.

The response to P fertilization was characterized by calculating the relative DM yield for each combination of site and year [START_REF] Colwell | The estimation of phosphorus fertilizer requirements of wheat in southern New South Wales by soil analysis[END_REF], as follows:

Relative DM yield = (DM yield in control plots / maximum DM yield) × 100 (8)

The maximum DM yield was the yield of P-fertilized plots at sites with only one P rate (FR-ERC, FR-GRA, and RO-DAM) or the yield of plots that received the highest P rate at sites with several P rates (CA-LEV and CH-LES). To describe the relationship between the relative DM yield and PNI, a linear-plateau model was used, as follows:

Relative DM yield = a + b × PNI if PNI < critical PNI (9)

Relative DM yield = plateau of relative DM yield if PNI > critical PNI (10) where a and b are fitted parameters.

The linear-plateau model was calculated by minimizing the residual sum of squares between the observations and regression estimates (least square) using Microsoft® Excel's Generalized Reduced Gradient nonlinear solver.

Results

Forage yield response to the grassland P nutrition status

DM yield response to P fertilization varied greatly among sites. P fertilization increased DM yield significantly in 2 out of 4 years at CH-LES, 1 out of 3 years at RO-DÂM, and 3 out of 9 years at FR-ERC, but had no effect at CH-LEV or FR-GRA. On control plots (P0), the mean PNI among years was 87% at CA-LEV and 94% at FR-GRA; these values were representative of adequate P nutrition for growth. Consequently, mean relative DM yields among years were high, with values of 98% at CA-LEV and 96% at FR-GRA, and remained constant even when the PNI increased due to P fertilization (Fig. 1a). For the three sites with a positive response to P fertilization, however, the PNIs on control plots were less than 80%, with mean values among years of 61% at CH-LES, 66% at FR-ERC, and 48% at RO-DÂM. These values indicated that grassland growth was P-limited (relative DM yields of 77%, 80%, and 57%, respectively) (Fig. 1a). The linear plateau model satisfactorily described the relationship between relative DM yield and PNI for the five sites (Fig. 1a). The critical PNI value, corresponding to the inflection point of the linear-plateau curve, was 92%. Below this threshold, the increase in DM yield was proportional to that in PNI (Fig. 1). We observed a very significant positive association between the average values of PNI and RDMY for each combination site-treatment (p=9.88e-05, R 2 =0.88), despites a moderate sample size (nine site-treatment combinations).

Nevertheless, this association is not observed within each site-treatmenent (Fig. 1b); indeed, the sign of the slope of the regression slope of PNI on relative DMY is negative in five out of nine site-treatment combinations; moreover, if one limits themself to significant associations (p<0.05), regression slope is negative for two out of three site-treatment combinations, and positive for one out of three.

Soil plant available P

The CP values varied among the fertilization treatments, with the control treatment (P0)

having significantly lower CP than the P-fertilized treatments at each site (Table 4). At sites where several P fertilizer rates were applied (CA-LEV and CH-LES), CP was highest at the highest P rate.

For control treatments, mean CP varied from 0.042 mg L -1 at FR-ERC to 0.38 mg L -1 at FR-GRA and between 0.18 mg L -1 , at FR-ERC, and 3.55 mg L -1 at FR-GRA for fertilized treatments (Table 4).

As for Cp, mean Olsen-P always differed significantly between control and fertilized plots;

at CH-LES, site Olsen-P was highest for the highest fertilizer rate. For control treatments, mean

Olsen-P varied from 10.3 mg kg -1 at FR-ERC to 14.5 mg kg -1 at FR-GRA and, from 10.3 mg kg -1 for FR-ERC to 71.6 mg kg -1 at FR-GRA for fertilized treatments (Table 4).

Data obtained from sorption-desorption and isotope dilution procedures showed that, for each site, the total amount of diffusive oPion in soils (Pr) increased as CP and time increased (Fig. 3); the magnitude of the response differed among sites. For similar CP ranges of less than 1 mg P L -1 , Pr measured in the FR-ERC soil was twice that measured in the CA-LEV soil, regardless of the time (t) (Fig. 3). The entire dataset served to estimate the parameters of Eq. ( 7) (Table 3). For each site, except RO-DÂM, the entire CP dataset (Table 2; Table 4) and the parameter estimates of the Freundlich kinetic equation (Table 3) were used to calculate (Qw + Pr) values for increasingly long periods of diffusion. For each value of t, a general relationship was built between (Qw + Pr) and PNI (data not shown); the best fit was obtained for a diffusion time t of 60 minutes (Pr60min) (Fig. 2c).

Like CP, the mean amount of diffusive P (Qw + Pr60min) varied at each site with treatments (Table 4). When several P fertilizer rates were applied (CH-LES and CA-LEV), the maximum (Qw + Pr60min) was measured for the highest P fertilizer rate. For each site, differences between control and fertilized plots were always significant.

For control treatments, mean (Qw + Pr60min) varied from 18.8 mg kg -1 at CH-LES to 22.3 mg kg -1 at CA-LEV and, from 21.5 mg kg -1 for CH-LES (P1) to 84.3 mg kg -1 at FR-GRA for fertilized treatments (Table 4).

Relationship between P nutrition index and soil plant available P

As for SPAP, the over years cumulative effects of P fertilization regimes induced a wide range of PNI at each site (Table 4; Fig. 2). Each SPAP tested generally displayed a positive and significant (P<0.05) relationship with PNI. Thus, any increase in soil P status improved grassland P nutrition status (Fig. 2 a, b andc). Compared to considering only CP (Fig. 2a), considering the pool of diffusive P (Fig. 2b) as well improved the reliability of the PNI response curve in the 0-5 cm soil horizon: R 2 = 0.65 (P< 0.05) for CP and R 2 =0.70 (P< 0.05)

for Qw + Pr60min. We obtained a similar response pattern for the relationship between the Olsen P stock in the 0-5 cm soil horizon (kg ha -1 ) and PNI with R 2 =0.85 (P<0.05) (Fig. 2b).

The threshold of 92% for PNI (Fig. 1b) served to calculate the critical SPAP values which value were 13.5 kg P ha -1 and 12.9 kg ha -1 for, respectively (Qw + Pr60min) and Olsen P.

Discussion

Relationship between relative dry matter yield and P nutrition Index

A general relationship between relative DM yield and PNI was obtained by combining data sets from five long-term experiments at contrasting sites with different response patterns to P fertilization (Fig. 1 a). For CA-LEV and FR-GRA, no significant yield response to P fertilization was observed throughout the experiments. At both sites, mean PNI values greater than 80%, for control treatment plots, indicated that P did not limit growth [START_REF] Duru | A nitrogen and phosphorus herbage nutrient index as a tool assessing the effect of N and P supply on the dry matter yield of permanent pastures[END_REF]; consequently, mean relative DM yields remained close to 100% and did not vary. At both sites, soil reserves provided enough P to meet grassland requirements and to maintain optimal sward P nutrition for nine years at CA-LEV and seventeen years at FR-GRA. At CH-LES, FR-ERC, and RO-DÂM, PNI values less than 80%, for control treatment plots, indicated that growth was P-limited [START_REF] Duru | A nitrogen and phosphorus herbage nutrient index as a tool assessing the effect of N and P supply on the dry matter yield of permanent pastures[END_REF]. Soil reserves did not provide enough P to meet grassland requirements for optimal growth and, consequently, a significant yield response to P addition was observed. At CH-LES site, with four P rates tested, we observed a similar response pattern to P; PNI increased in proportion with fertilizer rate between 0 and 26 kg P ha - 1 , and, consequently, relative DMY. At that site, relative DM response cannot be exclusively ascribed to P nutrition since fertilization rates differed in both P and K amounts. We cannot exclude the hypothesis that relative DMY responded to K as well; however, K impact should be limited as reported by [START_REF] Duru | Diagnostic de la nutrition minérale de prairies permanentes au printemps[END_REF].

At the start of the experiments, the initial SPAP value exceeded the critical value of 25 mg Olsen P kg -1 at FR-GRA (Table 1) identified by [START_REF] Poulton | Comparison of fertilizer phosphorus recommendations in Ireland and England and Wales[END_REF]. The experimental site of CA-LEV was chosen because of its expected positive response to P fertilization [START_REF] Bélanger | Phosphorus and nitrogen relationships during spring growth of an aging timothy sward[END_REF]. Its soil (Mehlich-3 extraction; 0-15 cm) contained 54 kg available P ha -1 (Table 1) and was considered poor in P according to local recommendations (Conseil des Productions

Végétales du Québec, 1996). During the experiment at FR-GRA, SPAP measured in the P0 treatment remained above the critical value despite large negative annual P budget (-35 kg ha -1 ; [START_REF] Stroia | Etude de fonctionnement de l'écosystème prairial en conditions de nutrition N et P sub-limitante ; application au diagnostic de nutrition[END_REF]. At CA-LEV, SPAP measured in the P0 and P15 treatments first decreased and then remained relatively stable over time, despite negative annual P budgets (-5 and -20 kg ha -1 , respectively) [START_REF] Messiga | Soil test phosphorus and cumulative phosphorus budgets in fertilized grassland[END_REF]. At FR-ERC and CH-LES, SPAP values laid below 25 mg

Olsen P kg -1 at the start of the experiments, and they decreased over time as cumulative P budgets became more negative [START_REF] Messiga | Soil test phosphorus and cumulative phosphorus budgets in fertilized grassland[END_REF].

For similar mean annual P outputs and budgets [START_REF] Messiga | Soil test phosphorus and cumulative phosphorus budgets in fertilized grassland[END_REF], soils were less resilient at FR-ERC than at CA-LEV and FRA-GRA, where productivity was not affected after not applying P for nine and seventeen years, respectively. These results confirmed that the long-term impact of stopping P fertilization on grassland productivity varied greatly for European upland grasslands [START_REF] Marriott | Long-term impacts of extensification of grassland management on biodiversity and productivity in upland areas[END_REF]) and that legacy P can contribute greatly to grassland nutrition [START_REF] Sattari | Residual soil phosphorus as the missing piece in the global phosphorus crisis puzzle[END_REF].

The significant relationship between relative DM yield and PNI demonstrated that PNI captures differences in DM yield between P fertilized and non-fertilized plots and that, under P limitation, the decrease in DM yield is directly proportional to the decrease in the grassland P nutrient status measured with PNI. The overall critical PNI value, corresponding to the inflection point of the linear-plateau curve, was 92%. This value was close to PNI = 100% which theoretically represents the boundary between P-limited and non-P-limited growth conditions according to [START_REF] Thélier-Huché | L'analyse d'herbe: un outil pour le pilotage de la fertilisation phosphatée et potassique des prairies naturelles et temporaires[END_REF]. The difference between this threshold (92%) and the statistical value (100%) given by [START_REF] Duru | N and P-K status of herbage: Use for diagnosis of grasslands[END_REF] and [START_REF] Thélier-Huché | L'analyse d'herbe: un outil pour le pilotage de la fertilisation phosphatée et potassique des prairies naturelles et temporaires[END_REF] is likely due to the smallest dataset that they used to determine the critical P-dilution curve or to establish the relationship between relative yield and PNI.

This study provides the first evidence that the relative DM yield of a P-limited sward, , is a direct function of its P status assessed with PNI, within different site-treatments. These results validate the PNI approach as an adequate tool for a posteriori assessment of grassland P nutrition. They extend conclusions of studies conducted on grasslands at local scales [START_REF] Duru | Pédoclimat, fertilisation et croissance des prairies permanentes au printemps I. Variabilité de la nutrition minérale[END_REF][START_REF] Duru | A nitrogen and phosphorus herbage nutrient index as a tool assessing the effect of N and P supply on the dry matter yield of permanent pastures[END_REF][START_REF] Duru | N and P-K status of herbage: Use for diagnosis of grasslands[END_REF]Liebisch et al., 2013).

Under P-limited nutrition status, relative DM yield was also shown to be a direct function of the PNI in canola (Brassica napus L.) [START_REF] Cadot | Critical plant and soil phosphorus for wheat, maize, and rapeseed after 44 years of P fertilization[END_REF] and maize (Zea Mays L.) [START_REF] Cadot | Critical plant and soil phosphorus for wheat, maize, and rapeseed after 44 years of P fertilization[END_REF][START_REF] Gagnon | Validation and use of critical phosphorus concentration in maize[END_REF].

While we observed a high positive association between average PNI and average relative DM measured within the nine site-treatment clusters (Fig. 1a), a similar relation was not observed for each site-treatment; relationships were positive for some site-treatments and negative for others (Fig. 1b). We considered that there were variables, other than PNI, that came into account to explain inter annual relative DM variability for a given site-treatment.

Since interactions between N and P nutrition control forage response to fertilization [START_REF] Duru | N and P-K status of herbage: Use for diagnosis of grasslands[END_REF], we tested to what extend NNI and KNI, for CH-LES site, could explain relative DM yield variability between years.

For each CA-LEV and FR-GRA treatments, there was no significant relationship between NNI and PNI, between relative DM and NNI; including NNI as a co variable did not improve none of the model obtained with PNI (Fig. 1b). No significant relationship was obtained either when testing the relationships for the three CA-LEV data sets pooled together.

At site level, these results confirmed that for both sites and treatment optimal N and P nutrition allowed optimal biomass production. On the other hand, negative relationships between relative DMY and PNI at CA-LEV P0 and CA-LEV P1 (Fig. 1b) could result from P luxury consumption and accumulation which increased as relative DM decreased.

For CH-LES, there was no significant relationship between NNI and PNI, relative DMY and NNI and between relative DMY and KNI for any of the treatments. Including NNI or KNI as co variable did not improve the model between relative DM and PNI for CH-LES P0 (Fig. 1b). Finally, when pooling the three CH-LES data sets, we got a significant relationship between relative DMY and NNI (R 2 =0.44; p=0.018); best fit was obtained with PNI as a single variable (R 2 =0.65; p=0.0016). This result demonstrated that, under P and N limited conditions, inter annual relative DM variability was better explained by PNI than NNI.

For FR-ERC, there was a significant relation between NNI and PNI (R 2 =0.49; p=0.0353) which confirmed that, under P limitation, improving P nutrition has a positive and significant effect on herbage nutrient status as reported by [START_REF] Duru | A nitrogen and phosphorus herbage nutrient index as a tool assessing the effect of N and P supply on the dry matter yield of permanent pastures[END_REF]. Introduction of NNI as a co variable with PNI (Fig. 1b) improved the relationship (R 2 =0.76; p=0.0135). Finally, best fit of the data was obtained with a simple linear model with NNI (R 2 =0.75; p=0.0024. Under P limited and non N limited conditions, NNI explained better variations in relative DM than PNI;

grassland response to P limitation varies according to NNI. For a given PNI, relative DM is higher, the higher NNI.

The results showed that P effect on relative DM was different according to the grassland nutritional status. At CH-LES site, with both N and P limitation, increasing PNI improved the sward efficiency for N conversion in biomass. P supply has a direct impact on forage growth.

On the opposite, at FR-ERC site, where P was limiting growth but not N, increasing PNI improved sward N nutrition status and consequently relative DM.

These results confirmed at multi annual scale that interaction between N and P controls forage response to P fertilization [START_REF] Duru | A nitrogen and phosphorus herbage nutrient index as a tool assessing the effect of N and P supply on the dry matter yield of permanent pastures[END_REF][START_REF] Jouany | Biological phosphorus cycling in grasslands: interactions with nitrogen[END_REF].

Relationship between P nutrition index and soil plant available P

Positive relationships between PNI and each SPAP indicator (Fig. 2) indicates that the plant nutritional status of grassland swards increases with increasing soil P status. This result extends the results of studies conducted at a regional scale by [START_REF] Duru | Diagnostic de la nutrition minérale de prairies permanentes au printemps[END_REF] to a wider range of soil and climate conditions. The response curve between PNI and each SPAP indicator could be used to determine the critical SPAP value required to obtain a PNI value of 92% (i.e. the PNI needed to reach maximum relative DM yield) (Fig. 1a). When considering all sites except RO-DÂM together, the critical CP value was 0.26 mg P L -1 (Fig. 2a), but it varied among sites, from a minimum of 0.12 mg P L -1 at FR-ERC to a maximum of 0.50 mg P L -1 at FR-GRA. This variability was due to large differences among sites in the ability of the soil solid phase to supply the soil solution with oPion, which is controlled by soil physicochemical characteristics (Table 1). The FR-ERC soil, with a clay-loam texture and high P-buffer capacity due to high contents of Fe and Al oxide, and hydroxide [START_REF] Stroia | Dynamics of diffusive soil phosphorus in two grassland experiments determined both in field and laboratory conditions[END_REF], had the lowest critical CP value. In contrast, the sandy soil at FR-GRA had a low P-buffer capacity and thus the highest critical CP value [START_REF] Stroia | Dynamics of diffusive soil phosphorus in two grassland experiments determined both in field and laboratory conditions[END_REF]. Morel et al. (2021) reported similar soil-specific responses of maize to P, measuring the lowest critical CP in Fe and Al oxide-rich soil and the highest critical CP in sandy soil.

The stronger relationship observed between PNI and (Qw + Pr60min) compared to that with CP alone (Fig. 2c) demonstrated that assessing SPAP as the amount of soil diffusive oPion, captured the specific effects of soil type on P-ion mobility better than CP did. These results generalized results obtained for annual crops by [START_REF] Morel | Transfer of phosphate ion between soil and solution. Perspectives in soil testing[END_REF]2021), to grassland soils.

However, our study differed on two points. First, the time of diffusion (t) that minimized variance in the relationship between PNI and (Qw + Pr) (Fig. 2c) was 60 minutes (Pr60min), much shorter than 1360 minutes for annual crops (Morel et al., 2021). The critical (Qw + Pr60min) value for grasslands was 13.5 kg P ha -1 (Fig. 2c), twice as large as that for maize (7.9 kg P ha -1 ; Morel et al. (2021)). These differences may be due to the soil depth considered when measuring the (Qw + Pr60min) stock: 0-5 cm for grasslands vs. the plowed layer (0-25 cm) for maize.

The assumption that maize obtains P from the plowed layer is reasonable since this layer contains 80% of maize root biomass [START_REF] Li | The long-term effects of tillage practice and phosphorus fertilization on the distribution and morphology of corn root[END_REF] and the soil horizons below it contribute little to crop P nutrition. This assumption is more questionable for permanent grasslands, in which soil horizon below 5 cm may contribute greatly to sward P nutrition. This is more likely to occur in non-fertilized grasslands with a soil with a low P status [START_REF] Fort | Grassland root functional parameters vary according to a community-level resource acquisition: conservation trade-off[END_REF].

SPAP tests could be compared only for three sites where the three indicators were measured (CH-LES, FR-ERC, and FR-GRA; Table 2). Olsen P explained more of the variation in PNI (R 2 = 0.85; P <0.05; Fig. 2b) than the (Qw + Pr60min) stock (R 2 = 0.70; P<0 .05; Fig. 4a) or CP (R 2 = 0.66; P < 0.05; Fig. 4b) did. The NaHCO3 extraction with the Olsen procedure represented soil-type-specific effects on oPion mobility better than CP (Fig. 2a) or (Qw + Pr60min) (Fig. 2c) did. These results for grasslands, however, differ from those for maize of [START_REF] Morel | Transfer of phosphate ion between soil and solution. Perspectives in soil testing[END_REF], who reported that (Qw + Pr) stock explained the yield response better than

Olsen P for three contrasting soil types.

We hypothesize that the better fit observed for Olsen P is due to its ability to extract organic P, which is partly mineralized when measuring phosphate ions. This is particularly likely to occur when the test is performed for grassland soils rich in organic matter (Bowman and Cole, 1978). [START_REF] Tate | Temporal variations in some plant and soil P pools in two pasture soils of widely different P fertility status[END_REF] demonstrated that soil organic P pool in grassland soils can contribute greatly to grassland P nutrition. In contrast, the (Qw + Pr) stock includes only the inorganic P pool.

Expressed on a stock basis, the Olsen P critical value was 12.9 kg P ha -1 (Fig. 2b) which was close to the (Qw + Pr60min) critical value of 13.5 kg P ha -1 (Fig. 2c).

The comparison of response curves showed that, for both Olsen P and (Qw + Pr), correlations were stronger when the PNI was related to SPAP measured in the 0-5 cm soil horizon instead of the 0-10 cm horizon (data not shown). This result confirms that surface soil horizons, where high nutrient concentrations [START_REF] Kidd | Impacts of 120 years of fertilizer addition on a temperate grassland ecosystem[END_REF] foster intensive exploitation and acquisition of resources by roots [START_REF] Fort | Grassland root functional parameters vary according to a community-level resource acquisition: conservation trade-off[END_REF], contribute greatly to grassland P nutrition.

Strengths and limits of nutrient index approach for Phosphorus diagnosis in agro ecosystems

Our results confirmed that PNI was a reliable indicator of the level of satisfaction of plant's P needs from soil. Over large soil P gradients, a significant relationship existed between grassland P nutrition status, evaluated with PNI, and SPAP, assessed according to conventional soil tests. Situations exist in agro systems where nutrition index approach could be used in association with soil analysis, or be an alternative to, when soil analysis is difficult to perform.

Our study demonstrated that PNI presents some assets for improving P status diagnosis for permanent grasslands where soil analysis is difficult to perform. Taking a representative soil sample is tough because of nutrients stratification and non-uniform vertical repartition of nutrients (; this issue specially applies to P that is not mobile in soils [START_REF] Messiga | Soil nutrients and other major properties in grassland fertilized with nitrogen and phosphorus[END_REF].

Moreover, little soil references are available for permanent grasslands, compare to cropping systems, and thresholds responses based on soil analysis are rarely available. Likewise, nutrition indices could be implemented for P diagnosis in cropping system where non-uniform nutrients repartition makes soil sampling more tedious (no tillage, Conservation agriculture, precision farming) and where available soil thresholds and references are unsuitable..

In other situations, PNI allows specifying, or even correct, soil diagnosis as it is the case for CA-LEV site in this study. Although initial soil P status was diagnosed as limiting for growth, based on soil P test, our study demonstrated that forage P nutrition status was adequate on control treatment plots and its conclusion questioned soil based diagnosis, or at least the method used.

On an other hand, this approach presents some limits since the 'critical' P concentrations (Eq. 4) that serves for PNI calculations (Eq. 3) were more an approximation of critical P concentration than critical concentration sensu stricto as defined in Justes et al. (1994). As a consequence, P critical value (Eq. 4) was higher and PNI lower than expected. This bias results in underestimation of forage P status diagnosis and, as a consequence, a risk of excess P supply in subsequent fertilization recommendations.

In order to improve the reliability of the nutrient index approach for P diagnosis, a more precise critical curve is necessary. In that purpose, the interaction between N and P must be investigated on P deficient grasslands, by combining different P and N doses in order to identify precisely the true critical P concentration.

Conclusions

This study of five long-term grassland experiments conducted on contrasting soil types and climates demonstrated that a direct and general relationship exists between PNI and the forage yield of the first growth cycle. Forage yield increased linearly as sward P nutrition status, assessed by the PNI, increased up to nearly 100%. This critical threshold differentiated Plimited growth from that with adequate P nutrition. Significant positive relationships between PNI and three SPAP indicators confirmed that the soil P status influences sward P nutrition status. The Olsen P extraction procedure provided the best fit with PNI, with a critical value of 12.9 kg ha -1 .

The study confirmed the potential of using plant analysis and nutrient indices for P diagnosis in forage systems and making fertilizer P recommendations. Our study highlights the utility of long-term fertilization experiments in which highly P-depleted soils (i.e. control plots with no P applied) offer the opportunity to test grassland responses to P fertilization and set more reliable 545 and precise critical curves. a v: total amount of the diffusive soil P after 1 minute when C P is 1 mg P L -1 . b w: the increase in Pr as a function of C P .

c p: the increase in Pr as a function of t.

Fig. 1 .Fig. 4 .

 14 Fig. 1. Relationship between relative dry matter yield (RDMY) and Phosphorus nutrition index (PNI) a) the average RDMY as a function of the average PNI, with averages computed over all years for each site-treatment combination; C represents the critical PNI (%) b) RDMY measurements as a function of PNI measurements, for each site-treatment combination. The line corresponds to the linear regression of PNI on RDMY, and p is the Fisher p-value. a

Table 1 .

 1 Climate, soil, and management characteristics of the five experimental sites; NA = no data available

	Site	CA-LEV	CH-LES	FR-ERC	FR-GRA	RO-DÂM
	Country	Canada	Switzerland	France	France	Romania
	Community	Lévis	Les Verrières	Ercé	Gramond	Dâmbovicioara
	Location	46°47' N 71°07' W	46°54'N 6°29' E	42°50' N 1°17' E 44°16' N 2° 22' E	45°24'N 25°14'E
	Climate type (Köppen-Geiger)	Continental humid	Continental humid	Temperate humid Temperate humid	Continental humid
	Elevation (m)	65	1100	660	607	1204
	Mean annual temperature (°C)	4.0	5.8	12.7	11.0	4.3
	Mean annual rainfall (mm)	692	1400	1079	960	895
	Soil horizon (cm)	0-15	0-5	0-5	0-5	0-5
	Soil type (US taxonomy)	Fragihumod	Cambisol	Alfisol	Inceptisol	Rendollic eutrocryepts
	Soil bedrock	Limestone	NA	Alluvium	Micashist	Limestone
	pH water	5.8	5.5	5.9	5.5	6.2
	Clay (g kg -1 )	NA	290	251	214	663
	Loam (g kg -1 )	NA	470	509	220	309
	Sand (g kg -1 )	NA	240	250	566	28
	Total soil C (g kg -1 )	26.0	40.6	55.2	36.8	195.5
	C: N ratio	NA	NA	10.0	11.2	17.6
	Cation Exchange Capacity (cmol+kg -1 )	NA	NA	18.6	9.7	38.8
	Exchangeable Ca (cmol+kg -1 )	NA	NA	15.7	8.3	35.8
	Exchangeable Mg (cmol+kg -1 )	NA	NA	1.6	1.0	1.9
	Exchangeable K (cmol+kg -1 )	NA	NA	0.2	0.2	0.4
	Exchangeable Na (cmol+kg -1 )	NA	NA	0.1	0.1	0.1

Table 3 .

 3 Mean (and standard error) estimates of parameters v, w and p of the Freundlich kinetic equation (P r = v × C P w × t p ).

	Site	Soil sampling year Soil horizon
			(cm)	v a	w b	p c
	CA-LEV	2006	0-15	6.95 (0.95) 0.38 (0.05)	0.371 (0.024)
	CH-LES	2008	0-5	6.54 (0.59) 0.48 (0.05)	0.428 (0.013)
	FR-ERC	2007	0-5	14.19 (0.46) 0.44 (0.01)	0.419 (0.006)
	FR-GRA	2007	0-5	9.62 (0.39) 0.36 (0.02)	0.273 (0.006)

Table 4 .

 4 Mean Phosphorus nutrition Index (PNI), soil solution oPions concentration (Cp), Olsen extracted Phosphorus (Olsen-P) and diffusive P (Qw + Pr60min) ; NA = no data available.

	Site	Treatment	PNI 1		Cp		Olsen-P		(Qw + Pr60min)
			(%)		mg P l -1		mg P kg -1 soil		mg P kg -1 soil
	CA-LEV	P0	83	a 2	0.3	a	NA	NA	22.3	a
		P1	95	b	0.59	ab	NA	NA	31.5	ab
		P2	101	bc	1	bc	NA	NA	41.3	bc
		P3	106	c	1.37	c	NA	NA	49.2	c
	CH-LES	P0	51	a	0.18	a	12.5	a	18.8	a
		P1	86	b	0.24	ab	16.7	a	21.5	ab
		P2	90	bc	0.32	b	23	b	24.1	b
		P3	99	c	0.34	b	32.3	c	25	b
	FR-ERC	P0	59	a	0.042	a	10.3	a	19.8	a
		P1	102	b	0.18	b	45.3	b	39.4	b
	FR-GRA	P0	89	a	0.38	a	14.5	a	22	a
		P1	127	b	3.55	b	71.6	b	84.3	b
	1 Measured on the soil sampling date (

Table 2 )

 2

Different letters in a given column for a given site indicate significant differences (P<0.05)

Acknowledgments

We thank Danielle Mongrain (Canada), Luc Stévenin (Switzerland), Éric Leroux and Denis Vialan (France), for their technical assistance. We also express our gratitude to Bernard Jeangros (Agroscope, Switzerland) and Vasile Cardașol (Research and Development Institute for Meadows, Romania) who initiated long-term field experiments, and to Marcel Fort for providing access to the long-term experiment in Ercé, France. The authors thank Sandra Plancade for help in data analysis. Long-term field experiments were supported by Agriculture and Agri-Food Canada, Agroscope (Switzerland), Research and Development Institute for Meadows, Romania, and the « Flexibilité stoechiométrique dans les agroécosystèmes » project of the French National Institute for Agriculture, Food and Environment (INRAE). The first author was supported by a grant from the Soutien à la Mobilité Internationale program of the National Polytechnic Institute of Toulouse.

Writing -review and editing.

Declaration of competing interest

The authors declare no conflict of interest of any kind that could have influenced the work reported in this paper.

RDMY (%)

PNI (%)

Supplementary materials

Table S1 Mean (and standard error) of dry matter (DM; t ha -1 ), nitrogen concentration (N; mg g -1 ), and phosphorus concentration (P; mg g 3.3 (0.1) 16.9 (0.4) 2.2 (0.1) P17 4.0 (0.4) 18.1 (0.4) 2.4 (0.1) P26

3.8 (0.2) 14.4 (0.1) 1.2 (0.1) Table S3 Mean (and standard error) of dry matter (DM; t ha -1 ), nitrogen concentration (N; mg g -1 ), and phosphorus concentration (P; mg g -1 ) measured at the first harvest at FR-ERC site from 1999-2018 (n=4) Year Treatment DMY N P t ha -1 mg g -1 mg g -1 1999 P0 3.2 (0.2) 28.9 (0.3) 2.4 (0.