William Galland

Florence Piola

Alexandre Burlet

Céline Mathieu

Mélisande Nardy

Sophie Poussineau

Leslie Blazère

Jonathan Gervaix

Sara Puijalon

Simon Laurent

Laurent Simon

Feth El Zahar Haichar

Feth El

Zahar Haichar
email: zahar.haichar@univ-lyon1.fr

F Z Haichar

Biological denitrification inhibition (BDI) in the field: A strategy to

Keywords: biological denitrification inhibition, field experiment, nitrate, denitrifiers, lettuce, rhizosphere, plant traits, productivity

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

INTRODUCTION

Nitrogen is one of the factors limiting plant growth [START_REF] Lebauer | Nitrogen limitation of net primary productivity in terrestrial ecosystems is globally distributed[END_REF]. It is the most important nutrient used to enhance agricultural yields. Thus, the development of plants depends on the processes linked to the nitrogen (N) cycle, which orchestrates the transformation of nitrogen into all its forms including ammonium (NH 4 + ) and nitrate (NO 3 -), the most plant-assimilable forms of nitrogen. The competition between plant roots and microorganisms for these two N forms is intense [START_REF] Kuzyakov | Competition between roots and microorganisms for nitrogen: mechanisms and ecological relevance[END_REF]. Microorganisms often out-compete plant roots, but some plant species are able to bypass this competition by taking control of the N in their rhizosphere through the production of secondary metabolites that inhibit N cycle processes [START_REF] Knops | Mechanisms of plant species impacts on ecosystem nitrogen cycling[END_REF][START_REF] Chapman | Plants actively control nitrogen cycling: uncorking the microbial bottleneck[END_REF][START_REF] Subbarao | Biological nitrification inhibition (BNI) activity in sorghum and its characterization[END_REF][START_REF] Bardon | Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration[END_REF]. For example, Brachiaria humidicola, by exuding some secondary metabolites (brachialactone), inhibits the nitrification process [START_REF] Subbarao | Evidence for biological nitrification inhibition in Brachiaria pastures[END_REF].

Nitrification is responsible for the conversion of ammonium to nitrate, which is rapidly converted into N 2 O (a greenhouse gas) by denitrification and/or leached in soils [START_REF] Di | Nitrate leaching in temperate agroecosystems: sources, factors and mitigating strategies[END_REF]. Denitrification, mostly carried out by bacteria, is the main form of nitrogen loss in most soils [START_REF] Van Der Salm | Estimation of nitrogen losses via denitrification from a heavy clay soil under grass[END_REF][START_REF] Radersma | Assessing denitrification and N leaching in a field with organic amendments[END_REF]. Denitrification leads first to nitrate reduction, which is associated with two homologous enzymes, namely, transmembrane nitrate reductase (Nar) and periplasmic nitrate reductase (Nap), which are encoded by the narG and napA genes, respectively (the latter is not present in all denitrifiers). Depending on the microorganisms, nitrite reduction to nitric oxide (NO) involves two nitrite reductases, one coupled with copper (encoded by nirK) and the other coupled with cytochrome cd1 (encoded by nirS). The transformation of NO into N 2 O (dinitrogen monoxide) involves NO reductase, which is a transmembrane enzyme encoded by the norB gene [START_REF] Wallenstein | Environmental controls on denitrifying communities and denitrification rates: insights from molecular methods[END_REF]. Finally, N 2 O reductase, which transforms N 2 O into dinitrogen (N 2 ), is a periplasmic enzyme encoded by the nosZ gene [START_REF] Khalil | Emissions de N2O par nitrification et dénitrification à l'échelle de la motte de sol: effet de la structure du sol, de l'aération et des activités microbiennes[END_REF].

However, this last step leading to the emission of N 2 , which can be fixed by some plants, is not present in all denitrifiers [START_REF] Galloway | Nitrogen cycles: past, present, and future[END_REF]. In European agro-ecosystems, losses due to N 2 O emissions account for 59% of the N loss of the system [START_REF] Oenema | Integrated assessment of promising measures to decrease nitrogen losses from agriculture in EU-27[END_REF].

Recently, [START_REF] Bardon | Evidence for biological denitrification inhibition (BDI) by plant secondary metabolites[END_REF] demonstrated that the invasive species Fallopia spp. can inhibit denitrification activity in soils through the release of procyanidins, a phenomenon termed biological denitrification inhibition (BDI). Procyanidins specifically inhibit membranebound NO 3 reductase, inducing enzymatic conformational changes through membrane disturbance [START_REF] Bardon | Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration[END_REF]. This strategy leads to a reduction in N 2 O emissions from soil of up to 95% [START_REF] Bardon | Evidence for biological denitrification inhibition (BDI) by plant secondary metabolites[END_REF]. In addition, it has been shown that the addition of procyanidins to the soil under experimental conditions in mesocosms leads to a 6-fold increase in the amount of nitrate compared to untreated soil, which reduces denitrification activity without affecting either the respiration or mineralization activities of soil microbial communities [START_REF] Bardon | Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration[END_REF]. This strategy could make nitrate more available in the soil, which may then be used by plants for their growth.

So far, the extent to which procyanidins can affect plant growth and nutrition via BDI under field conditions has not been investigated. Our hypothesis is that the addition of procyanidins to cultivated soils under field conditions induces BDI, with an increase in available nitrate and therefore a gain in productivity. To test our hypothesis, field experiments were carried out on cos or romaine lettuce crops (Lactuca sativa) at the SERAIL experimental station (Brindas, 69126 Rhône, France). Different concentrations of procyanidins were tested in the lettuce fields: 0, 8, 42, 83 and 210 kg ha -1 . Soil denitrification enzyme activity (DEA), nitrate concentration, above-and below-ground lettuce traits (shoot and root fresh masses, specific leaf area, biomechanical traits of leaves, anthocyanin and flavonoid contents of leaves and N content of tissues) and the abundance of all bacteria and denitrifiers were measured in lettuces treated or untreated with procyanidins.

MATERIALS AND METHODS

Plant growth and experimental design

A 202 m 2 field (composed of 24 plots of 1.4 m x 6 m (8.4 m 2 )) located at the SERAIL experimental station (Brindas, 69126 Rhône, France) (45°43'46.4"N 4°43'37.1"E) was used for growing romaine lettuces (Lactuca sativa var Lotus RZ), following a Fisher system of 4 plots per treatment [START_REF] Preece | RA Fisher and experimental design: a review[END_REF]. Lettuce seedlings were planted in three rows spaced 0.45 m apart and each lettuce spaced 0.45 m apart, for a total of 39 lettuces per plot (156 lettuces per treatment). Six treatments were considered: unplanted soil, soil with lettuce without procyanidin addition, and 4 lettuce crops treated with procyanidins at 8, 42, 83 and 210 kg ha -1 . These concentrations are based on those used in our previous studies in vitro [START_REF] Bardon | Evidence for biological denitrification inhibition (BDI) by plant secondary metabolites[END_REF][START_REF] Bardon | Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration[END_REF]. Firstly, we considered the dry weight of soil in the field at 10 cm of depth corresponding to lettuce seedling roots implantation. Secondly, we measured a surface relative to this weight, and then, we estimated the weight of 10 cm of soil per hectare. Finally, we calculated how much kg ha -1 of procyanidins must be added to the plots to obtain the equivalent of 0.01, 0.05, 0.1 and 0.25 mg of procyanidins per gram of soil.

The entire experiment was watered the first week after planting with 3 mm of water every day. For the next 10 days, 8 mm of watering was done per day. From then until the end of the experiment, 12 mm of watering was done every two days. Procyanidins were added 2 weeks after planting (stage 7-9 leaves) on a soil whose nitrate had been brought up to the ZENIT grid standard, i.e. 45 kg-NO 3 -ha -1 [START_REF] Despujols | Control of nitrate level in autumn greenhouse lettuce[END_REF]. The commercial procyanidins (Laffort TANIN VR GRAPE ® , Bordeaux, France) were applied in aqueous solution (standard water) by 2 nozzle spray booms, such as to give 500 L/ha or 0.42 L per plot, between the rows of lettuce, and the soil was then hoed. The site was watered (with 8 mm) just after the addition of procyanidins.

Measurement of lettuce traits

Measurement of lettuce mass, specific leaf area (SLA) and flavonoid levels in leaves

After 6 weeks of growing, the lettuces were harvested and 12 shoots only per plot were used to determine the fresh weight of the aerial parts. In addition, 4 whole lettuces per plot were used to determine their mass and to recover the root system and the three leaves of the fourth crown for further analysis.

Each fresh lettuce was weighed on a balance (± 0.5 g). The root system was washed with distilled water and weighed with a precision balance (± 0.001 g), dried at 68°C for 24 h and weighed again on the same balance in order to determine dry mass.

The three leaves from the 4th leaf crown were scanned using Winfolia software (Regent Instruments Inc., Canada), weighed on a precision balance (± 0.001 g), and then used for biomechanical measurements (see point 2.2). The cut-out parts for biomechanical measurements were put back with the initial weighed material and finally dried at 68°C for 24 h and weighed again. The SLA was calculated by dividing the surface area by the dry mass (cm 2 g -1 ). Each week, measurements of flavonoid and anthocyanin levels were made on the 4th leaf of 4 lettuces per plot using Dualex™ (FORCE-A, Orsay, France) technology.

Leaf mechanical properties

One piece of tissue was cut from each leaf (on 4 leaves per plot), for biomechanical measurements. Leaf toughness was measured using 'punch and die' tests, which consist in punching a hole through the leaf lamina. Tests were performed on a universal testing machine (Instron 5942, Canton, MA, USA) using a device consisting of a flat-ended cylindrical steel rod (punch, 2.0 mm diameter) mounted onto the moving head of the testing machine, and a stationary base with a sharp edged hole with a 0.1 mm clearance according to [START_REF] Foucreau | Effect of leaf litter characteristics on leaf conditioning and on consumption by G ammarus pulex[END_REF]. The punch moved down at a constant speed of 10 mm s -1 , without any friction in the hole. The leaves were positioned to avoid primary and secondary veins.

The force applied to the leaf and the displacement were both recorded simultaneously at 10

Hz. Leaf thickness (±0.01 mm) was measured with a digital thickness gauge avoiding major veins. The specific work to punch (called specific toughness, J m -3 ) was calculated as the area under the force-displacement curve corrected by the area of the punch and the leaf thickness [START_REF] Aranwela | Methods of assessing leaf-fracture properties[END_REF].

Plant N content

Total N concentration was measured from 4 leaves and 4 root systems per plot (16 per treatment), using 1.5 mg of ground root or leaf material enclosed in tin capsules, using a Flash 1112 Elemental Analyzer (Thermo Scientific, Waltham, MA, USA). Calibration was performed using aspartic acid (10.52% N) and birch leaf standards (2.12% N; Elemental Microanalysis, Okehampton, UK) interspersed with the samples and used as quality control.

Leaf and/or root N content represent the percentage of N in leaf and/or root dry mass.

Denitrification Enzyme Activity (DEA)

Denitrification enzyme activity (DEA) was measured from pooled root-adhering soil (RAS) retrieved from 4 lettuces per plot and from 4 samples of bulk soil, according to [START_REF] Guyonnet | The effects of plant nutritional strategy on soil microbial denitrification activity through rhizosphere primary metabolites[END_REF]. First, 5 g of soil was placed in 150 ml airtight plasma-flasks sealed with rubber stoppers. In each flask, air was removed and replaced with a He/ C 2 H 2 mixture (90: 10, v/v) to create anoxic conditions and inhibit N 2 O-reductase. A nutritive solution (1 ml) containing glucose (0.5 mg of C-glucose g -1 of dried soil), glutamic acid (0.5 mg of C-glutamic acid g -1 of dried soil) and potassium nitrate (50 mg of N-KNO 3 g -1 of dried soil) was added to the soil.

N 2 O levels during incubation at 28°C were measured each hour for 6 h. The slope of the linear regression (R 2 on average greater than 0.98) was used to estimate DEA as the N 2 O produced (g -1 h -1 ). Gases (CO 2 and N 2 O) were measured with a gas chromatograph coupled to a micro-catharometer detector (µGC-R3000; SRA instruments).

Nitrate concentrations in soil

NO 3 -was extracted every week over time (6 extractions per week) from the planted rhizospheric soils (20 cm from the base of 4 lettuces per plot and pooled) and from the unplanted soils. Nitrate was extracted from 5 g eq. of dried soil supplemented with 20 ml of a solution with 0.01 M of CaCl 2 [START_REF] Houba | Soil analysis procedures using 0.01 M calcium chloride as extraction reagent[END_REF]. Briefly, soil suspensions were shaken at 140 rpm for 2 h at 10°C. The suspension was filtered (0.22 µm) and the NO 3 -concentration was quantified using an ionic chromatograph ICS-900 (Thermo Scientific Dionex, Sunnyvale, California, USA).

Effect of procyanidins on the NarG of nitrate reducing bacteria

To determine whether procyanidins act specifically on the nitrate reductase (NarG) enzyme of denitrifiers, we used two nitrate reducers (but not denitrifier) strains: Escherichia coli MG1655 and Corynebacterium glutamicum ATCC 13032, and a denitrifying strain:

Pseudomonas brassicacearum NFM421. The three strains were grown in 20 ml of Lysogeny Broth (LB) medium supplemented with KNO 3 (20 mM) and procyanidin at 0.01 mg ml -1 , or water as a control. The experiments were performed in plasma flasks sealed with a rubber stopper. To ensure anaerobic conditions, the air was removed as in denitrification measurements. Plasma flasks were inoculated with each strain independently in triplicate at Optical Density (OD) 0.1 and incubated at 28°C for P. brassicacearum, 37°C for E. coli and 30°C for C. glutamicum, with agitation (140 r.p.m.). Growth (OD measurement) and counts on Petri dishes (LB medium) were done from 1 ml of recovered medium at t0, 2, 4, 25 and 30 hours of growth. The count was made using an automatic counter (Scan 1200, Grosseron, Coueron, France) from the 1 ml of medium recovered each time, and diluted by a factor of 10 4 . Cultured petri dishes were incubated at the same temperature as previously stated for each strain.

Quantification of total bacteria and denitrifier abundance

Total DNA was extracted from 0.5 g of RAS for three treatments (unplanted soil, soil with lettuce, soil with lettuce given 210 kg of procyanidins per hectare), in triplicate using the Fast DNA Spin Kit for Soil (MP), according to the manufacturer's protocol. The amount of DNA extracted was estimated using a Quant-iT PicoGreen® dsDNA Assay kit. The amounts of bacteria and denitrifiers were quantified using real-time quantitative PCR (q-PCR) with primers targeting the 16S rRNA and nirK/nirS genes, as described previously by [START_REF] Bru | Determinants of the distribution of nitrogen-cycling microbial communities at the landscape scale[END_REF]. For nirK, the amplification was performed using the primers nirK876 (5'-ATYGGCGGVCAYGGCGA-3') and nirK1040 (5'-GCCTCGATCAGRTTRTGGTT-3') [START_REF] Henry | Quantification of denitrifying bacteria in soils by nirK gene targeted realtime PCR[END_REF]. The 20 µl final reaction volume contained SYBRgreen PCR Master Mix (QuantiTect SYBRgreen PCR kit, Qiagen, Courtaboeuf, France), 1 µM of each primer, 400 ng of T4gp32 (MPbiomedicals, Illkvich, France) and 5 ng of extracted DNA. Thermal cycling was as follows:

15min at 95°C; 6 cycles at 95°C for 15s, 63°C for 30s, with a touchdown of -1°C by cycle, 72°C

for 30s; 40 cycles at 95°C for 15s, 58°C for 30s and 72°C for 30s. For nirS, the amplification was performed using the primers nirSCd3aF (5'-AACGYSAAGGARACSGG-3') and nirSR3cd (5'-GASTTCGGRTGSGTCTTSAYGAA-3') [START_REF] Throbäck | Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE[END_REF]. The 25 µl final reaction volume contained SYBR green PCR Master Mix (as above), 1 µM of each primer, 400 ng of T4gp32 (MPbiomedicals, Illkvich, France) and 12.5 ng of extracted DNA. Thermal cycling was as follows: 15min at 95°C; 6 cycles at 95°C for 15s, 59°C for 30s with a touchdown of -1°C by cycle, 72°C for 30s and 80°C for 30s; 40 cycles at 95°C for 15s, 54°C for 30s, 72°C for 30s and 80°C for 30s). For 16S rRNA, the amplification was performed using the primers 519F (5'-CAGCMGCCGCGGTAANWC-3') and 907R (5'-CCGTCAATTCMTTTRAGTTT-3') [START_REF] Lane | Nucleic acid techniques in bacterial systematics[END_REF]. The 20 µl final reaction volume contained Mix SYBR® Green Master (Roche Diagnostics, Penzberg, Germany), 1µM of each primer and 5 ng of extracted DNA. Thermal cycling was as follows: 10 min at 95°C; 40 cycles at 95°C for 15s, 63°C for 30s and 72°C for 30s; Then 95°C for 1 s; 65°C for 60 s, 68°C for 20s and a continuous increase to 97°C to determine the melting point and finally 10 s at 40°C for cooling. The standard curves for nirK and nirS qPCR were generated by amplifying 10-fold dilutions (10 7 -10 2 ) of a linearized plasmid containing the nirK gene of Sinorhizobium meliloti 1021 and nirS gene of Pseudomonas stutzeri Zobell DNA (GenArt, Invitrogen, Lifetechnologies, Regensburg, Germany). The standard curves for 16S rRNA qPCR were generated by amplifying 10-fold dilutions (10 8 -10 2 ) of the standard DNA pQuantAlb16S plasmid [START_REF] Zouache | Chikungunya virus impacts the diversity of symbiotic bacteria in mosquito vector[END_REF] for 16S rRNA. Melting curve analysis confirmed the specificity of amplification and amplification efficiencies for 16S rRNA, nirK and nirS genes were higher than 90%.

Root development of lettuce seedlings on Knop agar

Lettuce seeds (Lactuca sativa var Lotus RZ) were disinfected according to [START_REF] Bressan | Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots[END_REF], then cultured on Petri dishes (12 x 12 cm) with 100 ml of Knop agar (4mM Ca(NO 3 ) 2 ;

1.8 mM KH 2 PO 4 ; 3.4 mM KCl; 1 mM MgSO 4 ; 1 µM ZnSO 4 ; 14 µM MnCl 2 ; 70 µM H 3 BO 3 ; 10 nM CoCI 2 ; 0.5 µM CuSO 4 ; 0.2 µM Na 2 MoO 4 ; 50 µM FeNa-EDTA; Agar concentration: 0.8%) [START_REF] Hornschuh | Epiphytic bacteria associated with the bryophyte Funaria hygrometrica: effects of Methylobacterium strains on protonema development[END_REF]. Commercial procyanidins (Laffort, Bordeaux, France) were added with a 0.2 µm filter after Knop agar autoclaving. Three conditions were tested, one control condition without the addition of procyanidins and two conditions with 0.1 and 0.2 mg of procyanidins per ml of Knop agar, based on [START_REF] Bardon | Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration[END_REF]. For each condition, three Petri dishes containing 9 seeds (3 replicates and 27 pseudoreplicates) were used. Each Petri dish was grown in phytotron (SANYO, Osaka, Japan) at 22°C, with the day/night period set at 16 h/8 h. After 14 days of growth, the fresh mass of all roots was measured with a precision balance ± 0.0001g. Root development (root length and root surface) was analysed using

WinRhizo software (Regent Instruments Inc., Canada), then placed in an oven at 68°C for 24 h to obtain the dry mass.

Data and statistical analyses

The differences on microbiological and lettuce morphological traits (n ≤ 4) between treatments was tested non-parametrically with a Wilcoxon test. Linear regression was performed to test the functional relationship between the procyanidin concentrations and the response variables. The significance of this regression was tested by Bravais-Pearson test. The difference of lettuce morphological traits (n 16) between treatments was determined using an ANOVA (analysis of variance) followed by a post-hoc Tukey HSD test.

Similarly, the normality (Shapiro test) and the homoscedasticity of variance (Fischer test) of all the variables were tested. For nitrate concentrations data, we compared the concentrations between treatments for each date. In addition, the absence of plot effect was tested on all traits using two-way ANOVA and mixed model. The effect of procyanidins on lettuce seeds was tested using an two-way ANOVA. All the analyses were done using R project software (v. 3.5.0).

RESULTS

Denitrification activity and biological denitrification inhibition

The denitrification activity of microbial communities colonizing the RAS of lettuces treated with 210 kg ha -1 of procyanidins was significantly lower (p-value = 0.023) than that of microbial communities of untreated lettuces. No significant differences in denitrification activity were observed between the RAS from treated lettuces with 8, 42 or 83 kg ha -1 of procyanidins and that from untreated lettuces (Fig. 1A). In addition, the BDI at 210 kg ha -1

was not significantly different from that at other concentrations. The decrease in denitrification activity at 210 kg ha -1 of procyanidins represents a BDI of approximatively 27.17% (p-value = 0.023) (Fig. 1B). In addition, untreated lettuce did not induce BDI compared to unplanted soil as they both presented the same level of denitrification activity.

Moreover, the CO 2 emissions under these conditions did not differ between treatments (data not shown). Interestingly, the biological denitrification inhibition showed a significant dose-response relationship to procyanidins (Fig. 1C).

Effect of procyanidins on NarG of nitrate-reducing bacteria

As shown in Figure 2, the addition of procyanidins, at 0.01 mg ml -1 , to E. coli and C. glutamicum cultures did not affect their growth compared to untreated cultures. However, P. brassicacearum growth was significantly lower (p-value = 0.044 for OD, p-value = 0.037 for enumeration) after the addition of procyanidins.

Abundance of total and denitrifying bacteria

The abundance of denitrifying bacteria was represented by the sum of the copy numbers of the nirS and nirK genes (Fig. 3). The results obtained showed significant differences (p-value = 0.045) among the three treatments (Fig. 3A). The total proportion of denitrifiers from the RAS of lettuces treated with 210 kg ha -1 of procyanidins was 4.5 times lower than that from the RAS of untreated lettuces. However, the total abundance of bacteria, represented by the copy number of 16S rRNA genes per gram of dry soil, did not show any significant variation among the different treatments (Fig. 2B). The proportion of denitrifying bacteria (Fig. 3C), represented by the ratio (nirK + nirS)/ARNr16S, in the RAS of lettuces treated with procyanidins was 5-fold lower (p-value = 0.046) than that in the RAS of untreated lettuces.

Effect of soil procyanidin addition to soil on nitrate concentrations

In the plant treatments with or without the addition of procyanidins, nitrate levels tended to decrease over time (Fig 4). This decrease was greater in the planted soils than in the unplanted soils. The planted soils contained less nitrate than the unplanted soils (p-value = 0.034). In addition, there was no difference in nitrate concentration each week between treatments amended with procyanidins (Fig. 4).

Effect of procyanidin addition on lettuce traits

The fresh mass of the shoot and root parts of lettuces tended to increase with the addition of 0 to 210 kg ha -1 procyanidins (Figs. 5 A andB). Indeed, the fresh mass of the shoot and root parts showed a significant dose-response relationship to procyanidins (p-value = 0.003 for the shoot mass and 0.0016 for the root mass) (Fig. 5C andD). The shoot fresh mass of the lettuces treated with 210 kg ha -1 procyanidins was significantly (16%) higher per lettuce (on average 120 g) (p-value = 0.021) than that of untreated lettuces (Fig. 5A). Similarly, the root fresh mass of lettuces treated with procyanidins at 83 kg ha -1 and 210 kg ha -1 was 39% (pvalue = 0.023) and 63% (p-value = 0.002) higher per lettuce, respectively, than the root fresh mass of untreated lettuces (Fig. 5B). In addition, the root dry mass of lettuces treated with procyanidins at 83 kg ha -1 and 210 kg ha -1 was significantly greater by 52% (p-value = 0.048) and 97% (p-value = 0.028) per lettuce, respectively, than the root dry mass of untreated lettuces. Moreover, the anthocyanin and flavonoid levels, as well as the SLA (cm 2 g -1 ), did not differ between procyanidin-treated and untreated lettuces (Figs. S1 andS2), and the specific toughness (J m -3 ) did not differ among the three leaves of the 4 th crown of treated and untreated lettuces (Fig. 6).

Nitrogen concentration in plants

Overall, the nitrogen content in the tissues (shoot and root) did not differ between treatments and was approximatively 3% in shoots and 1.7% in roots (Fig. 7). However, by relating this percentage of nitrogen in the plant to the mass gain of treated lettuces, we obtain an average gain of 3.6 grams of nitrogen per lettuce treated at a concentration of 210 kg ha -1 .

Effect of procyanidins on root development in vitro

To test whether the addition of procyanidins had a direct impact on root system development, we measured the length and surface area of fresh lettuce roots in Knop agar with or without added procyanidins (Fig. 8). The results showed no significant differences in the fresh mass of young lettuce roots grown in Knop agar with or without added procyanidins (Fig. 8A). The dry weight was slightly lower at 0.2 mg ml -1 procyanidins than under other conditions (Fig. 8B); however, this decrease was not significant. Similarly, no significant differences were observed in the length (Fig. 8C) and root surface area (Fig. 8D) of plants grown with or without the addition of procyanidins.

DISCUSSION

In this study, we found that the addition of 210 kg ha -1 procyanidins to field-grown lettuce crops lead to a significant gain in productivity, resulting from BDI.

The addition of procyanidins under field conditions causes BDI and changes in denitrifier abundance

Denitrification is an inducible function that requires favourable conditions to be expressed: low oxygen concentration, easily available organic compounds as energy sources and the presence of nitrate. In plant rhizospheres, the roots of growing plants cannot only release carbon compounds via exudation but also modify the soil oxygen partial pressure and the nitrate concentration [START_REF] Lecomte | Diversifying anaerobic respiration strategies to compete in the rhizosphere[END_REF][START_REF] Achouak | Plant hosts control microbial denitrification activity[END_REF]. In our case, the addition of procyanidins led, as expected, to a significant decrease in the denitrification activity of lettuce RAS treated with procyanidins at 210 kg ha -1 , which corresponded to a BDI of approximately 27.17% (Fig. 1B). Although denitrification enzyme activity did not decrease under 8, 42 and 83 kg ha -1 of procyanidin amendment (Fig. 1A), the biological denitrification inhibition showed a significant dose-response relationship to procyanidins (R 2 =0.863, pvalue = 0.005) (Fig. 1C), which was consistent with the result of a previous study by [START_REF] Bardon | Evidence for biological denitrification inhibition (BDI) by plant secondary metabolites[END_REF]. Indeed, the same concentrations tested in vitro on the P. brassicacearum strain induced a significant decrease in DEA [START_REF] Bardon | Evidence for biological denitrification inhibition (BDI) by plant secondary metabolites[END_REF], which was observed in the field only at the largest concentration. This result could be explained by the fact that procyanidins at a low concentration may aggregate with soil particles and become less bioavailable.

However, at 210 kg ha -1 , the highest procyanidin concentration, procyanidins were more bioavailable in the soil.

Although denitrification by archaea [START_REF] Shoun | Denitrification by fungi[END_REF] and fungi [START_REF] Philippot | Denitrifying genes in bacterial and archaeal genomes[END_REF] cannot be excluded, procyanidin amendment was demonstrated to have an impact on denitrifying bacteria, such as the P. brassicacearum NFM421 strain. Indeed, [START_REF] Bardon | Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration[END_REF] demonstrated that procyanidins act specifically by inhibiting membrane-bound NO 3reductase, thereby inducing enzymatic conformational changes through membrane disturbance. However, some bacteria are not denitrifiers but do possess this enzyme (NarG), such as E. coli [START_REF] Taniguchi | Nitrate reductase of nitrate respiration type from E. COLI: I. Solubilization and purification from the particulate system with molecular characterization as a metalloprotein[END_REF] and C. glutamicum [START_REF] Nishimura | Anaerobic growth of Corynebacterium glutamicum using nitrate as a terminal electron acceptor[END_REF]. Our results showed that the nitrate reductase of these non-denitrifiers was not affected by procyanidins (Fig. 2). Procyanidins act specifically on the NarG of denitrifiers that contribute to N 2 O emissions. However, as other nitrate reducers are part of the rhizospheric microbial community, the narG gene cannot be used as marker for denitrifiers. Thus, we chose to target the nirK and nirS genes as markers for denitrifiers to assess the impact of procyanidins on denitrifiers. At 210 kg ha -1 of procyanidins, the proportion of denitrifying bacterial communities in the lettuce rhizosphere was reduced (Fig. 3A). The loss of the denitrification function is probably a disadvantage for the denitrifying bacterial community, which becomes less competitive, thus explaining their counter-selection in the rhizosphere. Denitrifiers can no longer use nitrate as an electron acceptor, which reduces their multiplication and their inclusion in the rhizosphere ecological niche, as observed in [START_REF] Dassonville | Niche construction by the invasive Asian knotweeds (species complex Fallopia): impact on activity, abundance and community structure of denitrifiers and nitrifiers[END_REF]. The fact that the total bacterial community abundance (Fig. 3B) and CO 2 release from the microbial community colonizing the RAS of treated lettuces did not change in the presence of procyanidins suggests that (i) other bacterial communities colonized the niches left vacant by denitrifying bacteria, as demonstrated by Jones and Hallin, ( 2010) and (ii) procyanidin addition did not have an antibacterial effect under field conditions, in contrast to the result of [START_REF] Mayer | Proanthocyanidins: target compounds as antibacterial agents[END_REF] and [START_REF] Lacombe | The antimicrobial properties of the lowbush blueberry (Vaccinium angustifolium) fractional components against foodborne pathogens and the conservation of probiotic Lactobacillus rhamnosus[END_REF], where procyanidins had antibacterial properties against some bacterial species.

The addition of procyanidins in the field modifies the soil nitrate concentration and increases lettuce growth.

In in situ microcosm experiments, [START_REF] Bardon | Biological denitrification inhibition (BDI) with procyanidins induces modification of root traits, growth and N status in Fallopia x bohemica[END_REF] observed nitrate conservation in unplanted soil treated with procyanidins compared to untreated soil. Indeed, BDI, which is induced by procyanidins, allows up to 6 x greater storage of nitrate in the soil [START_REF] Bardon | Biological denitrification inhibition (BDI) with procyanidins induces modification of root traits, growth and N status in Fallopia x bohemica[END_REF]. To limit nitrate leaching and make nitrate available for lettuce crops, we added procyanidins one week before the period when the lettuces needed the maximum amount of nitrate for growth (according to the ZENIT grid standard for lettuce). No differences were observed in soil nitrate levels between lettuces treated or untreated with procyanidins, regardless of the concentration (Fig. 4), most likely due to nitrate absorption by lettuce crops. The addition of procyanidins at all tested concentrations tended to induce better lettuce growth as significant dose-response relationship was observed between shoot and root mass and procyanidins concentrations (Fig. 5). This growth improvement was significant only at the highest concentration (210 kg ha -1 ), with an increase in the shoot of up to 16% and of the root of up to 63% (Fig. 5). At this concentration, denitrifiers appear to be inhibited enough to have a visible effect not only on denitrification activity but also on mass gains.

However, at the other concentrations, procyanidins are probably less bioavailable in soil, explaining why no denitrifier inhibition allowing a decrease in nitrate consumption by bacteria was observed. Furthermore, the nitrogen levels in the tissues did not differ between treated and untreated lettuces regardless of the concentration (Fig. 7), which means that there were no physiological changes in lettuces due to the addition of procyanidins that allowed greater nitrogen absorption by the lettuces. However, since lettuces tend to grow better in plots treated with procyanidins, the larger mass implies a greater nitrogen mass. For example, at 210 kg ha -1 procyanidins, we obtained up to 3.6 g of nitrogen on average. Indeed, the procyanidin treatment allowed nitrogen to be preserved in the form of nitrate via biological inhibition of denitrifying bacteria; hence, more nitrate became available for lettuces, allowing better growth. This finding is consistent with the greenhouse study performed on several lettuce varieties, where plant nitrogen use efficiency was not affected by the nitrate concentrations in the soil [START_REF] Urlić | Effect of NO3 and NH4 concentrations in nutrient solution on yield and nitrate concentration in seasonally grown leaf lettuce[END_REF]. SLA trait did not differ between treated and untreated lettuces, regardless of the concentration (Fig. S1), suggesting that treated lettuces did not change their resource allocation to leaf structure [START_REF] Evans | Photosynthetic acclimation of plants to growth irradiance: the relative importance of specific leaf area and nitrogen partitioning in maximizing carbon gain[END_REF]. Moreover, the increase in plant growth did not alter the leaf quality of the lettuce. Indeed, the levels of flavonoids and anthocyanins did not differ between treated and untreated lettuces (Fig. S2), which explains the absence of stress that would have caused an increase in these levels in treated lettuces, as observed for nitratedeficient wine grape and broccoli plants [START_REF] Cerovic | New portable optical sensors for the assessment of winegrape phenolic maturity based on berry fluorescence[END_REF][START_REF] Tremblay | The Dualex-a new tool to determine nitrogen sufficiency in broccoli[END_REF]. In addition, procyanidin amendment did not change the quality of the leaves as no changes in the applied force to pierce a leaf volume were observed (Fig. 6). This result suggests that the procyanidin amendment did not alter leaf vulnerability to potential herbivores and pests [START_REF] Clissold | Gross vs. net income: how plant toughness affects performance of an insect herbivore[END_REF], and did not induce a gustatory difference for consumers in the chewing quality of lettuces potentially placed on the market [START_REF] Lopez-Galvez | Quality changes in packaged salad products during storage[END_REF][START_REF] Rico | Extending and measuring the quality of fresh-cut fruit and vegetables: a review[END_REF].

Interestingly, we showed a larger gain in root mass (up to 63%) than in shoot mass (up to 16%) (Fig. 5). This root mass gain is clearly not due to the direct effect of procyanidins on root development but due to the BDI of denitrifiers and therefore to better availability of nitrate in the rhizosphere of lettuces treated with procyanidins. Indeed, the absence of differences between lettuce seedlings grown in sterile medium with and without procyanidins (Fig. 8) allows us to conclude that procyanidins are not a source of carbon and/or a phytohormone for root development. As the denitrifier distribution in soil is heterogeneous, nitrates available for plants due to denitrifier inhibition will present a heterogeneous spatial distribution in soil. To benefit from this nitrate, plants concentrate new and more root growth in the nitrate-containing zone, which could explain the gain in root mass, as already demonstrated for young lettuce plants submitted to a heterogeneous spatial distribution of nitrate in the root zone [START_REF] Burns | Short-and long-term effects of a change in the spatial distribution of nitrate in the root zone on N uptake, growth and root development of young lettuce plants[END_REF].

The procyanidin source used in this study was from the wine industry and is used to mature wines with a too-low tannin content [START_REF] Harbertson | Impact of exogenous tannin additions on wine chemistry and wine sensory character[END_REF]. The cost of this source of procyanidin is too high to be economically viable for field applications. We used it in our study to demonstrate, for the first time, that biological denitrification inhibition can occur in the field and can induce plant growth improvement. In the context of sustainable agriculture with economic issues, there are other sources of procyanidins that are less expensive and have a higher concentration of procyanidins, such as mimosa and quebracho bark and grape seeds [START_REF] Thompson | Simple 13C-NMR methods for quantitative determinations of polyflavonoid tannin characteristics[END_REF][START_REF] Gabetta | Characterization of proanthocyanidins from grape seeds[END_REF][START_REF] Vivas | Differentiation of proanthocyanidin tannins from seeds, skins and stems of grapes (Vitis vinifera) and heartwood of Quebracho (Schinopsis balansae) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and thioacidolysis/liquid chromatography/electrospray ionization mass spectrometry[END_REF][START_REF] Venter | Analysis of commercial proanthocyanidins. Part 1: The chemical composition of quebracho (Schinopsis lorentzii and Schinopsis balansae) heartwood extract[END_REF]. Future studies using these sources of procyanidins in the field and evaluating the benefits/costs of such applications are needed.

Conclusions

The addition of procyanidins in the field allows a decrease in denitrification activity, making nitrates available at ground level, which results in an increase in lettuce yields without changing their physiology. Based on the results of this study, we propose in the short-term the development of a more environmentally friendly method of sustainable agriculture by limiting fertilizer use and nitrogen losses in the soil while increasing plant growth and productivity. Procyanidin concentrations (kg.ha -1 )

Figure legends:

Figure S2

Figure

Figure 1 :

 1 Figure 1: Impact of procyanidins applied in the field on microbial denitrification activity.

Figure 2 :

 2 Figure 2: Effects of procyanidins (0.01 mg ml -1 ) on E. coli MG1655, Corynebacterium

Figure 3 :

 3 Figure 3: Effects of procyanidins (210 kg of procyanidins per hectare) applied in the field to

Figure 4 :

 4 Figure 4: Effect of procyanidins addition on soil nitrate level. Effects of different

Figure 5 :

 5 Figure 5: Effects of different concentrations of procyanidins (0, 8, 42, 83 and 210 kg of

Figure 6 :

 6 Figure 6: Effects of different concentrations of procyanidins (0, 8, 42, 83 and 210 kg of

Figure 7 :

 7 Figure 7: Nitrogen concentration in plants treated with procyanidins. Effects of different

Figure 8 :

 8 Figure 8: Effect of procyanidins on root development in vitro. Effects of different

Figure S1 :

 S1 Figure S1: Effects of different concentrations of procyanidins (0, 8, 42, 83 and 210 kg of

Figure S2 :

 S2 Figure S2: Effects of different concentrations of procyanidins (0, 8, 42, 83 and 210 kg of

Acknowledgements

This work was supported by Auvergne-Rhône-Alpes region (ARC 2016-2019) for thesis subvention. Quantitative PCR were performed at the DTAMB platform (FR BioEnviS, University Lyon1). Field experiments were performed at Serail station. Microbial activities were measured at the AME platform (UMR 5557) and C and N content were measured at Isotope Ecology platform (UMR 5023). We thank Laurent Philippot and Delphine Moreau for scientific discussions. The authors gratefully thank the editor Dr Kamlesh Jangid and reviewer for improving our manuscript.