WO2016146955A1 - Use of proanthocyanidins for limiting denitrification - Google Patents

Use of proanthocyanidins for limiting denitrification Download PDF

Info

Publication number
WO2016146955A1
WO2016146955A1 PCT/FR2016/050600 FR2016050600W WO2016146955A1 WO 2016146955 A1 WO2016146955 A1 WO 2016146955A1 FR 2016050600 W FR2016050600 W FR 2016050600W WO 2016146955 A1 WO2016146955 A1 WO 2016146955A1
Authority
WO
WIPO (PCT)
Prior art keywords
proanthocyanidin
proanthocyanidins
denitrification
use according
soil
Prior art date
Application number
PCT/FR2016/050600
Other languages
French (fr)
Inventor
Franck POLY
Florence PIOLA
Gilles Comte
Feth el Zahar HAICHAR
Clément BARDON
Original Assignee
Universite Claude Bernard Lyon I
Centre National De La Recherche Scientifique
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universite Claude Bernard Lyon I, Centre National De La Recherche Scientifique filed Critical Universite Claude Bernard Lyon I
Priority to EP16715023.4A priority Critical patent/EP3271312A1/en
Publication of WO2016146955A1 publication Critical patent/WO2016146955A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • C05G3/90—Mixtures of one or more fertilisers with additives not having a specially fertilising activity for affecting the nitrification of ammonium compounds or urea in the soil

Definitions

  • the present invention relates to the environmental field, and more specifically to a means of limiting the denitrificadon intervening in the environment, especially in the soil, and more particularly the crop,
  • Nitrogen is a limiting factor for plant growth (LeBauer & ⁇ reseder, 2008). The development of plants therefore strongly depends on the processes of the nitrogen cycle which condition the transformation of nitrogen in all its forms including the availability of NH and G3, which are the assimilable forms of nitrogen by plants.
  • denitrification mostly carried out by so-called denitrifying bacteria, which consists of the reduction of NO3 (nitrate) to H 2 Q and 2, is considered as the main biological pathway of loss of nitrogen.
  • denitrifying bacteria which consists of the reduction of NO3 (nitrate) to H 2 Q and 2
  • these losses represent 59% of the total losses of the system (Genema et al., 2009).
  • the gaseous forms of nitrogen, N3O and N3 ⁇ 4 generated by denitrification are released in the atmosphere and thus made inaccessible to plants.
  • DMPP dimethylpyrazophosphate
  • Root and rhizome extracts from Failopla spp. An invasive plant complex, have been described as reducing the denitrification process of soybeans (Bardon et al, 2014).
  • Bardon et al 2014 identified in this publication a number of metabolites present in the tested extract of Faiiopia spp. including (+) ofcechin, ⁇ + ⁇ - epiatechin., piceatanoi glucoside, resveratroloside, iemodine, physion, resveratrol and pictreide and show a correlation between catechin concentration in the extracts and ⁇ Inhibition of denitrifioation.
  • proanthocyanidins were responsible for the inhibition of the denervation observed with extracts of Faliopsa x bohemica.
  • these compounds do not affect the activity of the enzymes of respiration, an important function for the growth of soil microorganisms.
  • the present invention relates to the use of at least one proanthocyanidin to imitate denitrification caused by microorganisms by contacting said at least one proanthocyanidin with said microorganisms.
  • microorganisms that cause the denitrifying activity that is reduced in the context of ⁇ nvention are most often present in a medium.
  • the microorganisms When used according to (Invention, the microorganisms will then be present in the medium with the said at least one proanthocyanidin, in which case the proanthocyanidin will be placed in contact with the medium and, consequently, with the microorganisms to be killed.
  • the medium (with which proanthocyanidin is brought into contact, called the treated medium) may be a solid medium, such as soil, and in particular crop soil, compost, manure, soil, compost, sewage sludge, sediment or a liquid medium, such as an aqueous medium such as river water, water wastewater, wastewater, without this list being exhaustive.
  • the contacting of at least one proanthocyanidlne with the medium of interest. Can be performed by any suitable technique ,, ", particularly by applying the proanthocyanidlne on said medium or by mixing the proanthocyanidin with said medium.
  • Contacting time with the microorganisms to the effect of said microorganisms on ocyanidlnes prooant corresponds to the desired period for obtaining the effect limiting dénitrlficatlon.
  • one or more proanhocyanidine (s) are (are) used to limit the denitrlficatlon caused by microorganisms normally having the capacity to reduce O3 (nitrate present in the treated medium) in f3 ⁇ 40 and
  • microorganisms possess the genes coding for nitrate and nitrite reductases.
  • microorganisms include fungi such as Cylindrocarpan tonkmense ?
  • the contacting with the microorganisms is carried out so as to obtain a reduction in the denlthfication caused by the microorganisms having a denitrifying activity, or in other words so as to at least partially inhibit the denitrifying activity of the microorganisms.
  • reduction or inhibition is to be considered, with respect to the emission of 20 and 3 ⁇ 4 and / or the denitrilfication (transformation of nitrate O3 or more precisely N ⁇ 1 ⁇ 4 " into N 3 and f ) which would be obtained in the absence of contacting the microorganisms concerned with said proanthocyanidin.
  • the denitrlficatlon activity is obtained by measuring the emission of 1 ⁇ 2G -f H 2f d one hand in the presence and, on the other hand, the absence of proanthocyanldine, which allows to obtain a percent inhibition of the denitrification corresponding to (transmission 2 0 + H 2 in the presence of proanthocyanidin) / (emission 2O + Nfe absence of proanthocyanldine) * 100 .
  • the inhibition of denitrification by proanthocyanidines can in particular reach a maximum level of 100%, and in the context of the invention will be obtained the% dinhsbitlon denitrification, generally from 20 to 100 %, preferably 50 to 100%,
  • the technique described in Dambraville et al., 2006, and used in the examples, can be used to evaluate U 2 -U 2 uptake.
  • the amount of proanthocyanidin (s) used will be adjusted by those skilled in the art to achieve the desired effect.
  • proanthocyanidins also known as proanthocyanidic tannins, condensed tannins, or proanthocyanidols, are fiavonoids, and more specifically polymers of hydroxyflavan-3-ol.
  • the hydroxyflavan-5-ol monomers constituting the proanthocyanidines have the following structure (I). ):
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 which are identical or different, are chosen from hydrogen, hydroxyl, gallate, methyl or oside groups.
  • proanthocyanidins are polymers hydroxyffevan d f ⁇ 3 ⁇ Oi which two consecutive monomers are linked by a single covalent bond between two carbons, depending on the positions 4-6 or 4-8 (respective positions in both monomers), as shown in the formula corresponding to a dimer comprising a 4-8 linkage, followed by a 4-6 linkage:
  • R 2 ', R y and R 6', R r, R, R 3 ", R 4", R 5 "and R 6" may be the same groups or different groups representing hydrogen, a hydroxyl group a gallate group, a methyl group or a saccharide group,
  • the proanthocyanidins of type A are polymers of hydroxyflavan-3-ol of which two consecutive monomers are connected by two covalent bonds between two carbons, at position 2-7 and 4-8 (respective positions in the two monomers), illustrated in the case of a dimer by formula (III) below i
  • R 1, R 2 f R 3, R 4 R s and f 6; R r , R f 4 ' , R 5' and R 6 ' which may be the same groups or different groups, representing hydrogen, a hydroxyl group, a gailate group, a methyl group or an osidic group.
  • proanthocyanidins which can be used in the context of the invention consist of 2 to n monomers (hydroxyflavan ⁇ 3 ⁇ ol) connected by covalent bonds previously described, according to all the possible variations.
  • one or more proanthocyanidins chosen from among the dimer, irimer and tetramer will be used.
  • proanthocyanidins are polymers of (- / +) - catechin, (7 ⁇ ) epicatechin., (- / - f-) ⁇ gallocatechin and (- / ⁇ ) - epigallocatechin.
  • proanthocyanidins are found subgroups of molecules, such as, for example, prodelphinidines. (prodeiphinidoi) or procyanidins (procyanidol).
  • Procyanidins correspond to polymers of (- / -) ⁇ cathehine and / or ⁇ - / - * ⁇ ⁇ - epicatechin.
  • Proanthocyanidins are natural products which can, in particular, be obtained and purified from extracts of Faifopia spp., And in particular Falhpia x bohemka as evidenced by the inventors, or else from Vitis vinifera seeds, Pteridium spp. VMdum spp. Schfûops / s spp ,, Vicia spp v C / 5 spp., Quercus spp , many commercial proanthocyanldines are available, especially in LAF OT (Bordeaux, France), Organic Herb Inc. (Changsha , China) Nanjing Zelang Medical Technology Co., Ltd. (Jiangsu, China),
  • proanthocyanidins of type B and even more preferably procyanidins of type 8, in particular in the form of dimer, trimer or tetramer,
  • said at least one proanthocyanidin used is not in the form of an extract of Fafiopia spp., Containing proanthocyanidins, and in particular Faiiopia x bohemica, and / or said at least one proanthocyanidin. is used, in the absence of cathehine and / or epicatechin,
  • the proanthocyanidin (s) used may be applied to the medium containing the microorganisms whose denitrification effect is to be limited, or mixed with the latter, in the form of an aqueous solution or suspension or still in the form of a powder or granule,
  • At least one proanthocyanidin may, in particular, be used to promote plant growth on a culture soil, by reducing denitrification caused by microorganisms. Having a denitrifying activity, and in particular caused by denitrifying bacteria present in said soil Proanthocyanidins being natural products, their use in crops is in total respect with the environment Proanthocyanidins can therefore be used in organic farming.
  • At least one proanthoeyanidine is applied to a soi, and in particular a culture soil, for example by deposition or spraying on the ground.
  • said at least one proanthoeyanidine is used to limit denitrification caused by soil bacteria, such as Pseudo onas brassacacearum and / or Sac us cernas,
  • proanthocyanidin in the case of a solution of proanthocyanidin (s), it may be applied by spraying or any other appropriate means.
  • the soils that can be treated in the context of the invention include, inter alia, cultivation, market gardening, cereal growing, viticulture and more generally, soils where various plants of agronomic interest, including various plants or flowers , are grown.
  • the effect of the proanthocyanidin (s) used on denitrification will be obtained over a wide range of temperatures, especially at a soil temperature of 0 to 5 ° C. and with an optimum effect at a moisture content greater than 50% of the field capacity.
  • proanthocyanidin (s) to achieve the desired denitrification limitation will be applied to the soil.
  • the amount applied will be adapted by those skilled in the art, in particular, depending on the formulation and the soil moisture content. .
  • from 0.01 to 10 mg / g of dry sol, and preferably from 0.05 to 5 mg / g of dry sol, of proanthocyanidene (s) is applied.
  • These amounts are applicable to solid media, other than the self, mentioned above, in which the microorganisms could be present.
  • proanthocyanidins and in particular type B procyanidins, make it possible to reduce the process of bacterial denitrification of soils.
  • the results were obtained on strains of bacteria in culture, but also on ones of different natures (one of river edge and grassland soil) and containing communities of complex and different denitrifying bacteria,
  • proanthocyaoidin may be associated, in the context of the invention, with one or more other fertilizers (nitrogen or phosphated, for example).
  • At least one proanfhocyanidine may be applied to a culture soil, to promote the growth of said culture against denitrification caused by microorganisms, and in particular bacteria, present in said soi. Said application may be made without the joint application of a nitrogen fertilizer.
  • a composition in solid or liquid form will be integrated in said medium.
  • An effective amount of the ocyanidine proant (s) to achieve the desired denitrification limitation will be incorporated into said medium.
  • the amount incorporated will be adapted by those skilled in the art.
  • from 0.01 to 2.5 mg of proanthocyanidin (s) per milliliter of liquid medium, and preferably from 0.01 to 1 mg of proanthocyanidin (s) per milliliter of liquid medium will be incorporated.
  • Fig. I Percent inhibition (relative to control) on the denitrification by Pseudomons brassicacearum of a mixture of the major compounds present in the alkaline bohemia and the Faptapta x bo emica extract.
  • the vertical bars correspond to the variations obtained on the different tests.
  • the stars indicate the significant variations relative to the control (Tukey test, ⁇ ⁇ 0.05).
  • Figure 2 Chroroatograrnnie at 280 nm extract Faiiopia x bohemica showing proanthocyanidins and their monomers detected as well as their UV spectrum.
  • Figure 4 Percentage inhibition of denitrification and respiration of a) Pseudomonas brassileacearum and b) E cereus as a function of concentration of purified proanthocyanidins »Stars indicate significant differences in means with control without proanthocyanidins (Tukey test a ⁇ 0.05).
  • Figure S Percentage inhibition of denitrification of river (Ainii) and grassland ( ⁇ ' ) soils as a function of commercial proanthocyanidin concentration.
  • the stars indicate significant differences in means with control (Tukey test, ⁇ 0.05).
  • the extract was analyzed using a UHPLC 1290 infinity series apparatus (Agitate Technologies, Santa Clara, USA) coupled to a G4212A DAD diode array UV detector (Agitate Technologies, Santa Clara, USA) and Q-TGF 6530 series mass spectrometer (Agllent Technologies, Santa Clara, USA),
  • UV spectra were recorded between 190 and 600 nm and the 280 nm response was used for qualitative interpretations.
  • the ESI source was optimized as follows: Positive Ionization mode, 80/2000 m / z spectra scan, 3.0 kV capillary voltage, 70 V fragmentor, Collision-induced dissociation (CID) collision ) was set at 20 eV. Nitrogen was used as a gas nebiller with a flow of 12 l / min and a temperature of 310 ° C to 40 psi. Proanthocyanidins were identified in the extract by UV, HS and MS / MS spectra analysis using the MassHunter Qualitative Analyst software (Agitant Technologies, Santa Clara, USA), the spectra obtained being shown in Part II B).
  • the proanthocyanidins contained in the E x bohe ica extract were purified using a Sephadex LH20 column.
  • Sephadex LH 20 was previously equilibrated three times by wetting 25 g of Sephadex LH 20 with 100 ml of 80% ethanol and then removing the supernatant.
  • Extracted Sephadex was loaded into a 10 ml glass buchner.
  • Sephadex LH 20 was washed with ethanol. 95% until the absorbance of the water at 280 nro is close to zero.
  • proanthocyanidins purified from F. x bohemica extracts was tested at concentrations of 0.1; 0.05; 0.01 or 0 mg.ml- 1 on the denitrification and respiration of Pseudomanas brassca ⁇ aru NFH421 and Badllus ⁇ reus A19, two denltriflant bacteria frequently found in soybeans, Four this, 5 ml of MB liquid growth medium with 20 mM NO.sub.2.
  • Respiration measurements were performed in sealed plasma bottles by CO 2 accumulation assay . without replacing the atmosphere of the flasks.
  • the N 2 O and C0 2 emitted were measured every two hours for 48 hours using a microcatheter (pGO 30Q0 f SRA instruments, Marcy tolle, France).
  • proanthocyanidines commercial purified from grape seeds were used (Laffort, Bordeaux, France). Samples of two soils were collected at the Ainl sites (45 ° 57 ⁇ 53 ⁇ , 5 ⁇ 15'25 ⁇ ) and ⁇ (45 ° 48 ⁇ 5 " ⁇ , 5 ° 10 * 29" E) and sieved at 2 mm.
  • the déûitrifîcatlon rate was measured emission% G every two hours for 30 hours under anaerobic conditions with a micro- cathanobooks (P6C-R30 ⁇ 0 f SRA instruments., Marcy l'Etoile, France).
  • sols were suspended in 10 ml of 0.1M phosphate buffer (MaH 2 PG and NaHPO 3 pH 7.5) supplemented with glucose (0.5mg of C-glucose 1 ), glutamic acid ( 0.5 mg of glutamic C-atide.mr 1 ) and N £ 3 ⁇ 4 (20 mM).
  • 0.1M phosphate buffer MoH 2 PG and NaHPO 3 pH 7.5
  • proanthocyanidins on denitrification was measured for concentrations of 2.5; i; 0.5; 0.1; 0.05; 0.01 and 0 mg.ml * * in the end, adding water or proanthocyanidins to the soil suspensions.
  • the soil suspensions were stirred at 28 ° C throughout the experiment. All treatments were performed in four replicates.
  • a range of active proanthocyanidin concentrations on soil denitrification reported in g of dry soil was carried out.
  • the soil used for this experiment is the so-called Ain IL.
  • Ten grams of dry equivalent soil were incubated in a plasma bottle in the open air for? days at 28 ° C to field capacity, the equivalent of 10; 5; 1; 0.5; 0.1; 0.05; 0.01 and mg.
  • the soils were supplemented with a solution of 0.5 ml containing the equivalent of 50 ⁇ gN-NCh.g- 1 sol dry, 0.5 mg of C-aclde glutarnique.g "1 dry soil and 0.5 mg of C-glucose.g "1 self sec.
  • Proanthocyanidins detected by UHPLC in extract F, x bo EMICA are Type B procyanldines ranging from d mother téfcramère of catéchlne and epicatechin with a absorbaoce maximaie at 278 nm and molecular weights of 579.15, 867.21 and 1155.27 g mol -1 , as shown in Figures 2 and 3 (the spectrum of Fig 3 corresponds to the dimer, the spectrum of Figure 38 to the trimer and the spectrum of Figure 3C to the tetramer).
  • the potential nitritlcafion of the two soil types was measured in suspension (1 g of dry equivalent sol per 10 ml) without proanthocyanidins (control) and in the presence of commercial proanthocyanidins, concentrated to 0.5; 0.1; 0.05 and 0.01 g / cm 3 in phosphate buffer solution at soil pH 7.5 (NaHiPC 2 and NaHPG 2 , 12 ⁇ 20) (to limit pH changes during incubation) supplemented with (NH SGs (SO g.mL "1). soils were placed in 150 ml flasks plasma aerobically blocked with psrafilm, flasks were set stirring at 140 rpm at 28 e C" Each conditions were realized in 4 replicas.
  • Nltrifscation potential measurement (PBN "nltrifylng enzymatic actlvlty” ⁇ was performed by assaying the production of NC3 ⁇ 4 over time using an ionic dpf chromafographe Dionex DX 120 (Thermo Fisher Sclentific, Waltham, USA), in removing and filtering at 0.2 ⁇ m, 1 ml of suspension every 2 hours between 2 and 10 am
  • the measurement of potential nitridation corresponds to the extent enzymatlque pool present in the sample initially, and is expressed by the production pg N- C .g "dry soil. fi 1 between 2 and 10 hours.

Abstract

The invention relates to the use of at least one proanthocyanidin for limiting denitrification, consisting in the reduction of NO3 in N2O et N2, caused by microorganisms particularly present in an environment, by bringing said at least one proanthocyanidin into contact with said microorganisms.

Description

La présente invention concerne e domaine environnemental, et plus précisément un moye de limiter la dénitrificadon intervenant dans l'environnement, notamment dans les sois, et plus particulièrement les sois de culture,  The present invention relates to the environmental field, and more specifically to a means of limiting the denitrificadon intervening in the environment, especially in the soil, and more particularly the crop,
L'azote est un facteur limitant de la croissance des végétaux (LeBauer & ïreseder, 2008). Le développement des végétaux dépend donc fortement des processus du cycle de l'azote qui conditionnent ia transformation de l'azote sous toutes ses formes notamment la disponibilité en NH et G3, qui sont les formes assimilables de l'azote par les végétaux. Nitrogen is a limiting factor for plant growth (LeBauer & ïreseder, 2008). The development of plants therefore strongly depends on the processes of the nitrogen cycle which condition the transformation of nitrogen in all its forms including the availability of NH and G3, which are the assimilable forms of nitrogen by plants.
L'azote atmosphérique est fixé dans les sols et transformé en ammonium ( H4 ou plus précisément MH +) par des bactéries fixatrices, une autre source de Nf est l minéralisation des matières organiques azotées, puis la nitrif tion correspondant à la transformation de l'ammonium en nitrate (NO3, ou plus précisément NO3') suivie de ia réduction de O3 en N2O et Hz interviennent. Différentes publications ont étudié l'influence de tanins condensés ou de terpènes sur la minéralisation de l'azote et sur la nitrificatîon. Adamczyk Bartosz et al : « esponse of soi! C and transformations to condensed tannins and différent organic N~coodensed tannin complexes », voL 64, 8 janvier 20.13, pages 163-170, décrit que dans des échantillons de sols, l'addition de tanins condensés diminue la nitrificatîon, suggérant leur effet toxique et/ou précipitant des protéines, Ba!dwin I T et al : « Protein binding phenolics and the inhibition of nitrificatîon in subalpine balsam fir soils », vol. 15, nô4, 1er janvier 1983, pages 419-423, étudie les effets sur la nitrificatîon d'un extrait méthanoîique de sols de forêt. Il est conclu que les tanins condensés présents dans les extraits étudiés sont les composants présentant une activité inhibitrlce de la nitrificatîon la plus importante. Adamczyk Sylvvia et ai : « Potentiel response of soi! processes to d'sterpenes, triterpenes and tannins : Nitrificatîon, growfh of microorganisms and précipitation of proteins »t vol 67, 24 mars 2013, pages 47-52, décrit l'activité de terpènes et d'acide tannique, ainsi que de tanins condensés, en tant qu'inhibiteurs de la nitrificatîon dans des suspensions de sols avec un excès de MH4-N. R L Bradley et ak « Changes to minerai N cyciing and microblal communities in black spruce humus after additions of (MH4)2S04 and condensed tannins extracted from Kalmia angustifolia and basam fir », 1er août 2000, pages 1227-1240 rapporte que ies tanins condensés diminuent la minéralisation de l'azote, en se liant et en séquestrant ies sources organiques d'azote. Sur de l'humus non fertilisé., une diminution de la concentration en nitrates est observée, The atmospheric nitrogen is fixed in the soils and transformed into ammonium (H4 or more precisely MH + ) by fixing bacteria, another source of Nf is the mineralization of the nitrogenous organic matter, then the nitrification corresponding to the transformation of the nitrogen. ammonium nitrate (NO3, or more precisely NO3 ' ) followed by the reduction of O3 in N 2 O and Hz occur. Various publications have investigated the influence of condensed tannins or terpenes on nitrogen mineralization and nitrification. Adamczyk Bartosz et al: "Esponse of oneself! C and transformations to condensed tannins and different organic Necoodensed tannin complexes, vol. 64, January 8, 2013, pages 163-170, discloses that in soil samples, the addition of condensed tannins decreases nitrification, suggesting their toxic effect. and / or precipitating proteins, Ba! dwin IT et al: "Protein binding phenolics and inhibition of nitrification in subalpine balsam fir soils", vol. 15 n O 4 1 January 1983, pages 419-423, studied the effects on nitrification of a methanolic extract of forest soils. It is concluded that the condensed tannins present in the extracts studied are the components exhibiting the most important nitrification inhibitory activity. Adamczyk Sylvvia and have: "Potential response of self! to processes of sterpenes, triterpenes and tannins: nitrification, growfh of microorganisms and precipitation of proteins' t flight 67, March 24, 2013, pages 47-52, describes the activity of terpenes and tannic acid, as well as condensed tannins , as inhibitors of nitrification in soil suspensions with an excess of MH4-N. RL Bradley et al. "Changes to ore N cyciing and microblal communities in black spruce humus after additions of (MH4) 2S04 and condensed tannins extracted from Kalmia angustifolia and basam fir", August 1, 2000, pages 1227-1240 reports that condensed tannins decrease nitrogen mineralization, by binding and sequestering organic sources of nitrogen. On unfertilized humus, a decrease in nitrate concentration is observed,
Parmi les processus du cycle de l'azote, la dénitrtlcation,, réalisée en majorité par des bactéries dites dénitrifiantes, qui consiste en la réduction du NO3 (nitrate) en H2Q et 2, est considérée comme la principale voie biologique de perte d'azote des sols, Dans ies agrosystèmes européens, ces pertes représentent 59% des pertes totales du système (Genema et al., 2009), En effet, les formes gazeuses de l'azote, N3O et N¾ générées par dénitrification, sont libérées dans l'atmosphère et donc rendues inaccessibles aux plantes. Among the processes of the nitrogen cycle, denitrification, mostly carried out by so-called denitrifying bacteria, which consists of the reduction of NO3 (nitrate) to H 2 Q and 2, is considered as the main biological pathway of loss of nitrogen. In European agro-ecosystems, these losses represent 59% of the total losses of the system (Genema et al., 2009). Indeed, the gaseous forms of nitrogen, N3O and N¾ generated by denitrification, are released in the atmosphere and thus made inaccessible to plants.
De manière classique en agriculture, les pertes d'azote causées par la dénitrlficatlon et responsables de perte de rendement sont compensées par l'ajout d'engrais azotés. Toutefois, cet apport entraîne une augmentation d'émission de Η*0, gaz à effet de serre 380 fois plus puissant que le CC¾.  In a conventional way in agriculture, nitrogen losses caused by denitrification and responsible for yield loss are offset by the addition of nitrogen fertilizers. However, this contribution increases the emission of Η * 0, a greenhouse gas 380 times more potent than CC¾.
Certaines solutions, à base de composés de synthèse, tel que le diméthylpyrazoiphosphate (DMPP), ont été proposées pour limiter temporairement les pertes en inhibant ia nitrlrleation, Ces composés inhibent l'activité des bactéries nitrifiantes du sol et en conséquence limitent la transformation de l'ammonium (NHU) en nitrate (NO3).  Some solutions, based on synthetic compounds, such as dimethylpyrazophosphate (DMPP), have been proposed to temporarily limit losses by inhibiting nitrilation. These compounds inhibit the activity of nitrifying bacteria in the soil and consequently limit the transformation of the soil. ammonium (NHU) to nitrate (NO3).
Des extraits de racines e de rhizomes de Failopla spp un complexe de plante invasive, ont, quant à eux, été décrits comme réduisant le processus de dénitrlficatlon des sois (Bardon et al, 2014). Bardon et al 2014, ont identifié dans cette publication, un certain nombre de métabolites présents dans l'extrait testé de Faiiopia spp. dont la (+)ofcéchine, la {+}- épîcatéchine., le piceatanoi glucoside, le resveratroloside, iémodine, le physion, le resvératrol et du picéide et montrent une corrélation entre ia concentration en catéchine dans les extraits et ί Inhibition de la dénitrifioation. Root and rhizome extracts from Failopla spp., An invasive plant complex, have been described as reducing the denitrification process of soybeans (Bardon et al, 2014). Bardon et al 2014 identified in this publication a number of metabolites present in the tested extract of Faiiopia spp. including (+) ofcechin, {+} - epiatechin., piceatanoi glucoside, resveratroloside, iemodine, physion, resveratrol and pictreide and show a correlation between catechin concentration in the extracts and ί Inhibition of denitrifioation.
Par la suite, les inventeurs de la présente demande de brevet ont testé l'effet de la catéchine,, ainsi que de tous les composés identifiés dans cette publication (épicatéchine, resvératrol, picéi e, émodlne et physcion) sur la dénltrificatlon de souches bactériennes dénltrifsantes et ont constaté, de manière totalement inattendue, que la catéchine même additionnée aux autres molécules Identifiées comme majoritairement présentes dans l'extrait, n'avait pas d'effet sur la dén itrifkation,  Subsequently, the inventors of the present patent application have tested the effect of catechin, as well as all the compounds identified in this publication (epicatechin, resveratrol, picone, emodyn and physcion) on the denltrification of bacterial strains. denltrifsantes and found, in a totally unexpected way, that the catechine itself, added to the other molecules identified as mainly present in the extract, had no effect on denitrification,
Les Inventeurs ont alors mis en évidence que d'autres molécules, non encore identifiées dans cette publication, les proanthocyanidines étaient responsables de {Inhibition de la déni tification observée avec les extraits de Faliopsa x bohemica. De plus, ces composés n'affectent pas ou peu l'activité des enzymes de la respiration, fonction importante pour la croissance des microorganismes du sol.  The inventors then demonstrated that other molecules, not yet identified in this publication, proanthocyanidins were responsible for the inhibition of the denervation observed with extracts of Faliopsa x bohemica. In addition, these compounds do not affect the activity of the enzymes of respiration, an important function for the growth of soil microorganisms.
Dans ce contexte, la présente invention concerne l'utilisation d'au moins une proanthocyanîdîne pour {imiter la dénitrification causée par des microorganismes, par mise en contact de ladite au moins proanthocyanîdîne avec lesdits microorganismes.  In this context, the present invention relates to the use of at least one proanthocyanidin to imitate denitrification caused by microorganisms by contacting said at least one proanthocyanidin with said microorganisms.
Les mlcroorganismes à l'origine de l'activité dénitrifiante qui est réduite dans le cadre de { nvention sont le plus souvent présents dans un milieu. Lors de l'utilisation selon (Invention, les microorganismes seront alors présents dans le milieu avec ladite au moins proanthocyanîdîne. Dans ce cas, la proanthocyanîdîne sera mise en contact avec le milieu et, de ce fait, avec les microorganismes à obier. Dans certains cas, la mise en contact de la proanthocyanîdîne et des microorganismes se fera suite à un phénomène de diffusion de la proanthocyanîdîne dans ie milieu. Le milieu (avec lequel la proanthocyanîdîne est mise en contact, nommé milieu traité), peut être un milieu solide, tel qu'un sol, et en particulier un sol de culture, compost, fumier, iisier, terreau., boues de station d'épuration, sédiments ou encore un milieu liquide, tel qu'un milieu aqueux du type eau de rivière, eaux usées, eaux de station d'épuration, sans que cette liste soit limitative. Dans le cadre de [Invention, la mise en contact d'au moins une proanthocyanidlne avec le milieu d'intérêt., peut être réalisée,, par toute technique appropriée», notamment par application de la proanthocyanidlne sur ledit milieu ou par mélange de la proanthocyanidlne avec ledit milieu. Le temps de mise en contact avec les microorganismes permettant l'effet des prooant ocyanidlnes sur lesdïts microorganismes correspond à la durée souhaitée d'obtention de l'effet limitant la dénitrlficatlon. The microorganisms that cause the denitrifying activity that is reduced in the context of {nvention are most often present in a medium. When used according to (Invention, the microorganisms will then be present in the medium with the said at least one proanthocyanidin, in which case the proanthocyanidin will be placed in contact with the medium and, consequently, with the microorganisms to be killed. In this case, the contact between proanthocyanidin and microorganisms will be due to a proanthocyanidin diffusion phenomenon in the medium The medium (with which proanthocyanidin is brought into contact, called the treated medium) may be a solid medium, such as soil, and in particular crop soil, compost, manure, soil, compost, sewage sludge, sediment or a liquid medium, such as an aqueous medium such as river water, water wastewater, wastewater, without this list being exhaustive. Under [Invention, the contacting of at least one proanthocyanidlne with the medium of interest., Can be performed by any suitable technique ,, ", particularly by applying the proanthocyanidlne on said medium or by mixing the proanthocyanidin with said medium. Contacting time with the microorganisms to the effect of said microorganisms on ocyanidlnes prooant corresponds to the desired period for obtaining the effect limiting dénitrlficatlon.
Dans le cadre de l'invention, une ou plusieurs proanihocyanidine(s) ast(sont) utillsée(s) pour limiter la dénitrlficatlon causée par des microorganismes ayant normalement la capacité de réduire du O3 (nitrate présent dans le milieu traité) en f¾0 et f¾ (capacité qualifiée d'activité dénitrlfiante), En particulier, de tels microorganismes possèdent les gènes codant pour la nitrate et nitrite réductases. De tels microorganismes sont notamment des champignons tels que Cylindrocarpan tonkmense? Fusanum oxysporum ou des bactéries des genres Pseudonomas, Badliusf Burkholderia, Achromobacter, Ochmbadrum, Citmbac er, Paraeoceus, Micrococcus, telles que les espèces Pseudomonas brassicacearum, Pseudomonas stutzeii, Pseudomonas aicaligenes, Pseudomonas fiuoresœns, Burkholderia vietnamiensis, Achromobacter xyiomxfdans, Parawcws denitrihcans, Badilus œreus, connues pour être des bactéries dénitrifiantes. Ces bactéries sont le plus souvent présentes dans les sols, In the context of the invention, one or more proanhocyanidine (s) are (are) used to limit the denitrlficatlon caused by microorganisms normally having the capacity to reduce O3 (nitrate present in the treated medium) in f¾0 and In particular, such microorganisms possess the genes coding for nitrate and nitrite reductases. Such microorganisms include fungi such as Cylindrocarpan tonkmense ? Fusanum oxysporum or bacteria of the Pseudomonas genera Badlius f Burkholderia, Achromobacter, Ochmbadrum, Citmbac er Paraeoceus, Micrococcus, such as the species Pseudomonas brassicacearum, Pseudomonas stutzeii, aicaligenes Pseudomonas, Pseudomonas fiuoresœns, Burkholderia vietnamiensis, Achromobacter xyiomxfdans, Parawcws denitrihcans, Badilus oers, known to be denitrifying bacteria. These bacteria are most often present in soils,
La mise en contact avec les microorganisme est réalisée., de manière à obtenir une diminution de la dénlthfication causée par les microorganismes présentant une activité dénîtrîfiante, ou en d'autres termes de manière à inhiber au moins partiellement l'activité dénitrifiante des microorganismes. Dans le cadre de l'invention, une telle limitation, diminution ou inhibition est à considérer, par rapport à émission de 2O et ¾ et/ou à la dénitrlficatlon (transformation de nitrate O3 ou plus précisément N<¼" en N 3 et f ) qui serait obtenue en l'absence de mise en contact des microorganismes concernés avec ladite proanthocyanidlne. Pour évaluer le pourcentage d'inhibition de la dénltnflcation, l'activité de dénitrlficatlon est obtenue par mesure de l'émission de ½G -f H2f d'une part en présence et, d'autre part en l'absence, de proanthocyanldine, ce qui permet d'obtenir un pourcentage d'inhibition de la dénitrification correspondant à (émission de 20 + H2 en présence de proanthocyanidine) / (émission de 2O + Nfe en absence de proanthocyanldine) *100. Dans le cadre de (Invention, l'inhibition de la dénitrification par tes proanthocyanldi nés peut notamment atteindre un niveau maximal de 100%, et on obtiendra dans le cadre de l'invention on % dinhsbitlon de la dénitrification, en général de 20 à 100 %, de préférence de 50 à 100%, La technique décrite dans Dambraville et al. 2006,, et utilisée dans les exemples, peut être mise en oeuvre pour évaluer rémission de U?Q - U2> Les conditions de mise en contact et la quantité de proanthocyanidine(s) utilisées seront ajustées par l'homme du métier pour obtenir l'effet souhaité. The contacting with the microorganisms is carried out so as to obtain a reduction in the denlthfication caused by the microorganisms having a denitrifying activity, or in other words so as to at least partially inhibit the denitrifying activity of the microorganisms. In the context of the invention, such a limitation, reduction or inhibition is to be considered, with respect to the emission of 20 and ¾ and / or the denitrilfication (transformation of nitrate O3 or more precisely N <¼ " into N 3 and f ) which would be obtained in the absence of contacting the microorganisms concerned with said proanthocyanidin. To evaluate the percentage of inhibition of denlnflcation, the denitrlficatlon activity is obtained by measuring the emission of ½G -f H 2f d one hand in the presence and, on the other hand, the absence of proanthocyanldine, which allows to obtain a percent inhibition of the denitrification corresponding to (transmission 2 0 + H 2 in the presence of proanthocyanidin) / (emission 2O + Nfe absence of proanthocyanldine) * 100 . In the context of (Invention, the inhibition of denitrification by proanthocyanidines can in particular reach a maximum level of 100%, and in the context of the invention will be obtained the% dinhsbitlon denitrification, generally from 20 to 100 %, preferably 50 to 100%, The technique described in Dambraville et al., 2006, and used in the examples, can be used to evaluate U 2 -U 2 uptake. the amount of proanthocyanidin (s) used will be adjusted by those skilled in the art to achieve the desired effect.
Les proanthocyanidlnes, également nommées tanins proanthocyanidiqyes., tanins condensés, ou proanthocyanldols, sont des fiavonoïdes, et plus précisément, des polymères dfiydroxyflavan~3-ol, Les monomères hydroxyflavan-S-ol, constitutifs des proanthocyanldi nés, présentent la structure suivante (I) :  The proanthocyanidins, also known as proanthocyanidic tannins, condensed tannins, or proanthocyanidols, are fiavonoids, and more specifically polymers of hydroxyflavan-3-ol. The hydroxyflavan-5-ol monomers constituting the proanthocyanidines have the following structure (I). ):
Figure imgf000006_0001
dans laquelle R1, R2, R3, R4, R5 et R6, identiques ou différents., sont choisis parmi l'hydrogène, les groupements hydroxyie, gallate, méthyle ou osidique.
Figure imgf000006_0001
in which R 1 , R 2 , R 3 , R 4 , R 5 and R 6 , which are identical or different, are chosen from hydrogen, hydroxyl, gallate, methyl or oside groups.
Ces monomères sont donc plus ou moins hydroxylés sur les noyaux A et 8, allant de 0 a 3 groupements hydroxylés. Ces monomères, hydroxyflavan-3-ol, peuvent également être estérifiés par un ou des groupements gallate et/ou glycosyiés et/ou méthylés sur les noyaux A, 8 et C Il existe deux grands types de proanthocyanidines, qui peuvent tous deux être utilisés dans le cadre de l'invention, ces deux types étant définis par les liaisons entre monomères : les proanthocyanidines de type A et les proanthocyanidines de type 8, Les proanthocyanidines de type B, qui sont préférées dans le cadre de l'Invention, sont des polymères dfhydroxyffevan~3~ oî dont deux monomères consécutifs sont reliés par une seule liaison covaiente entre deux carbones, selon les positions 4-6 ou 4-8 (positions respectives chez les deux monomères), comme illustré sur la Formule correspondant à un dimère comprenant une liaison 4-8, suivie d'une liaison 4-6 : These monomers are therefore more or less hydroxylated on the rings A and 8, ranging from 0 to 3 hydroxyl groups. These monomers, hydroxyflavan-3-ol, may also be esterified with one or more gallate and / or glycosylated and / or methylated groups on the rings A, 8 and C There are two main types of proanthocyanidins, both of which can be used in the context of the invention, these two types being defined by the bonds between monomers: proanthocyanidins type A and proanthocyanidins type 8, proanthocyanidins type B which are preferred in the context of the invention, are polymers hydroxyffevan d f ~ 3 ~ Oi which two consecutive monomers are linked by a single covalent bond between two carbons, depending on the positions 4-6 or 4-8 (respective positions in both monomers), as shown in the formula corresponding to a dimer comprising a 4-8 linkage, followed by a 4-6 linkage:
Figure imgf000007_0001
Figure imgf000007_0001
(II)  (II)
avec R1, R2, R3, R4, R5 et R6, R\. R2', Ry et R6', Rr, R , R3", R4", R5" et R6", pouvant être les mêmes groupements ou des groupements différents, représentant l'hydrogène, un groupement hydroxyle, un groupement gallate, un groupement méthyle ou encore un groupement osidique, Les proanthocyanidines de type A sont des polymères dtiydroxyflavan- 3~ol dont deux monomères consécutifs son reliés par deux liaisons covalentes entre deux carbones, en position 2-7 et 4-8 (positions respectives chez les deux monomères), illustrées dans le cas d'un dimère par ia formule (III) ci-après i with R 1 , R 2 , R 3 , R 4 , R 5 and R 6 , R 1. R 2 ', R y and R 6', R r, R, R 3 ", R 4", R 5 "and R 6" may be the same groups or different groups representing hydrogen, a hydroxyl group a gallate group, a methyl group or a saccharide group, The proanthocyanidins of type A are polymers of hydroxyflavan-3-ol of which two consecutive monomers are connected by two covalent bonds between two carbons, at position 2-7 and 4-8 (respective positions in the two monomers), illustrated in the case of a dimer by formula (III) below i
Figure imgf000008_0001
Figure imgf000008_0001
(III) avec R1, R2 f R3, R4 f Rs et 6 ; Rr, R f 4', R5' et R6', pouvant être les mêmes groupements ou des groupements différents, représentant l'hydrogène, un groupement hydroxyie, un groupement gailate, un groupement mèthyl ou encore un groupement osidique. (III) with R 1, R 2 f R 3, R 4 R s and f 6; R r , R f 4 ' , R 5' and R 6 ' , which may be the same groups or different groups, representing hydrogen, a hydroxyl group, a gailate group, a methyl group or an osidic group.
Les proanthocyanidines pouvant être utilisées dans le cadre de Hnventîon sont constituées de 2 à n monomères (hydroxyflavan~3~ol) reliés par des liaisons covalentes précédemment décrites, selon toutes les déclinaisons possibles. En particulier, on utilisera dans ie cadre de l'invention une ou plusieurs proanthocyanidines., choisies parmi les dîmères, irimères et tétramères.  The proanthocyanidins which can be used in the context of the invention consist of 2 to n monomers (hydroxyflavan ~ 3 ~ ol) connected by covalent bonds previously described, according to all the possible variations. In particular, within the scope of the invention, one or more proanthocyanidins chosen from among the dimer, irimer and tetramer will be used.
Les proanthocyanidines les plus connues sont des polymères de (-/+)- catéchine, (7÷}-épicatéchine., {-/-f-)~gallocatéchine et (-/÷)- épigallocatéchine. Dans le groupe des proanthocyanidines sont retrouvés des sous-groupes de molécules, comme., par exemple,, les prodelphinidines (prodeiphinidoi) ou tes procyanidines (procyanidol). Les procyanidines correspondent à des polymères de (-/ -)~catéehine et/ou de {-/-*■}- épicatéchine. Les prodelphinidines des polymères de (-/- )~galfocatéchine et (~/-f }-épIgalfocaté hlne, Par {-·/+}, on désigne lisomère {+ ) ou {Isomère {-} ou un mélange de ces isomères» The most well known proanthocyanidins are polymers of (- / +) - catechin, (7 ÷) epicatechin., (- / - f-) ~ gallocatechin and (- / ÷) - epigallocatechin. In the group of proanthocyanidins are found subgroups of molecules, such as, for example, prodelphinidines. (prodeiphinidoi) or procyanidins (procyanidol). Procyanidins correspond to polymers of (- / -) ~ cathehine and / or {- / - * ■ } - epicatechin. The prodelphinidins of (- / -) ~ galfocatechin polymers and (~ / -f) -epigalfocaté hlne, Par {- · / +}, denotes {+) or {Isomer {-} or a mixture of these isomers »
Les proanthocyanîdines sont des produits naturels qui peuvent, notamment, être obtenus et purifiés à partir d'extraits de Faifopia spp, et en particulier de Falhpia x bohemka comme mis en évidence par les inventeurs, ou encore a partir de pépins de Vitis vinifera, de Pteridium spp., VMdum spp., Schfûops/s spp,, Vicia sppv C /5 spp., Quercus spp,, De nombreuses proanthocyanldines commerciales sont disponibles, notamment chez LAF O T (Bordeaux, France), Organic Herb Inc. (Changsha, Chine) Nanjing Zelang Médical Technology Co., Ltd. (Jiangsu, Chine), Proanthocyanidins are natural products which can, in particular, be obtained and purified from extracts of Faifopia spp., And in particular Falhpia x bohemka as evidenced by the inventors, or else from Vitis vinifera seeds, Pteridium spp. VMdum spp. Schfûops / s spp ,, Vicia spp v C / 5 spp., Quercus spp ,, many commercial proanthocyanldines are available, especially in LAF OT (Bordeaux, France), Organic Herb Inc. (Changsha , China) Nanjing Zelang Medical Technology Co., Ltd. (Jiangsu, China),
Dans le cadre de [Invention., on utilisera, de préférence, des proanthocyanîdines de type B, et de manière encore plus préférée des procyanidines de type 8, en particulier sous la forme de dimère, trimère ou tétramère,  In the context of [the invention, use will preferably be made of proanthocyanidins of type B, and even more preferably procyanidins of type 8, in particular in the form of dimer, trimer or tetramer,
Selon des modes de réalisation particuliers de l'invention, ladite au moins proanthocyanidine utilisée ne se présente pas sous la forme d'un extrait de Fafiopia spp, contenant des proanthocyanîdines^ et en particulier de Faiiopia x bohemica, et/ou ladite au moins proanthocyanidine est utilisée, en l'absence de catéehine et/ou d'épicatéchine,  According to particular embodiments of the invention, said at least one proanthocyanidin used is not in the form of an extract of Fafiopia spp., Containing proanthocyanidins, and in particular Faiiopia x bohemica, and / or said at least one proanthocyanidin. is used, in the absence of cathehine and / or epicatechin,
Dans le cadre de l'invention, la ou les proanthocyanîdines utilisées peuvent être appliquées, sur le milieu contenant les microorganismes dont l'effet dénitrif nt est à limiter, ou mélangées à ce dernier, sous la forme d'une solution ou suspension aqueuse ou encore sous la forme d'une poudre ou granulé,  In the context of the invention, the proanthocyanidin (s) used may be applied to the medium containing the microorganisms whose denitrification effect is to be limited, or mixed with the latter, in the form of an aqueous solution or suspension or still in the form of a powder or granule,
Les modes de réalisation dans lesquels les microorganismes sont présents dans un sol vont maintenant être décrits, de manière plus détaillée. Dans le cadre de l'invention, au moins une proanthocyanidine pourra, notamment, être utilisée pour favoriser la croissance végétale sur un sol de culture, par réduction de la dénitrification causée par les microorganismes présentant une activ té dénitriftante, et en particulier causée par tes bactéries dénitrifiantes, présents dans ledit sol Les proanthocyanidines étant des produits naturels., leur utilisation dans des cultures est en total respect avec l'environnement Les proanthocyanidines peuvent donc être utilisées en agriculture biologique. Embodiments in which microorganisms are present in soil will now be described in more detail. In the context of the invention, at least one proanthocyanidin may, in particular, be used to promote plant growth on a culture soil, by reducing denitrification caused by microorganisms. Having a denitrifying activity, and in particular caused by denitrifying bacteria present in said soil Proanthocyanidins being natural products, their use in crops is in total respect with the environment Proanthocyanidins can therefore be used in organic farming.
Dans le cadre de l'utilisation selon l'invention sur un sol, au moins une proanthoeyanidine est appliquée sur un soi, et notamment un sol de culture, par exemple par dépôt ou pulvérisation sur la terre.  In the context of the use according to the invention on a soil, at least one proanthoeyanidine is applied to a soi, and in particular a culture soil, for example by deposition or spraying on the ground.
En particulier, ladite au moins proanthoeyanidine est utilisée pour limiter la dénitrlfication causée par des bactéries du sol, telles que Pseudo onas brassîcacearum et/ou Sac us cernas,  In particular, said at least one proanthoeyanidine is used to limit denitrification caused by soil bacteria, such as Pseudo onas brassacacearum and / or Sac us cernas,
Dans le cas d'une solution de proanthocyanidine(s), celle-ci pourra être appliquée par pulvérisation ou tout autre moyen approprié. Les sols qui pourron être traités dans le cadre de llnvention sont, notamment, des sois de culture, de maraîchage, de culture céréaiière, de viticulture et plus généralement, des sols où des végétaux divers d'intérêt agronomique, notamment des plantes ou fleurs diverses, sont cultivés.  In the case of a solution of proanthocyanidin (s), it may be applied by spraying or any other appropriate means. The soils that can be treated in the context of the invention include, inter alia, cultivation, market gardening, cereal growing, viticulture and more generally, soils where various plants of agronomic interest, including various plants or flowers , are grown.
L'effet de la ou des proanthocyanidines utilisées sur la dénitriflcation sera obtenue, sur une large gamme de températures, notamment à une température du sol de 0 à 5Q C et avec un effet optimum à un taux d'humidité supérieur à 50% de la capacité au champ.  The effect of the proanthocyanidin (s) used on denitrification will be obtained over a wide range of temperatures, especially at a soil temperature of 0 to 5 ° C. and with an optimum effect at a moisture content greater than 50% of the field capacity.
Une quantité efficace de proanthocyanldine{s) pour obtenir la limitation de la dénitriflcation souhaitée sera appliquée sur le sol, La quantité appliquée sera adaptée, par l'homme du métier, notamment, en fonction de la formulation et du taux d'humidité du sol. En particulier, de 0.01 à 10 mg/g de sol sec, et de préférence de 0,05 à 5 mg/g de soi sec, de proanthocyanidlne(s) est appliqué. Ces quantités sont applicables à des milieux solides, autres que le soi, précédemment cités, dans lesquels les microorganismes pourraient être présents»  An effective amount of proanthocyanidin (s) to achieve the desired denitrification limitation will be applied to the soil. The amount applied will be adapted by those skilled in the art, in particular, depending on the formulation and the soil moisture content. . In particular, from 0.01 to 10 mg / g of dry sol, and preferably from 0.05 to 5 mg / g of dry sol, of proanthocyanidene (s) is applied. These amounts are applicable to solid media, other than the self, mentioned above, in which the microorganisms could be present.
Les résultats présentés dans le cadre de llnvention mettent en évidence que les proanthocyanidines, et en particulier les procyanldines de type B, permettent de réduire le processus de dénitriflcation bactérien des sols. Les résultats ont été obtenus sur des souches de bactéries en culture., mais également sur des soi de natures différentes (un de bord de rivière et un sol de prairie) et contenant des communautés de bactéries dénitrifiantes complexes et différentes, The results presented in the context of the invention show that proanthocyanidins, and in particular type B procyanidins, make it possible to reduce the process of bacterial denitrification of soils. The results were obtained on strains of bacteria in culture, but also on ones of different natures (one of river edge and grassland soil) and containing communities of complex and different denitrifying bacteria,
L'utilisation selon { nvention permet donc de limiter les pertes d'azote par dénitrif ation et, par là même, les émissions de gaz à effet de serre et, également, de réduire la nécessité d'utiliser des engrais azotés en agriculture. Néanmoins, l'utilisation de proanthocyaoidine{s) peut être associée, dans le cadre de l'invention, à un ou plusieurs autres fertilisants (azoté ou phosphaté par exemple).  The use according to the invention thus makes it possible to limit nitrogen losses by denitrification and, consequently, greenhouse gas emissions, and also to reduce the need to use nitrogen fertilizers in agriculture. Nevertheless, the use of proanthocyaoidin (s) may be associated, in the context of the invention, with one or more other fertilizers (nitrogen or phosphated, for example).
Dans le cadre de l'invention, au moins une proanfhocyanidine pourra être appliquée sur un sol de culture, pour favoriser la croissance de ladite culture en lutisnt contre la dénitrification causée par ies microorganismes, et en particulier les bactéries, présents dans ledit soi. Ladite application pourra être faite, sans application conjointe d'un engrais azoté.  In the context of the invention, at least one proanfhocyanidine may be applied to a culture soil, to promote the growth of said culture against denitrification caused by microorganisms, and in particular bacteria, present in said soi. Said application may be made without the joint application of a nitrogen fertilizer.
Concernant maintenant, les modes de réalisation dans lesquels les microorganismes sont présents dans un milieu liquide., une composition sous forme solide ou liquide sera intégrée dans ledit milieu. Une quantité efficace de proant ocyanidine(s) pour obtenir la limitation de la dénitrification souhaitée sera Incorporée dans ledit milieu, La quantité incorporée sera adaptée,, par l'homme du métier. A titre d'exemple, de 0,01 à 2,5 mg de proanthocyanidine(s) par Millilitre de milieu liquide, et de préférence de 0,01 à 1 mg de proanthocyanidine(s) par Millilitre de milieu liquide sera incorporé.  As regards now, the embodiments in which the microorganisms are present in a liquid medium, a composition in solid or liquid form will be integrated in said medium. An effective amount of the ocyanidine proant (s) to achieve the desired denitrification limitation will be incorporated into said medium. The amount incorporated will be adapted by those skilled in the art. By way of example, from 0.01 to 2.5 mg of proanthocyanidin (s) per milliliter of liquid medium, and preferably from 0.01 to 1 mg of proanthocyanidin (s) per milliliter of liquid medium will be incorporated.
Les exemples ci-après, en référence aux Figures annexées, permettent dillustrer l'Invention, mais n'ont aucun caractère limitatif.  The examples below, with reference to the appended figures, illustrate the invention, but are not limiting in nature.
Figyre I : pourcentage d'inhibition (relatif par rapport au contrôle) sur la dénitrification par Pseudomonss brassicacearum d'un mélange des composés majoritaires présents dans Faliopïa x bohemica et de /'extrait Faffapta x bo emîca. Les barres verticales correspondent aux variations obtenues sur les différents essais, Les étoiles indiquent ies variations significative par rapport au contrôle (test de Tukey; α<0,05). Fîspre 2 ; Chroroatograrnnie à 280 nm de S'extrait Faiiopia x bohemica présentant les proanthocyanidines et leurs monomères détectés ainsi que leur spectre UV. Fig. I: Percent inhibition (relative to control) on the denitrification by Pseudomons brassicacearum of a mixture of the major compounds present in the alkaline bohemia and the Faptapta x bo emica extract. The vertical bars correspond to the variations obtained on the different tests. The stars indicate the significant variations relative to the control (Tukey test, α <0.05). Figure 2; Chroroatograrnnie at 280 nm extract Faiiopia x bohemica showing proanthocyanidins and their monomers detected as well as their UV spectrum.
Figuras 3A à 3€ : Spectres de masse ESï positifs des proanthocyanidines de type 8 retrouvées dans Fallopîa x bohemica (Dlmères (F gyre 3A)f Trimères (Figure 38), Tétrarnères (Figura 3£). Figuras 3A to 3 €: ESi positive mass spectra of proanthocyanidins of type 8 found in Fallopila x bohemica (Dlmers (F gyre 3A) f Trimers (Figure 38), Tetramers (Figure 3 £).
Figure 4 : Pourcentage d'Inhibition de la dénitrïflcatlon et de la respiration de a) Pseudomonas brassîeacearum et b) E cereus en fonction de la concentration en proanthocyanidines purifiées» Les étoiles indiquent les différences significatives de moyennes avec le contrôle sans proanthocyanidines (Test de Tukey; a<0,05).  Figure 4: Percentage inhibition of denitrification and respiration of a) Pseudomonas brassileacearum and b) E cereus as a function of concentration of purified proanthocyanidins »Stars indicate significant differences in means with control without proanthocyanidins (Tukey test a <0.05).
Figure S: Pourcentage d'inhibition de la dénitrîficatîon des sols de bord de rivière (Ainiî) et de prairie (Αΐηδ') en fonction de la concentration en proanthocyanidines commerciales. Les étoiles indiquent les différences significatives de moyennes avec le contrôle (Test de Tukey; <0.05). Figure S: Percentage inhibition of denitrification of river (Ainii) and grassland (Αΐηδ ' ) soils as a function of commercial proanthocyanidin concentration. The stars indicate significant differences in means with control (Tukey test, <0.05).
Figure 6 ; Pourcentage d'Inhibition de la dénitrification du sol de prairie Ainl i après 7 jours d'Incubation (à la capacité au champ) e présence de différentes quantités de proanthocyanidines commerciales. Les étoiles indiquent les différences significatives de moyennes avec le contrôle (Test de Tukey; a<0.05). Figure 6; Percentage Inhibition of denitrification prairie soil AINL i after 7 days of incubation (to field capacity) e presence of different amounts of commercial proanthocyanidins. The stars indicate significant differences in means with control (Tukey's test, a <0.05).
F g re 7 t Pourcentage d'inhibition de la nifrifieation potentielle dans le sol de Ain il (triangle) et celui de A!n6 (rond) en fonction de la concentration en proanthocyanidines. Les barres indiquent des erreurs standards.  F g re 7 t Percentage inhibition of potential nifrification in the soil of Ain il (triangle) and that of A! N6 (round) as a function of proanthocyanidin concentration. The bars indicate standard errors.
L MATES IELS ET MÉTHODES  L MATES IELS AND METHODS
A)- SOURCE BIOLOGIQUE DES PROANTHOCYANIDINES  A) - BIOLOGICAL SOURCE OF PROANTHOCYANIDINES
- PREPARATION DES EXTRAITS DE FÂLLQPÎA SPP,  - PREPARATION OF EXTRACTS OF FALLY SPP,
Des composés ont été extraits de racines et de rhizomes du génotype F. x bohemica (Chrtek et Chrtkovâ), Les rhizomes collectés sur le terrain ont été plantés en pots dans du compost ( lasmann 153/sand, 80/20), Les rhizomes ont été mis en culture pendant six mois dans une serre, sous lumière artificielle à 21 °C Les rhizomes et les racines produits ont été récoltés, nettoyés à l'eau et lyophilisés. Soixante-cinq grammes de rhizomes et racines lyophilisées broyés à l'aide d'un TissueLyser il bead mil! (Qlagen Venlo, Netherlands) ont été mis en suspension dans 750 ml d'un mélange eau : méthanol (50/50; v/v) et placés aux ultrasons pendant 30 min. Après fîltration à l'aide d'une pompe à vide, l'éluat a été collecté, et une autre extraction a été réalisée sur le résidu avec 2 x 500 mL de méthanol pur, Tous les éluats ont ensuite été mélangés et séchés, Puis, ces extraits secs ont été re~suspendu$ dans un mélange eau ; méthanol (50/50; v/v) à la concentration de 10 mg ml'1. Compounds were extracted from roots and rhizomes of the genotype F. x bohemica (Chrtek and Chrtkov). The rhizomes collected in the field were planted in pots in compost (lasmann 153 / sand, 80/20). cultured for six months in a greenhouse, under artificial light at 21 ° C The rhizomes and roots produced were harvested, cleaned with water and freeze-dried. Sixty-five grams of rhizomes and lyophilized roots ground using a TissueLyser it bead millet! (Qlagen Venlo, Netherlands) were suspended in 750 ml of water: methanol (50/50, v / v) and sonicated for 30 min. After filtration using a vacuum pump, the eluate was collected, and another extraction was carried out on the residue with 2 x 500 mL of pure methanol. All the eluates were then mixed and dried. these dry extracts were re ~ suspended in a water mixture; methanol (50/50, v / v) at a concentration of 10 mg ml -1 .
- IDENTIFICATION DES PROANTHOCYÀNIDI ES CONTENUS DANS L'EXTRAIT DE E X 80 EMICA ;  - IDENTIFICATION OF PROANTHOCYANITIS CONTAINED IN THE EXTRACT OF E X 80 EMICA;
L'extrait a été analysé à l'aide d'un appareillage d'UHPLC 1290 ïnfinity séries (Agitent Technologies, Santa Clara, USA) couplé à un détecteur UV â barrette de diodes G4212A DAD (Agitent Technologies, Santa Clara, USA) et on spectromètre de masse Q-TGF 6530 séries (Agllent Technologies, Santa Clara, USA),  The extract was analyzed using a UHPLC 1290 infinity series apparatus (Agitate Technologies, Santa Clara, USA) coupled to a G4212A DAD diode array UV detector (Agitate Technologies, Santa Clara, USA) and Q-TGF 6530 series mass spectrometer (Agllent Technologies, Santa Clara, USA),
La séparation des composés a été réalisée sur une colonne Porosheli 120 EC-C18 (2,7 pmf 3,0 x 100 mm, Agllent Technologies, Santa Clara, USA) à une température de 60°C. La phase mobile était un gradient linéaire de 0,4% d'acide acétique dans de l'eau (solvant A) et acétonltrile (soîvant B). La phase mobile passait à travers la colonne à un flux de 0,6 ml/min. Le gradient linéaire était de G à 1,5 min 2% solvant B; .1,5 à 22 min 2% â 17% solvant B; 22 à 41 min 17% â 1.00% solvant B; 41 à 41,5 min 100% solvant B, Le système a été préalablement équilibré avant chaque échantillon, Le volume d'Injection était de ΙμΙ. Separation of compounds was performed on a column Porosheli 120 EC-C18 (2.7 f 3.0 pm x 100 mm, Agllent Technologies, Santa Clara, USA) at a temperature of 60 ° C. The mobile phase was a linear gradient of 0.4% acetic acid in water (solvent A) and acetonitrile (solvent B). The mobile phase passed through the column at a flow rate of 0.6 ml / min. The linear gradient was from G to 1.5 min 2% solvent B; 1.5 to 22 min 2% to 17% solvent B; 22 to 41 min 17% to 1.00% solvent B; 41 to 41.5 min 100% solvent B, the system was previously balanced before each sample, the injection volume was ΙμΙ.
Les spectres UV ont été enregistrés entre 190 et 600 nm et la réponse à 280 nm a été utilisée pour les interprétations qualitatives. La source ESI a été optimisée de la manière suivante : mode Ionisation positive, scan des spectres de m/z 80 à 2000, voltage du capillaire 3,0 kV, fragmenteur 70 V, la collision Induite de dissociation (collislon-induced dissociation ; CID) a été fixée à 20 eV. L'azote a été utilisé comme nébuilseur en gaz avec un flux de 12 l/min et: une température 310 °C à 40 psi» Des proanthocyanidines ont été identifiées dans l'extrait par analyse des spectres UV, HS and MS/MS, en utilisant le logiciel MassHunter Qualitative Analyste (Agitent Technologies; Santa Clara, USA), les spectres obtenus étant présentés dans la partie II B). UV spectra were recorded between 190 and 600 nm and the 280 nm response was used for qualitative interpretations. The ESI source was optimized as follows: Positive Ionization mode, 80/2000 m / z spectra scan, 3.0 kV capillary voltage, 70 V fragmentor, Collision-induced dissociation (CID) collision ) was set at 20 eV. Nitrogen was used as a gas nebiller with a flow of 12 l / min and a temperature of 310 ° C to 40 psi. Proanthocyanidins were identified in the extract by UV, HS and MS / MS spectra analysis using the MassHunter Qualitative Analyst software (Agitant Technologies, Santa Clara, USA), the spectra obtained being shown in Part II B).
- PURIFICATION DES PROANTHOCYANIDINES. DE E x SOHEMICA  - PURIFICATION OF PROANTHOCYANIDINES. FROM E x SOHEMICA
Les proanthocyanidines contenues dans l'extrait de E x bohe ica ont été purifiées â l'aide d'une colonne de Sephadex LH20. Le Sephadex LH 20 a été préalablement équilibré trois fois en humidifiant 25 g de Sephadex LH 20 avec 100 ml d'éthanol 80% et en retirant ensuite le surnageant L'extrait E x bohemica (100 ml) concentré à 10 mg mi'1 a ensuite été ajouté au Sephadex LH 20 équilibré et agité pendant: 3 min. Le Sephadex chargé en extrait a été chargé dans un Buchner en verre de 10Q ml Le Sephadex LH 20 a été lavé avec de i'éthano! 95% jusqu'à ce que j'absorbance de i'éiuat à 280 nro soit proche de zéro. Le Sephadex LH 20 a ensuite été lavé avec de l'acétone 50% jusqu'à ce que le Sephadex soit totalement lavé, L'éluat d'acétone 50% récupéré a été évaporé à l'aide d'un évaporateur rotatif. Les proanthocyanidines sèches, ainsi purifiées ont par la suite été mises en solution à 10 mg.mf1 dans de l'eau, cette solution étant nommée par la suite proanthocyanidines purifiées. Cette solution contient les dlmères, trimères et tétramères identifiés sur les Figures 3A et 38, mais pas les monomères catéchine et épicatéchine, The proanthocyanidins contained in the E x bohe ica extract were purified using a Sephadex LH20 column. Sephadex LH 20 was previously equilibrated three times by wetting 25 g of Sephadex LH 20 with 100 ml of 80% ethanol and then removing the supernatant. The extract E x bohemica (100 ml) concentrated at 10 mg ml -1 then added to the equilibrated Sephadex LH and stirred for 3 min. Extracted Sephadex was loaded into a 10 ml glass buchner. Sephadex LH 20 was washed with ethanol. 95% until the absorbance of the water at 280 nro is close to zero. Sephadex LH 20 was then washed with 50% acetone until the Sephadex was completely washed. The 50% acetone eluate recovered was evaporated using a rotary evaporator. The dried proanthocyanidines, thus purified, were subsequently dissolved in 10 mg.mf 1 in water, this solution being subsequently called purified proanthocyanidines. This solution contains the dimer, trimers and tetramers identified in FIGS. 3A and 38, but not the monomers catechin and epicatechin,
s)- SOURCE COMMERCIALE DES PROANTHOCYA DÎ ES  s) - COMMERCIAL SOURCE OF PROANTHOCYA DIES
îl s'agit de proanthocyanidines purifiées à partir de pépins de raisins (Bîotan, Laffort, Bordeaux, France). Une analyse par UHPLC a permis de vérifier que ces proanthocyanidines n'étaient pas en mélange avec de la catéchine, ni avec de ί 'épicatéchine,  It is proanthocyanidins purified from grape seeds (Bîotan, Laffort, Bordeaux, France). UHPLC analysis made it possible to verify that these proanthocyanidines were not mixed with catechin or with epicatechin,
IL EFFET DES PROANTHOCYANIDINES SUR LA DENITRÎFÏCÀTÎO :  IT EFFECT OF PROANTHOCYANIDINES ON DENITRIDY:
A)- TEST DE L'EFFET DES COMPOSES MAJORITAIRES CONTENUS A S LES EXTRAITS DE E X BOHEMICA  A) - TESTING THE EFFECT OF THE MAJORITY COMPOUNDS CONTAINED IN THE EXTRACTS OF E X BOHEMICA
L'effet de la catéchine, l 'épicatéchine, le picéide, le resvératrol, rémodine et le physeion, Identifiés par Bardon et al (2014), comme les composés majoritaires des extraits de Failopia x bohemica, ont été testés en mélange sur l'activité de dénitrificstion de Pseudomonas brass/cacearvm, comme décrit dans le paragraphe suivant, A partir de produits commerciaux purs (Slgma-Âl rlch, Saint Louis, USA), tous ces composés ont été préparés en mélange dans une solution méthanol/eau (v/v, 50/50} à la concentration retrouvée dans les extraits de Faliopia x bohemica. Les concentrations de ces composés exposés aux bactéries dans le milieu sont de 0,0037 mg ml*1 catéchine, 0,0057 mg mi"1 épicatéchine, 0,022 mg ml"1 picéide., 0,0007 mg mi"1 resvèratrol, 0,0015 mg ml*1 émodine et 0,0047 mg ml"1 physcion), The effect of catechin, epicatechin, picteide, resveratrol, remodin and physeion, identified by Bardon et al (2014), as the major components of the extracts of Failopia x bohemica, were tested in mixture on the denitrification activity of Pseudomonas brass / cacearvm, as described in the following paragraph. From pure commercial products (Slgma-Aldch, Saint Louis, USA), all these compounds were prepared as a mixture in a methanol solution / water (v / v, 50/50) at the concentration found in the extracts of Faliopia x bohemica The concentrations of these compounds exposed to bacteria in the medium are 0.0037 mg ml * 1 catechin, 0.0057 mg mi 1 epicatechin, 0.022 mg ml- 1 pictide, 0.0007 mg of 1- resverratrol, 0.0015 mg ml- 1 emodin and 0.0047 mg ml- 1 physcion),
B>- SOUCHES BACTERIENNES DEN TiRiFI ANTES  B> - BACTERIAL STRAINS DEN TiRiFI ANTES
L'effet des proanthocyanidines purifiées à partir des extraits de F. x bohemica a été testé à des concentrations de 0,1 ; 0,05 ; 0,01 ou 0 mg.ml"1 sur la dénitriflcation et la respiration de Pseudomanas brasscaœaru NFH421 et Badllus œreus A19, deux bactéries dénltriflantes fréquemment rencontrées dans les sois, Four cela, 5 ml de milieu de croissance liquide MB avec 20 mM de NOs, contenant les différentes concentrations en proanthocyanidines,. a été inoculé à une densité optique de (D.O.) ~ 0 L Les mesures de dénitrification ont été réalisées dans des flacons plasma scellés par un bouchon en caoutchouc, par le dosage de l'accumulation du f¾Q, Ceci a été réalisé après avoir remplacé l'air des flacons par un mélange d'hélium et d'acétylène (He/ ^Hs; 90/10), Chacun des traitements a été réalisé en quatre répllcats. L'acétylène a pour fonction de bloquer la transformation de N20 en 2f la mesure de l'émission de f¾G étant alors reconnue comme représentative de émission de 20 et H2 (Dambreville et al. 2006). The effect of proanthocyanidins purified from F. x bohemica extracts was tested at concentrations of 0.1; 0.05; 0.01 or 0 mg.ml- 1 on the denitrification and respiration of Pseudomanas brasscaœaru NFH421 and Badllus œreus A19, two denltriflant bacteria frequently found in soybeans, Four this, 5 ml of MB liquid growth medium with 20 mM NO.sub.2. , containing the different concentrations of proanthocyanidins, was inoculated at an optical density of (OD) ~ 0 L The denitrification measurements were carried out in plasma bottles sealed by a rubber stopper, by the assay of the accumulation of f¾Q This was done after replacing the air in the flasks with a mixture of helium and acetylene (He / Hs, 90/10). Each of the treatments was carried out in four replicates. to block the transformation of N 2 0 in 2f the measurement of f émissionG emission being then recognized as representative of emission of 2 0 and H 2 (Dambreville et al., 2006).
Les mesures de respiration ont été réalisées dans des flacons plasma scellés par dosage de Accumulation de CO?. sans remplace l'atmosphère des flacons. Respiration measurements were performed in sealed plasma bottles by CO 2 accumulation assay . without replacing the atmosphere of the flasks.
Le NjO et le C02 émis ont été mesurés toutes les deux heures pendant 48 heures à l'aide d'un microcatharomètre (pGO 30Q0f SRA instruments,. Marcy tolle, France). The N 2 O and C0 2 emitted were measured every two hours for 48 hours using a microcatheter (pGO 30Q0 f SRA instruments, Marcy tolle, France).
C)- COMMUNAUTES COMPLEXES DE SOLS EN SUSPENSION  C) - COMPLEX COMPLEXES OF SOIL IN SUSPENSION
Afin de tester l'effet des proanthocyanidines sur la dé itrification de communautés complexes présentes dans le sol,, des proanthocyanidines commerciales purifiées à partir de pépins de raisins ont été utilisées (Laffort, Bordeaux, France). Des échantillons de deux sols ont été récoltés sur les sites Ainlî (45°57ί53ίί ; 5Ό15'25Έ) et ΑΙηβ (45°48Ί5"Ν; 5°10*29"E), puis tamisés à 2 mm, Le taux de déûitrifîcatlon a été mesuré par émission de %G toutes les deux heures pendant 30 heures en anaérobiose avec un mîcro- cathanomètre (p6C-R30Û0f SRA instruments., Marcy l'Etoile, France). Un gramme de sol équivalent sec a été placé dans un flacon plasma de !50mi scellé par un bouchon en caoutchouc. L'atmosphère du flacon a été remplacée par un mélange He/CiH?. mixture (90/10 v/v). Les sols ont été mis en suspension dans 10 mi de tampon phosphate Ô.J.M (MaH^PG et NaHPO^ pH-7,5) supplémenté de glucose (0,5mg de C-giucose.mr1), d'acide glutamique (0,5mg de C-atide glutarnique.mr1) et de N£¾ (20 mM). L'effet des proanthocyanidlnes sur la déni rification a été mesuré pour des concentrations de 2,5; i; 0,5; 0,1; 0,05; 0,01 et 0 mg.ml** au final, en ajoutant de l'eau ou des proanthocyanidlnes aux suspensions de sols» Les suspensions de sol on été maintenues en agitation à 28°C tout au long de l'expérience. Tous les traitements ont été réalisés en quatre réplicats. In order to test the effect of proanthocyanidins on the deification of complex communities present in the soil , proanthocyanidines commercial purified from grape seeds were used (Laffort, Bordeaux, France). Samples of two soils were collected at the Ainl sites (45 ° 57 ί 53 ίί , 5 Ό 15'25Έ) and ΑΙηβ (45 ° 48Ί5 "Ν, 5 ° 10 * 29" E) and sieved at 2 mm. the déûitrifîcatlon rate was measured emission% G every two hours for 30 hours under anaerobic conditions with a micro- cathanomètre (P6C-R30Û0 f SRA instruments., Marcy l'Etoile, France). One gram of dry equivalent sol was placed in a 50mm plasma flask sealed with a rubber stopper. The atmosphere of the flask was replaced by a He / CiH mixture ? . mixture (90/10 v / v). The sols were suspended in 10 ml of 0.1M phosphate buffer (MaH 2 PG and NaHPO 3 pH 7.5) supplemented with glucose (0.5mg of C-glucose 1 ), glutamic acid ( 0.5 mg of glutamic C-atide.mr 1 ) and N £ ¾ (20 mM). The effect of proanthocyanidins on denitrification was measured for concentrations of 2.5; i; 0.5; 0.1; 0.05; 0.01 and 0 mg.ml * * in the end, adding water or proanthocyanidins to the soil suspensions. The soil suspensions were stirred at 28 ° C throughout the experiment. All treatments were performed in four replicates.
D)- EFFETS DES PROA THOCVA ÎDINES SUR UNE COMMUNAUTÉ COMPLEXE DE SOL RAPPORTES EN MASSE DE PROANTHOCYANÏDÎNES PAR RAPPORT A UNE MASSE DE SOL SEC  D) - EFFECTS OF PROA THOCVA ÎDINES ON A COMPLEX FLOOR COMPLEX REPORTED IN MASS OF PROANTHOCYANIDINES IN RELATION TO A MASS OF DRY SOIL
Une gamme de concentrations en proanthocyanidlnes actives sur la dénitrification du sol rapportée en g de sol sec a été réalisée. L sol utilisé pour cette expérience est le soi Ain IL Dix grammes de sol équivalent sec ont été incubés en flacon plasma à l'air libre pendant ? jours à 28°€ à la capacité au champ, L'équivalent de 10; 5; 1; 0,5; 0,1; 0,05; 0,01 et mg. g"1 de sol sec de proanthocyanidlnes commerciales ont été apportés lors de humidification à l capacité au champ du sol. Chaque traitement a été réalisé en quatre réplicas. Après 7 jours, la mesure de dénitrlfieation des sols traités a été réalisée par mesure d'émission de i¾û en anaérobiose toutes les 30 min pendant 6 heures à l'aide d'un mîcrocatharomètre (pG€- 3000, SRA instruments, Marcy l'Etoile, France), Les flacons plasma on été scellés par un bouchon en caoutchouc, l'atmosphère des flacons s été remplacée par de l'hélium à l'aide d'un banc dlnertage. Les sols ont été supplémentés d'une solution de 0,5 ml contenant l'équivalent de 50 pgN- NCh.g"1 sol sec, 0,5 mg de C-aclde glutarnique.g"1 de sol sec et 0,5 mg de C-glucose.g"1 de soi sec.A range of active proanthocyanidin concentrations on soil denitrification reported in g of dry soil was carried out. The soil used for this experiment is the so-called Ain IL. Ten grams of dry equivalent soil were incubated in a plasma bottle in the open air for? days at 28 ° C to field capacity, the equivalent of 10; 5; 1; 0.5; 0.1; 0.05; 0.01 and mg. g "1 of commercial proanthocyanidin solids were applied during humidification to the soil field capacity Each treatment was carried out in four replicas After 7 days, the denitrification measure of the treated soils was carried out by measuring emission of i¾u anaerobically every 30 min for 6 hours using a microcatharometer (pG € - 3000, SRA instruments, Marcy l'Etoile, France), the plasma bottles were sealed by a rubber stopper, l atmosphere of the flasks has been replaced by helium using a bench. The soils were supplemented with a solution of 0.5 ml containing the equivalent of 50 μgN-NCh.g- 1 sol dry, 0.5 mg of C-aclde glutarnique.g "1 dry soil and 0.5 mg of C-glucose.g "1 self sec.
IIL RESULTATS IIL RESULTS
A)- EFFET DES COMPOSES MAJORITAIRES SUR LA DENITRIFÎCAÏION DE A) - EFFECT OF MAJORITY COMPOUNDS ON THE DENITRIFICATION OF
PSEUDQMQNÂS 8RA$STCACEARUM PSEUDQMQNÂS 8RA $ STCACEARUM
La dénitrlficatlon de Pseudamo s èrassicacearu n'est pas Inhibée par 1e mélange des composés majoritaires (catéchlne., épicatéchïne,, picéide, resvératrol, émodine et physcion), présents dans les extraits de Faiïopia x ùohemÎcs, alors qu'un effet inhibiteur marqué est obtenu avec l'extrait Failopia x ho emiœ pour une même concentration de ces composés, comme illustré dans la Figure I. Il apparaît donc que l'activité inhibitrice obtenue n'implique pas ces composés,. The dénitrlficatlon of Pseudamo s èrassicacearu is not Inhibited by first mixing the major compounds (catéchlne., Epicatechin ,, piceid, resveratrol, emodin and physcion) present in extracts Faiïopia x ùohemÎcs, whereas a marked inhibitory effect is obtained with the extract Failopia x ho emiœ for the same concentration of these compounds, as shown in Figure I. It thus appears that the inhibitory activity obtained does not involve these compounds ,.
8)- IDENTIFICATION DES PROANTHOCYANIDINES CONTENUES DANS LES EXTRAITS DE F. X BOHEMICÂ  8) - IDENTIFICATION OF PROANTHOCYANIDINES CONTAINED IN THE EXTRACTS OF F. X BOHEMICÂ
Les proanthocyanidines détectées par UHPLC dans l'extrait de de F, x bo emîca sont des procyanldines de type B allant du d mère au téfcramère de catéchlne et d'épicatéchine avec une absorbaoce maximaie à 278 nm et des masses moléculaires de 579,15, 867,21et 1155,27 g mol"1, comme présenté dans les Figures 2 et 3 (le spectre de la Figyre 3Â correspond au dlmère, le spectre de la Figure 38 au trimère et le spectre de la Figure 3C au tétramère). Proanthocyanidins detected by UHPLC in extract F, x bo EMICA are Type B procyanldines ranging from d mother téfcramère of catéchlne and epicatechin with a absorbaoce maximaie at 278 nm and molecular weights of 579.15, 867.21 and 1155.27 g mol -1 , as shown in Figures 2 and 3 (the spectrum of Fig 3 corresponds to the dimer, the spectrum of Figure 38 to the trimer and the spectrum of Figure 3C to the tetramer).
c)- EFFET DES PROANTHOCYANÏDINES PURIFIÉES SUR LA DENÈTRÈFICATIGN c) - EFFECT OF PURIFIED PROANTHOCYANIDINES ON DENETRAFICATIGN
DES BACTERIES DEN ÎXR l FI AN T E S BACTERIA DENIXR l FI AN T E S
II apparaît que les proanthocyanidines purifiées inhibent significativement la dénitrlfication de Pseudomonas brassicacearu et B, cereust comme cela ressort des résultats présentés Figure 4, Pour les deux souches bactériennes,, la dénitrification est affectée jusqu'à 80%. La réponse à l'inhibition est dose-dépendante entre 0,01 et 2,,5 mg.mi . D)- EFFET DES PROANTHOCYÂNIDINES COMMERCIALES SUR LA DENITRIFICATION DES SOLS EN SUSPENSION It appears that the purified proanthocyanidins significantly inhibit dénitrlfication Pseudomonas brassicacearu and B cereus t as the results shown in Figure 4, for the two bacterial strains ,, denitrification is affected up to 80%. The response to inhibition is dose-dependent between 0.01 and 2.5 mg. D) - EFFECT OF COMMERCIAL PROANTHOCYANIDINES ON SOIL DENITRIFICATION IN SUSPENSION
Il a également été observé que les proanthocyânidines commerciales testées inhibent significativement la dénit if catlon des deux sols en suspension (Figure S). L'inhibition est dose-dépendante avec un maximum d'Inhibition atteint dès 0,5 mg.mi"1 pour le sol Άίηί! et 1 nig. mi i pour le soi Ain6. Le maximum d'inhibition est de 85% pour le soi Ain il et de 80% pour le sol Αίηδ. Le plus faible effet significatif mesuré était à une concentration de 0,01 mg.mî1, It has also been observed that commercial proanthocyanidins tested significantly inhibit the denitiation of the two suspended soils (Figure S). The inhibition is dose-dependent with a maximum of inhibition reached in 0.5 mg.mi "1 for soil Άίηί! And 1 nig. Mi i for self Ain6. The maximum inhibition was 85% for 80% for soil Αίηδ. The lowest significant effect measured was at a concentration of 0.01 mg.ml 1 ,
E)~ EFFETS DES PROANTHOCYÂNIDINES SUR UNE COMMUNAUTÉ COMPLEXE DE SOL RAPPORTES EN MASSE DE PROANTHOCYANIDINES PAR RAPPORT A UNE MASSE DE E) ~ EFFECTS OF PROANTHOCYANIDINES ON A COMPLEXITY OF SOIL COMPLEX IN THE MASS OF PROANTHOCYANIDINES IN RELATION TO A MASS OF
SOL SEC DRY SOIL
Les proanthocyânidines commerciales inhibent significativement la dénitrification du sol Âînll après 7 jours de contact avec lesdites proanthocyânidines (Figure S), L'Inhibition est dose-dépendante avec un maximum de 100% dinhibition atteint à 5 mg.g'1 de sol sec. Le plus faible effet significatif était à une concentration de ô,i mg.g" , de sol sec, Commercial proanthocyanidins significantly inhibit denitrification Âînll soil after 7 days of contact with said proanthocyanidins (Figure S), the inhibition is dose-dependent with a maximum of 100% of inhibition reached 5 mg.g '1 dry soil. The smallest significant effect was at a concentration of 0.1 mg / g , of dry soil,
F)- EFFET DES PROANTHOCYÂNIDINES SUR LA TFJFÎCATÎON  F) - Effect of proanthocynidines on the diet
La nitritlcafion potentielle des deux types de sols (AM I. et Αίηβ) a été mesurée en suspension (1 g de sol équivalent sec pour 10 ml) sans proanthocyânidines (témoin) et en présence des proanthocyânidines commerciales, concentrées à 0,5 ; 0,1 ; 0,05 et 0,01 g.rni"*, dans une solution tampon phosphate au pH des sols 7,5 (NaHiPC? et NaHPG», 12Η20) (pour limiter les variations de pH au court de l'incubation) supplémentée en (NH^SGs (SO g.mL"1). Les sols ont été placés dans des flacons plasma de 150 ml en aérobiose bouchés par du psrafilm, Les flacons ont été mis à agiter à 140 rpm à 28eC« Chacune des conditions a été réalisée en 4 réplicas. The potential nitritlcafion of the two soil types (AM I. and Αίηβ) was measured in suspension (1 g of dry equivalent sol per 10 ml) without proanthocyanidins (control) and in the presence of commercial proanthocyanidins, concentrated to 0.5; 0.1; 0.05 and 0.01 g / cm 3 in phosphate buffer solution at soil pH 7.5 (NaHiPC 2 and NaHPG 2 , 12Η20) (to limit pH changes during incubation) supplemented with (NH SGs (SO g.mL "1). soils were placed in 150 ml flasks plasma aerobically blocked with psrafilm, flasks were set stirring at 140 rpm at 28 e C" Each conditions were realized in 4 replicas.
La mesure de nltrifscation potentielle (NEA : « nltrifylng enzymatic actlvlty »} a été réalisée en dosant la production de NC¾ au cours du temps à l'aide dfun chromafographe ionique Dionex DX 120 (Thermo Fisher Sclentific, Waltham, USA), en prélevant et en filtrant à 0,2 pm, 1 ml de suspension toutes les 2 h entre 2 et lOh. La mesure de nitrtficatîon potentielle correspond à la mesure du pool enzymatlque présent dans l'échantillon initialement, et est exprimée par la production de pg de N- C .g" de sol sec. fi1 entre 2 et 10 heures. Nltrifscation potential measurement (PBN "nltrifylng enzymatic actlvlty"} was performed by assaying the production of NC¾ over time using an ionic dpf chromafographe Dionex DX 120 (Thermo Fisher Sclentific, Waltham, USA), in removing and filtering at 0.2 μm, 1 ml of suspension every 2 hours between 2 and 10 am The measurement of potential nitridation corresponds to the extent enzymatlque pool present in the sample initially, and is expressed by the production pg N- C .g "dry soil. fi 1 between 2 and 10 hours.
Les résultats sont présentés Figyre 7. Ces résultats montrent qu'aux mêmes concentrations testées sur ia dénitrification, les proanthocyanidines n'ont pas d'effet significatif sur la nitrifeation. The results are presented Figyre 7. These results show that in the same concentrations tested ia denitrification, proanthocyanidins do not have a significant effect on the nitrifeation.
Références bibliographiques : Bibliographical references:
Bardon G, Piola F, Belivert F,, Halchar F.Z, Comte G;. Meiffren G, Pommier Tf Pu'rjaion S,, Tsafack N, Pdy F, 2014. Evidence for bioiogical denitrification Inhibition (BDI) by plant seconda ry metaboiites, New Phytologlst n/a-n/a, Bardon G, Piola F, F ,, Belivert Halchar FZ, Count G; . Meiffren G, Apple Tree T f Pu 'rjaion S, N Tsafack, Pdy F, 2014. Evidence for bioiogical denitrification inhibition (BDI) by plant seconda ry metaboiites, New Phytologlst n / an / a,
Heiistrom f Hattlla PH. 2008. HPLC détermination of extrada bie and unextractable proanthocyan dins in plant matériels. Journal of Agriculture! and Food Chemistry 56(17): 7617-7624. Heiistrom f Hattlla PH. 2008. HPLC determination of extraneous and unextractable proanthocyanins in plant material. Journal of Agriculture! and Food Chemistry 56 (17): 7617-7624.
Lacombe A, McGivney C, Tadepalli S, Son XH,. Wu VCH. 2013. The effect of American cranberry (Vacdnîum macrccarpon) constituants on the growth inhibition, membrane iniegrty, and injury of Escherichîa coli 0157 :H7 and Usteria monoeytogenes In comparison to Lactobaci ius rhamnosu5. Food Microblotogy 34(2): 352-359.  Lacombe A, McGivney C, Tadepalli S, Son XH ,. Wu VCH. 2013. The effect of American cranberry (Vacdnum macrccarpon) constituents on the inhibition inhibition, membrane iniegrty, and injury of Escherichia coli 0157: H7 and Usteria monoeytogenes In comparison to Lactobaci ius rhamnosu5. Food Microblotogy 34 (2): 352-359.
Lacombe A, Wu VCHf Whfêe 3,. Tadepalli S, André EE. 2012, The antimicrobia! properties of the iowbush blueberry (Vacdnîum angustifoiium) fractions! comportants against foodbome pathogens and the consen/atlo of probiooc Lactobacllius rhamnosus. Food MîcrobioEogy 30(1): 124-131, Lacombe A, Wu VCH f Whfêe 3 ,. Tadepalli S, André EE. 2012, The antimicrobia! properties of the blueberry iowbush (Vacdnîum angustifoiium) fractions! Behaviorists against foodborne pathogens and the consen / atlo of probiotoc Lactobacillus rhamnosus. Food MicrobioEogy 30 (1): 124-131,
LeBauer DS, Treseder KK. 2008. Nitrogen limitation of net primary productivity in terrestriai ecosystems is globally distnbuted, Eco ogy 89(2): 371-379.  LeBauer DS, Treseder KK. 2008. Nitrogen limitation of net primary productivity in terrestrial ecosystems is globally distnbuted, Eco ogy 89 (2): 371-379.
Oenema O, Witzke HP,, Klimont Z, Lessciien f Veithof GL. 2009, Integrated assessment of promislng measures to decrease nitrogen !osses from agriculture in EU-27, Agriculture Ecosystems â Environment 133(3-4): 280-288. Christophe DambrevsilefStéphanie Ha te^Chnstop e Nguyen/Tftierry Morvan, Jean-Ciaude Germon, LaurentPhilippot 2006. Structure and activlty of the denstnfying community in a maize-cropped ise d fertiiized ith composted pig manure or ammonium nitrate FEHS MICROBIGLOGY ECOLOGY, Volume; 56; 1:119-131 Oenema O, Witzke HP ,, Klimont Z Lessciien f Veithof GL. 2009, Integrated Environmental Assessment of Agriculture in Agriculture, Agriculture Ecosystems at Environment 133 (3-4): 280-288. Christophe Dambrevsile f ^ te Stephanie Ha Nguyen Chnstop e / Tftierry Morvan, Jean-Ciaude Albacore LaurentPhilippot 2006. Structure and activlty denstnfying of the community in a maize-cropped ise of composted pig manure fertiiized ith gold ammonium nitrate FEHS MICROBIGLOGY ECOLOGY, Volume; 56; 1: 119-131

Claims

REVENDICATIONS
1 - Utilisation d'au moins une proanthocyanidlne pour limiter la dénitrifîcation, consistant en la réduction de NO3 en 2O et N2, causée par des microorganismes, par mise en contact de ladite au moins proanthocyanidlne avec lesdlts microorganismes, 1 - Use of at least one proanthocyanidlne to limit denitrification, consisting in the reduction of NO3 2O and N 2, caused by microorganisms, by contacting said at least proanthocyanidlne lesdlts with microorganisms,
2 ~ Utilisation selon la revendication I caractérisée en ce que ladite au moins proanthocyanidlne est une proanthocyanidlne de type 8>  2 ~ Use according to claim I characterized in that said at least one proanthocyanidin is a type 8 proanthocyanidin>
3 - Utilisation selon la revendication 1 ou 2 caractérisée en ce que ladite au moins proanthocyanidlne est constituée exclusivement de monomères choisis parmi la catéehine et épicatéchlne.  3 - Use according to claim 1 or 2 characterized in that said at least proanthocyanidine consists exclusively of monomers selected from cathehine and epicatenol.
4 - Utilisation selon Tune quelconque des revendications 1 à 3 caractérisée en ce que ladite au moins proanthocyanidlne est un dlmère, trimère ou tétramère.  4 - Use according to any one of claims 1 to 3 characterized in that said at least one proanthocyanidine is a dimer, trimer or tetramer.
5 - Utilisation selon Tune quelconque des revendications 1 à 4 caractérisée en ce que les microorganismes sont présents dans un milieu dans lequel ladite au moins proanthocyanidlne est: présente.  5 - Use according to any one of claims 1 to 4 characterized in that the microorganisms are present in a medium in which said at least proanthocyanidine is: present.
6 ~ Utilisation selon la revendication 5 caractérisée en ce que le milieu est un sol et Tau moins une proanthocyanidlne est appliquée sur ledit soi, par exemple par dépôt ou pulvérisation,  6 ~ Use according to claim 5 characterized in that the medium is a sol and Tau at least one proanthocyanidine is applied to said soi, for example by deposition or spraying,
7 - Utilisation selon la revendication 6 caractérisée en ce que de 0,01 à 10 mg/g de sol sec., et de préférence de 0,5 à 5 mg/g de sol sec, de proanthocyanidîne(s) est appliqué sur ledit soL  7 - Use according to claim 6 characterized in that from 0.01 to 10 mg / g of dry sol., And preferably from 0.5 to 5 mg / g of dry sol, of proanthocyanidine (s) is applied to said ground
S - Utilisation selon l'une quelconque des revendications 1 à 7 caractérisée en ce que ladite au moins proanthocyanidlne est utilisée pour limiter la dénMcation causée par des bactéries dénitrifiantes., en particulier choisies parmi Pseudomonas brassicaœarum, Pseudomonas stuizen, Pseudomonas afcaiigenes, Pseudomonas ffuoœsœns, Butkholderîa vietnamiensis, Ac ro ohac er xylosoxidans, Paracoccus denitrifîcans, Badifus cereus,  S - Use according to any one of Claims 1 to 7, characterized in that the said at least one proanthocyanidin is used to limit the denmation caused by denitrifying bacteria, in particular chosen from Pseudomonas brassicaœarum, Pseudomonas stuizen, Pseudomonas afcigenes, Pseudomonas fufuosens, Butkholderia vietnamiensis, Acroxylosoxidans, Paracoccus denitriflcans, Badifus cereus,
§ - Utilisation selon l'une quelconque des revendications 1 à 8 caractérisée en ce que ladite au moins proanthocyanidine ne se présente pas sous la forme d'un extrait de Fallopia spp. contenant des proanthocyanidines. 10 - Utilisation selon l'une quelconque des revendications i à 9 caractérisée en ce que ladite au moins proanthocyanidine est utilisée, en l'absence de catéehine et/ou d'épicatéchlne. - Use according to any one of claims 1 to 8 characterized in that said at least one proanthocyanidin is not in the form of an extract of Fallopia spp. containing proanthocyanidins. 10 - Use according to any one of claims i to 9 characterized in that said at least proanthocyanidin is used in the absence of cathehine and / or epicatenol.
PCT/FR2016/050600 2015-03-19 2016-03-17 Use of proanthocyanidins for limiting denitrification WO2016146955A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP16715023.4A EP3271312A1 (en) 2015-03-19 2016-03-17 Use of proanthocyanidins for limiting denitrification

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR1552258 2015-03-19
FR1552258A FR3033790B1 (en) 2015-03-19 2015-03-19 USE OF PROANTHOCYANIDINES TO LIMIT DENITRIFICATION

Publications (1)

Publication Number Publication Date
WO2016146955A1 true WO2016146955A1 (en) 2016-09-22

Family

ID=53274619

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/FR2016/050600 WO2016146955A1 (en) 2015-03-19 2016-03-17 Use of proanthocyanidins for limiting denitrification

Country Status (3)

Country Link
EP (1) EP3271312A1 (en)
FR (1) FR3033790B1 (en)
WO (1) WO2016146955A1 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107858311A (en) * 2017-12-07 2018-03-30 江西省农业科学院畜牧兽医研究所 A kind of Achromobacter xylosoxidans X10 and its application
US9975817B2 (en) 2015-07-13 2018-05-22 Pivot Bio, Inc. Methods and compositions for improving plant traits
CN110257291A (en) * 2019-06-25 2019-09-20 北京大学 One plant of achromobacter for being resistant to nickel ion toxicity and its application
CN113234626A (en) * 2021-05-13 2021-08-10 安徽省农业科学院水产研究所 Strain with heterotrophic nitrification-aerobic denitrification function and application thereof
US11479516B2 (en) 2015-10-05 2022-10-25 Massachusetts Institute Of Technology Nitrogen fixation using refactored NIF clusters
US11565979B2 (en) 2017-01-12 2023-01-31 Pivot Bio, Inc. Methods and compositions for improving plant traits
US11678667B2 (en) 2018-06-27 2023-06-20 Pivot Bio, Inc. Agricultural compositions comprising remodeled nitrogen fixing microbes
US11946162B2 (en) 2012-11-01 2024-04-02 Massachusetts Institute Of Technology Directed evolution of synthetic gene cluster

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000159669A (en) * 1998-11-26 2000-06-13 Mikoo:Kk Urease inhibitor, medicine and food composition containing the inhibitor and measurement of urease activity
EP2653038A1 (en) * 2012-04-16 2013-10-23 OK Biotech ApS Plant residues from Sapotaceae trees and uses thereof
US20140190225A1 (en) * 2004-10-04 2014-07-10 Agresearch Limited Altering animal waste composition

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000159669A (en) * 1998-11-26 2000-06-13 Mikoo:Kk Urease inhibitor, medicine and food composition containing the inhibitor and measurement of urease activity
US20140190225A1 (en) * 2004-10-04 2014-07-10 Agresearch Limited Altering animal waste composition
EP2653038A1 (en) * 2012-04-16 2013-10-23 OK Biotech ApS Plant residues from Sapotaceae trees and uses thereof

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
ADAMCZYK BARTOSZ ET AL: "Response of soil C and N transformations to condensed tannins and different organic N-condensed tannin complexes", APPLIED SOIL ECOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 64, 8 January 2013 (2013-01-08), pages 163 - 170, XP028977307, ISSN: 0929-1393, DOI: 10.1016/J.APSOIL.2012.12.003 *
ADAMCZYK BARTOSZ, RESPONSE OF SOIL C AND N TRANSFORMATIONS TO CONDENSED TANNINS AND DIFFÉRENT ORGANIC N-CONDENSED TANNIN COMPLEXES, vol. 64, 8 January 2013 (2013-01-08), pages 163 - 170
ADAMCZYK SYLWIA ET AL., POTENTIAL RESPONSE OF SOIT PROCESSES TO DITERPENES, TRITERPENES AND TANNINS : NITRIFICATION, GROWTH OF MICROORGANISMS AND PRÉCIPITATION OF PROTEINS, vol. 67, 24 March 2013 (2013-03-24), pages 47 - 52
ADAMCZYK SYLWIA ET AL: "Potential response of soil processes to diterpenes, triterpenes and tannins: Nitrification, growth of microorganisms and precipitation of proteins", APPLIED SOIL ECOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 67, 24 March 2013 (2013-03-24), pages 47 - 52, XP028544794, ISSN: 0929-1393, DOI: 10.1016/J.APSOIL.2013.02.009 *
AINO SMOLANDER ET AL: "Nitrogen transformations in boreal forest soils-does composition of plant secondary compounds give any explanations?", PLANT AND SOIL ; AN INTERNATIONAL JOURNAL ON PLANT-SOIL RELATIONSHIPS, KLUWER ACADEMIC PUBLISHERS, DO, vol. 350, no. 1 - 2, 12 August 2011 (2011-08-12), pages 1 - 26, XP019989866, ISSN: 1573-5036, DOI: 10.1007/S11104-011-0895-7 *
BALDWIN I T ET AL., PROTEIN BINDING PHENOLICS AND THE INHIBITION OF NITRIFICATION IN SUBALPINE BALSAM FIR SOITS, vol. 15, no. 4, 1 January 1983 (1983-01-01), pages 419 - 423
BALDWIN I T ET AL: "Protein binding phenolics and the inhibition of nitrification in subalpine balsam fir soils", SOIL BIOLOGY AND BIOCHEMISTRY, PERGAMON, OXFORD, GB, vol. 15, no. 4, 1 January 1983 (1983-01-01), pages 419 - 423, XP023671423, ISSN: 0038-0717, [retrieved on 19830101], DOI: 10.1016/0038-0717(83)90006-8 *
BARDON C; PIOLA F; BELLVERT F; HAICHAR F.Z; COMTE G; MEIFFREN G; POMMIER T; PUIJALON S; TSAFACK N; POLY F: "Evidence for biological dénitrification inhibition (BDI) by plant secondary metabolites", NEW PHYTOLOGIST: N/A-N/A, 2014
CHRISTOPHE DAMBREVILLE; STÉPHANIE HALLET; CHRISTOPHE NGUYEN; THIERRY MORVAN; JEAN-CLAUDE GERMON; LAURENTPHILIPPOT: "Structure and activity of the denitrifying community in a maize-cropped field fertilized with composted pig manure or ammonium nitrate", FEMS MICROBIOLOGY ECOLOGY, vol. 56, no. 1, 2006, pages 119 - 131
HELLSTROM JK; MATTILA PH: "HPLC détermination of extractable and unextractable proanthocyanidins in plant materials", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, vol. 56, no. 17, 2008, pages 7617 - 7624
LACOMBE A; MCGIVNEY C; TADEPALLI S; SUN XH; WU VCH: "The effect of American cranberry (Vaccinium macrocarpon) constituents on the growth inhibition, membrane integrity, and injury of Escherichia coli 0157:H7 and Listeria monocytogenes in comparison to Lactobacillus rhamnosus", FOOD MICROBIOLOGY, vol. 34, no. 2, 2013, pages 352 - 359
LACOMBE A; WU VCH; WHITE J; TADEPALLI S; ANDRE EE: "The antimicrobial properties of the lowbush blueberry (Vaccinium angustifolium) fractional components against foodborne pathogens and the conservation of probiotic Lactobacillus rhamnosus", FOOD MICROBIOLOGY, vol. 30, no. 1, 2012, pages 124 - 131
LEBAUER DS; TRESEDER KK: "Nitrogen limitation of net primary productivity in terrestrial ecosystems is globally distributed", ECOLOGY, vol. 89, no. 2, 2008, pages 371 - 379
OENEMA O; WITZKE HP; KLIMONT Z; LESSCHEN JP; VEITHOF GL: "Integrated assessment of promising measures to decrease nitrogen losses from agriculture in EU-27", AGRICULTURE ECOSYSTEMS & ENVIRONMENT, vol. 133, no. 3-4, 2009, pages 280 - 288
R L BRADLEY ET AL., CHANGES TO MINERAI N CYCLING AND MICROBIAL COMMUNITIES IN BLACK SPRUCE HUMUS AFTER ADDITIONS OF (NH4)2S04 AND CONDENSED TANNINS EXTRACTED FROM KALMIA ANGUSTIFOLIA AND BASAM FIR, 1 August 2000 (2000-08-01), pages 1227 - 1240
R L BRADLEY ET AL: "Changes to mineral N cycling and microbial communities in black spruce humus after additions of (NH4)2SO4 and condensed tannins extracted from Kalmia angustifolia and balsam fir", SOIL BIOLOGY & CHEMISTRY, 1 August 2000 (2000-08-01), pages 1227 - 1240, XP055244185, Retrieved from the Internet <URL:http://ac.els-cdn.com/S0038071700000390/1-s2.0-S0038071700000390-main.pdf?_tid=7032da5e-d170-11e5-8d4b-00000aab0f27&acdnat=1455271835_bd9db0293a9cbcc19736192d96659524> [retrieved on 20160125], DOI: 10.1016/S0038-0717(00)00039-0 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11946162B2 (en) 2012-11-01 2024-04-02 Massachusetts Institute Of Technology Directed evolution of synthetic gene cluster
US9975817B2 (en) 2015-07-13 2018-05-22 Pivot Bio, Inc. Methods and compositions for improving plant traits
US10384983B2 (en) 2015-07-13 2019-08-20 Pivot Bio, Inc. Methods and compositions for improving plant traits
US10556839B2 (en) 2015-07-13 2020-02-11 Pivot Bio, Inc. Methods and compositions for improving plant traits
US10919814B2 (en) 2015-07-13 2021-02-16 Pivot Bio, Inc. Methods and compositions for improving plant traits
US10934226B2 (en) 2015-07-13 2021-03-02 Pivot Bio, Inc. Methods and compositions for improving plant traits
US11739032B2 (en) 2015-07-13 2023-08-29 Pivot Bio, Inc. Methods and compositions for improving plant traits
US11479516B2 (en) 2015-10-05 2022-10-25 Massachusetts Institute Of Technology Nitrogen fixation using refactored NIF clusters
US11565979B2 (en) 2017-01-12 2023-01-31 Pivot Bio, Inc. Methods and compositions for improving plant traits
CN107858311B (en) * 2017-12-07 2020-11-24 江西省农业科学院畜牧兽医研究所 Achromobacter xylosoxidans X10 and application thereof
CN107858311A (en) * 2017-12-07 2018-03-30 江西省农业科学院畜牧兽医研究所 A kind of Achromobacter xylosoxidans X10 and its application
US11678667B2 (en) 2018-06-27 2023-06-20 Pivot Bio, Inc. Agricultural compositions comprising remodeled nitrogen fixing microbes
US11678668B2 (en) 2018-06-27 2023-06-20 Pivot Bio, Inc. Agricultural compositions comprising remodeled nitrogen fixing microbes
US11963530B2 (en) 2018-06-27 2024-04-23 Pivot Bio, Inc. Agricultural compositions comprising remodeled nitrogen fixing microbes
CN110257291B (en) * 2019-06-25 2021-07-23 北京大学 Achromobacter capable of tolerating nickel ion toxicity and application thereof
CN110257291A (en) * 2019-06-25 2019-09-20 北京大学 One plant of achromobacter for being resistant to nickel ion toxicity and its application
CN113234626A (en) * 2021-05-13 2021-08-10 安徽省农业科学院水产研究所 Strain with heterotrophic nitrification-aerobic denitrification function and application thereof

Also Published As

Publication number Publication date
FR3033790B1 (en) 2018-05-04
EP3271312A1 (en) 2018-01-24
FR3033790A1 (en) 2016-09-23

Similar Documents

Publication Publication Date Title
WO2016146955A1 (en) Use of proanthocyanidins for limiting denitrification
Bremner et al. Inhibition of nitrification in soil by allelochemicals derived from plants and plant residues
Adamczyk et al. The contribution of ericoid plants to soil nitrogen chemistry and organic matter decomposition in boreal forest soil
Linskens et al. The release of free amino acids from germinating pollen
Mascarua-Esparza et al. Acetylene reduction and indoleacetic acid production by Azospirillum isolates from Cactaceous plants
Spiteri et al. Artefactual origins of cyclic AMP in higher plant tissues
Kim et al. Effect of fertilization on free amino acid concentrations in black spruce and jack pine containerized seedlings
Domenech et al. Bacillus spp. and Pisolithus tinctorius effects on Quercus ilex ssp. ballota: a study on tree growth, rhizosphere community structure and mycorrhizal infection
Kuo et al. Isoxazolin-5-ones and amino acids in root exudates of pea and sweet pea seedlings
Pankhurst et al. Effectiveness of Lotus Root Nodules: II. RELATIONSHIP BETWEEN ROOT NODULE EFFECTIVENESS AND ‘IN VITRO’SENSITIVITY OF FAST-GROWING LOTUS RHIZOBIA TO FLAVOLANS
Thomas Phytotoxic metabolites produced by Fusarium nygamai from Striga hermonthica
Vázquez et al. The dynamics of different organic and inorganic phosphorus fractions in soils from the south of Santa Fe province, Argentina
BOWEN et al. The occurrence and effects of red clover necrotic mosaic virus in red clover (Trifolium pratense)
Nakaji et al. Growth and nitrogen availability of red pine seedlings under high nitrogen load and elevated ozone
Driessche et al. Total and soluble nitrogen in Douglas fir in relation to plant nitrogen status
Castro et al. Lowest effective and optimum poultry manure dose for reclaiming burnt soils: pot experiments
Omar PNITROGEN-FIXING ABILITIES OF SOME CYANOBACTERIA IN SANDY LOAM SOIL AND EXUDATE EFFICIENCY ON RICE GRAIN GERMINATION
Bertagnolli et al. Some physiological responses of Chlorella pyrenoidosa to 2, 4-dichlorophenoxyacetic acid
Popoola et al. Effect of water stress on infection by species of honey fungus (Armillaria mellea and A. gallica)
Garcia et al. Fractionation and characterization of humic substance fractions with different molecular weights, obtained from animal wastes
Iwasaki et al. Extracellular accumulation of aromatic compounds by a photosynthetic bacterium
Ibrahim et al. An attempt towards standardization of the production process of dawadawan botso (a fermented condiment)
Al‐Yousif et al. Influence of boron concentrations on some meta‐bolites of date palm and sorghum seedlings
CN116478238B (en) Lipopeptide compositions for promoting root nodule growth
EP3898938B1 (en) Biofertilizing bacterial strain

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16715023

Country of ref document: EP

Kind code of ref document: A1

REEP Request for entry into the european phase

Ref document number: 2016715023

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE