WO2018229450A1

WO2018229450A1 - Extract of knots from trees belonging to the genus pinus as fungicide against oomycetes infections

Info

Publication number: WO2018229450A1
Application number: PCT/FR2018/051429
Authority: WO
Inventors: Tristan RICHARD; Jean-Michel Merillon; Julien GABASTON; Marie-France CORIO-COSTET; Benoît BIAIS; Jean-Claude Izard
Original assignee: Universite de Bordeaux; Institut Polytechnique De Bordeaux; École Nationale Supérieure Des Sciences Agronomiques De Bordeaux Aquitaine (Bordeaux Sciences Agro); Institut National De La Recherche Agronomique Inra; Actichem
Priority date: 2017-06-15
Filing date: 2018-06-15
Publication date: 2018-12-20
Also published as: FR3067592B1; FR3067592A1

Abstract

The present invention relates to the use of an extract of knots from trees belonging to the genus Pinus for the treatment of a pathogenic infection due to at least one oomycete. The invention also relates to the extracts obtained by extraction of knots from trees belonging to the genus Pinus, the process for obtaining same, and also the use thereof as fungicide and/or algicide and especially for the treatment of a pathogenic infection due to at least one oomycete.

Description

EXTRACT OF TREE NODES BELONGING TO THE PINUS GENUS AS A FUNGICIDE AGAINST OOMYCETATE INFECTIONS

TECHNICAL AREA

The present invention relates to the use of an extract of tree nodes belonging to the genus Pinus for the treatment of a pathogenic infection due to at least one oomycete. It also relates to extracts obtained by extraction of tree nodes belonging to the genus Pinus, their method of production, and their use, as a fungicide and / or algicide, Pinus, preferentially for the treatment of a pathogenic infection due to to oomycetes.

STATE OF THE ART

Today there is a need to turn to a more environmentally friendly and consumer friendly agriculture. This resolution results in a process of reduction of chemical inputs. The Ecophyto 2018 plan will impose in particular a halving of the pesticides used for vine cultivation, 80% of which consist of fungicides. These products, such as the Bordeaux mixture (copper-based treatment), aim to stem the infection of vineyards with various diseases, including late blight.

Downy mildew is the generic name for a series of fungal diseases affecting many plant species, but of epidemic proportions in some economically important crops, such as grapes, tomatoes, potatoes, lettuce or squash such as melon. These pathogenic infections are due to oomycetes. Oomycetes are non-photosynthetic organisms. Given their morphological similarities, they have long been classified as a group close to fungi. The biflagellate spores, the metabolism or the non chitinous wall of oomycetes bring them closer to the algae; they are classified in Stramenopiles, close to brown algae.

Oomycetes Plasmopara viticola, Phytophthora infestans, Pseudoperonospora cubensis or Bremia lactucae may be mentioned.

Plasmopara viticola is responsible for the downy mildew of the vine. This obligatory endoparasite develops within the vine leaves (appearance of "oil stains") and bunches causing significant damage within the vineyards.

Late blight of tomato and potato is caused by Phytophthora infestans. The feet of tomatoes are stained with black, first the leaves, the stems then the fruits. For the potatoes, translucent spots appear on the leaves, then they take an oily appearance and have a blackish necrotic center with a livid margin. Soon, the leaf spots meet and the attacked leaf group curls up.

Pseudoperonospora cubensis causes in many cases damaging damage to various Cucurbitaceae (squash) such as cucumber, melon, watermelon, zucchini.

It produces oily leaf spots, well defined by the veins of the leaves. The spots later show a gray-brown felting then the leaves turn yellow and dry out. Bremia lactucae is an oomycete that parasitizes about 230 plants belonging to the asteraceae family and more particularly lettuce.

Many studies on different plants have highlighted the antifungal character of stilbenes.

The vine was one of the most studied plants on this subject. On the one hand, it has been demonstrated that in the face of an attack of a fungus (Botrytis cinerea), the vine produced in vivo many stilbenes such as resveratrol or viniferine (oligomers) making it possible to reduce the mycelial growth and the germination of the fungi. spores (JEANDET et al., 2002). On the other hand, complex stilbenes from vine shoots can inhibit in vitro the growth of Plasmopara viticola and several fungi involved in wood diseases in grapevines (LAMBERT et al., 2012, SCHNEE et al, 2013). . The use of vine shoots is, however, expensive.

More and more studies are also focusing on conifer resistance to fungi. Pine produces Pinosylvine, a monomeric stilbene, which allows the conifer to defend itself (CELIMENE et al., 1999, LEE et al., 2005).

Currently, the main agent for controlling downy mildew is Bordeaux mixture. Indeed, copper is a product of preventive contact. It has a fungicidal effect due to the destruction of mildew spores. In Europe, Bordeaux mixture is permitted within certain limits (Regulation (EC) No 889/2008). Since ¹ January 2006, you must not exceed 6 kg / ha / year on average over 5 years. AFSSA recommends an annual limit of 4kg / ha / year and projects that this limit be strict per year and not on average over several years. There is therefore a regulatory reduction in the use of Bordeaux mixture. Copper has adverse effects on soils through its accumulation and is toxic to humans with acute effects. European regulations impose a maximum tolerated value in agricultural soils of 150 mg / kg of dry matter. Following the Grenelle Environment and the second National Environmental Health Plan, the Ecophyto 2018 plan, the Ministry of Agriculture wants to significantly reduce the use of pesticides. One of the goals is to divide their uses by half. There are also other chemical treatments: contact products (mancozebe, folpel ...), penetrating products (dimethomorph, cymoxanil ...), systemic products (phosethyl aluminum or metalaxyl). However, these products are affected by resistances. It is therefore important to have new families to diversify the portfolio of anti-mildew products. In addition, regulatory and consumer pressures are making it increasingly necessary to develop more environmentally friendly treatments.

The development of alternative or complementary strategies to the use of pesticides as well as Bordeaux mixture for the treatment of late blight is a major economic challenge for viticulture in particular and agriculture in general.

There is a need to produce antifungals and algicides alternative to chemicals, and also to reduce the use of copper. OBJECT OF THE INVENTION

The present invention relates to the use of an extract of tree nodes belonging to the genus Pinus comprising stilbenes, flavonoids and lignans for the treatment of a pathogenic infection due to at least one oomycete. Advantageously, the use of an extract of tree nodes belonging to the genus Pinus makes it possible to inhibit the growth of downy mildew and such an extract has a much higher activity than that of a vine shoot extract known for its activity. inhibitor of downy mildew.

Advantageously also, the use of pine nodes makes it possible to recover the waste of the paper industry.

The present invention also relates to an extract obtained by extraction of tree nodes belonging to the genus Pinus, said extract comprising at least one stilbene, at least one flavonoid and at least one lignane and its use as a fungicide and / or algicide, and more particularly for the treatment of a pathogenic infection due to at least one oomycete. DETAILED DESCRIPTION

The present invention relates to the use of an extract of tree nodes belonging to the genus Pinus comprising stilbenes, flavonoids and lignans for the treatment of a pathogenic infection due to at least one oomycete. The term "tree node" in the sense of the present invention, the structures where the branches are inserted at the level of the trunk of trees.

Typically, the tree from which the extract is obtained belongs to the genus Pinus, and will be preferentially selected from the following species:

Aleppo pine or Jerusalem pine, {Pinus halepensis)

Bosnian pine (Pinus heldreichii, syn. Pinus leucodermis)

Calabrian pine or Turkish pine or Persian pine (Pinus brutia)

Canary Pine (Pinus canariensis)

Pine cembro, arolle, alvier, (Pinus cembrd)

Macedonian pine or Balkan pine (Pinus peuce) Maritime pine or Landes pine (Pinus pinaster)

Mountain pine or mugo pine, recumbent pine (Pinus mugo)

Black pine (Pinus nigrd)

Parasol pine or pinion pine, pine pine (Pinus pinea)

Scots pine (Pinus sylvestris)

Pinus contorta

Pinus sibirica

Monterey pine (Pinus radiata)

Incense pine (Pinus taeda)

Marsh pine (Pinus palustris)

Gray Pine (Pinus banksiana)

Pinus caribae

Pinus tecunumanii. Preferably, the species of the tree will be Pinus sylvestris or Pinus pinaster, preferably Pinus pinaster.

These pine nodes are by-products of the unutilized pine industry and can be recovered from the paper industry's waste. Typically, the oomycete will be selected from Plasmopara viticola, Phytophthora infestans, Pseudoperonospora cubensis or Bremia lactucae.

Preferably, the oomycete is Plasmopara viticola. In one embodiment, the extract according to the invention further comprises at least one phenolic acid.

The stilbenes of the tree node extract belonging to the genus Pinus comprise one or more compounds selected from pinosylvin, its monomethyl ester and pterostilbene or a mixture thereof. The flavonoids of the tree node extract belonging to the genus Pinus comprise one or more compounds selected from taxifolin, dihydrokaempferol, pinocembrin and pinobanskin or a mixture thereof.

The phenolic acids of the extract according to the invention comprise one or more compounds selected from ferulic acid and caffeic acid or a mixture thereof. The lignans of the extract according to the invention comprise one or more compounds chosen from nortrachelogenin, pinoresinol, matairésinol, secoisolariciresinol and isolariciresinol or a mixture thereof.

The stilbene content of the extract of tree nodes belonging to the genus Pinus according to the invention is between 1 and 20%, preferably between 3 and 15%, and even more preferably between 4 and 10%.

The flavonoid content of the extract of tree nodes belonging to the genus Pinus according to the invention is between 1 and 20%, preferably between 3 and 15%, and even more preferably between 4 and 10%.

The phenolic acid content of the extract of tree nodes belonging to the genus Pinus according to the invention is between 0.02 and 1%, preferably between 0.03 and 0.5%, and even more preferably, between 0.04 and 0.3%.

The lignan content of the extract of tree nodes belonging to the genus Pinus according to the invention is between 5 and 40%, preferably between 6 and 25%, and even more preferably between 8 and 16%. Typically, the extract of tree nodes belonging to the genus Pinus comprises a stilbene content of between 1 and 20%, preferably between 4 and 10%, a content of flavonoids between 1 and 20%, preferably between 4 and 10%, a lignan content of between 5 and 40%, preferably between 8 and 16% and a phenolic acid content of between 0.02 and 1%, preferably between 0.04 and 0.04. and 0.3% by weight.

Typically, the tree nodes belonging to the genus Pinus make it possible to obtain by extraction, an amount of extract (weight / weight) of between 0.03 and 20%, preferably between 1 and 15%, and even more preferably between 5 and 10%.

The contents of phenolic acids, stilbenes, flavonoids and lignans were measured by high performance liquid chromato graphy (HPLC), ultra high performance liquid chromatography (UHPLC-DAD-MS) and / or resonance nuclear magnetic resonance (NMR). These are contents in weight. Typically, tree node extract of the genus Pinus can be obtained by hot water extraction, subcritical water extraction, by extraction with a water / organic solvent mixture, by extraction with a mixture of hydroalcoholic solvents. Preferably, said extract is obtained by extraction with a water / organic solvent mixture, the organic solvent being chosen from ethanol, acetone or methanol.

Preferably, said extract is a hydro alcoholic extract, the alcohol being preferably chosen from ethanol and methanol.

In a preferred embodiment, the extraction will be carried out with an ethanol-water mixture. Preferably, the volume proportions of the ethanol / water mixture are 85/15. According to one embodiment, the extract of tree nodes belonging to the genus Pinus used comprises at least the following compounds:

pinosylvin;

pinosyl mono methyl ether;

the pinocembrine

pinobanskin

pinoresinol

nortrachelogenin;

isolariciresinol;

the matairésinol.

In a preferred embodiment, the tree node extract belonging to the Pinus genus used comprises at least the following compounds: pinosylvin;

pinosyl mono methyl ether;

pterostilbene;

ferulic acid;

caffeic acid;

pinocembrine;

pinobanskin;

taxifolin;

dihydrokaempferol,

matairésinol;

nortrachelogenin;

pinoresinol;

isolariciresinol;

secoisolariciresinol. The pinosilin content of the extract according to the invention is between 0.5 and 10%, preferably 1 and 7.5%, and even more preferably between 1.5 and 5% by weight. The content of pinosylvin mono methyl ether of the extract according to the invention is between 0.5 and 10%, preferably 1 and 7.5% and even more preferably between 2 and 6% by weight.

The pterostilbene content of the extract according to the invention is between 0.025 and 0.5%, preferably 0.05 and 0.4% and even more preferably between 0.10 and 0.3% by weight.

The ferulic acid content of the extract according to the invention is between 0.005 and 0.4%, preferably 0.0075 and 0.3% and even more preferably between 0.01 and 0.2% by weight.

The caffeic acid content of the extract according to the invention is between 0.005 and 0.4%, preferably 0.0075 and 0.3% and even more preferably between 0.01 and 0.2% by weight.

The pinocembrin content of the extract according to the invention is between 0.5 and 12%, preferably 1.5 and 10% and even more preferably between 2.5 and 8% by weight. The pinobanskin content of the extract according to the invention is between 0.5 and 7%, preferably 0.75 and 6% and even more preferably between 1 and 4% by weight.

The taxifolin content of the extract according to the invention is between 0.01 and 0.5%, preferably 0.02 and 0.3% and even more preferably between 0.03 and 0.2% by weight. . The dihydrokaempferol content of the extract according to the invention is between 0.02 and 2%, preferably 0.04 and 1.5% and even more preferably between 0.06 and 1.25% by weight.

The mesinsinol content of the extract according to the invention is between 0.15 and 6%, preferably 0.25 and 5% and even more preferably between 0.5 and 3.5% by weight. The nortrachelogénine content of the extract according to the invention is between 3 and 35%, preferably 4 and 20% and even more preferably between 5 and 15% by weight.

The pinoresinol content of the extract according to the invention is between 0.25 and 8%, preferably 0.5 and 6% and even more preferably between 0.75 and 5% by weight.

The isolariciresinol content of the extract according to the invention is between 0.05 and 3%, preferably 0.10 and 2% and even more preferably between 0.25 and 1.50% by weight.

The secoisolariciresinol content of the extract according to the invention is between 0.05 and 3%, preferentially 0.10 and 2% and even more preferably between 0.25 and 1.50% by weight.

Typically, the tree node extract belonging to the genus Pinus comprises a pinosylvin content of between 0.5 and 10%, preferably 1.5 and 5%, a pinosylvin mono methyl ether content of between 0.5 and 10%, preferably 2 and 6%, a pterostilbene content of between 0.025 and 0.5%, preferentially 0.10 and 0.3%, a ferulic acid content of between 0.005 and 0.4%, preferably between 0, 01 and 0,2%, including caffeic acid content between 0.005 and 0.4%, preferably between 0.01 and 0.2%, a pinocembrin content of between 0.5 and 12%, preferably 2.5 and 8%, a pinobanskin content of between 0.5 and 7%, preferably 1 and 4%, a taxifoline content of between 0.01 and 0.5%, preferably 0.03 and 0.2%, a dihydrokaempferol content of between 0.02 and 2%, preferably 0, 06 and 1.25%, a matinsinol content of between 0.15 and 6%, preferentially 0.5 and 3.5%, nortrachelogenin content of between 3 and 35%, preferably 5 and 15%, a pinoresinol content between 0.25 and 8%, preferably 0.75 and 5%, an isolariciresinol content of between 0.05 and 3%, preferably 0.25 and 1.50%, a secoisolariciresinol content of between 0.05%. and 3%, preferably 0.25 and 1.50%.

The contents were measured by high performance liquid chromatography (HPLC), UHPLC-DAD-MS ultra-high performance liquid chromatography and / or NMR nuclear magnetic resonance. These contents are contents by weight.

The tree node extract belonging to the genus Pinus according to the invention used comprises a quantity of null or non-zero resins.

For the purposes of the present invention, the term "resin" means non-polar natural compounds, mainly lipids, triglycerides, fatty acids, sterols, diterpenes, etc. present in conifers such as those belonging to the genus Pinus. Typically, the resin content is between 0 and 25%, preferably 2 and 20%, and preferably between 5 and 15% by weight.

The resin content can be measured by any technique known to those skilled in the art. For illustrative purposes, weighing is given. This content is a weight content. The resins can participate in the antifungal activity of the tree node extracts according to the invention. Resin content also has an impact on the ease of manipulation of the extracts, especially in the formulation and / or analysis steps thereof.

Advantageously, those skilled in the art will be able to determine the appropriate resin content according to the desired use.

The present invention also relates to the use of an extract of tree nodes belonging to the genus Pinus in association with a fungicide and / or an algicide and / or an adjuvant, as fungicide and / or algicide, preferentially for the treatment of a pathogenic infection due to at least one oomycete.

Typically, oomycete will be selected from Plasmopara viticola, Phytophthora infestans, Pseudoperonospora cubensis or Bremia lactucae.

Typically, the fungicide will be selected from a copper product such as Bordeaux mixture, dithiocarbamate, dicarboximide, phosphite, cyanoimidazole, carboxylic acid amide, acetamide, phenylamide and benzamide.

Typically, the adjuvant may be a terpenes extract.

Typically algicide may be selected from copper sulfate, bromine, chlorine, cobalt and Bordeaux mixture.

Preferentially, algicide will be the Bordeaux mixture. The present invention also relates to an extract obtained by extraction of tree nodes belonging to the genus Pinus, and comprising at least the following compounds:

pinosylvin;

pinosyl mono methyl ether;

pinocembrine.

More specifically, the extract of tree nodes belonging to the genus Pinus, comprises at least the following compounds:

- pinosylvin;

pinosyl mono methyl ether;

pinocembrine;

pinobanskin;

matairésinol;

- nortrachelogenin;

pinoresinol;

the isolariciresinol.

In another preferred embodiment, the tree node extract belonging to the genus Pinus comprises at least the following compounds: pinosylvin;

pinosyl mono methyl ether;

pterostilbene;

- ferulic acid;

caffeic acid;

pinocembrine;

pinobanskin;

taxifolin;

- dihydrokaempferol,

matairésinol; nortrachelogenin;

pinoresinol;

isolariciresinol;

secoisolariciresinol.

The contents of each of the various compounds in the extract are preferably as defined above.

The extract according to the invention further comprises ferulic acid, caffeic acid, secoisolariciresinol, taxifolin, dihydrokaempferol and / or pterostilbene.

In one embodiment, the extract is a hydroalcoholic extract.

Preferably, the hydroalcoholic solvent mixture is an ethanol / water mixture, and even more preferably, the volume of ethanol relative to that of water is greater than 50%, preferably 85%.

The invention also relates to a composition comprising an extract according to the present invention. It also relates to a composition comprising the combination of an extract according to the present invention and at least one fungicide and / or at least one algicide.

Typically, the fungicide will be selected from a copper product such as Bordeaux mixture, dithiocarbamate, dicarboximide, phosphite, cyanoimidazole, carboxylic acid amide, acetamide, phenylamide or benzamide. Typically the algicide may be selected from copper sulfate, bromine, chlorine, cobalt and Bordeaux mixture. Preferably, the algicide will be the Bordeaux mixture.

In one embodiment, the composition may further comprise an adjuvant. Typically, the adjuvant may be a terpenes extract.

Typically, the extract according to the present invention may be used alone or in combination with at least one fungicide and / or algicide, as a fungicide and / or algicide, preferably for the treatment of a pathogenic infection due to at least one oomycete, such as than Plasmopara viticola, Phytophthora infestans, Pseudoperonospora cubensis or Bremia lactucae, and preferentially Plasmopara viticola.

The present invention also relates to a fungicidal or algicidal composition comprising an extract of tree nodes belonging to the genus Pinus as defined in any one of the preceding embodiments.

The invention also relates to a process for obtaining a hydroalcoholic extract obtained by extraction of tree nodes belonging to the genus Pinus and comprising at least the steps of:

i. take knots of trees belonging to the genus Pinus;

ii. extracting said ground tree nodes with a mixture of hydroalcoholic solvents preferably composed of an ethanol / water mixture; An additional filtration step may be carried out after step ii. The purpose of this filtration is to separate the hydro alcoholic extract from the pine knot powder.

Typically, the filtration will be carried out using depth filter (cotton) or screen filter (membrane, sieve) by sedimentation or by centrifugation.

The temperature of the ethanol / water mixture will be greater than 20 ° C., preferably approximately 60 ° C. The volume of ethanol relative to that of water is greater than 50%, preferably 85%.

In one embodiment, the extract obtained in step ii. will also undergo the following steps:

iii. concentrating said extract obtained in step ii. ;

iv. lyophilized.

In other embodiments, the extract obtained in step ii. will undergo one or more purification steps, so as to remove at least a portion of the resins present.

Thus, and in another embodiment, the process for obtaining a hydroalcoholic extract obtained by extracting tree nodes belonging to the Pinus genus will further comprise at least the steps such as: iii. concentrating, for example at 1/10 of the extract obtained in stage ii;

-decantation to obtain two phases, one comprising a supernatant, the other comprising the resins

recovering the supernatant and removing resins;

iv. Evaporate and lyophilize said supernatant obtained in step iii. to obtain a purified extract. In one embodiment, additional steps prior to step iv. may be carried out, such as concentration, for example 1/10 of the extract obtained in step ii. ; and or

adding an ethanol / water mixture; and or

decanting to obtain two phases, one comprising a supernatant, the other comprising the resins;

Preferably, the volume proportions of the ethanol / water mixture added are 60/40.

The addition of the ethanol / water mixture promotes the formation of two phases.

According to another embodiment, the additional steps prior to step iv. may be performed, such as: concentration, for example 1/10 said extract obtained in step ii. ; adding an ethanol / water mixture, for example, the volume proportion is 60/40;

recover the supernatant and remove the resins;

concentration, for example at 1/2 of the supernatant obtained in the preceding step;

adding an ethanol / water mixture, for example, the volume proportion is 50/50;

decanting to obtain two phases, one comprising a supernatant, the other comprising the resins; The addition of the ethanol / water mixture promotes the formation of two phases.

In another embodiment, the extract of tree nodes belonging to the genus Pinus may be obtained by extraction with hot water.

Typically, the extraction with hot water can be carried out with water at a temperature of 95 ° C for 2 hours, with stirring.

In yet another embodiment, the tree node extract belonging to the Pinus genus may be obtained by subcritical water extraction. These knots are crushed and the powder is extracted with water at a temperature of between 50 and 250 ° C. under a pressure of between 10 and 150 bars. A co-solvent may or may not be added, preferably ethanol, at a concentration up to 40%. In yet another embodiment, the extract of tree nodes belonging to the genus Pinus may be obtained by extraction with a water / organic solvent mixture in which the ethanol is replaced by acetone or methanol, following the same steps as before. EXAMPLES

Other advantages, aims and particular features of the present invention will emerge from the examples which will follow, facts, for an explanatory and non-limiting purpose.

In Examples 1 to 6 which follow, the extract used is an extract of Pinus pinaster knots for its use in the treatment of a pathogenic infection due to Plasmopara viticola In Example 7, an extract of Pinus pinaster knots. has been used for the treatment of pathogenic infections caused by Phytophthora infesting on potato (Solanum tuberosum) Phytophthora infesting on tomato {Solanum lycopersicum)

Pseudoperonospora cubensis on melon (Cucumis melo)

Bremia lactucae on lettuce (Lactuca sativa)

The characterization of the hydro-alcoholic extracts of pine knot was carried out by HPLC, UHPLC-DAD-MS and NMR.

- HPLC analysis

HPLC analyzes were performed on an Agilent 1260 Technologies HPLC chain equipped with a C18 column (250mm x 4.6mm, 5μιη), a DAD diode array detector and driven by Varian ™ software for pumps and Agilent ™ for the detector and samples part. The solvents used are water / TFA 0.025% as solvent A and acetonitrile / TFA 0.025% as solvent B. The elution gradient was set up so that solvent B was 10% from 0 to 4 min, 20% to 20min, 30% to 30min to 40min, 45% to 60min and 100% to 61 to 70min, with a flow rate of 1ml / min. - preparative HPLC

In order to collect the different molecules to allow their structural identification, preparative HPLC was performed. The separation of the compounds was made here using a Varian Pro Star HPLC chain equipped with an Agilent Zorbax SB-C18 PrepHT column (250mm x 21.2mm, 7pm). Solvent A (water / TFA0.025%) and solvent B (acetonitrile / TFA0.025%) are involved in a gradient which allows to have 0min to 5min 30% of solvent B, 30 to 44min 40% of solvent. B, from 45 to 50 min 100% of B and from 52 to 58 min 30% of B with a flow rate of 15 ml / min.

UHPLC-MS analysis UHPLC-MS analyzes were performed on an Agilent Technologies 1290 Series UHPLC instrument equipped with an Agilent Zorbax SB-C18 column (100mm x 2.1mm, 1.8μιτι) coupled to an "ion trap" mass spectrometer Bruker ™ Daltonics Esquire 6000 with Electrospray Interface (ESI). The UHPLC conditions use a solvent A (water / formic acid 0.010%) and a solvent B (acetonitrile / formic acid 0.010%) with a gradient starting at 10% solvent B from 0 to 1.7 min then 20% B to 3, 4min, 30% B from 5.1 to 6.8min, 35% B to 8.5min, 60% B to 11.9min, 100% B from 15.3 to 17min then 10% B to 17.3min. The UV spectrum was recorded over a wavelength range of 200 to 400 nm. Detection in mass spectrometry was carried out in negative mode.

NMR Analysis The NMR analyzes were carried out on a Bruker Ultrashield 600 MHz NMR spectrometer with a TXI probe. The deuterated solvents used are acetone d6 or methanol d4. The extracts are solubilized in 200 μl of solvent and then deposited in NMR tubes for analysis. - Resin content measurement by weighing

solvents

Organic solvents used in handling and analysis are of analytical quality and come from Fischer (Illkirch, France) and VWR (Fontenay-sous-Bois, France).

Extractions on raw materials are carried out using industrial grade ethanol.

Measurement of anti-mildew effects in Examples 1 to 6 1. Biological materials

Material (Plasmopara viticola)

The anti-mildew tests were conducted on mildew {Plasmopara viticola) of strain ANN. This strain was initially collected from Ugni-Blanc grape vines in a Charentes vineyard in 2015 (New Aquitaine). Provided by the UMR SAVE of the INRA of Villenave d'Ornon (France), the strain is cultivated in culture chamber and transplanted each week on a freshly picked vine leaf (Cabernet-S auvignon).

Plant material The anti-mildew tests are performed on leaf discs made from Cabernet Sauvignon vine leaves. These vine plants are produced in a greenhouse on the INRA site.

2. Pretreatment and artificial inoculation of Plasmopara viticola

As a first step, Cabernet Sauvignon vine leaves (4-5 leaves from the apex) are harvested from the greenhouse and washed. Leaf discs were cut with a punch and eight discs were placed per box, each containing a filter and 4 mL of sterilized water. For the extracts, an aqueous mother solution was prepared at 1% final ethanol (first in pure ethanol for better dissolution). Based on the established concentration range (0.05; 0.1; 0.2; 0.3; 0.5; 0.8; 1 g / L), daughter solutions were prepared from the initial solution and supplemented with a 1% solution of ethanol. The extracts were then sprayed onto the leaf discs using an Eco-spray and the boxes were placed in the dark to allow the opening of the stomata and thus the penetration of the extracts. In a second step, between 15 and 18 hours after the application of the extracts, the boxes were dried in order to inoculate the mildew. With the help of a wet brush, the spores were harvested and placed in a tube placed in ice containing 2 ml of water. Using a Malassez cell, the number of spores per milliliter was counted and then a dilution was made to have a solution at 15.10 spores / mL (4 mL of final volume). Three drops of 15μί each were deposited on each disk, ie 360μί per box. The boxes were again placed in the dark to allow the opening of the stomata and thus the entry of zoospores.

In a third time, after 15 hours of inoculation, the drops of inoculum were dried (the spores being returned to the stomata). The boxes were then placed in a culture chamber for seven days. After this incubation time, the dishes are visually analyzed with a notation based on the width and thickness of the sporangia. From the growth of downy mildew and its ratio to the control (without treatment), the percentage of inhibition is then calculated by difference.

EXAMPLE 1 Extraction of tree nodes belonging to the genus Pinus according to a first extraction method, characterization and anti-mildew effect of the extract thus obtained

1. Extraction A hydro-alcoholic extraction (10 liters, ethanol / water mixture at 85-15% (v / v) at 60 ° C) on 1 kg of ground Pinus pinaster knots in powder form was carried out.

The extract was filtered. Concentration followed by freeze-drying yielded the final extract of pine knots.

135 grams of extract are thus obtained for 1 kg of dry pine knots.

2. Characterization by HPLC, UHPLC-DAD-MS and NMR

The pine node extract was analyzed by HPLC, UHPLC-DAD-MS and NMR. The contents of compounds are shown in Table 1.

TABLE 1

mg POH / g mg POH / kg node extracted% (m / m)

pine extract ferulic acid 0.30 0.03 40.55 caffeic acid 0.26 0.03 35.01 isolariciresinol 3.04 0.30 410.55 secoisolariciresinol 5.08 0.51 686.07 nortrachelogenin 66.23 6 , 62 8941,27 pinoresinol 11,35 1,14 1532,53 matairésinol 7,91 0,79 1067,43 pinobanskin 13,13 1,31 1772,42 pinocembrin 36,35 3,64 4907,50 taxifoline 0.40 0 , 04 53.73 dihydrokaempferol 0.94 0.09 126.33 pinosyline 18.40 1.84 2483.36

SME 30.02 3.00 4052.56 pterostilbene 1.56 0.16 210.85

Total 194.96 19.50% 26320.18

POH = Polyphenols

SME = pinosylvine mono methyl ether

Thus, according to this first method of extracting tree knots of the genus Pinus, it is possible to obtain about 26.3 grams of polyphenols and 109 grams of resins per 1 kg of dry pine knots.

3. Anti-mildew effect The anti-mildew activity was tested as described above and is reported in Table 2.

TABLE 2

Concentrations required to inhibit 50% of late blight growth (IC ₅₀ ) and 100% inhibition (IC ₁₀ o) for pine knot extract

EXAMPLE 2 Extraction of tree nodes belonging to the genus Pinus according to a second extraction method and characterization of the extract thus obtained

1. Extraction A hydroalcoholic extraction (10 liters, ethanol / water mixture at 85-15% (v / v) at 60 ° C) on 1 kg of pinus pinaster knots crushed in powder form was carried out.

The extract was filtered.

2. Primary Purification A 1/10 concentration (between 800 mL and 1 L remaining) was performed to reveal a precipitate.

The extract was allowed to settle in order to obtain the formation of two phases, one corresponding to the supernatant and the other to the resin.

Resin was removed and the supernatant was recovered.

Evaporation of the solvents was done to reduce the volume of extract and lyophilization to obtain the final extract of pine knots.

75 grams of extract are thus obtained for 1 kg of dry pine knots.

3. Characterization by HPLC, UHPLC-DAD-MS and NMR The pine node extract was analyzed by HPLC, UHPLC-DAD-MS and NMR. The contents of compounds are shown in Table 3.

TABLE 3 mg POH / kg node mg POH / g extract extract% (m / m) pine ferulic acid 0.51 0.05 38.14 caffeic acid 0.44 0.04 32.83 isolariciresinol 5.33 0.53 399.96 secoisolariciresinol 6.33 0.63 474.68 nortrachelogenin 113.16 11.32 8486.72 pinoresinol 17.41 1.74 1306, 05 matairésinol 8.84 0.88 662.96 pinobanskin 16.11 1.61 1209.82 pinocembrin 42.29 4.23 3172.01 taxifolin 0.76 0.08 57.05 dihydrokaempferol 3.06 0.31 229, 51 pinosylvin 26.69 2.67 2002.03

SME 32.69 3.29 2451.72 pterostilbene 1.86 0.19 139.37

275.50 27.55% 20662.85

POH = Polyphenols

SME = pinosylvine mono methyl ether

Thus, and according to this first method of extracting tree nodes of the genus Pinus, it is possible to obtain about 21 grams of polyphenols and 54 grams of resins per 1 kg of dry pine knots.

4. Anti-mildew effect Anti-mildew activity is reported in Table 4.

TABLE 4

Example 3 Characterization of a hydro alcoholic extract of a pine knot according to a third extraction process 1. Extraction

A hydroalcoholic extraction (10 liters, ethanol / water mixture at 85-15% (v / v) at 60 ° C) on 1 kg of pinus pinaster knots crushed in powder form was carried out.

The extract was filtered.

2. Primary purification

A 1/10 concentration (between 800 mL and 1 L remaining) was carried out to reveal a precipitate.

Between 800 mL and IL of a 60-40% (v / v) ethanol / water mixture was added to promote the formation of two phases.

The resin was removed and the supernatant was recovered.

18 grams of extract are thus obtained for 1 kg of dry pine knots.

3. Characterization by HPLC, UHPLC-DAD-MS and NMR The pine node extract was analyzed by HPLC, UHPLC-DAD-MS and NMR. The contents of compounds are shown in Table 5. TABLE 5

H = Polyphenols SME = pinosylvine mono methyl ether

Thus, according to this first method of extracting tree knots of the genus Pinus, it is possible to obtain about 6.3 grams of polyphenols and 11.7 grams of resin per 1 kg of dry pine knots.

4. Anti-mildew effect Anti-mildew activity is reported in Table 6.

TABLE 6

Example 4 Characterization of a hydro alcoholic extract of a pine knot according to a fourth extraction process

1. Extraction

The extract was filtered. 2. Primary purification

The resin was removed and the supernatant was recovered.

3. Secondary purification

A second concentration at 1/2 (between 400 mL and 500 mL remaining) was performed in order to reappear a precipitate.

500 mL of a 50-50% (v / v) ethanol / water mixture was added to promote the formation of two phases.

The extract was allowed to settle in order to obtain the formation of two phases, one corresponding to the supernatant and the other to the resin. The resin was removed and the supernatant was recovered.

Evaporation of the solvents was done to reduce the volume of extract and lyophilization to obtain the final pine knot extract. 14 grams of extract are thus obtained for 1 kg of dry pine knots. 4. Characterization by HPLC, UHPLC-DAD-MS and NMR

The pine node extract was analyzed by HPLC, UHPLC-DAD-MS and NMR. The contents of compounds are shown in Table 7.

TABLE 7 mgPOH / g extract% mgPOH / kg extracted knot (m / m) pine ferulic acid 0.840 0.08 0.01 caffeic acid 1.161 0.12 15.96 isolariciresinol 11.419 1.14 156.89 secoisolariciresinol 11.371 1.14 156.23 nortrachelogenin 152.273 15.23 2092.24 pinoresinol 17.265 1.73 237.22 matairésinol 12.553 1.26 172.48 pinobanskin 13.368 1.34 183.67 pinocembrin 23.761 2.38 326.48 taxifolin 1.381 0.14 18, 98 dihydrokaempferol 5,497 0,55 75,53 pinosyl 15,665 1,57 215,24

SME 16.793 1.68 230.73 pterostilbene 0.912 0.09 12.53 Total 284,259 28.43% 3905.72

POH = Polyphenols

SME = pinosylvine mono methyl ether

Thus, according to this first method of extracting tree nodes of the genus Pinus, it is possible to obtain about 4 grams of polyphenols and 10 grams of resins per 1 kg of dry pine knots.

5. Anti-mildew effect Anti-mildew activity is reported in Table 8.

TABLE 8

EXAMPLE 5 Comparison of the anti-mildew effects of a hydro-alcoholic extract of pine knot with an alcoholic extract of vine shoot 1. Extraction

Tree nodes of the genus Pinus were extracted according to the method as described in Example 2. A hydroalcoholic extraction (10 liters, ethanol / water mixture at 85-15% (v / v) at 60 ° C) on 1 kg of pinus pinaster knots crushed in powder form was carried out. The extract was filtered.

A 1/10 concentration (between 800 mL and 1 L remaining) was carried out to reveal a precipitate. The extract was allowed to settle in order to obtain the formation of two phases, one corresponding to the supernatant and the other to the resin.

Resin was removed and the supernatant was recovered. Evaporation of the solvents was done to reduce the volume of extract and lyophilization to obtain the final extract of pine knots.

2. Characterization by HPLC, UHPLC-DAD-MS and NMR The pine node extract was analyzed by HPLC, UHPLC-DAD-MS and NMR. These analyzes show that pine nut extract is mainly rich in lignans (nortrachelogenin, pinoresinol, matairésinol, isolaricirésinol, and secoisolaricirésinol), in flavonoids (pinocembrin and pinobanskin) and in stilbenes (pinosylvine and pinosylvine mono methyl ether). The contents of compounds are shown in Table 9.

TABLE 9

0.04 caffeic acid

isolariciresinol 0.53

secoisolariciresinol 0.63

nortrachelogenin 11,32

pinoresinol 1.74

matairésinol 0.88

pinobanskin 1.61

pinocembrin 4,23

0.08 taxifolin

dihydrokaempferol 0.31

pinosylvin 2,67

SME 3,27

Pterostilbene 0.19

Total 27.55%

SME = pinosylvine mono methyl ether

3. Comparison of anti-mildew effects

The anti-mildew activity is reported in Table 10. A vine shoot extract is used as a reference. The extract of hydroalcoholic shoots is marketed by the company Actichem (France). These results show that the pine nut extract has a much higher activity than that of the vine extract.

TABLE 10

100% inhibition (in

IC ₅₀ extract (in mg / 1)

mg / L) extract from shoots of

210,800

vine

extract of pine knots 62 500 Pine nut extracts inhibit late blight growth with an IC ₅₀ of 62 mg / L.

With an ICso more than three times lower than that of the vines, the extract of pine knots thus has an interesting anti-mildew capacity.

As the level of purification increases, the resin content of the extract decreases, as well as the content of some of the most active molecules such as pinosylvin and pinocembrin. Inhibitory concentrations 50 and 100 (IC ₅₀ and IC ₁₀ 0) are higher. This increase in the necessary concentrations, however, does not affect the anti-mildew activity of the extracts and the skilled person will advantageously determine the adequate resin content for each extract and the level of purification. Example 6: Anti-mildew effects of the purified molecules of the extracts

Some polyphenols present in the pine knots have been purified and tested for their anti-mildew activity. The standards of pinosylvin, pinosylvin monomethyl ether, nortrachelogenin, pinoresinol, isolariciresinol, matairésinol, pinocembrin and pinobanskin were isolated in the laboratory.

The results are shown in Table 11.

TABLE 11

Development of late blight Mobility of zoospores

Compounds IC ₅₀ (in μΜ) CI ₅ o (in mg / 1) IC ₅₀ (in μΜ) CI ₅ o (in mg / 1)

Pinosylvine 23 5 34 7

pinosylvin

18 3 23 5 monomethyl ether Pinocembrine 19 6 22 6

Pinobanskine 137 37 141 39

Matairésinol 101 36 81 29

Nortrachelogenin 259 97 264 99

Pinoresinol 269 96 296 106

Isolariciresinol 551 198 223 80

Pinosylvine, Pinosylvine monomethyl ether and Pinocembrin have the best anti-mildew activity.

Example 7 Effects of a tree node extract on a pathogenic infection caused by Phytophthora infestons, Pseudoperonospora cubensis or Bremia lactucae

The tree node extract belonging to the genus Pinus obtained according to the extraction and characterization method of Example 1, corresponding to 135 g of extract per kg of dry pine knots, was tested on other oomycetes. on young plants under glass. Oomycetes and plants are:

Phytophthora infestans on the potato (Solanum tuberosum) Phytophthora infestans on the tomato (Solanum lycopersicum)

Pseudoperonospora cubensis on melon (Cucumis melo)

- Bremia lactucae on lettuce (Lactuca sativa)

The extract was prepared at two concentrations 0.5 g / L and 1 g / L, in aqueous solution with 1% final ethanol. Two applications of the solutions were carried out using a spray jet spray, up to the limit of runoff, upper and lower face of the leaves, on the plants at one week intervals.

Inoculation of the oomycete is carried out 1 to 2 days after the second application of the solutions. For each modality, 12 plants are used. A control is carried out under the same conditions with a solution without extract.

The infection surface on the leaves is evaluated visually, 1 week after inoculation, then a second time corresponding to 2 weeks after inoculation.

From the growth of downy mildew and its ratio to the control (without treatment), the percentage of inhibition is then calculated by difference.

The results are shown in Table 12.

TABLE 12

Percent Growth Inhibition (+ SD) of the different Mildews at 1 week after inoculation (7 AI) and 2 weeks after inoculation (14 AI) by application of Pine Nut Extract at 0 , 5g / L (E 0.5) and lg / L (E 1)

IFAP: Phytophthora infestations (inoculated at 48,000 spores / mL) on potato (Solanum tuberosum). Solanum tuberosumau had 6 leaves (stage 6 ^th sheet) at the nodes of the extract by treating trees and 8 sheets (stage 8 ^th sheet) when the second evaluation of the disease (14ai). PIT: Phytophthora infestans (inoculated at 40 000 spores / mL) on tomato (Solanum lycopersicum). Solanum lycopersicum had 3 leaves (stage ^3e leaf) during treatment with the extract nodes of trees and 7 sheets (stage 7 ^th sheet) when the second evaluation of the disease (14ai).

PCM: Pseudoperonospora cubensis (inoculated at 40,000 spores / mL) on melon (Cucumis melo). Cucumis melo had three leaves (stage 3 ^rd leaf) during treatment with the extract of tree nodes and leaf 9 ^(9th stage "sheet ¹⁶⁾ when the second evaluation of the disease (14ai).

BLL: Bremia lactucae on lettuce (Lactuca sativa)

*% leaf area infested with the control without treatment.

The pine nut extract shows a very good efficacy on the tomato and potato late blight, and a more moderate efficacy on the melon blight, but in the presence of a very strong infestation as demonstrated by the percentage of leaf area infested with control melon without treatment.

Regarding lettuce, infestation with downy mildew was insufficient to determine percent inhibition. However, mildew attack points were observed on the control leaves, and not on the plants treated with the 0.5 and 1g / L extract, at the time 14 days after inoculation. This result indicates that the extract is also effective against Bremia lactucae. Advantageously, the extract of tree nodes belonging to the genus Pinus according to the present invention has a good anti-mildew activity and allows the treatment of pathogenic infections due to oomycetes.

Claims

1. Non-therapeutic use of tree node extract belonging to the genus Pinus comprising stilbenes, flavonoids and lignans for the treatment of a pathogenic infection due to at least one oomycete.

2. Non-therapeutic use of an extract according to claim 1, wherein oomycete is selected from Plasmopara viticola, Phytophthora infestans, Pseudoperonospora cubensis or Bremia lactucae.

3. Non-therapeutic use of an extract according to any one of claims 1 to 2, wherein the extract further comprises at least one phenolic acid.

4. Non-therapeutic use of an extract according to any of claims 1 to 3, wherein the stilbenes comprise one or more compounds selected from pinosylvin, its monomethyl ester and pterostilbene or a mixture thereof.

5. Non-therapeutic use of an extract according to any one of claims 1 to 4, wherein the flavonoids comprise one or more compounds selected from pinocembrin, pinobanskin, taxifolin and dihydrokaempferol or a mixture thereof.

The non-therapeutic use of an extract according to any one of claims 1 to 5, wherein the phenolic acids comprise one or more compounds selected from ferulic acid and caffeic acid or a mixture thereof.

7. Non-therapeutic use of an extract according to any one of claims 1 to 6, wherein the lignans comprise one or more compounds selected from nortrachelogenin, pinoresinol, matairésinol, secoisolaricirésinol and isolaricirésinol or a mixture of them.

8. Non-therapeutic use of an extract according to any one of claims 1 to 7, wherein the stilbene content is between 1 and 20%, preferably 4 and 10% by weight, the lignan content is 5 and 40%, preferably 8 and 16% by weight, the content of flavonoids is between 1 and 20%, preferably 4 and 10% by weight, and the phenolic acid content is between 0.02 and 1%, preferably between 0.04 and 0.3%. in weight

9. Non-therapeutic use of an extract according to any one of claims 1 to 8, characterized in that said extract is a hydro alcoholic extract.

10. Non-therapeutic use according to any one of claims 1 to 9, wherein the extract comprises a non-zero amount of resins.

11. Tree node extract belonging to the genus Pinus, said extract comprising at least the following compounds:

pinosylvin;

pinosyl mono methyl ether;

pinocembrine;

- pinobanskin;

matairésinol;

nortrachelogenin;

pinoresinol;

the isolariciresinol.

12. Extract according to claim 11 characterized in that said extract is a hydro-alcoholic extract.

13. Non-therapeutic use of an extract according to claim 11 or 12 for the treatment of a pathogenic infection due to at least one oomycete.

14. Composition comprising an extract according to claim 11 or 12.

15. A composition comprising an extract according to claim 11 or 12 and a fungicide and / or algicide.