EP4068965A1

EP4068965A1 - Use of date saccharides alone or in combination with polyphenols to protect plants against pathogens

Info

Publication number: EP4068965A1
Application number: EP20819668.3A
Authority: EP
Inventors: Fethi HAKKAR
Original assignee: Mytamar GmbH
Current assignee: Mytamar GmbH
Priority date: 2019-12-02
Filing date: 2020-12-01
Publication date: 2022-10-12
Also published as: CA3160242A1; WO2021110648A1; CN115151134A; US20230015964A1; MX2022006625A

Abstract

The present invention relates to the use of an extract of water-soluble saccharides from fruits of the date palm Phoenix dactylifera for protecting plants against pathogens. In particular, according to the invention, such an extract is used for stimulating the defense and resistance reactions of the plant for treatment or prevention, in particular in order to induce an activity that elicits the defense mechanisms.

Description

USE OF DATE SACCHARIDES ALONE OR IN MIXTURES WITH POLYPHENOLS TO PROTECT PLANTS AGAINST PATHOGENS

TECHNICAL AREA

The present invention belongs to the technical field of agronomic or agricultural protection products and relates to the use of an extract of saccharides, or of a mixture of extracts of saccharides and polyphenols, obtained from fruits of the date palm Phoenix dactylifera for protect plants against pathogens, in particular to stimulate the natural defenses of plants and develop their resistance against pathogens.

Plants are regularly attacked by pathogens with the consequences of major damage to crops, or even total loss of crops. Grapevine downy mildew, for example, is a disease caused by an oomycete, Plasmopara viticola, which causes many losses every year and which is likely to appear all over the world. Prevention is necessary and requires the establishment of regular treatments throughout the year. Other pathogens, such as fungi or insects, are also feared in agronomic and horticultural environments.

The fight against these pathogens often involves the use of products from the chemical industry to set up the defense and resistance reactions of the plant necessary to maintain the crop and the yield. However, if such treatments are effective, the impact of these products on the environment and the health of consumers is worrying. Furthermore, such treatments are not compatible with organic farming.

Thus, for several years now, the use of substances of natural origin as protective agents for plants against pathogens has been the subject of research. These substances can be of plant, animal or mineral origin. They generally have a short lifespan, which limits their presence in food products and in the environment. They can act in different ways, for example by stimulating the natural defenses of the plant or by acting directly on a population of pathogens to inhibit its growth and development.

As compounds acting by triggering the plant's defense mechanisms, mention will be made, for example, of laminarin extracted from brown algae or else grape pomace extract.

Plants do not have an immune system equivalent to that of humans and animals, however they are able to set up defense mechanisms which can be induced by compounds acting as signals on the defense genes to trigger the synthesis of anti-pathogenic molecules or molecules capable of structurally strengthening the plant. Such compounds are said to be elicitors.

Thus, if a plant is brought into contact with an elicitor compound before it comes into contact with a pathogen, the plant will be somewhat immune to that pathogen.

The elicitors are therefore extremely interesting in the fight against plant pathogens. DISCLOSURE OF THE INVENTION

Thus, the need to offer compounds of natural origin, respectful of the environment, crops and health, for the protection of plants, is today at the heart of concerns. In particular, there is a need to identify and provide such compounds which exhibit activity for stimulating the defense system of plants against pathogens and for inhibiting the growth and development of pathogens.

It is in this context that the applicant company worked and demonstrated that compounds derived from fruits of the date palm Phoenix dactylifera could protect plants against pathogens, in particular could induce and stimulate the defense reactions of plants against pathogens.

Thus, the present invention relates to the use of an extract of water-soluble saccharides obtained from fruits of the date palm Phoenix dactylifera for the protection of plants against pathogens.

In particular according to the invention, such an extract is used for stimulating the defense and resistance reactions of the plant for treatment or prevention, in particular for inducing an eliciting activity of the defense mechanisms.

Dates can be considered as an interesting source of compounds with various biological activities. In addition, a significant part of the production of dates is transformed into derived products, such as jams, juices and syrups. Therefore, a large amount of date kernels and unmarketed fruit is produced as waste which can be recovered on the basis of its high content of bioactive compounds such as polysaccharides.

In fact, a saccharide extract in the context of the invention can comprise a quantity of saccharides greater than or equal to 40%, 50% or even 60%, by weight of the extract.

According to one characteristic of the invention, the saccharides contain rhamnose, arabinose, fucose, xylose, mannose, galactose, galacturonic acid, glucose and / or glucuronic acid.

Saccharides, within the meaning of the invention, are monosaccharides, oligosaccharides or polysaccharides. Preferably, they will be polysaccharides.

Polysaccharides are organic compounds that are abundant in nature. They are homo or heteropolymers formed by the linear or branched chain of oses.

The polysaccharides found in dates are cellulose, pectin and hemicelluloses. These polysaccharides are part of the category of dietary fiber and are divided into two groups: soluble fibers, or water soluble, including pectins and some hemicelluloses, and insoluble fibers, insoluble in water, including cellulose and certain other hemicelluloses .

The saccharides, according to a preferred embodiment of the invention, are naturally water-soluble polysaccharides or a mixture of such polysaccharides with hydrolysates of naturally non-water-soluble polysaccharides.

In fact, the extraction carried out on the date fruits makes it possible to obtain a fraction enriched in soluble and insoluble polysaccharides. The initially insoluble polysaccharides can be upgraded within the framework of the invention once made water-soluble, in particular after the implementation of a method of enzymatic or chemical hydrolysis, for example by acid hydrolysis.

According to one embodiment of the invention, the saccharide extract contains, by weight: at least 20% mannose, at least 7% arabinose, at least 6% glucose, at least 5% galactose.

This saccharide extract is generally obtained from the kernel of date fruits.

According to one embodiment of the invention, the saccharide extract contains, by weight: at least 40% galacturonic acid, at least 7% arabinose, at least 3% xylose.

This saccharide extract is obtained from the skin and pulp tissues of date fruits.

As the following examples will demonstrate, the polysaccharides extracted from dates have a very high inducing effect on the natural defenses of plants. In particular, they make it possible to induce the genes most representative of the PR (Pathogenesis Related) proteins involved in plant defense reactions. They are therefore good agents for stimulating the natural defense reactions of plants.

PR proteins accumulate in the tissues of a plant in response to biotic or abiotic stress, or following the application of a product that induces the defenses. Each PR protein has its own function. They accumulate at a local and systemic level and their presence is an indicator of the establishment of defense mechanisms by the plant.

PR1 proteins would be involved in the sequestration of sterols. They are thought to have an anti-fungal and anti-oomycete action, by inhibiting the growth and germination of spores, in particular against downy mildew and gray rot in tomatoes (Niderman et al., 1995; Garnish et al., 2016). They would also have a favorable action in drought resistance (Liu et al., 2013).

PR2 proteins have a glucanase (b-1,3-glucanase) action, responsible for the degradation of glucan, a major polysaccharide constituent of the wall of fungi (Benhamou, 2009). PR2 is induced in response to the salicylic acid pathway. PR2 is a protein that may also be involved in tolerances to abiotic stresses such as drought (Liu et al., 2013).

PR3 proteins have chitinase activity. They have a direct antimicrobial effect through the degradation of the fungal wall and an indirect effect through the possible release of fragments inducing defense genes (Benhamou, 2009).

The PR4 proteins showed in particular ribonuclease and chitinase activity. Studies in wheat have suggested anti-fungal activity against Fusarium culmorun (Bertini et al., 2009; Caruso et al., 2001; Filipenko et al., 2013). Another study reported possible antifungal activity in corn (Bravo et al., 2003). PR4 is a protein that appears to be activated by both SA and JA pathways (Bertini et al., 2003; Wang et al., 2011).

PR5 proteins are “Thaumatin like” proteins with activity against oomycetes but also against Verticillium, Fusarium, and necrotrophic fungi. This protein also helps induce drought tolerance (Liu et al., 2013). PR8 proteins exhibit antibacterial action through lysosomal and antifungal activity, catalyzing hydrolysis of chitin from fungal walls and inhibiting hyphal growth. By virtue of their degradation action, they are thought to be at the origin of the production of endogenous elicitors, thus triggering the establishment of defense reactions within the attacked plant (Métraux et al., 1988).

The PR14 proteins are lipid transfer proteins expressed in young leaves and appear to have a role in the transport of cutin monomers; they are associated with the assembly of cutin and wax (Sels et al., 2008). They have antimicrobial properties, for example against Pseudomonas, Fusarium, Pythium and Botrytis (Kido et al., 2010).

According to one embodiment of the invention, the extract also contains at least 5%, by weight, of proteins, preferably at least 10%, more preferably between 5 and 15%.

According to an advantageous embodiment of the invention, the extract of water-soluble saccharides is a mixture with an extract of polyphenols also obtained from fruits of the date palm Phoenix dactylifera.

The mixture will be in the form of a composition.

Polyphenols are antioxidant compounds specific to the plant kingdom, and endowed with various biological activities demonstrated by numerous studies described in the scientific literature. Mention will in particular be made of their antioxidant, anti-inflammatory, antimicrobial, antiviral or even anticancer activity.

Advantageously according to the invention, the saccharides / polyphenols ratio is between 30/70 and 70/30, expressed by weight.

Polyphenols are present in all organs of the plant. They present a very wide variety of structures. Thus, they are divided into different families:

Phenolic acids, such as derivatives of hydroxy-benzoic acid and derivatives of cinnamic acid,

Flavonoids such as flavan-3-ols, including catechins and tannins, and flavones, including flavonols.

The date palm fruit consists of three tissues: the stone, the pulp and the skin. It also evolves according to four stages of maturity: Hababouk, Blah (Kimri), Besser (Khalal) and Tamar. The selection of the fruit according to one of these stages, and according to the tissues, makes it possible to obtain different results in terms of polyphenol content.

Advantageously, the polyphenol extract contains up to 80% polyphenols, by weight relative to the total weight of the purified dry extract.

Preferably, the polyphenol extract contains at least 95%, more preferably at least 97%, even more preferably at least 99% of condensed tannins, by weight relative to the total weight of polyphenols.

The tannins condensed in the extract according to the invention are oligomers or polymers of pro-anthocyanidins.

The extract can be more or less concentrated in polyphenols depending on the tissue and the stage of maturity but also depending on the extraction method. These choices can be guided by the examples which follow. According to one embodiment of the invention, an effective amount of the extract of saccharides, or of the mixture of extracts of saccharides and of polyphenols, supplied to the plants is at least 0.01 g per liter, preferably of at least 0.1 g per liter, more preferably at least 0.7 g per liter, for a contribution in liquid form, or at least 10 g per hectare, preferably at least 100 g per hectare for a intake in solid form.

The effective amount should be sufficient to induce defense and resistance mechanisms and will vary from plant to plant or depending on the method of treatment. This amount can be easily determined by a person skilled in the art.

According to one embodiment of the invention, the saccharide extract, or the mixture of extracts of saccharides and polyphenols, is applied to the whole plant, to the leaves, to the flowers, to the roots, to the fruits, to the seeds, to seedlings, soil, solid or liquid culture medium, culture material.

According to one embodiment of the invention, the application is carried out by sprinkling, irrigation, spraying, soaking or injection.

According to one characteristic of the invention, the plants are agronomic, ornamental, aromatic or medicinal plants, in particular, the plants are fruit plants, in particular vines, vegetable plants, flowers, trees, shrubs.

According to another characteristic of the invention, the plant pathogens are fungi, bacteria, viruses, nematodes, parasitic plants, protozoa or insects, in particular Plasmopora viticola.

In fact, as the examples which follow will demonstrate, the mixture of extracts of saccharides and of polyphenols has been particularly effective against the pathogen responsible for downy mildew of the vine.

The invention also relates to a method of protecting a plant against pathogens, in particular for activating defense and resistance reactions of the plant, the method comprising application to said plant, or in the environment of the plant. , an effective amount of an extract of water-soluble saccharides obtained from fruits of the date palm Phoenix dactylifera alone or as a mixture with an extract of polyphenols obtained from this same date palm, the extracts being as described above and applied in the same way as mentioned above previously.

The invention also relates to a plant protection product containing an effective amount of a saccharide extract obtained from fruits of the Phoenix dactylifera date palm alone or as a mixture with a polyphenol extract from this same date palm, the extracts being as described above.

According to one embodiment, the plant protection product further comprises at least one other component such as a solvent, a surfactant, an emulsifier, a dispersing agent, a filler, a fertilizing compound or a plant protection compound.

Advantageously, the plant protection product is in liquid form, in gel form, in powder or granule form.

DETAILED EXPOSURE OF EMBODIMENTS The characteristics of the invention mentioned above, as well as others, will emerge more clearly on reading the following description of exemplary embodiments.

Plant material:

All varieties of dates can be used. Fruits can be selected according to one of the following four stages of maturity: Hababouk (stage 1), Blah (Kimri) (stage 2), Besser (Khalal) (stage 3), Tamar (stage 4). The tissues, that is, the skin, the pulp and the nucleus, are separated from each other. After a grinding operation, the fabrics are dried and powdered. It should be noted that in stage 1 of ripening, the fruit is studied entirely without separation of the tissues because the stone is not yet formed.

Example 1

Obtaining water-soluble polysaccharide extracts

Saccharide extracts can be obtained from tissue powders according to a process consisting in first extracting the alcohol-insoluble matter (MIA) and then, in a second step, in purifying the water-soluble saccharides, in particular the polysaccharides , from this material. Of course, other extraction methods can be implemented. a) Obtaining alcohol-insoluble matter:

MIA is obtained using the process below:

• Extraction step: the date powder is dispersed in a solution of ethanol (96% + 1% HCl) (V / P) brought to the boil beforehand. After stirring, the mixtures are immediately cooled and placed at 4 ° C overnight,

• Filtration step,

• Ethanol rinsing step (65% + 1% HCl),

• Filtration step,

• Rinsing step with acetone,

• Drying in an oven at 45 ° C,

• Obtaining a powder containing polysaccharides.

It should be noted that depending on the tissues and the stage of ripeness of the fruit, the percentages of alcohol insoluble matter vary but can reach almost 90%, by weight of fresh matter. b) Process for the purification of water-soluble polysaccharides from MIA:

• Agitation of the powders obtained in (a) in water acidified with 1% acetic acid,

• Centrifugation,

• Lyophilization of the supernatant to obtain a fraction containing soluble fibers in the form of a powder, • The residue, ie the fraction containing the insoluble fibers, is passed in an oven at 45 ° C for drying. c) Determination of the mass composition of the monosaccharides of MIAs and of soluble and insoluble polysaccharide fractions obtained from MIAs:

The determination of the mass composition of neutral and acidic monosaccharides is carried out by gas chromatography coupled with mass spectrometry (in English Gas chromatography-mass spectrometry or GC-MS). This analysis is carried out on the MIAs obtained from the nuclei and on the soluble fractions obtained from the MIAs of skin, pulp and nucleus tissues as well as on the insoluble fractions obtained from the MIAs of nuclei.

The analyzes by GC-MS of the simple sugars constituting the polysaccharide structures of AIMs show us a great variability of the quantities of polysaccharides according to the fractions resulting from pulp, skin or stones.

The results are given in Table 1 below in which the units are expressed in pg / mg.

Table 1

Rha: rhamnose; Macaw: arabinose; Fuc: fucose; Xyl: xylose; Man: mannose; Gai: galactose; GalA: galacturonic acid, GIc: glucose, GIcA: glucuronic acid.

MIA: fraction of Alcohol-Insoluble Matter from the nucleus; FSN: fraction of soluble polysaccharides obtained from MIA of nuclei; FSC soluble polysaccharide fraction obtained from pulp and skin MIA; FISN: fraction of insoluble polysaccharides obtained from MIA of nuclei.

Thus, the fraction of soluble polysaccharides extracted from nuclei (FSN) is characterized by a polysaccharide content of nearly 50%, by weight. The fraction of soluble polysaccharides extracted from pulps and skins (FSC) is for its part characterized by a polysaccharide content of more than 66%, by weight.

These polysaccharides are composed of monosaccharides of rhamnose, arabinose, fucose, xylose, mannose, galactose, galacturonic acid, glucose and glucuronic acid.

In the fraction originating from the FSN nucleus, mannose is the monosaccharide which is found in greater quantity. There is also a significant amount of glucose. This testifies to the presence of polysaccharides from the glucomannan family. In the fraction derived from FSC tissues and skin, it is galacturonic acid which is in significant quantity. The presence in this fraction of galactose and xylose testifies to the presence of pectin-type polysaccharides.

The fraction of insoluble polysaccharides obtained from MIA of FISN cores is also rich in monosaccharides, in particular in mannose. Mannose forms about 90% of the weight by weight of the insoluble fiber fraction. This testifies to the presence of polysaccharides of the galactomannan family. Thus, it may be advantageous, from this fraction of insoluble fibers, to prepare a soluble fraction of monosaccharides (see Example 4) which can be used in the context of the applications targeted by the invention. d) Determination of the mass protein composition of the soluble and insoluble polysaccharide fractions obtained from the AIMs of nuclei and tissues (pulp and skin):

The method for determining the protein concentration of the MIA, FSC, FISN and FSN fractions is based on the determination of nitrogen and carbon by combustion of samples of each of these fractions, in the form of lyophilized powder, according to the method well known to Dumas. The results are reported in Table 2.

Table 2

The value in brackets corresponds to the confidence interval a 0.05% with n = 3; ¹ conversion using the factor 6.25 according to standard IS016634

Depending on the samples, the contents vary from 40 to 50% for carbon and from 1.0 to 2.2% for nitrogen. These data make it possible to estimate protein contents varying from 6 to 14% depending on the samples. The FISN fraction with an estimated protein content of almost 14% is significantly richer in protein than the other fractions. There is no significant difference between the estimated protein contents for the FSN and FSC fractions.

Example 2

Obtaining polyphenol extracts:

Polyphenols can be obtained from tissue powders according to a process for extracting and purifying polyphenols essentially comprising the following steps: solid-liquid extraction using a solvent, preferably polar, and recovery of an extract containing polyphenolic compounds, purification of the polyphenolic compounds of the extract by a solid phase chromatography method, for example in reverse phase or by ion exchange, recovery of a purified extract concentrated in polyphenolic compounds.

The implementation is as follows:

10 g of fruit tissue powder are brought into contact with 80 ml of hexane for 15 min., With stirring, then filtered, during a first extraction. The residue obtained is subjected to a second extraction by bringing it into contact with 80 ml of hexane for 15 min., With stirring and then filtered. The residue obtained is recovered and then dried under nitrogen for 1 h. This step is optional. The dry residue from the extraction is brought into contact with 30 ml of polar solvent for 15 min., With stirring, then filtered, during a first extraction. The polar solvent is composed of ethanol / water / acetic acid in a ratio of 50 to 80 ethanol: 1 to 10 acetic acid: qs in water (V / V / V) (hydroalcoholic solvent). For example, a ratio of 80: 19: 1 (Vol. Ethanol / Vol. Water / Vol. Acetic acid /) is suitable. In a variant, a solvent composed of acetone / water / acetic acid in a ratio of 50 to 80 acetone: 1 to 10 acetic acid: qs water (V / V / V) (hydroacetonic solvent). For example, a ratio of 50: 49: 1 can be used.

The extract obtained, named El, is stored, and the residue obtained is subjected to a second extraction by bringing into contact with 30 ml of the same solvent for 15 min., With stirring and then filtered. The extract obtained, named E2, is stored and the residue obtained is recovered and subjected to a third extraction by bringing into contact with 30 ml of the same solvent for 15 min., With stirring and then filtered. The extract obtained, named E3, is kept. The extracts E1, E2 and E3 are mixed. Filtration is carried out in order to recover the supernatant and then the phenolic compounds in the extract are concentrated by evaporation under vacuum.

The extracts are centrifuged at 4500 rev / min for 10 min at 4 ° C in order to recover the supernatant which will be used for the Sep Pack C18 cartridge (sold by the company Waters) for the implementation of a solid phase extraction ( SPE).

SPE cartridges containing 5 g of C18 gel (Sep Pack C18 cartridge sold by the company Waters) are conditioned in ethanol (20 mL) then equilibrated with acidified water (40 mL) before the aqueous extracts of each sample (40 ml), previously centrifuged at 4500 rpm for 10 min at 4 ° C. Rinsing is carried out with 40 mL of acidified water (at 2% acetic acid), then the retained fraction is eluted with 40 mL of ethanol / water / acetic acid mixture (50 to 80 ethanol: 1 to 50 acetic acid: qsp water), preferably 50: 49: 1 (V / V / V). The column is then washed with 30 ml of ethanol to remove the very hydrophobic polyphenols which can still be fixed.

In a variant, the purification can be carried out using an FPX 66 ion exchange column (Rohm & Haas France SAS).

Concentration of the extracts is carried out by evaporation of the solvent and a purified fraction of polyphenols is obtained. The fraction is then dried to obtain a polyphenol powder.

The extracts obtained contain up to 80% polyphenols, by weight relative to the total weight of the purified dry extract. In addition, the extracts contain at least 95%, more preferably at least 97%, even more preferably at least 99% of condensed tannins, by weight relative to the total weight of polyphenols.

Example 3

Preparation of a composition containing an extract of water-soluble polysaccharides and an extract of polyphenols

70 mg of soluble fibers previously extracted are dissolved in 200 ml of water acidified with 0.1% formic acid. 70 mg of previously extracted polyphenols are dissolved in 50 mL of water acidified with 0.1% formic acid.

The two solutions are mixed, stirred and then centrifuged at 4500 rpm for 10 min. The supernatant is recovered and then dried.

The mixtures to be studied are prepared by soluble fiber-tannin fractions (50/50 w: w).

Example 4

Obtaining and analysis of a hydrolyzate of polysaccharides initially insoluble in water and obtained from fruits of the date palm Phoenix dactylifera a) Obtaining:

The objective is to prepare fractions of soluble saccharides rich in oligomeric mannoses from the fraction containing insoluble polysaccharides FISN obtained from MIA.

Using a beta-mannanase bacterial or fungal, in particular beta-mannanase derived from Aspergillus Niger including beta-mannanase purified û'Aspergillus Niger marketed under the tradename Gamanase ^® (NOVO-NORDISK, Denmark).

The unit of beta-mannanase is defined as being the quantity of beta-mannanase which releases, from locust bean gum, a quantity of reducing sugars equivalent to 1 micromole of mannose per minute, at pH5 and at 30C.

Beta-mannanase can be immobilized by covalent bonding to the surface of a traditional support, or else immobilized by polymerization after having been adsorbed to the surface of a traditional support. The liquid extract can then be hydrolyzed at a temperature of 40-70 ° C with the immobilized enzymes.

Identical incubation conditions were used with each b-mannanase (McCleary, B. V., 1979). The insoluble fraction FISN (20 ml, 0.5% w / v, no buffer) was incubated with b-mannanase (0.8 pkat on carob galactomannan; 0.4 pkat on soluble mannan). Aliquots (4 ml) were taken at 20 minute, 1, 6 and 18 hour intervals, heated to denature b-mannanase activity, lyophilized and readjusted to 2% by weight carbohydrate. Aliquots (20 µl) were applied to plates prepared twice with n-PrOH-EtOH-H2O (7: 1: 2). The spots were visualized by spraying 5% H2SO4 in EtOH and heating at 110 ° C for approximately 10 min.

This technique made it possible to dissolve 99.5% of the fraction containing insoluble fibers. b) Analysis: determination of the mass composition of monosaccharides by GC-MS in the insoluble fractions hydrolyzed by the enzymatic route

The analyzes by GC-MS of the polysaccharide structures of the hydrolyzed and solubilized insoluble fractions show the richness of this fraction in mannose. This mannose obviously comes from polysaccharides of the galactomannan and glucomannan family since this soluble part is rich in glucose and galactose. The sugars identified are mannose (Man); galactose (Gai); a trisaccharide with a galactose / mannose ratio of 1: 2 (Gal-Man2); a tetrasaccharide with a galactose / mannose ratio of 1: 3 (Gal-Man3); and a pentasaccharide with a galactose / mannose ratio of 1: 4 (Gal-Man4). The results are reported in Table 3 and the units in pg / mg.

Table 3

Example 5

Demonstration of the effectiveness of water-soluble polysaccharides derived from fruits of the date palm Phoenix dactylifera, in stimulating the natural defenses of wheat.

Aim: the objective is to evaluate, under semi-controlled conditions, the inducing effect of the extract containing water-soluble polysaccharides (obtained according to Example 1 and named hereafter “Polysaccharides”) on the expression of 7 genes of PR proteins, markers of defense mechanisms, using the qPFD ^® tool (described in the patent application WO2011 / 161388) on wheat plants.

Biological material

The wheat plants were produced from seeds of the soft wheat variety ALIXAN. For each modality, 30 grains of wheat were sown in 3 pots (2 repetitions / modality) then placed under controlled conditions for 1 week (25 ° C constant, 16h photoperiod), until the emergent F2 leaf stage. The young plants were then transferred to a climatic chamber dedicated to experimentation.

Tested and control products

The following modalities were compared (Table 4).

Table 4

In order to facilitate the adhesion of the candidate product on the wheat plants, Tween 20 was added to each candidate product, at a concentration of 0.05%, ie 25 μl per 50 ml of product.

Tusk markers used

The 7 genes of the PR (Pathogenesis Related) proteins below have been selected as being representative of the major PR proteins involved in plant defense.

PR-1: Pathogenesis-related protein 1

PR-2: Pathogenesis-related protein 2 (glucanases)

PR-4: Pathogenesis-related protein 4 (hevein-like) PR-5: Pathogenesis-related protein 5 (thaumatin-like, osmotin) PR-8: Pathogenesis-related protein 8 (class III chitinase)

PR-14: Pathogenesis-related protein 14 (lipid transfer protein) PR-15: Pathogenesis-related protein 15 (oxalate oxidase)

Protocol

Two repetitions of the entire experiment were performed, as well as two quantitative PCR analyzes.

The tests are carried out in a climatic chamber, under controlled conditions (21 ° C day / 19 ° C night, 16h photoperiod) on wheat plants (2/3 leaf stage), randomized in blocks of 7 pots.

The products of each modality (2 pots) are applied twice, with the exception of the WATER control which is applied once only (OJ). The candidate product and the SDP control are applied 4 days apart, on D-4 and on D0. Each modality is then treated on D1 with hydrogen peroxide (H202) to simulate a pest attack. All treatments are carried out up to the limit of runoff, with a sprayer connected to compressed air.

For each modality, ten leaf fragments (approx. 1 cm) are taken by combining 5 F2 leaves from 5 different plants, on the following dates:

- JO: initial sample on 5 plants that have not received any treatment

- D2: sample 2 days after treatment

- D3: sample 3 days after treatment

These samples are placed in liquid nitrogen and then stored at -80 ° C. until the extraction of the RNAs.

RNA extraction, reverse transcription and quantitative PCR

The RNAs were extracted from the leaf tissues collected, using the NucleoSpin RNA Plant kit (Macherey-Nagel). The yield and the quality of the extracted RNAs were evaluated using a spectrophotometer (Nanodrop ND-1000). The RNAs were then reverse-transcribed into cDNA and the expression levels of 7 PR protein genes were monitored (3 technical repeats) by quantitative PCR (SYBR Green intercalating agent) using the qPFD ^® tool ( “Low Quantitative Density Chip” developed by INRA). The relative expression levels were calculated using the 2 ^DDa method: these are expressions relative to the OJ sample (before treatment) at each sample time, normalized by the geometric mean of the relative expressions of 3 genes reference (TuB, Actin, GAPDH). These relative expressions are transformed into log2 to give the same weight to the inductions and repressions of the genes.

Results

Relative expression density map of PR protein genes Table 5 below shows the density map of the relative expressions of the 7 genes of the PR proteins. The expression levels of the 7 genes, transformed into log2, are represented by a density map. This makes it possible to have a global view of the induction profile and to directly visualize the differences in the levels of expression of the marker genes between the treated samples, after having subtracted the variations in expression of the EAU control. Log2 variation scale (relative expression): the more the value extends in the positive, the stronger the induction.

Table 5

The SDP control has a very strong capacity for inducing the PR1, PR4 and PR5 genes, 2 and 3 days after the second treatment (D2). It also shows a moderate induction of the PR8 gene on date D2. The induction profile of the SDP control in this test corresponds to the known effects of this product. The test is therefore validated.

The polysaccharides show a strong capacity for inducing the PR1, PR4 and PR5 genes, 2 days after the second treatment (D2). The PR14 gene is also induced at a high level, 2 days after the last treatment (D2).

Fold changes PR protein genes

The “Fold change” values of the 7 genes of the PR proteins, for each of the treatment modalities (candidate product and SDP control), on the sampling dates D2 and D3, are detailed in Tables 6 and 7 below.

The Fold change corresponds to the ratio of the level of expression of a gene for a treatment modality (SDP control, candidate product) compared to the EAU control, without log2 transformation and averaged for the 2 repeats. Fold change values greater than or equal to 2 represent moderate to strong inductions.

Table 6 Table 7

Two days after the treatment, the SDP control very strongly induces the PR1, PR4 and PR5 genes, at a level 253 to 1515 times higher than the WATER control. The PR8 and PR14 genes are induced respectively 4 and 3 times more than with the EAU control. The polysaccharides exhibit a strong capacity for inducing the PR1, PR4, PR5 and PR14 genes, at a level 7 to 40 times higher than the EAU control.

Three days after the treatment, the SDP control exhibits a strong capacity for inducing the PR1, PR4 and PR5 genes, 100 to 170 times higher than the EAU control. The polysaccharides strongly induce the PR1, PR4 and PR5 genes, respectively 18, 78 and 85 times more than the EAU control.

Conclusions:

The polysaccharides derived from the fruits of the date palm Phoenix dactylifera have a high capacity for inducing PR protein genes in wheat. They therefore have an inducing effect of the defense genes on the wheat crop and can be used as elicitors.

Example 6

Demonstration of the effectiveness of an extract of water-soluble polysaccharides from fruits of the date palm Phoenix dactylifera, in stimulating the natural defenses of the vine.

Aim: The objective is to confirm on vine plants what was observed on wheat plants in the previous example.

Biological material

The vines were produced from seeds of the Chardonnay grape variety. They were cultivated for 9 to 10 weeks in a greenhouse under semi-controlled conditions (19 ° C, 16 hours of daylight) then selected at the 4 - 6 leaf stage and transferred to a greenhouse dedicated to experimentation.

Tested and control products

The following modalities were compared (Table 8).

Table 8

In order to facilitate the adhesion of the candidate products on the vine plants, Tween 20 was added to each candidate product, at a concentration of 0.05%, ie 25 μl for 50 ml of product.

Tusk markers used

The 7 PR protein genes mentioned in Example 4: PR-1; PR-2; PR-4; PR-5, PR-8; PR- 14; PR-15.

Protocol

Each block of each modality is treated twice, with the exception of the WATER indicator which is applied once only (J0). The candidate products are applied 4 days apart (D-4 / D0) and the SDP control at 6-day intervals (D-6 / D0). Each modality is then treated on D1 with hydrogen peroxide (H202) to simulate an attack by pests and diseases. All the treatments are carried out on the 2 leaf faces, up to the limit of runoff, with a sprayer connected to compressed air.

For each modality, 8 leaf discs (6 mm diameter) are taken by combining 4 developed leaves from 4 different plants, on the following dates:

- D0: initial sample from 5 plants that have not received any treatment,

- D2: sample 2 days after treatment,

- D3: sample 3 days after treatment.

These samples are placed in liquid nitrogen and then stored at -80 ° C. until the extraction of the RNAs. RNA extraction, retro-transcription and quantitative PCR The procedure is identical to that of Example 4.

Results

Relative expression density map of PR protein genes

Table 9 below shows the density map of the relative expressions of the 7 genes of the PR proteins.

Table 9

The SDP control exhibits a strong capacity for inducing PR protein genes, with the exception of the PR14 gene, 2 days after the second treatment (D2). On the other hand, it no longer shows induction of the PR protein genes, 3 days after the second treatment (D3). The profile and the high level of induction on D2 of the SDP control in this test correspond to the known effects of this product under our conditions. The test is therefore validated.

The polysaccharides show a strong inducing capacity of the PR protein genes, 2 days after the second application (D2).

Fold changes PR protein genes

The “Fold change” values of the 7 genes of the PR proteins, for each of the treatment modalities (candidate products and SDP control), on the sampling dates D2 and D3, are detailed in Tables 10 and 11 below.

Table 10

Table 11

Two days after the treatment, the SDP control induces the PR2, PR3, PR4, PR5, PR8 and PR14 genes, at a level 2 to 27 times higher than the WATER control. The PR1 gene is the most induced, 158 times more than with the EAU control. The polysaccharides exhibit a capacity for inducing the PR5 and PR14 genes, at a level 3 times higher than the EAU control. They strongly induce the PR1, PR3, PR4 and PR8 genes, at a level 5 to 36 times higher than the EAU control. The PR2 gene is the most induced, 50 times more than with the EAU control.

Three days after the treatment, the SDP control exhibits a low capacity for inducing the PR14 gene, at a level 1.5 times higher than the EAU control. It does not induce the other PR protein genes.

The polysaccharides induce the PR3 gene at a level 3 times higher than the EAU control.

Conclusions:

The polysaccharides derived from the fruits of the date palm Phoenix dactylifera have a high capacity for inducing PR protein genes in grapevine. They therefore have an inducing effect of the defense genes on the wheat crop and can be used as elicitors. Example 7

Demonstration of the effectiveness of a mixture of extract of water-soluble polysaccharides and extract of polyphenols from fruits of the date palm Phoenix dactylifera in the protection of plants against downy mildew of the vine

Aim: The study is being carried out in order to determine the effectiveness of protection of a mixture of water-soluble polysaccharides and polyphenols against downy mildew (Plasmopara viticola) of the vine (Vitis vinifera), at three different doses, in preventive or curative application . The tests are carried out on vine discs under controlled conditions.

Methods: In this study, mixtures of water-soluble polyphenols and polysaccharides (weight ratio of 50/50) are applied at different doses (0.01%; 0.1%; 0.7%) (g / L) as a preventive measure , that is to say 24 hours before the plant is contaminated with Plasmopara viticola, and for curative purposes, that is to say 24 hours after the plant has been contaminated.

As controls, water and Bordeaux mixture (reference product) are also applied 24 hours before or 24 hours after contamination of the plant by Plasmopara viticola.

Table 12 illustrates the application modalities.

Table 12

Plant material: vine seedlings are obtained from seeds of the Chardonnay grape variety.

Plasmopara viticola: Leaves 5 cm in diameter with large sporulation spots were frozen. The strain is then multiplied just before the experiment on vine plants, in order to have a fresh inoculum.

Culture: the seedlings are cultivated for 6 weeks under controlled conditions (25 ° C, 16h photoperiod) then selected at the 4-leaf stage and transferred to a dedicated climatic module for experimentation at the time of inoculation (21 ° C day, 19 ° C night, 16h photoperiod). Treatment: the products are applied by spraying using a sprayer connected to compressed air up to the point of runoff on both leaf faces. The treatments were applied as a preventive measure, at -24h before inoculation, on plants, or curative, at +24h on discs.

Tinoculum production and inoculation: the leaves with dense sporulation are rinsed with reverse osmosis water and the suspension is filtered. The final concentration of the inoculum is adjusted to the concentration of 5.10 ⁴ in sp / ml. Leaf discs are made using a cookie cutter at the rate of 8 discs per Petri dish. The inoculum is then applied to the underside of the discs with a fine droplet sprayer. The dishes are kept at room temperature until reading.

Method of analysis: the results are read between 6 and 9 days after inoculation using a quantitative scale for the severity of the disease varying from 0 to 100% of sporulation covering the surface of the leaf discs.

The severity (intensity) of the disease is calculated by averaging the scores (%) obtained by modality. The incidence (frequency) of the disease is calculated and corresponds to the percentage of plants affected by late blight. Finally, the percentage of protection is calculated by the formula ((Water witness score-X score / Water witness score) x 100) from the severity scores.

The data were analyzed with the XLSTAT software with an ANOVA. The statistical analysis used to differentiate the means is the Fisher Minimum Significant Difference (LSD) procedure. It indicates statistically significant differences at the 95% confidence level.

This analysis was carried out on the ratings of the severity scores. In the case of a non-parametric test, the Kruskal-Wallis test is used; this test is a nonparametric equivalent of one-way ANOVA. This nonparametric test is based on the comparison of the confidence intervals of the median on a Tukey boxplot representation.

Results: the incidence and severity of late blight on the leaf discs inoculated artificially under controlled conditions and the percentages of protection are indicated in Table 13 below.

Table 13 According to these data, in preventive application, a severity score is observed which is significantly lower than the corresponding treated water control for doses at 0.1 and 0.7% of the mixture of water-soluble polyphenols and polysaccharides, respectively 73 and 35%. , with a positive dose effect. The degree of protection obtained for the mixture according to the invention at a dose of 0.7% is high (61%).

In curative application, a severity score is also observed which is significantly lower than the corresponding treated water control for the doses at 0.01%, 0.1% and 0.7% of the mixture according to the invention, which are respectively 79 , 80 and 71%, with a positive dose effect. The degree of protection obtained for the mixture at the dose of 0.7% is 30%.

In conclusion: we observe that the mixture has the ability to induce natural defense reactions in the plant and to protect it before it is attacked by the pathogen but also to induce defense mechanisms in treatment when the plant is in contact with the pathogen.

The combination of an extract containing polysaccharides and an extract containing polyphenols is therefore very useful in the protection of plants against pathogens. Indeed, such a mixture, once applied to the plant, will be able to act over the long term, both in prevention, by inducing defense and protection mechanisms, in particular by an elicitor activity, and in treatment. It is therefore possible to formulate a crop care product with a broad spectrum of activity.

Claims

1) Use of an extract of water-soluble compounds obtained from fruits of the date palm Phoenix dactylifera, the extract containing at least 30%, by weight, of water-soluble saccharides, for the protection of plants against pathogens.

2) Use according to claim 1, wherein the extract additionally contains at least 5%, by weight, of proteins, preferably at least 10%, more preferably between 5 and 15%.

3) Use according to claim 1 or 2, for stimulating the defense and resistance reactions of the plant for treatment or prevention, in particular for inducing an eliciting activity of the defense mechanisms.

4) Use according to one of the preceding claims, in which the extract contains naturally water-soluble polysaccharides or a mixture of such polysaccharides with hydrolysates of naturally insoluble polysaccharides.

5) Use according to one of the preceding claims, wherein the saccharides contain rhamnose, arabinose, fucose, xylose, mannose, galactose, galacturonic acid, glucose and / or l ' glucuronic acid.

6) Use according to one of the preceding claims, wherein the extract contains, by weight: at least 20% mannose, at least 7% arabinose, at least 6% glucose, at least 5% galactose.

7) Use according to one of the preceding claims, wherein the extract contains, by weight: at least 40% galacturonic acid, at least 7% arabinose, at least 3% xylose.

8) Use according to one of the preceding claims, in which the extract of water-soluble saccharides is mixed with an extract of polyphenols obtained from fruits of the date palm Phoenix dactylifera.

9) Use according to claim 8, wherein the ratio between the saccharide extract and the polyphenol extract is between 30/70 and 70/30, expressed by weight.

10) Use according to one of claims 5 to 6, wherein the polyphenol extract comprises at least 95%, more preferably at least 97%, even more preferably at least 99% of condensed tannins, by weight relative to weight total polyphenols.

11) Use according to one of the preceding claims, in which the effective amount of the saccharide extract, or of the mixture of extracts of saccharides and of polyphenols, supplied to the plants, is at least 0.01 g per liter , preferably at least 0.1 g per liter, more preferably at least 0.7 g per liter, for a contribution in liquid form, or at least 10 g per hectare, preferably at least 100 g per hectare for a supply in solid form.

12) Use according to one of the preceding claims, in which the saccharide extract, or the mixture of extracts of saccharides and of polyphenols, is applied to the whole plant, to leaves, flowers, roots, fruits, seeds, seedlings, soil, solid or liquid growing medium, growing material.

13) Use according to one of the preceding claims, wherein the application is carried out by sprinkling, irrigation, spraying, soaking or injection.

14) Use according to one of the preceding claims, wherein the plants are agronomic plants, in particular wheat, ornamental, aromatic or medicinal, in particular fruit plants, in particular vines, vegetable plants, flowers, trees, shrubs.

15) Use according to one of the preceding claims, wherein said plant pathogens are fungi, bacteria, viruses, nematodes, parasitic plants, protozoa or insects, in particular Plasmopora viticola.

16) Method of protecting a plant against pathogens, in particular for activating defense and resistance reactions of the plant, characterized in that it comprises application to said plant, or in the environment of the plant , an effective amount of an extract of water-soluble saccharides obtained from fruits of the date palm Phoenix dactylifera alone or as a mixture with an extract of polyphenols from this date palm, said composition being as described in one or other of claims 1 to 15.

17) Plant protection product containing an effective amount of a saccharide extract from fruits of the Phoenix dactylifera date palm, alone or as a mixture with an extract of polyphenols from the same date palm, said extracts being as described in one or the other of claims 1 to 15.

18) Plant protection product according to claim 17, further comprising at least one other component such as a solvent, a surfactant, an emulsifier, a dispersing agent, a filler, a fertilizing compound or a plant protection compound.

19) Plant protection product according to one of claims 17 or 18, characterized in that it is in liquid form, in gel form, in powder or granule form.