ES2331784B1 - COMPOSITION FOR PHYTOPHARMACEUTICAL APPLICATION INTENDED TO STIMULATE THE NATURAL DEFENSES OF THE PLANTS. - Google Patents

Abstract

Compositions for phytopharmaceutical application intended to stimulate the natural defenses of plants, in particular of the vine. In particular, the invention aims at compositions containing oligosaccharides secreted by a strain of pathogenic or non-pathogenic fungus from plants or obtained at start from it.

Description

Composition for phytopharmaceutical application intended to stimulate the natural defenses of plants.

The present invention aims at compositions for phytopharmaceutical application intended for stimulate the natural defenses of plants, in particular of the vine In particular, the invention aims at compositions containing oligosaccharides secreted by a strain of pathogenic or non-pathogenic fungus of plants or obtained at start from it.

Throughout their development, the plants are subjected to continuous aggressions by their environment. Without However, they are often able to resist naturally to these external aggressions thanks to the presence or the activation of defense mechanisms (Hammond-Kosak and Jones, 1996). So, although some of these mechanisms are constitutive and provide a barrier physical and chemical to an aggression, others are induced only after an attack by a harmful element.

As soon as a plant detects a pathogen, it starts one of the most effective natural defense systems: the hypersensitivity response. At the level of the pathogen penetration site, this reaction, rapid and violent, is translated by the death of the first infected cells and the appearance of a small necrotic area, thus isolating the attacked cells from the rest of the plant (Dangl et al . , 1996; Lamb and Dixon, 1997). The activation of this response depends on a specific recognition of the pathogen by the host plant. In fact, the attacked plant recognizes by means of a protein (receptor) some molecules produced by the pathogen, called eliciters. In the plant, the genes that code for the receptor proteins are called resistance genes (R genes), and for the pathogen, the genes that code for the elicitor molecules are called avirulence genes (Avr gene): it is then treated in this case of a gene by gene relationship or R-Avr relationship (Hammond-Kosak, 1996). Other molecules, qualified as general eliciters, are also capable of initiating (in a less specific way than the elicitors mentioned above) a defense reaction of the host plant. These are most commonly oligosaccharides released by the pathogen (exogenous elicitors) or the plant cell (endogenous elicitors) (Scheel and Parker, 1996).

These oligosaccharides are divided into 4 classes: oligoglycans, oligochitin and oligochitosan of fungal origin; and oligogalacturonids of plant origin. The oligoglycans are composed of β-glucose residues linked in 3-,
6- and 3,6-. Chitin is a linear polymer of 4--linked N-acetyl-β-glucosamine residues, which represents the major constituent of the mycelium wall of fungi; Chitosan is formed by the same deacetylated residues. The oligogalacturonides are released by the degradation of the pectic compounds of the plant cell wall (homogalacturonanos) after the attack of the pathogen; they are composed of α-galacturonic acid residues linked in 4-. Oligosaccharides should be most frequently composed of a number of residues between 4 and 15, to have an elicitor action (Côté et al ., 1994).

This intense defense reaction is followed by continuation by the implementation of defense mechanisms in neighboring cells of the infected area, which implies diffusion of a signal, is the local reaction. Finally, there is an issue of various warning signs to all other organs of the plant, which allows the plant to react more quickly and more effectively during a new aggression, there is talk of systemic reaction

         \ vskip1.000000 \ baselineskip
      

During these reactions, three can be activated categories of defense systems:

-

the formation of a healing epidermis and the reinforcement of the walls (lignification, etc.) (Dai et al ., 1995);

-

the synthesis of defense proteins or "Pathogenesis Related" (PR), proteins discovered in 1970 in tobacco. These PR proteins include, for example, protease inhibitors (Ryan, 1992), hydrolytic enzymes, such as chitinases or β-1,3-glucanases (Derckel et al ., 1996; Robinson et al ., 1997; Kraeva et al ., 1998; Salzman et al ., 1998; Renault et al ., 2000); Y

-

the synthesis of secondary metabolites of phytoalexin type. Between the secondary metabolites, have already been characterized more than 300 phytoalexins They belong to a wide spectrum of chemical classes different, among which you can mention the coumarins, the benzofurans, terpenes, alkaloids, and certain polyphenols (Smith, 1996).

         \ vskip1.000000 \ baselineskip
      

The start-up of plant defense reactions involves a whole panoply of transduction signals that lead to rapid induction of defense gene expression. Thus, recognition of the pathogen by the host plant will activate a whole cascade of signals in the attacked cells, such as protein phosphorylation by kinase proteins, ionic species flows (Ca2 +), species formation reactive oxygenates (Coté and Hahn, 1994; Shibuya et al ., 1996; Benhamou, 1996).

In addition, the attacked cells are capable of produce alarm signals transmitted to nearby cells (local reaction) as well as the entire plant, thus generating, as the reaction phenomenon has been stated in the previous paragraph systemic

The most studied systemic resistance mechanism is the phenomenon of SAR or "systemic acquired resistance". The term SAR has been defined by Ross in 1961. It describes the emergence of resistance of a plant as a result of an attack by a pathogen, both in infected parts and in healthy parts of the plant. It usually develops after the appearance of necrotic lesions around the site of inoculation. This localized hypersensitivity response restricts the pathogen at and around the infection site, and it seems that the plant becomes more resistant to aggressions by various organisms (Ryals et al ., 1996). It is in 1966 when Ross developed the idea of the existence of signal molecules that, at low concentrations, would be able to activate defense mechanisms in tissues distant from the infected area.

Three types of molecules can intervene in the plants as an alarm signal at the intra and intercellular levels, to short or long distance: salicylic acid, ethylene and Jasmonatos

On the vine, the signaling mechanisms involved in the expression of defense reactions are not yet well known. However, the synthesis of the three types of defense molecules (lignin, defense proteins and phytoalexins) is found. In particular, the role of phytoalexins is played in particular by a family of original compounds: polyphenols (Deloire et al ., 2000). Present in more or less large quantities in all the organs of the plant, phytoalexins can be induced at the level of the leaves and berries. This type of induction is designated by the term elicitation.

         \ vskip1.000000 \ baselineskip
      

Elicitation factors (or elicitors) They may have different origins. It can be:

-

biotic elicitation, for example during an attack by a pathogen such as Botrytis cinerea , agent of the gray rot (Jeandet et al ., 1995; Bavaresco et al ., 1997), Plasmopara vitícola, agent of mildew (Dercks and Creasy, 1989) or Phomopsis viticola , responsible for excoriosis (Hoos and Blaich, 1990).

-

Abiotic elicitation through environmental factors such as UV, temperature, light, asphyxiation (Jeandet et al ., 1997; Langcake and Pryce, 1977b; Douillet-Breuil et al ., 1999), aluminum chloride (Adrian et al ., 1996) or ozone (Sarig et al ., 1996).

         \ vskip1.000000 \ baselineskip
      

During an elicitation, some polyphenols can be induced, and in particular some stilbenes such as trans- resvera-
Troll, trans- piceid, ε-viniferin, δ-viniferin and pterostilbene, at the level of leaves and berries (Soleas et al ., 1997; Jeandet et al ., 2004). This de novo biosynthesis property of stilbenes in response to stress, and in particular after an attack by a pathogen, suggests that these molecules could play a role as a natural defense medium for plants.

This function of defense molecules is corroborated by certain studies that seem to indicate a close correlation between the level of natural resistance of the plant and its ability to synthesize these molecules. For example, Langcake and McCarthy (1979) have demonstrated a relationship between the resistance of certain species of the genus Vitis to Botrytis cinerea or Plasmopara viticola and their ability to biosynthesis of stilbenes (resveratrol and viniferins). In addition, Dercks and Creasy (1989) have shown that Plasmopara viticola resistant species produce five times more phytoalexins than sensitive species. Also, inside the vinifera species there are some more or less tolerant to the attack by fungi according to their ability to produce phytoalexins.

It is also interesting to observe in other plants the acquisition of resistance to certain fungal diseases, possible after the introduction of the gene that codes for stilbene synthase. Thus, alfalfa plants ( Medicago sativa L.) transformed with the peanut stilbene synthase gene ( Arachis hypogeae ) have a resistance to Phoma medicaginis infection that is correlated with the appearance of leaf-level spruce (Hipskind and Palva, 2000). Similarly, transgenic tobacco, tomato or rice plants that have incorporated a gene that codes for grapevine stilbene synthase are less sensitive to infection with Botrytis cinerea, Phytophtora infestans and Pyricularia oryza , respectively (Thomzick et al ., 1997; Stark-Lorenzen et al ., 1997; Hain et al ., 1993).

More recently, Coutos-Thévenot et al . (2002) have obtained vine plants transformed by the stilbene synthase gene ( Vst 1) located under the influence of an inducible promoter. In these plants, the accumulation of trans-resveratrol is very clearly increased (by a factor of 200) and these transgenic vines have a very high resistance to Botrytis cinerea .

From document FR 2 836 011 a known procedure for the stimulation of the natural defenses of plants comprising the application to said plants of an agent elicitor or sensitizer that is capable, upon contact with plants, to prevent preventively in these, the activation of the genes of defense on the product application site. This procedure use an effective amount of a oligo-? -3-3-glucan, prepared from bacteria, algae or cereals.

However, these oligosaccharides have 20 to 33 ossical units, which limits their penetration at the level of leaves. On the other hand, seaweeds are extracted from seaweed, which is an exhaustible source, and that may contain some residues of Marine contamination. In addition, they are chemically modified by sulfation.

The inventors have developed a technology innovative and controlled, which guarantees quality and authenticity of the products. It's about mushroom growing in large volumes, which respond to the needs of the industry, in particular:

-

the absence of solvent and waste,

-

the homogeneity of the substrates,

-

the continuous production,

-

the total absence of contaminants,

-

the standardized and reproducible production in terms of quality and The concentration of metabolites.

         \ vskip1.000000 \ baselineskip
      

In a completely surprising way, the inventors of the present application have discovered that it could directly incorporate the culture filtrate, after autoclave treatment, in a phytopharmaceutical composition. This procedure presents in particular the advantage of providing a Useful and innovative alternative to conventional extractions by solvents, and thus avoid producing toxic effects on the floor In the case of an ethanol extraction, as presented in example 1, the drawbacks are minimal since it is a natural solvent, usually produced by some yeasts from glucose, and found in wine or in Beer. The objective of the present invention is to propose a alternative procedure to the use of chemical products respecting thus the environment and the consumer. In addition, the elicitation of natural defenses of the plants allows to limit the dose and the number of treatments

Another aspect of the invention is level of the mode of action that is preventive. In effect, this new class of elicitors allows to simulate an attack of pathogens at the level of plant cells. These will initiate a natural mechanism of defense that will cause a hypersensitivity reaction, but also an acquired systemic resistance, this at least once, which will allow to prevent the attack even more effectively later. Very low amounts of elicitors are sufficient to sensitize the cytoplasmic membrane. Thus, the threshold of detection of elicitors by plants can reach a value 10-10 molar or even lower.

A first aspect of the present invention is refers to a composition for phytopharmaceutical application, characterized in that it includes as an active principle the less an oligosaccharide secreted by a strain of pathogenic fungus or not plant pathogen or obtained from it.

Through the expression "composition phytopharmaceutical "means to designate according to the present invention any composition that relates to the protection of the plants or harvested products.

The term "oligosaccharides" means designate according to the present invention substances formed by several bear molecules (mostly 4 to 15) linked through some osidic links.

The term "fungus" is understood to designate according to the present invention all known fungal species. Preferably, the fungus is selected from Botrytis cinerea, Phomopsis viticola and Eutypa lata , and mixtures thereof.

Obviously, the active ingredients that are they can use in the composition according to the invention they can come from mixtures of fungal cultures obtained from different genres.

According to the present invention, the active ingredient can be obtained from an in vitro culture of plant pathogenic fungi by recovering the culture medium and the mycelium, without separation. The whole crop, that is the culture medium and the mycelium, is then used to obtain the active ingredient.

In addition, the active ingredient according to the invention can also be obtained from an in vitro culture of pathogenic fungi from plants by separation of the culture medium from the mycelium.

The active substance is then obtained from of the culture medium or from the mycelium.

In the first case, which is the simplest for an industrial exploitation, the active principle is constituted by an acellular fraction of the culture medium. Through the expression "acellular fraction" means a fraction without whole cells, but it may contain some cell fragments as well as some molecules initially present in the body mobile.

This culture medium can be purified eventually, the purification being carried out by precipitation of an aqueous filtrate from the culture medium by means of a solvent, preferably alcohol.

In the second case, the active substance is obtained by extraction and lysis of mycelial cells.

Thus, for example, a procedure for obtaining of a mushroom extract characterized in that the mushroom extract it is a culture filtrate and / or a mycelium preparation, it comprises the following stages:

to)

fungus culture in liquid medium for 3 to 28 days

b)

separation of the culture filtrate and of the mycelium

C)

culture filtrate recovery obtained in stage b) and, optionally, autoclave treatment of this culture filtrate, and / or

d)

mycelium recovery obtained in the stage b) and preparation of a mycelium preparation.

In the case where the active substance is obtained only from the culture medium, the procedure does not It comprises step d) but may optionally comprise the following stages:

?

after stage c), a step e) of isolation of a polysaccharide fraction from culture filtrate obtained in step c). This stage c) can be perform by precipitation with ethanol,

?

a stage f) of Iophilization of the polysaccharide fraction isolated in step e),

?

the polysaccharide fraction that it is obtained in stage e), and eventually lyophilized in stage f), is dissolved again in water and then, optionally, treated in autoclave in order to obtain a solution comprising some oligosaccharide fragments.

In the case where the active substance is obtained only from the mycelium, the procedure does not include the stage c), and the preparation of the mycelium preparation in the step d) consists of an aqueous extraction, an acid hydrolysis, an enzymatic hydrolysis and / or an autoclave treatment from of the mycelium recovered in step d).

In the case where the active substance is obtained from the mycelium and the culture medium, the The procedure includes steps c) and d), as well as eventually, the optional stages mentioned.

In vitro fungal cultures that can be used according to the invention can be obtained by any method known in the prior art. In particular, fungal cultures can be carried out in a liquid culture medium, in Erlenmeyer bottles or in bioreactors, in particular in fermenters.

Through the expression "culture medium" means to designate, according to the present invention, any means of crop generally known to the person skilled in the art. Sayings media can be solid, liquid or semi-solid. By way of preferred, the culture medium is a liquid medium.

As a solid culture medium, the 2% Malt-Agar and Potato Dextrose Agar medium, and as medium of liquid culture the medium Czapek-Dox.

In addition, the composition according to the present invention can be treated in autoclave.

Preferably, the active substance of the Composition according to the present invention is in liquid form.

According to the present invention, the composition is presented in the form of a solution, a concentrate or a lyophilized powder

According to the present invention, the composition is use as a preventive or curative title.

Another aspect of the invention relates to the use of the composition according to the present invention to stimulate the natural defense reactions of plants against aggression Present or future.

According to the present invention, aggression is an attack by a pathogen, preferably a fungus. Preferably, the fungus is Botrytis cinerea, Phomopsis viticola, Eutypa lata, Erisyphe necator (formerly called Uncinula necator ), Phoma uvicola, Phomopsis viticola, Phaeoacremonium sp., Phytophtora sp. or in particular Phytophtora infestans or Phytophtora parasitica .

According to the present invention, the natural defense reactions of plants are the production by the leaves and / or the fruits of defense molecules such as phytoalexins, such as for example the polyphenols of the vine, particularly the stilbenes, more particularly trans- and cis -piceids, their aglycones ( trans- and cis -resveratrol), pterostilbene or oligomers such as ε-and-delta-viniferins. In addition, the expression of certain genes is clearly increased, such as those encoding stilbene synthase, glucanases and chitinases.

According to the present invention, the plants are select from among the vine, fruit trees, crops of orchard, cereals and oilseeds, protein crops, Tobacco and ornamental plants. Preferably, the plant chosen is the vine, the tomato or the potato.

Another aspect of the invention relates to a Plant treatment procedure for stimulation of natural defenses to plant pathogens, characterized in that it is applied, in particular to the sheets or to the fruits or seeds or seeds, preferably leaves, an effective amount of a composition according to the present invention.

In the process according to the present invention, the following plants are treated: the vine, the fruit trees, the garden crops, cereals, oilseeds, protein crops, tobacco, ornamental plants, preferably the vine, the tomato or the potato.

In the process according to the present invention, plants are treated to fight against pathogens, preferably fungi, even more preferably Botrytis cinerea, Plasmopara viticola, Eutypa lata, Uncinula necator; Phoma uvicola, Phomopsis viticola, Phaeoacremonium sp., Phytophtora infestans or Parasitic Phytophtora .

Preferably, plants are treated for fight against mildews and powdery mildews.

Even more preferably, the method according to the present invention allows treating the vine against Uncinula necator (responsible for the powdery mildew) and Plasmopara viticola (responsible for mildew), and treating the tomato and potato against Phytophtora sp. (responsible for mildew).

The process according to the present invention allows to prevent or treat diseases such as gray rot, powdery mildew, mildew, excoriosis, black gangrene, eutipiosis, mottled fruit trees, peach leprosy, seproriosis, rust, coals, cavities, anthracnose, necrosis of the neck or tinder. From preferred way, these diseases are gray rot, the powdery mildews. Even more preferred these diseases are the mildew and the mildew of the vine, or the mildew of the Tomato and potato.

The following list shows the main diseases (listed from 1 to 11), as well as fungi responsible (in italics) and the affected plants (between parentheses), for which the procedure according to the present Invention would allow a treatment or prevention:

1 - Oídios

Erysiphe necator = Uncinula necator (vine)

Erysiphe graminis (cereals)

Podosphera leucotricha (arboriculture, fruit trees)

Sphaerotheca panosa (rose bushes)

         \ vskip1.000000 \ baselineskip
      

2 - Mildius

Plasmopara viticola (vine)

Plasmopara helianthi (sunflower)

Phytophthora infestans (potato)

Phytophthora parasitica (tomato)

         \ vskip1.000000 \ baselineskip
      

3 - Spotted fruit trees

Venturia inaequalis (apple tree)

Venturia pyrine (pear tree)

         \ vskip1.000000 \ baselineskip
      

4 - Peach Leprosy Taphrina deformans

         \ vskip1.000000 \ baselineskip
      

5 - Septoriosis

Septoria nodorum and Septoria tritici (cereals)

Septoria sp. (rape)

         \ vskip1.000000 \ baselineskip
      

6 - Royas

Puccinia graminis, Puccinia recondita and Puccinia striiformis (cereals)

         \ vskip1.000000 \ baselineskip
      

7 - Coals

Ustilago tritici (wheat)

Ustilago maydis (corn)

Ustilago hordei (barley)

         \ vskip1.000000 \ baselineskip
      

8 - Caries

Tilletia tritici, Tilletia caries and Tilletia foetida (cereals)

         \ vskip1.000000 \ baselineskip
      

9 - Neck necrosis

Phoma lingam (cruciferous, rapeseed)

Rhizoetonia solani (potato)

Scierotinia sclerotiorum (tomato)

         \ vskip1.000000 \ baselineskip
      

10 - Anthracnose

Colletotrichum gloeosporioides (pea)

Colletotrichum lindemuthianum (green bean)

         \ vskip1.000000 \ baselineskip
      

11 - Gray rot

Botrytis cinerea (vine)

         \ vskip1.000000 \ baselineskip
      

In practice, the plants of which wants to stimulate natural defenses during an attack by a pathogen or in view of a future attack, with the help of a composition, in particular of an aqueous solution that is you must apply on the leaves, the fruits or on the seeds, and that It also includes the usual vehicles and constituents of this type of composition, one or several wall oligosaccharides of fungi as defined above, present at some concentrations that depend on the desired result, that is a immediate stimulation or potentiation of defenses.

The treatment is usually done from of the first vegetative stages of the plant, eventually in several successive applications, advantageously by spray.

The precise moment of the application (s) It will be chosen in particular according to the treated plant. He Vector vehicle is usually water. However it is possible resort, instead of water, to a vector selected from the group comprising vegetable oils, agents surfactants, solvents, dispersing agents and / or ones solid charges

The present invention will be described in greater detail. detail with the help of the following examples and compositions, illustrating the invention without limiting in any way its ambit.

Legend of the figures

Figure 1: Styloben content in the leaves of plants (vine) treated by elicitor F241.

Figure 2: Efficiency on the mildew of the grapevine F241 product tested at two doses. Pollution 2 days later of the treatment and observation on the treated leaves.

Figure 3: Accumulation of the mRNAs of the genes that code for the enzymes of the biosynthesis pathway of stilbenes (A) and PR-proteins (B and C) in the leaves of plants ( Vitis vinifera ) treated by elicitor F241.

Figure 4: Effectiveness on tomato mildew and of the product F241 potato at a dose of 25%. Pollution 2 days after treatment and observation of the infestation on The leaves treated 7 days after contamination.

Examples Example 1 Production procedure of new elicitors

Stage one

Preparation of fungal cultures

The first stage for the development of fungal cultures in vitro consists in selecting the species that produces the substances sought. It is now admitted that within the same species, there is a variability in the production capacities of a given metabolite, partly of genetic origin. When possible, this variability should therefore be exploited, selecting the best genotype, that is, the most productive for the metabolite sought.

The selected fungus strain ( Botrytis cinerea for example) is grown on a solid medium (2% Malt-agar) for 10 days at 23 ± 2 ° C with a photoperiod (16 h day / 8 h dark) until Obtaining conidia. Then, the conidia are suspended in water with 0.05% Tween 20. Aliquots of this conidial suspension are added to the modified Czapek-Dox culture medium (3 g / l of NaNO 3, 3 g / l of KH 2 PO 4, 0.5 g / l of KCl, 0.5 g / l of MgSO4, 0.01 g / l of FeSO4, 5 g / l of tartaric acid, 20 to 50 g / l of D-glucose or sucrose, pH adjusted to 3 , 2 by KOH) previously sterilized by autoclaving, at a final concentration of 2.5.10 4 conidia / ml of medium. The cultures are then placed in a bioreactor of 5 to 20 liters, at 23 ± 2 ° C with a maximum agitator rotation speed of 300 rpm, and a maximum air flow of 5 vvm, for a maximum 35 days

Stage 2

Preparation of oligosaccharides

After separation of the mycelium and the culture medium by filtration on a blutter cloth
(50 µm) or centrifugation, the filtrate is recovered and subjected to an autoclave treatment at 115-120 ° C for 40 to 60 minutes depending on the volume treated. After this treatment, the filtrate, called F241, can be used in this way to treat the plants, or it can be lyophilized and then put back into solution just before its
use.

Stage 3

Drying or atomization or lyophilization of extracts

This stage is advantageously performed at a temperature below 60 ° C, for example between -60 ° C and 50 ° C, with the Help of a freeze dryer. In this case, they are obtained, after the lyophilization, approximately 1 to 5 grams of dry powder per liter of cultivation Then, the powder obtained is crushed with the help of a crusher, preferably with the help of a crusher type mortar, pearls or blades.

Stage 4

Mixing and / or incorporation into one or some excipients and / or others active ingredients for the preparation of the composition for use phytopharmaceutical

The isolated oligosaccharides are dosed by colorimetry thanks to the phenol-sulfuric acid reaction (Dubois et al ., 1956), and then diluted in distilled water or in adjuvants.

         \ vskip1.000000 \ baselineskip
      

Example 2 Stimulation of the natural defenses of vine plants treated by an elicitor from the culture filtrate (F241)

The tests have been carried out on seedlings of grapevine vine Cabernet Sauvignon (CS), and produced in greenhouse from cuttings of a bud. A few lots of 6 plants for the study of each modality of an essay. The Foliar levels have been identified at the time of treatments and contamination with the help of an indicator colored arranged under the third extended sheet. In first instead, whole plants of the CS varieties have been treated by aqueous compositions containing 0.1% Triton, and comprising elicitor F241 at a concentration of 20% (v / v) that It is the optimal dose for elicitor activity.

A usual input mode is used by spray by means of a jet spray bank projected and at a constant pressure. The application is done on The whole vegetable. The pressure at which the Spraying depends on the volume to contribute. A car of spraying, equipped with 5 nozzles (1 upper center, 2 right sides, 2 left sides) move over a guide rail at a constant speed. The same plants Iota are treated simultaneously and perfectly controlled. The application volume of the considered elicitor is 600 l / ha.

Forty-eight hours after the treatment of the seedlings, a batch is selected, and the leaves are collected. Then, they are dried with the help of a lyophilizer (Virtis Uni-Trap 10-100). The dust obtained after crushing with the help of a type crusher mortar, extracted with methanol, at a rate of 10 ml per 200 mg of dry matter at 4 ° C. After centrifugation (10 min. 3,500 rpm), 8 ml of metallic leaf extract are evaporated with the Speed Empty The sample is collected in 1 ml of MeOH / H2O (30/70, v / v) and then it is chromatographed on a mini-column of grafted silica C18 (SPE). The conditioning of the cartridges is  performed by successive washes with MeOH (2 ml), with 50% MeOH (2 ml), and with distilled water (6 ml). After the deposit of the sample, stilbenes are eluted with 5 ml of MeOH / H2O (80/20, v / v). The eluate obtained is injected directly into HPLC on a reverse phase silica column (C18). Stilbenes are detected by fluorescence, with an excitation wavelength of 300 nm and an emission wavelength of 390 nm, which correspond to the optimal wavelengths for stilbenes. The stilbene contents of the sheets are calculated in relation to to calibration curves made with pure compounds.

The results obtained are shown in the Figure 1.

         \ vskip1.000000 \ baselineskip
      

Example 3 Study of the resistance of vine seedlings treated by elicitor F241 to a Plasmopara viticola infection

A batch of treated plants is dried by elicitor at room temperature, and then put back in the greenhouse until the moment of its contamination 48 hours more late. The fungal material consists of a set of sporocistos of good viability that come from a strain of mildew or of reference newspaper, sensitive to the main families fungicides used today.

The sporocyst suspension is prepared in water permutated kept at low temperature in order to block the release of zoospores until the moment of inoculation. By washing the infected leaves, the sporulations of the fungus and are suspended in permutated water. Be perform a titration of the inoculum concentration with the help of a Malassez cell. The optimal concentration sought is 50,000 sp / ml.

For a plant, they are sprayed approximately 10 ml of sporocyst suspension. The inoculum preparation is develops just before pollution. Each plant is handled in isolation and is contaminated at all levels of the leaves. Plants of the same condition are then incubated in some isolation enclosures. A vaporization of water inside these individual enclosures will allow moisture saturation permanent very favorable for the development of the disease.

All plants are incubated after their pollution in a room heated to 21 ± 2 ° C, and illuminated 14 hours per day. The plants remain in these conditions for 8 days At the end of this period, an entry is made of the damage of the fungus on each plant and for each state foliar.

The annotation is based on a visual observation of the intensity of fungus damage divided into two elements: the surface colonized by the fungus and the intensity of the sporulation A scale between 0 and 100% is used (per 5% step).

Each sheet is observed individually. The Foliar states noted in the case of plants are those that correspond to the fourth level of sheet at the time of treatment and of the following (F4; F3; F2; F1, N1, N2, N3, etc.). A ligature of color arranged under the third sheet extended at the time of treatment allows to easily locate the different levels of sheet without any mistake.

First, the average of the damage noted on the 6 plants of the same lot. The plant whose damage is the furthest from this mean (attack strong or low). The average of the 5 most homogeneous plants is then calculate and give the damage rate for each condition of the essay. Then, the efficiency (E) of each modality is calculated of the test with reference to the batch of untreated plants of the test according to the formula:

E = {[(T-D) / T] * 100}

in which T is the average grade of attack on untreated plants, and D observed on plants treated with the dose d.

An analysis of variance allows comparison of the results and give, if necessary, the differences between conditions studied.

The results are represented in Figure 2. The Figure 2 represents the attack intensities as well as the efficiencies noted on the different batches of plants (vine) treated or not by elicitor F241.

Approximately 43% of plant area is attacked by mildew after treatment with the elicitor at 2% dose. On the contrary, the damages are very low in the plants treated with this same product at a dose of 20% (less than  1% damage)

In parallel, the index of mRNA expression of different genes that code for some enzymes involved in plant defense reactions by quantitative real-time PCR, thanks to the use of specific primers

The results represented in Figure 3 show that elicitor F241 induces in the leaves of plants ( Vitis vinifera ), from the first hours after treatment, a clear increase in the expression rate of the genes that code for the enzymes of the pathway of biosynthesis of stilbenes, such as phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS), by a factor of 300 to 600, as well as those encoding PR (Pathogenesis-Related) proteins such as chitinases (CHIT), polygalacturonase inhibitor protein (PGIP) and serine proteinase inhibitor (PIN) by a factor of 5 to 60.

         \ vskip1.000000 \ baselineskip
      

Example 4 Study of the resistance of tomato and potato plants treated by elicitor F241 with an infection with Phytophtora sp.

The results represented in figure 4 show that infestation rates are very low over plants treated with elicitor F241 (infestation intensity less than 3% for both plants), with an attack frequency of only 29% for tomato.

Claims (19)

1. Composition characterized in that it comprises as an active principle at least one oligosaccharide from an in vitro culture of pathogenic fungi of the plants selected from Botrytis cinerea, Phomopsis viticola and Eutypa lata , and mixtures thereof, by recovering the medium of culture and mycelium, without separation, and eventually purification of the culture medium and mycelium, for phytopharmaceutical application. 2. Composition according to claim 1, characterized in that the active ingredient is obtained from an in vitro culture of pathogenic fungi from plants by separation of the culture medium from the mycelium. 3. Composition according to claim 2, characterized in that the active ingredient is constituted by an acellular fraction of the culture medium. 4. Composition according to claim 3, characterized in that the culture medium is purified by precipitation of an aqueous filtrate of the culture medium by alcohol. 5. Composition according to claim 2, characterized in that the active ingredient is obtained after extraction and lysis of mycelium cells. 6. Composition according to one of claims 1 to 5, characterized in that the culture medium is a liquid medium. 7. Composition according to one of claims 1 to 6, characterized in that it is autoclaved. 8. Composition according to one of claims 1 to 7, characterized in that the active ingredient is in lyophilized or atomized form. 9. Composition according to any one of claims 1 to 8, characterized in that it is in the form of a solution, a concentrate, or a lyophilized powder. 10. Use of a composition according to one of the claims 1 to 9, for preventive or curative purposes. 11. Use of the composition according to claim 10, to stimulate natural defense reactions of the plants against a present or future aggression. 12. Use according to claim 11, characterized in that the aggression is an attack by a pathogen, preferably a fungus. 13. Use according to claim 12, characterized in that the fungus is Botrytis cinerea, Phomopsis viticola or Eutypa lata . 14. Use according to any of claims 11 to 13, characterized in that the natural defense reactions of the plants are the production by the leaves and / or the berries of the plants of defense molecules such as polyphenols, particularly stilbenes, more particularly the trans and cis piceids, their aglycones, the trans and cis resveratrol, the pteroestilbenes or the ε and δ viniferins and / or the production of PR proteins. 15. Use according to any of claims 11 to 14, characterized in that the plants are selected from among the vine, fruit trees, orchard crops, cereals and oilseeds, protein crops, tobacco and ornamental plants, preferably the Vine, tomato and potato. 16. Procedure of treatment of plants for the stimulation of natural defenses to plant pathogens, characterized in that it applies, in particular to leaves or fruits or seeds or seeds, preferably to leaves, an amount effective of a composition according to one of claims 1 to 9. 17. Process for treating plants according to claim 16, characterized in that the following plants are treated: the vine, fruit trees, orchard crops, cereals, oilseeds, protein crops, tobacco, ornamental plants, preferably the vine, the tomato and the potato. 18. Method of treating plants according to any of claims 16 or 17, characterized in that plants are treated to fight against pathogens, preferably fungi, more preferably still Botrytis cinerea, Plasmopara viticola, Eutypa lata, Uncinula necator, Phoma uvicola , Phomopsis viticola, Phaeoacremonium sp ., Or Phytophtora sp . 19. Method of treatment of plants according to any of claims 16 to 18, characterized in that it allows to prevent or treat diseases of plants such as gray rot, mildew, mildew, excoriosis, black gangrene, eutipiosis, or Tinder