EP2453745A2 - Antifungal compounds and use thereof - Google Patents

Abstract

The present invention relates to an antifungal composition including at least two compounds selected from among the group consisting of cysteine, salicylic acid, iron(II) sulfate and fosetyl-aluminum, as well as to a method for treating a plant, infected or not infected by a pathogen. Specifically, the present invention relates to an antifungal composition for treating vineyards.

Description

 ANTIFUNGAL COMPOSITIONS AND USE

DESCRIPTION Technical field

 The present invention relates to an antifungal composition and a method of treating a plant infected with a pathogen. More particularly, the present invention relates to an antifungal composition for the treatment of grapevines.

In the description below, references in square brackets ([]) refer to the list of references presented at the end of the text.

State of the art

 [0003] Antimicrobial compositions have been known for several decades. These are compositions that kill microbes. Among these antimicrobial compositions some kill for example fungi and are called antifungal. There are different types of antifungals: the so-called "natural" antifungals, these are compounds or compositions derived from plants, from fruits (for example grapefruit); and antifungals produced by chemical synthesis.

[0004] A large number of antifungal molecules are known in the state of the art. Mention may be made, for example, of amorolfine hydrochloride, amphotericin B, benzoic acid, benzyl hydroxybenzoate, bifonazole, boric acid, bromoxyquinone, buclosamine, caprylic acid and chlormidazole hydrochloride. chlorobutanol, chlorquinol, ciclopiroxolamine, clofenoxide, cloprimazole edisilate, clotrimazol, clotrimazole chloride, dequanilium chloride, dimazol hydrochloride, econazole nitrate, fenticonazole nitrate, etc. The known antifungal compositions can be used in the agronomic field, for example for the prevention or treatment of pathologies such as Esca, Eutypiose, mildew, powdery mildew.

In the agricultural field, the vine is a plant that requires a massive use of phytosanitary treatments to stem many diseases. Thus, 30% of the tonnage of fungicides consumed in the European Union is used for viticulture, which represents only about 1% of cultivated areas (Daire et al., 2002 (Ref 1)). These treatments are due to the high parasite pressure on the vineyards because only varieties of Vitis vinifera of European origin, which are much more susceptible to diseases, are allowed in most wine-growing regions and vineyards are located. in temperate and humid zones favorable to the development of pathogenic fungi responsible for diseases.

The antifungal compositions of the state of the art can also be used in the food industry, for example in dairy compositions to prevent and / or inhibit the development of fungi in yoghurt etc. ; in the veterinary field, for example by topical application, intravenous administration to sheep, domestic animals, for example cats, dogs, etc., and in the medical field, for example for the treatment of mycoses, by topical application, intravenous injection, subcutaneous injection etc.

One of the problems associated with the use of fungicides is the appearance of resistance phenomena. Indeed, the massive use of fungicides leads to the appearance of "resistant" as observed for antibiotics. These resists require a continuous search for new compounds that can inhibit the growth of fungi.

In addition, it is well known that fungicides with a single site of action (fungicide unisite) such as benzimidazoles (Benlate (trademark)), promote the development of resistance, while fungicides with multiple sites of action (multisite fungicides) such as dithiocarbamates (Ferbam (trademark)) can limit the occurrence of the phenomenon of resistance.

Another known disadvantage of fungicides is their toxicity. Indeed, it is known that the fungicides used in the agricultural field can be toxic towards insects, for example bees, but also with respect to aquatic organisms. This is for example the Zirane (trademark).

Finally, because of their toxicity, some fungicides have been removed from commerce, for example sodium arsenite. This compound has a broad spectrum, with efficacy against four families of pathogenic fungi (Ascomycetes, Basidiomycetes,

Deuteromycetes and Oomycetes). It has been used for the treatment and prevention of Esca, a complex pathology caused by several Ascomycetes and Basidiomycetes fungi that infect woody plants, such as vines. This compound has been banned because it is carcinogenic and toxic to humans. There are currently no compounds for the treatment of Esca.

Salicylic acid (AS) is a molecule involved in the defense reactions of the plant. Initially, aspirin, a derivative of AS, has been shown to induce SAR (Systemic Acquired Resistance) and the synthesis of PR proteins (the pathogenesis associated or "pathogenesis related"). (White, 1979 (Ref 2)), mimicking the action of a natural benzoic compound acting as a defense signal (Van Loon, 1983 (Ref 3)). It was not until 1990 that AS was shown to be an essential element in the induction of SAR (Malamy et al., 1990 (Ref 4), Métraux et al., 1990 (Ref 5). )). AS stimulates the production of glucanases and chitinases that are transported to the distal parts of the plant where they degrade the wall of fungal or bacterial cells (Kessmann et al., 1996 (Ref. 6)). AS also has antifungal properties with respect to Eutypa lata. This action is pH dependent and the threshold value is estimated at 0.1 mM at pH 4.1 (Amborabé et al., 2002 (Ref 7)).

The use of AS has been considered as an elector against pathogens in crop protection (Regnault-Roger C, 2006 (Ref 8)). AS has a phloem and xylem system on castor seedlings. ¹⁴ C labeled AS and 10 μM concentration were detected in phloem sap at a concentration of 100 μM and xylem sap at 0.67 μM after 4 hours of absorption (Rocher et al., 2006). (Ref 9)). The AS concentration factor in castor phloem is of the order of 10, for pHs between 4.6 and 5.0 (Rocher, 2004 (Ref. 10)).

 AS analogs have also been synthesized: 2,6-dichloroisonicotinic acid (INA) and acibenzolar-S-methyl (ASM) which is a benzothiadiazole (see formulas (II) and (III)). )). These molecules are capable of inducing SAR (Dong, 1998 (Ref 11)).

The INA was quickly abandoned because of its phytotoxicity. In contrast, the efficacy of ASM has been shown in many annuals (cereals, potato, tomato) to different pathogens such as fungi, viruses and bacteria (Van Toor et al. ., 2001 (Ref 12)). It was introduced in 1996 as an activator of defenses for the control of powdery mildew in cereals. Germany and Switzerland (Ruess et al., 1996 (Ref 13)) and is also used against pyraculariosis of rice and various mildews (Ishii et al., 1999 (Ref 14), Leroux, 2003 (Ref 15)). It also has antibacterial and antiviral activities. On perennial plants, few studies have been conducted (Brisset et al., 2005 (Ref. 16)). ASM has been marketed under the trade name 'Bion' but in practice this molecule has not shown the expected efficacy (Alabouvette et al., 2003 (Ref. 17)). NNA and ASM have been shown to not directly induce defense responses but lower the response threshold to a fungal elicitor (Conrath et al., 2002 (Ref 18)).

 Currently, the mode of application used consists of injecting salts of salicylate into the vine stock to control Eutypa lata and Chondostereum purpureum (Spiers and Ward, 2001 (Ref 19)). The injection of salicylic acid in the form of potassium salts (18.5 g / L) in the trunk of ceps cv. Sauvignon showed an interesting action with regard to Esca (45.4% efficiency), the percentage of re-expression of symptoms being much lower for the treated plants than for the control plants. However, the efficacy of the product does not persist since eighteen months after its application, the number of Escha expressing stocks is identical in the control group and in the treated batch (Larignon and Molot, 2004 (Ref. 20)).

Cysteine is an amino acid that has a thiol group (formula (IV)) and is present in most proteins. Although poorly represented, its presence in the composition of proteins is very important, especially because it makes it possible to form disulfide bridges.

 Cysteine may be involved in defense phenomena since it has been shown that during tomato root infection with Verticillium dahliae, cysteine, sulfate and glutathione concentrations increase in tissues (Williams et al., 2002 (Ref 21)).

 Various studies have shown that cysteine has antifungal activity against certain fungi. Thus, cysteine, applied at concentrations of 0.9 and 9 mM, inhibits the development of Inonotus obliquus (Kahlos and Tikka, 1994 (Ref 22)). Comparable results were obtained with Eutypa lata, the pathogen of grape Eutypiasis. Cysteine inhibits mycelial development and also induces the death of Eutypa lata (Octave et al., 2005 (Ref 23)). It has also been shown that cysteine inhibits spore germination of $ Alternaria cassiae, Alternaria crassa, and Alternaria macrospora (Daigle and Cotty, 1991 (Ref. 24)).

Cysteine has another action since it causes, in Inonotus obliquus and Eutypa lata, an ergosterol release (Kahlos and Tikka, 1994 (Ref 22)); Octave et al., 2005 (Ref 23)) which is a sterol of fungal origin known to be a nonspecific elicitor capable of activating plant defense reactions (Granado et al., 1995 (Ref 24); 2000 (Ref 26), Amborabé et al., 2003 (Ref 27), Luini 2003 (Ref 28), Rossard 2003 (Ref 29)). The activity of cysteine is strongly related to its structure. Indeed, a structural modification or a change in the chemical function -SH abolishes its effectiveness. Leaf discs and leaves isolated from different plant species exposed to L-cysteine emit SH ₂ , a volatile compound with potentially antifungal activity (Sekiya et al., 1982 (Ref 30)). A Exogenous supply of cysteine on poplar leaf discs increases the content of glutathione and α-glutamylcysteine, two molecules involved in the protection of cells against active forms of oxygen in defense reactions (Noctor et al., 1996). (Ref 31)

However, cysteine alone is not sufficient in fungicidal use to the extent that even if it has an inhibitory activity of growth of pathogen in-vitro, its in-vivo activity has not been demonstrated.

The importance of iron in the relationships between various pathogenic microorganisms (bacteria, fungi) and their hosts has been highlighted in many studies (Weinberg, 1966 (Ref 32)). In the Botrytis cinerea I Vicia faba interaction, iron chelators such as EDTA (ethylene diamine tetraacetic acid) and DHBA (dihydroxy benzoic acid) increase the aggressiveness of conidia of Botrytis cinerea. The addition of ferric sulfate (Fβ2 (SO ₄₎ 3) inoculum treated with these chelators that reduces aggression (Brown and Swinburne, 1982 (Ref 33)). The presence of iron sulphate inhibits the formation and development of necrotic lesions caused by Botrytis fabae and Botrytis cinerea on detached leaves of Vicia faba (Vedie and Le Normand, 1984 (Ref 34)).

 In viticulture, iron sulfate is brought to the vineyard to fight against ferric deficiencies responsible for chlorosis. The fight against chlorosis is done by a contribution at the level of the ground or at the level of the leaves. It should be noted that the recommended soil intake is 1 kg / foot with 10 L of solvent (10%) (water or vinasse) whereas in foliar spray, iron sulfate is used at 1% for water volume of 300

L / ha.

 Fosetyl-aluminum (aluminum tris-o-ethylphosphonate), initially tested as antiperspirant, showed a protective effect vis-à-vis the grape downy mildew and is used against many species of

Phytophthora. It inhibits the germination of sporangia and the penetration of mycelium in the plant. It also limits the mycelial development and sporulation of Plasmopara viticola, the agent of downy mildew (Gouot, 2006 (Ref 35)). Phosphonates do not show fungicidal or fungistatic activity when used alone but inhibition of growth of Phaeomoniella chlamydospora is noted when associated with resveratrol (Mazullo et al., 2000 (Ref. 36)).

 The plant decomposes fosetyl-aluminum phosphorous acid and ethanol phosphonic acid during the absorption

(Leconte et al., 1988 (Ref 37)) (see Figure V).

 Fosetyl -aluminium stimulates the plant's defense system. As a preventative, it potentiates the plant's defense reactions and prepares the cells for subsequent attack by a pathogen. Phosphonates act on the phosphatic metabolism of fungi and generate more elicitors production (Leroux, 2000 (Ref 38)). This hyperelicitation causes an increased synthesis of signal molecules of the jasmonic acid, ethylene and salicylic acid type and an accelerated transcription of the defense genes resulting in the synthesis of phytoalexins (resveratrol, ε-viniferine), phenolic compounds, PR proteins such as chitinases and glucanases (Dercks and Creasy, 1989 (Ref 39), Lacombe, 2003 (Ref 40), Molina et al., 1998 (Ref 40), Nemestothy and Guest, 1990 (Ref 42), Serrano et al. , 1994 (Ref 43)).

Fosetylaluminium and phosphonic acid are ambimobile systemic products (Leconte et al., 1988 (Ref 37), Leroux, 2000).

(Ref 38); Leroux, 2003 (Ref 44)). The transport of fosetyl-aluminum, studied on cotyledons of Ricinus communis L., is similar to that of sucrose in the plant. It is detected in the phloem exudate 30 min after application. The maximum absorption occurs at pH 5.0. It is also detected in the root exudates of Lycopersicon esculentum Mil. and Persea indica L. in hydroponics (Ouimette and Coffey, 1990 (Ref. 45)).

 Various studies on the effect of fosetyl aluminum on vines with Esca have been carried out. Greenhouse trials have shown that five treatments made with fosetyl aluminum (Aliette 80 WP) on vines aged 2 years and then inoculated with Phaeomoniella chlamydospora and Phaeoacremonium aleophilum reduce the area of necrosis in the wood compared to untreated vines. Both fungi are isolated after treatment (Di Marco et al., 2000 (Ref 46), Mazullo et al., 2000 (Ref 47), Di Marco and Osti, 2005 (Ref 48)). Experiments combining fosetyl-aluminum with mancozeb, cimoxanil and / or copper oxychloride and carried out in the vineyard lead to a decrease in the number of vines expressing leaf symptoms. On the other hand, fosetyl-aluminum does not affect the number of dead feet (Di Marco and Osti, 2005 (Ref. 48)). These results demonstrate that this compound is not fungicidal against fungi involved in wood diseases.

There is therefore a real need to find new fungicidal compositions overcoming these defects, disadvantages and obstacles of the prior art, in particular a method for controlling the amounts of fungicides administered, to reduce costs, improve the effectiveness of fungicides to completely inhibit the development of pathogens. This also limits the amount of different active ingredients used to reduce the toxicity and environmental impact of fungicides. Description of the invention

The present invention specifically aims to meet the needs and disadvantages of the prior art by providing antifungal composition.

 The present invention relates to an antifungal composition comprising at least two of the compounds selected from the group consisting of cysteine, salicylic acid, iron (II) sulfate and fosetyl-aluminum.

The inventors have discovered, surprisingly and unexpectedly, that the composition of the invention has antifungal activity which is much greater than those of the state of the art and thus makes it possible to have an antifungal effect with a composition with amounts of weak compounds.

In addition, the inventors have demonstrated that the compounds used in the composition of the invention have a synergistic action, which can also reduce the appearance of "resistant".

Preferably, the composition of the invention comprises at least three compounds selected from the group consisting of cysteine, salicylic acid, iron (II) sulfate and fosetyl-aluminum.

 Even more preferably, the composition of the invention comprises cysteine, salicylic acid, iron (II) sulphate and fosetyl-aluminum.

 In the present "antifungal" means a molecule that acts against pathologies caused by fungi, bacteria, phytoplasmas, viruses, yeasts, preferably fungi and / or yeasts.

 [0037] Preferably, the composition is a fungicidal and / or fungistatic composition.

According to the present invention, by "fungicide" is meant a composition which is capable of causing the death of fungi, bacteria, phytoplasmas, viruses, yeasts, preferably fungi and / or yeasts.

 According to the present invention, "fungistatic" means a composition which is capable of stopping the growth of fungi bacteria, phytoplasmas, viruses, yeasts, preferably fungi and / or yeasts.

 In the present patent, "cysteine" is understood to mean a natural α-amino acid or a chemically synthesized amino acid which has a sulfhydryl or thiol (SH) group. For example, it may be L-cysteine of formula (IV) below:

D-cysteine, an amino acid derived from cysteine, for example acetylcysteine, carbocysteine or carboxymethylcysteine, or any other molecule derived from cysteine and which retains a cysteine radical.

According to the invention, the concentration of cysteine in the composition of the invention may be between 0.01 and 150 mM. Preferably, the concentration of cysteine is between 1 to 120 mM, even more preferably between 4 and 65 mM.

In the present text, "salicylic acid" means 2-hydroxybenzoic acid or a derivative thereof. For example, salicylic acid can be synthesized naturally, for example extracted from white willow bark or by chemical synthesis, for example by Kolbe (or Kolbe-Schmitt) reaction.

According to the invention, the concentration of salicylic acid in the composition of the invention may be between 0.01 and 4 mM. Preferably, the concentration of salicylic acid may be between 0.1 and 3 mM, more preferably between 0.4 and 1.5 mM. In the present text, "iron (II) sulphate" means the compound of formula FeSO ₄ or a derivative thereof. For example, it can act hydrated iron sulphate (FeSO ₄ , nH ₂ O), iron sulfate heptahydrate (FeSO ₄ JH ₂ O), or other ferrous salts such as iron chloride (FeCl ₂ ), iron acetate (Fe (CH ₃ CO ₂ ) ₂ ).

 According to the invention, the concentration of iron (II) sulfate in the composition of the invention may be between 0.01 and 160 mM. Preferably, the concentration of iron (II) sulphate may be between 3 and 120 mM, more preferably between 4 and 65 mM.

In the present "fosetyl-aluminum minium" is understood to mean ethyl hydrogen phosphonate aluminum, or aluminum tris (ethylphosphonate), or aluminum tris-O-ethylphosphonate, C ₆ Hi ₈ AIO ₉ P ₃ .

According to the invention, the concentration of fosetyl aluminum in the composition of the invention may be between 0.01 and 81 mM. Preferably, the concentration of fosetylaluminium may be between 1 and 55 mM, more preferably between 2 and 30 mM.

According to the invention, the upper limits of amounts of the different products have been determined during phytotoxicity tests. At these quantities of the first symptoms on the leaves appear. The ranges of values mentioned above are those that are recommended so that there are no symptoms on the leaves in general. These amounts may be slightly different or even higher depending on the sensitivity of the plant. The skilled person will easily know how to determine the quantities.

 According to the invention, the pH of the composition of the present invention can be modified to be acidic.

According to the invention, the modification can be carried out by any means known to those skilled in the art, for example by adding an acid solution, for example HCl, H ₂ CO ₃ . According to the invention, the pH of the composition may be, for example less than 7, for example from 4 to 6, preferably from 4.5 to 5.

 A pH of the composition may advantageously make it possible to further increase the antifungal efficacy.

According to the invention, the pH of the composition may be determined by any method known to those skilled in the art, for example by means of a pH meter.

 According to the invention, the composition may be for agricultural, veterinary and / or medical use.

According to the invention, the composition may be in liquid form, an emulsion, an ointment, a foam, a paste, a powder or a gel.

 The composition of the invention may be manufactured by any method known to those skilled in the art. It may be for example a simple mixture, leading preferably to a homogeneous composition.

 The invention also relates to a method of treating a plant comprising applying the composition of the present invention.

 The invention also relates to a method of treatment and / or preventive treatment comprising the application of the composition of the present invention to a plant, infected with a pathogen and / or on a plant that presents a risk of infection. infection with a pathogen.

 The application is as defined below.

 In the present "plant", it is understood any living plant fixed in the ground and whose upper part blooms in the air or in fresh water. For example, it may be woody plants, for example indigenous woody species, vines, for example vines Aligoté, Auxerrois, Cabernet Franc, Cabernet Sauvignon, Carmenère, Chardonnay, Colombard, Folignan, Folle Blanche, Gamay, Gewurztraminer, Grenache, White Jurançon, Malbec, Merlot, Meslier Saint

François, Montils, Muscadelle, Muscadet, Petit Verdot, Pinots, Poulsard, Riesling, Sauvignon, Savagnin, Select, Sémillon, Sylvaner, Syrah, Tokay, Trousseau, Ugni blanc.

 According to the invention by "treatment", it is understood, for example at least one application of the composition of the invention, for example in a form as described above, capable of preventing or stopping an infection, for example by stopping the growth of the pathogen and / or death of the pathogen.

 According to the invention "preventive treatment", it is understood for example at least one application of the composition of the invention, for example in a form as described above, capable of preventing or stopping an infection, for example by avoiding the occurrence of infection by a pathogen, by arresting the growth of the pathogen and / or death of the pathogen.

In the present invention by "pathogen" is meant an agent selected from the group consisting of fungi, bacteria, phytoplasmas, viruses and yeasts. For example, they may be fungi Phaeomoniella chlamydospora, Phaeocremonium aleopholilum, Fomitiporia mediterranea, Strereum hirsutum, Phellinus igniarius, Eutypa lata, Botryosphaeria obtusa, Neofusicoccum parvum, Botryosphaeria dothidea, Botryosphaeria stevensii, Phomopsis viticola, Plasmopara viticola, Botrytis cinerea, Erysiphe necator. Preferably, the fungi Phaeomoniella chlamydospora and Phaeocremonium aleopholilum.

According to the method of the invention, the application of the composition of the invention may be carried out by any means known to those skilled in the art. For example, the application of the composition can be carried out by sprinkling the plant, watering, misting, painting, immersion, dusting.

According to the invention, the application of the composition can be carried out with various devices, for example with any type of agricultural sprayer known to those skilled in the art. The skilled person will be able to easily determine the type of sprayer that can be used, for example it may be a towed pneumatic sprayer.

According to the invention, the application of the composition can be programmed, for example via an automatic programming device with a daily, weekly, monthly application.

 According to the invention, the application of the composition can be carried out in order to maintain a constant amount of composition at the plant level, for example the application can be carried out at least once a month, once per month. week, or once a day. Those skilled in the art will easily adapt the application of the composition according to the plant and / or the constant amount to maintain.

Other advantages may still appear to those skilled in the art on reading the examples below, illustrated by the accompanying figures, given for illustrative purposes.

Brief description of the figures

 FIG. 1 shows a diagram representing the mycelial growth in mm on a solid medium (YNB, 50 mM glucose, 40 mM glutamine (GIn), 10 mM MES, pH 5.5) as a function of the incubation time. More particularly, this diagram shows the kinetics of the growth of Phaeomoniella chlamydospora (Pch) and Phaeoacremonium aleophilum (Pal).

 FIG. 2 shows a diagram showing the mycelial growth in mm of Phaeomoniella chlamydospora at 18 days (A) and Phaeoacremonium aleophilum at 12 days (B) on a solid medium (YNB, 50 mM glucose, 40 mM glutamine, HEPES 10 mM, pH 4.2) as a function of the concentration of cysteine (Cys) in the medium.

FIG. 3 shows a diagram representing the mycelial growth in mm of Phaeomoniella chlamydospora at 18 days (A) and Phaeoacremonium aleophilum at 12 days (B) on a solid medium (YNB, 50 mM glucose, 40 mM glutamine, HEPES 10 mM, pH 4.2) as a function of the presence of different sulphates in the medium at the concentration of 40 mM: ammonium sulphate (NH ₄ 2 (SO ₄ )), potassium (K ₂ SO ₄ ), magnesium (MgSO ₄ ), iron (FeSO ₄ ) and copper (CuSO ₄ ).

FIG. 4 shows a diagram showing mycelial growth in mm of Phaeomoniella chlamydospora at 18 days (A) and Phaeoacremonium aleophilum at 12 days (B) on a solid medium (YNB, 50 mM glucose, 40 mM glutamine, HEPES 10 mM, pH 4.2) as a function of the concentration of iron sulphate (FeSO ₄ ) in the medium.

FIG. 5 shows a diagram representing mycelial growth in mm of Phaeomoniella chlamydospora at 18 days (A) and Phaeoacremonium aleophilum at 12 days (B) on a solid medium (YNB, 50 mM glucose, 40 mM glutamine, HEPES 10 mM, pH 4.2) as a function of different concentrations of iron sulphate (FeSO ₄ ), iron chloride (FeCl ₂ ) and iron acetate (Fe (CH ₃ CO ₂ ) ₂ ) in the medium.

FIG. 6 shows a diagram showing mycelial growth in mm of Phaeomoniella chlamydospora at 18 days (A) and Phaeoacremonium aleophilum at 12 days (B) on a solid medium (YNB, 50 mM glucose, 40 mM glutamine, HEPES 10 mM, pH 4.2) as a function of different concentrations of salicylic acid (AS) in the medium.

FIG. 7 is a diagram showing the mycelial growth in mm of Phaeomoniella chlamydospora at 18 days (A) and Phaeoacremonium aleophilum at 12 days (B) on a solid medium (YNB, 50 mM glucose, 40 mM glutamine, HEPES 10 mM, pH 4.2) as a function of different fosetyl-aluminum concentrations in the medium.

[0076] FIG. 8 is a diagram showing the effect of the association of cysteine with different salicylic acid concentrations on mycelial growth in mm of Phaeomoniella chlamydospora at 18 days (A) and Phaeoacremonium aleophilum at 12 days (B). on a solid medium (YNB, 50 mM glucose, 40 mM glutamine, 10 mM HEPES, pH 4.2). More particularly, the different conditions incubation are 5 mM cysteine (Cys) and different concentrations of salicylic acid (AS), used alone (I) or associated (Ma).

FIG. 9 is a diagram showing the effect of the combination of cysteine and salicylic acid with different concentrations of iron sulfate on mycelial growth in mm of Phaeomoniella chlamydospora at 18 days (A) and Phaeoacremonium aleophilum at 12 days (B) on a solid medium (YNB, 50 mM glucose, 40 mM glutamine, 10 mM HEPES, pH 4.2). More particularly, the different incubation conditions are 5 mM cysteine (Cys), 0.5 mM salicylic acid (AS) and various iron sulphate (FeSO ₄ ) concentrations, used alone (I) or combined with 2 (II) or 3 (III).

FIG. 10 is a diagram showing the effect of the combination of cysteine, salicylic acid, iron sulfate and fosetyl aluminum on mycelial growth in mm of Phaeomoniella chlamydospora at 21 days (A). ) and Phaeoacremonium aleophilum at 13 days (B) on a solid medium (YNB, 50 mM glucose, 40 mM glutamine, 10 mM HEPES, pH 4.2). More particularly, the different incubation conditions are 5 mM cysteine (Cys), 0.5 mM salicylic acid (AS), 5 mM iron sulfate (FeSO ₄ or Fe) and 2.7 mM fosetyl- aluminum (Fos Al), used alone (I) or associated with 2 (II), 3 (III), or 4 (IV).

FIG. 11 is a diagram showing the effect of the combination of cysteine, salicylic acid, iron sulfate and fosetyl-aluminum on mycelial growth in mm of Phaeomoniella chlamydospora at 18 days (A). ) and Phaeoacremonium aleophilum at 12 days (B) in a liquid medium (YNB, 50 mM glucose, 40 mM glutamine, 10 mM HEPES, pH 4.2). More particularly, the different incubation conditions are 5 mM cysteine (Cys), 0.5 mM salicylic acid (AS), 5 mM iron sulfate (FeSO ₄ or Fe) and 2.7 mM fosetyl- aluminum (Fos Al or Fos), used alone (I) or combined with 2 (II), 3 (III), or 4 (IV).

FIG. 12 is a diagram showing the effect of the combination of cysteine, salicylic acid, iron sulphate and fosetyl-aluminum on spore germination of Phaeomoniella chlamydospora (A) and Phaeoacremonium aleophilum (B) in liquid medium (YNB, glucose 50 mM, glutamine 40 mM, HEPES 10 mM, pH 4.2) after 5 days and observed by an OD modification at 595 nm. More particularly, the different incubation conditions are 5 mM cysteine (Cys), 0.5 mM salicylic acid (AS), 5 mM iron sulfate (FeSO ₄ or Fe) and 2.7 mM fosetyl- aluminum (Fos Al or Fos), used alone (I) or combined with 2 (II), 3 (III), or 4 (IV).

FIG. 13 shows a reversibility curve of the mycelial growth in mm of Phaeomoniella chlamydospora (A, B and C) and of Phaeoacremonium aleophilum (A 'and B') treated with various molecules of the invention during 8 (A). , A '), (B, B') and 21 (C) days and after passage through a nutrient medium without antifungal molecules (YNB, 50 mM glucose, 40 mM glutamine, 10 mM HEPES, pH 4.2). More particularly, the different incubation conditions are 5 mM cysteine (Cys), 0.5 mM salicylic acid (AS), 5 mM iron sulfate (FeSO ₄ or Fe) and 2.7 mM fosetyl- aluminum (Fos Al or Fos), associated with 2 (II), 3 (111), or 4 (IV).

FIG. 14 shows a diagram representing the mycelial growth in mm of different strains of Phaeomoniella chlamydospora at 18 days (A) and Phaeoacremonium aleophilum at 12 days (B) as a function of the presence of 5 mM cysteine (Cys) 0.5 mM salicylic acid (AS), 5 mM iron sulfate (FeSO4) and 2.7 mM fosetyl aluminum (Fos Al) on a solid nutrient medium (YNB, 50 mM glucose, 40 mM glutamine). , 10 mM HEPES, pH 4.2).

FIG. 15 shows photographs of vine leaves two weeks after foliar treatment with different compositions of the invention on Cabernet Sauvignon cuttings (A and B) and after four treatments, carried out 14 days apart, on white Ugni cuttings (C, D, E, F). More particularly, this diagram makes it possible to verify the innocuity of the invention on the vine. A and C: untreated cuttings, D: Treatment with cysteine (5 mM), salicylic acid (0.5 mM) and wetting agent (Etaldyne 95, Fertiligen), E: treatment with iron sulfate (5 mM), fosetyl-aluminum (2.7 mM) and a wetting agent (Etaldyne 95, Fertiligen), F: treatment with cysteine (5 mM), salicylic acid (0.5 mM), iron sulfate (5 mM), fosetyl aluminum (2.7 mM) and a wetting agent (Etaldyne 95, Fertiligène).

 Figure 16 shows the prints of shoots from cuttings inoculated with pathogens (Phaeomoniella chlamydospora) and performed 14 days after 1, 2, 3 or 4 treatments of said cuttings in the presence of different compositions of the invention. More particularly, the fingerprints are revealed with antibodies to P. chomydospora. A: uninoculated cuttings, B: untreated cuttings, C: cuttings treated with salicylic acid (0.5 mM), cysteine (5 mM) and a wetting agent (Etaldyne 95, Fertiligen), D: treated cuttings with iron sulphate (5 mM), fosetyl aluminum (2.7 mM) and a wetting agent (Etaldyne 95, fertilizer), E: cuttings treated with iron sulphate (5 mM), fosetyl aluminum (2 , 7 mM), salicylic acid (0.5 mM), cysteine (5 mM) and a wetting agent (Etaldyne 95, Fertiligen), +: positive control corresponding to 5 ng of proteins (F2) of Phaeomoniella chlamydospora and arranged for each revelation.

[0085] Figure 17 shows the prints of shoots from cuttings inoculated with a pathogen (Phaeoacremonium aleophilum) made 14 days after 2 or 4 treatments of said cuttings in the presence of different compositions of the invention. B: untreated cuttings, C: cuttings treated with salicylic acid (0.5 mM), cysteine (5 mM) and a wetting agent (Etaldyne 95, Fertiligenic), D: cuttings treated with iron sulfate ( 5 mM), fosetyl aluminum (2.7 mM) and a wetting agent (Etaldyne 95, Fertiligen), E: cuttings treated with iron (5 mM) sulfate, fosetyl-aluminum (2.7 mM), salicylic acid (0.5 mM), and cysteine (5 mM) and a wetting agent (Etaldyne 95, Fertiligen). +: positive control corresponding to 5 ng of proteins (F2) of Phaeoacremonium aleophilum and arranged for each revelation.

EXAMPLES

In the examples below, the various measurements or experiments were performed according to the protocols described below. Biological materials and culture conditions

Fungal materials

 Strains of Phaeomoniella chlamydospora W. Gams, Crous and Phaeoacremonium aleophilum W. Gams, Crous, MJ. Wingfield, L. Mugnaï used have been isolated and referenced in the framework of the European project "Mastery of the Esca and respect for the environment". They were provided by P. Larignon (ITV of Nîmes) and J. -P. Péros (INRA of Montpellier). Among the different strains, two were mainly used in our research: PC-PC 37 and PA-PC 24. These strains were isolated at the Fougerat foundation in the town of Graves (16), from vine cv. Ugni white with Esca. Table 1 presents the references of the strains used as well as the commune and grape variety on which they were isolated.

Table 1: Reference and origin of the different strains used

 (Borie et al., 2002 (Ref 49))

 [0088] Other fungi were also used to test the specificity of the antibodies: Alternaria sp., Botryosphaeήa obtusa, Neofusicoccum parvum, Botrytis cinerea, Cladosporium sp., Cylindrocarpon destructans, Epicoccum sp., Eutypa lata, Fusaήum sp., Paecilomyces sp., Penicillium sp., Pestalotia sp., Phoma sp., Phomopsis viticola, Pullularia sp., Trichoderma atroviride, Trichoderma harzianum and Verticillium chlamydosporum. Strains were identified and provided by P. Larignon (I.F.V. Rhone-Mediterranean Pole).

In vitro culture conditions of fungi.

Composition of the basic nutrient medium

 The fungi were cultured on a totally synthetic medium consisting of YNB (Yeast Nitrogen Base without amino acid and ammonium sulfate; DIFCO 233520) at 1.7 g / L, glucose at 50 mM.

(Sigma G5767) and 40mM glutamine (Sigma P0380). The pH was adjusted to 5.5 or 4.2 for the test of molecules, and the medium is autoclaved

15 min at 110 ^° C.

 Solid medium

 The above medium was solidified with agar at 20 g / L (Sigma

A1296). The strains were grown in a 9 cm diameter petri dish in the dark at 25 ° C, an average temperature favorable for the development of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum. The strains were stored as mycelial disks in sterile water in the dark at 4 ° C.

 Liquid medium

The cultures in liquid medium were made in Erlenmeyer flask containing 300 ml of the medium defined above, inoculated with three agar plates 1 cm in diameter on which the inoculum developed.

Biological activity test

 The tests with the different molecules in solid medium (nitrogen sources, carbon sources and antifungal molecules) were made in 6-well boxes of 35 mm diameter. The medium was buffered to pH 4.2 with 10 mM HEPES for the antifungal molecules. The molecules to be tested were added before solidification of the medium. The medium was inoculated with a 4 mm diameter mycelial disk from a solid mother culture.

Tests on the effect of antifungal molecules on the germination of spores were made in 96-well plate. The basic nutrient medium (YNB or "Yeast Nitrogen Base"), 50 mM glucose, 40 mM glutamine, 10 mM HEPES pH 4.2) is autoclaved for 15 min at 110 ^° C. The various compounds to be tested have been added to the medium. cooled. 200 μL of the test medium are deposited per well and inoculated with 50,000 spores. The microplates were stirred (160 rpm) at 22 ° C and in the dark. The absorbance was measured at 595 nm every two or three days using a microplate reader (Sunrise reader, Tecau). Before reading the absorbance, the plate was stirred 5 s in order to homogenize the contents of the wells. Measurement of growth parameters of fungi

In solid medium

 The diameter of the development of the fungus was measured using a ruler graduated every two days for one month.

In liquid medium Three parameters were monitored during the development of fungi in a liquid medium: the dry mass of mycelium, the pH of the culture medium and the concentration of secreted proteins in the medium.

At the time chosen, the liquid culture medium was centrifuged for 30 min at 20000 xg at 4 ° C to pellet the mycelium and spores. The pellet obtained after centrifugation was dried in an oven at 60 ^° C. for 24 hours in order to determine the dry mass of the fungus. The supernatant was recovered and passed through a filter whose pores are 0.22 μm in diameter (Whatman) in order to completely eliminate the spores and the remaining mycelium. The pH of the supernatant was then measured using a pH meter (pH 526, WTW). The protein concentration was measured according to the technique of Bearden (1978) (Ref 50). This technique consists in adding 100 μl of Triton X-100 at 0.1% (w / v) to x μL containing the proteins to be assayed, x corresponds to 100 μL for the determination of the proteins contained in the mushroom culture medium, 10 μL for the determination of the proteins of fraction F2. After 10 min of incubation, the volume is supplemented to 1 mL with milliQ water. 1 ml of Bearden reaction medium is added. The reading of the absorbance is carried out at 595 nm after 10 minutes of waiting. The standard range is performed with serum bovine albumin (BSA).

 Cultivation conditions of plant material

 The vines Vitis vinifera L. were white Ugni (variety mainly used for the production of Cognac since it represents 95% of the encépagement in white) and Cabernet Sauvignon (black grape variety).

The plants were obtained by cuttings from vines harvested from the vineyard. For this, a cutting formed of two nodes separated by an internode was removed. The lower bud was removed using a pruner. The cuttings thus obtained were placed in a pot containing potting soil supplemented with pozzolan and was watered every three days with water. When the roots and leaves have been sufficiently developed (4 leaves), the cuttings are then watered with a nutritive solution composed of potassium nitrate, monoammonium phosphate, monopotassium phosphate, magnesium sulphate, calcium nitrate, trace elements (B, Cu, Mn, Mo, Zn) and Séquestrène (registered trademark) (Syngenta). Plants were illuminated 16 h per day mainly with natural light and sodium vapor lamps (Philips SON-T, 423 W) when the amount of light radiation was too low. The average temperature was 22 ° C ± 2 ° C and the humidity was 70% ± 10%.

Inoculation of cuttings

 The cuttings were inoculated selectively in a cavity 1 cm deep made in the apical part, with a 4 mm diameter agar disc on which developed the mycelia of Phaeomoniella chlamydospora (strain PC-PC37). or Phaeoacremonium aleophilum (strain PA-PC 24). Hydrophilic cotton was then deposited on the agar plate and the whole was surrounded by parafilm and then placed in the ground. Treatment on cuttings

 The cuttings were treated by foliar spraying until runoff. The molecules tested were L-cysteine (Sigma), salicylic acid (Sigma), iron sulfate FeSθ4 heptahydrate (7H2O) (Sigma) and fosetyl aluminum (Aliette 80, KB garden). A wetting agent (Etaldyne 95, Alkylphenoloxyethylene, Fertiligen) was used at a concentration of 0.5 mL / L to optimize the contact area between the composition and the sheet. The composition was sprayed on the plant at pH 4.5.

Plants in the vineyard

The plants used in natural conditions came from a plot of Ugni white planted in 1974 located in Grande Champagne. The vines were driven in double flat Guyot. The rows were separated by 3 m and the stock of 1, 3 m. The density was 2564 ceps per hectare.

Biochemical methods

Antibody synthesis

 Two syntheses of antibodies were carried out: one directed against the proteins secreted by Phaeomoniella chlamydospora and the other against the proteins secreted by Phaeoacremonium aleophilum. Two rabbits were immunized for each antibody synthesis. The secreted proteins in the culture medium were lyophilized and suspended in milliQ water and then the sample was passed on Sephadex G25 resin (PD 10, GE Healthcare) in order to eliminate the salts contained in the nutrient medium. . The fraction containing the proteins was lyophilized again and then suspended in PBS (saline buffer or "phosphate buffer saline") (4.4 mM NaH 2 PO 4, 16 mM Na 2 HPO 4, 300 mM NaCl, pH 7.4). . The proteins were assayed according to the Bearden technique. A quantity of 1 mg of proteins to be injected per rabbit was necessary in order to carry out the immunization.

The synthesis of antibodies was entrusted to the company Agrobio (La Ferté Saint Aubin). The protocol for obtaining the antibodies lasts 77 days. Four antigen injections of 250 μg of protein per rabbit are performed. Samples at D49, D68 and D77 (corresponding to the final sample) are taken.

The antibodies obtained during the final sample were then purified by protein affinity chromatography A. The A proteins have the property of interacting specifically with the Fc part of the mammalian immunoglobulins.

Hybridization of antibodies - dot-blot method This technique was used to detect circulating proteins in the plant through an antibody directed specifically against these proteins.

 [00106] A print of a shoot was made immediately after section, applying the sectioned area in contact with the membrane (HybondTM-ECLTM, GE Healthcare) for 10 seconds. Protein binding was performed by allowing the membrane to dry for 30 min at room temperature. The membrane was then rinsed for 5 min in 20 mM PBS pH 7.4 supplemented with 0.2% (v / v) Tween 20 and incubated overnight at 4 ° C in saturation buffer ( PBS 20 mM pH 7.4, milk protein 3%, Tween 20 0.2%) to block nonspecific sites. The rest of the protocol (antibody hybridization and revelation) was substantially identical to that of the western blot described above. The membrane was incubated for 120 minutes in contact with the primary antibody diluted in 1/250 saturation buffer. Three rinses of ten minutes with the saturation buffer were then made. The membrane was then incubated in the peroxidase-coupled secondary antibody, diluted 1/2000 with saturation buffer for 90 minutes. The membrane was rinsed for 10 minutes with the saturation buffer and then twice for 10 minutes with 20 mM PBS pH 7.4 milk protein 3% and finally twice for 5 minutes with 20 mM PBS pH 7.4.

Revelation

The revelation of the antigen-antibody complexes formed is carried out using a kit (ECL kit RPN 2106, GE Healthcare) containing the peroxidase substrate conjugated to the secondary antibody. The interaction between the substrate and the enzyme causes the emission of light capable of impressing a photographic film (Hyperfilm, GE Healthcare). The film was then treated with a developer (llford llfotec LC 29) diluted 1/10 and then with a fixative (llford Rapid Fixer) diluted 1/10. EXAMPLE 1 Test of compounds on Phaeomoniella chlamydospora and Phaeoacremonium aleophilum

1. Effect of sulfur molecules

 1.a. Effect of csetein

 The inventors have shown that cysteine slows the growth of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum (Figure 2). An identical observation has been observed with other fungi such as Inonotus obliquus (Kahlos and Tikka, 1994 (Ref22)), Alternaria sp. (Daigle and Cotty, 1991 (Ref. 24)), Botrytis cinerea (Leroux, 1994 (Ref. 51)) and in previous work on Eutypa lata (Amborabé et al., 2005 (Ref 52), Octave et al., 2005 (Ref 23)).

The effect of different concentrations of cysteine on the fungal growth of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum was studied. The results obtained are shown in Table 2 below and in Figure 2.

Table 2: Results Table for Cysteine

-: no inhibition; MNB: Basic nutrient medium These results demonstrate that the mycelial growth of the two fungi is inhibited by cysteine at concentrations of 5 mM and 10 mM despite the presence of another nitrogen source (40 mM glutamine) in the nutrient medium. The slowing down of mycelial development is therefore linked to an inhibitory effect of cysteine and not to a problem of assimilation by fungi.

 As demonstrated herein, cysteine alone inhibits only the development of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum at relatively high concentrations. The sensitivity of both fungi to cysteine is different.

1.b. Effect of sulphates

 Among the different nitrogen sources slowing the development of fungi, the inventors have observed that ammonium sulfate (40 mM), used as the sole nitrogen source, slows the growth of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum. (results not shown)

[00113] In order to know if other sulphates have the same property as ammonium sulphate, the sulphates of potassium, magnesium, ferrous iron (Fe ²⁺ ) and copper have been tested at a concentration of 40 mM. and added to the Basic Nutrient Medium (MNB).

The results are shown in Table 3 below and in Figure 3. Table 3: Table of results for ammonium sulphate ((NH _{4) 2} SO ₄ ), potassium (K ₂ SO ₄ ) and magnesium (MgSO ₄ ).

 -: no inhibition

Iron sulphate and copper sulphate completely inhibit the mycelial growth of Phaeomoniella chlamydospora and

Phaeoacremonium aleophilum at concentrations of 40 mM.

 Copper sulfate is already widely used (Bordeaux mixture) in viticulture to fight against various diseases (mildew, bacterial, bacterial necrosis).

 Nevertheless, the large use of copper (family of heavy metals) makes the soils more and more polluted (Brun and Geoffrion,

2003 (Ref 53)). In addition, copper sulphate can not be mixed with fosetyl aluminum. As a result, copper sulphate was not retained.

1 C. Effect of different concentrations of iron sulphate (FeSO4) on the mccelian growth

 The results are shown in Table 4 and in Figure 4 (A and B).

Table 4: Results for different concentrations of iron sulphate (FeSO ₄ ).

As demonstrated above, iron sulfate further inhibits the growth of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum at concentrations of 5 mM and 10 mM.

Sulfur does not seem to play a role in the effectiveness of the various sulphates since there is no inhibition of the growth of fungi in the presence of ammonium sulfate, potassium and magnesium. In order to know if the effectiveness of the molecule comes from iron, other molecules with iron in ferrous form (Fe ²⁺ ) were tested: iron chloride (FeCl2) and iron acetate (Fe (CH3CO2) 2) at concentrations of 5, 10 and 20 mM.

 [00121] Figure 5A shows that the growth of Phaeomoniella chlamydospora is inhibited in the presence of sulfate, chloride and iron acetate in a dose dependent manner. The inhibition caused by iron chloride is comparable to that obtained with iron sulfate irrespective of the concentration applied. The inhibition due to iron acetate is slightly decreased to 5 mM. Indeed, it is 40% while that obtained with iron sulfate is 65%. At the concentration of 10 mM, fungal development is completely inhibited with iron acetate, which is not the case with sulfate and iron chloride. At 20 mM, the growth of Phaeomoniella chlamydospora is totally inhibited irrespective of the ferrous molecule tested.

[00122] FIG. 5B shows that sulfate, chloride and iron acetate also inhibit the development of Phaeoacremonium aleophilum in a dose-dependent manner. At 5 mM, the growth of Phaeoacremonium aleophilum is more inhibited with iron sulfate than with chloride and iron acetate. At the concentration of 10 mM, the inhibition caused by chloride and iron sulfate is 75%. On the other hand, the inhibition observed with iron acetate is lower since it reaches 45%. At the concentration of 20 mM, the fungal development is totally inhibited with chloride and iron acetate. Phaeoacremonium aleophilum develops slightly in the medium containing iron sulphate.

As demonstrated above, the experiments carried out with various ferrous salts shows that the effectiveness of the molecule can therefore be attributed to the iron atom since all the ferrous molecules tested have the same capacity to inhibit mycelial development. Phaeomoniella chlamydospora and Phaeoacremonium aleophilum.

2. Effect of salicylic acid

The results presented in Figure 6 A show that salicylic acid does not inhibit the fungal development of Phaeomoniella chlamydospora at concentrations of 0.05 mM and 0.1 mM. At 0.5 mM, growth was inhibited by 10% and at 1 mM by 28%.

Salicylic acid therefore has little effect on the mycelial growth of Phaeomoniella chlamydospora. Higher concentrations of salicylic acid have not been tested because at high concentrations, phytotoxicity problems can be noted.

[00126] Figure 6 B shows that the development of Phaeoacremonium aleophilum is slightly inhibited to 0.05 mM. At 0.5 mM growth was inhibited by 50% and at 1 mM it was completely inhibited.

 As demonstrated in this experiment, salicylic acid alone weakly inhibits the development of Phaeomoniella chlamydospora and more strongly that of Phaeoacremonium aleophilum.

3. Effect of aluminum fosetyl

 Different fosetyl-aluminum concentrations were tested on the growth of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum (Figure 7).

The results presented in FIG. 7A show that fosetyl -aluminium has no effect on the growth of Phaeomoniella. chlamydospora at 0.3 mM. The mycelial development of Phaeomoniella chlamydospora is weakly inhibited at 1 mM (6%) and 1.7 mM (14%). At higher concentrations, the inhibition is greater: it reaches 46% at 2.7 mM and 86% at 3.4 mM.

[00130] Figure 7 B shows that fosetyl-aluminum has no effect at 0.3 mM and 1 mM on the growth of Phaeoacremonium aleophilum. A weak inhibition of the order of 6% is observed at the concentration of 1.7 mM. At higher concentrations growth is inhibited by 30% at 2.7 mM and 45% at 3.4 mM.

As demonstrated above, fosetyl-aluminum alone inhibits the mycelial growth of Phaeomoniella chlamydospora and more weakly that of Phaeoacremonium aleophilum. The sensitivity of Phaeoacremonium aleophilum to fosetyl fosetyl-aluminum is therefore lower than that of Phaeomoniella chlamydospora.

EXAMPLE 2 Test of the Fungicidal Activity of Examples of the Composition of the Invention on Phaeomoniella chlamydospora and Phaeoacremonium aleophilum

 In this example, for each molecule, the concentration used is that for which growth has been approximately 50% inhibited. Then, the molecules were associated with each other by two then by three and all together.

 An analysis of the synergy of the mixture of the different molecules was carried out. This analysis consists of comparing the efficiency of the experimentally observed mixture (E ') with the calculated theoretical efficiency.

(E) thanks to the formula of Colby (1967) (Ref 54), this knowing the effectiveness of each of the products.

 E = (x + y) - (x, y) / 100

E: theoretical efficiency percentage of the association of the ^1st product and the ^2nd product

x: percentage efficacy ¹ product are: efficiency percentage of the ^2nd product

 When there is a synergy of the products, the experimental efficiency (E ') of the mixture is greater than the calculated theoretical efficiency (E). 1. Cysteine / salicylic acid combination

 Inhibition was measured on a culture in solid medium. The medium and the culture conditions are described in the previous section.

 Cysteine, at a concentration of 5 mM, and salicylic acid, at different concentrations ranging from 0.05 mM to 1 mM, were tested alone or in combination to study their impact on mycelial growth.

 [00137] FIG. 8A shows that the growth of Phaeomoniella chlamydospora is inhibited by 98% (E ') in the presence of the 1mM cysteine / salicylic acid mixture, which is greater than the effect alone induced by each compound and addition of the effects produced by each compound separately. The calculation by the Colby formula (E = 60%) demonstrates that it is a synergistic effect.

 Similarly, for Phaeoacremonium aleophilum (FIG. 8B), the combination of the two cysteine / salicylic acid molecules makes it possible to reduce mycelial growth. This effect is in each case due to a synergistic effect of the molecules (E '= 35> E = 34 for cysteine / salicylic acid 0.05 mM, E' = 52> E = 45 for cysteine / salicylic acid 0.1 mM ;

E = 80%> E = 64% for 0.5 mM cysteine / salicylic acid; E = E = 100 for 1mM cysteine / salicylic acid).

The composition comprising cysteine and salicylic acid inhibits the growth of pathogens and has a synergistic effect.

2. Composition comprising iron sulfate, cysteine and salicylic acid

2.a. Cysteine / iron sulfate: The results presented in FIG. 9 show that the combination of cysteine and iron sulfate at 5, 10 and 20 mM makes it possible to reduce the growth of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum.

FIG. 9B shows that the inhibition of the growth of Phaeoacremonium aleophilum due to the combination of cysteine and iron sulfate (at the various concentrations tested) is greater than the inhibition caused by the compounds alone. A synergistic effect is observed in each case since the experimental efficiency (65%, 87%, 94%) is greater than the theoretical efficiency (55%, 86%, 93%) (FIG. 9B (Hb)).

2.b. Salicylic acid / iron sulfate:

 The composition comprising 0.5 mM salicylic acid with iron sulfate at 5, 10 and 20 mM completely inhibits the fungal development of both fungi irrespective of iron sulfate concentration. A synergistic effect was observed for each concentration with both fungi.

 For Phaeomoniella chlamydospora, the experimental efficiency E '(100%) is greater than the theoretical efficiency E which is 67% for the salicylic acid 0.5 mM / iron sulfate 5 mM, 94% for the 0.5 mM salicylic acid / 10 mM iron sulfate combination and 100% for the salicylic acid / 20 mM iron sulfate combination ((Figure 9 A (Hc)).

 For Phaeacremonium aleophilum, the experimental efficiency E '(100%) is greater than the theoretical efficiencies E which are respectively 69%, 91% and 95% for each of the associations with an increasing concentration of iron sulphate (FIG. 9 B (Hc)).

This example clearly demonstrates that the composition of the invention is an antifungal composition having a synergistic effect with results much higher than those observed with the compositions of the state of the art. 2.c. Iron sulphate / cysteine / salicylic acid:

 The experimental results of this example are presented in FIGS. 9A and 9B and demonstrate that the mixture of the three molecules at the studied concentrations (III) completely inhibits the mycelial growth of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum whatever the concentration of added iron sulphate.

These results confirm the synergistic effect of the composition of the invention.

3. Composition comprising fosetyl-aluminum, cysteine, salicylic acid, iron (IV) sulphate

 The compounds tested include 2.7 mM fosetyl aluminum, 5 mM cysteine, 0.5 mM salicylic acid and 5 mM iron sulfate. The molecules were tested associated by two, three or four. a) Inhibition of mushroom growth with compositions comprising two or three compounds

 Table 5 below and Figure 10 A (II and III) and B (II and III) represent the experimental results obtained on fungi grown on solid medium with different combinations.

Table 5: Table of results of different associations

The experimental results obtained presented in FIG. 11A (II and III) and B (II and III) demonstrate the total inhibition of the growth of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum in a liquid medium with the compositions comprising salicylic acid and iron sulphate.

 Certain combinations of molecules do not completely inhibit the fungal development of Phaeoacremonium aleophilum in a liquid medium (FIG. 11B (II and III)). Residual growth is observed for mixtures of salicylic acid / fosetyl-aluminum (3.5%), cysteine / salicylic acid / iron sulfate (2%), cysteine / salicylic acid / fosetyl-aluminum (0.5%), acid salicylic acid / iron sulfate / fosetyl-aluminum (10%).

The culture conditions of the fungus can therefore modify the effectiveness of the treatment applied with compositions comprising two or three compounds. b) Inhibition of fungal growth with compositions comprising four compounds As demonstrated in this experiment (FIG. 10A and B (IV) and FIG. 11A and B (IV)), the growth of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum in a liquid medium is totally inhibited in the presence of the 5mM cysteine mixture. 0.5 mM salicylic acid / 5 mM iron sulphate / 2.7 mM aluminum fosetyl in a liquid and solid medium.

 As demonstrated in this example, a composition according to the invention makes it possible to inhibit the growth of pathogens.

Example 3 Effect of the Composition on Spore Germination

The various molecules used alone or in a mixture were tested in order to know if the composition has an effect on the germination of the spores of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum.

[00156] FIG. 12A demonstrates that all the compositions tested have an inhibitory effect on germination.

 More particularly, mixtures of salicylic acid / fosetyl-aluminum (II), cysteine / salicylic acid / iron (III) sulphate, salicylic acid / iron sulphate / fosetyl-aluminum (III), cysteine / salicylic acid / sodium sulphate iron / fosetyl-aluminum (IV) completely inhibit spore germination of Phaeomoniella chlamydospora. On the other hand, it is not completely inhibited in the presence of the salicylic acid / iron (II) sulfate mixture, whereas its growth is completely inhibited in solid and liquid media. The salicylic acid / iron sulphate mixture thus seems to have mainly an effect on the growth of the mycelium and a lesser effect on the germination of the spores. In the same way, spore germination is not totally inhibited in the presence of cysteine / salicylic acid / iron (III) sulphate, whereas the mycelium develops weakly in a solid medium under the same conditions.

[00158] FIG. 12B shows that the spores of Phaeoacremonium aleophilum are completely inhibited in the presence of salicylic acid / iron sulfate, salicylic acid / fosetyl- aluminum (II), cysteine / salicylic acid / iron sulfate, cysteine / salicylic acid / fosetyl-aluminum (III), cysteine / salicylic acid / iron sulfate / fosetyl-aluminum (IV). Mycelial growth is totally inhibited with the same mixtures in solid medium and very strongly in liquid medium.

 Several mixtures of molecules totally or partially inhibit spore germination of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum.

 These results further demonstrate that the composition of the invention used during the period of sporulation of fungi could prevent new contaminations by inhibition of sporulation.

Example 4: Measurement of the fonqistatic effect or fungicidal effect of the composition

 In order to measure the fungicidal effect and / or the fungistatic effect of the composition, mycelial implants used to seed the nutrient medium containing the various test molecules in a solid medium were deposited on a nutritive medium without antifungal molecules after 8, 15 and 21 days of contact with the antifungal molecules according to the protocol described above.

 The resumption of growth indicates that the mixture has a fungistatic effect while zero growth indicates that the treatment has a fungicidal effect.

The results also make it possible to determine the necessary contact time between the fungus and the treatment so that the fungus is killed and the germination of the conidiospores is totally inhibited. This measurement was carried out by the growth of the fungus in the presence of various combinations and then after passing on a nutrient medium free of antifungal molecules after 8, 15 or

21 days of treatment. The results presented in Figure 13 A, B and C and in Table 7 below show the fungicidal or fungistatic effect of the compositions.

 After 8 days of contact (FIG. 13A) with the composition: salicylic acid / iron sulfate, salicylic acid / fosetyl-aluminum, iron sulfate / fosetyl-aluminum, cysteine / salicylic acid / fosetyl-aluminum, cysteine / Iron sulfate / fosetyl-aluminum, Phaeomoniella chlamydospora develops after passage over the medium without antifungal molecules. These different associations have a fungistatic effect.

In the case of cysteine / salicylic acid / iron sulfate, salicylic acid / iron sulfate / fosetyl-aluminum and cysteine / salicylic acid / iron sulfate / fosetyl-aluminum combinations, the passage of mycelial implants on a medium without molecules. antifungals does not restore the growth of the fungus. These compositions therefore have a fungicidal effect.

 After 15 days (FIG. 13B), the mycelial implants in contact with the iron sulfate / fosetyl-aluminum, cysteine / salicylic acid / fosetyl-aluminum, cysteine / iron sulfate / fosetyl-aluminum combinations respectively develop , 7 and 3 days after passage on a medium without molecules, which indicates that these associations have a fungistatic effect. Mycelial implants in contact with other mixtures (salicylic acid / iron sulfate, cysteine / salicylic acid / iron sulfate, salicylic acid / iron sulfate / fosetyl-aluminum, cysteine / salicylic acid / iron sulfate / fosetyl-aluminum) do not resume their development. These compositions therefore have a fungicidal effect.

13C shows that only the mycelium of Phaeomoniella chlamydospora in contact for 21 days with the cysteine / iron sulfate / fosetyl-aluminum mixture resumes its growth after passage on medium without antifungal molecules after a latency period of 7 days . All other tested associations have a fungicidal effect after 21 days of contact. [00169] Figures 13 A 'and B' show the growth of Phaeoacremonium aleophilum after contact with different combinations for 8 and 15 days and transfer to a medium lacking antifungal molecules. After 8 days of contact, the mycelial growth is totally inhibited in the presence of the following combinations: salicylic acid / fosetyl-aluminum, cysteine / salicylic acid / fosetyl-aluminum, salicylic acid / iron sulfate / fosetyl-aluminum, cysteine / salicylic acid / iron sulfate / fosetyl-aluminum. These mixtures have a fungicidal effect. On the other hand, the inhibition of growth observed with the salicylic acid / iron sulfate and cysteine / salicylic acid / iron sulfate mixtures is only maintained 5 and 7 days after the passage on a medium lacking antifungal molecules. After this time, the fungus resumes normal growth indicating that these associations have a fungistatic effect. After 15 days of treatment, the development of Phaeoacremonium aleophilum in the presence of the various combinations is totally inhibited.

 Part of the results are summarized in Table 6 below.

 Table 6: Fungicidal or fungistatic effects of different mixtures on the growth of Phaeomoniella chlamydospora and

Phaeoacremonium aleophilum after 8, 15 or 21 days of contact

EXAMPLE 5 Effect of the Composition on Different Strains of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum

The developed mixture was developed on a strain of Phaeomoniella chlamydospora (PC-PC 37) and a strain of Phaeoacremonium aleophilum (PA-PC 24).

 The composition was tested on other strains of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum in solid medium to ensure that it inhibits the growth of other strains of fungi grown in vitro.

 The strains and the conditions of realization of this experiment are presented in the material part and method previously described.

The different strains of Phaeomoniella chlamydospora tested were PC-PC 37, PC-PC 3, PC-PC 21, PC-PC 32.

In the presence of the cysteine / salicylic acid mixture, the inhibition of mycelial growth is 44% for PC-PC 37, 36% for PC-PC 3, 41% for PC-PC 21 and 27% for PC-PC 32. The treatment with the iron / fosetyl-aluminum sulfate combination totally inhibits the development of PC-PC 37 and PC-PC strains 3 whereas the development of the strain PC-PC 21 is inhibited by 96% and that of PC-PC 32 by 83%. The combination of the four molecules completely inhibits the mycelial growth of all strains of Phaeomoniella chlamydospora (Figure 14A).

 The various Phaeoacremonium aleophilum strains tested were PA-PC 24, PA-PC6, PA-PC20 PA-AQ 30. As shown in FIG. 14B, the growth of the various strains is strongly inhibited with cysteine / salicylic acid and iron sulfate / aluminum fosetyl and is completely inhibited with the mixture of the four molecules.

The results obtained demonstrate that the composition of the invention inhibits the growth of the fungus regardless of the strain tested.

Example 6: Verification of the Safety of Compositions on Cuttings

Compositions were tested in a mixture of two (5 mM cysteine / 0.5 mM salicylic acid and 5 mM iron sulfate / 2.7 mM fosetyl aluminum) or four on white Ugni cuttings and also on Cabernet Sauvignon cuttings.

The cuttings and the conditions of realization are identical to those presented previously.

 A wetting agent (Etaldyne 95, Fertiligen) was added to the mixture which was applied to the cuttings by foliar spraying.

No phytosanitary treatment was performed in parallel with these treatments.

The observation of cuttings was performed two weeks after treatment for cuttings of Cabernet Sauvignon (Figure 15). B) and two weeks after four treatments, made 14 days apart, for cuttings of Ugni blanc (Figures 15D, E, F).

The mixture of the four molecules causes a slight anthocyanin synthesis (FIG. 15B) in the older Cabernet Sauvignon leaves. This anthocyanin synthesis is characteristic of the activation of the secondary metabolism of the plant and does not correspond to a phytotoxic effect.

 As shown in FIG. 15, white Ugni cuttings show no sign of phytotoxicity whether for treatments made from a mixture of two (FIGS. 15D and 15E) or four molecules (FIGS. Figure 15F).

 The composition of the invention therefore makes it possible to inhibit mycelial growth without deleterious effect for the plant, unlike the compositions of the state of the art.

Example 7: Verification of the effectiveness of compositions on cuttings with the technique of fingerprinting on membrane and antibodies developed

 The antibodies and the implementation protocols of this example are described in the methods section.

 Four treatments were performed on inoculated cuttings at an interval of 14 days.

The first treatment was performed on cuttings inoculated for two and a half months. In order to evaluate the effectiveness of the treatment, the membrane imprint method was used. Fingerprints were performed 14 days after each treatment.

 For each mushroom, three lots of cuttings were made

 a batch of uninoculated cuttings (64 cuttings),

 a batch of untreated inoculated cuttings (32 cuttings per mushroom),

 a batch of cuttings inoculated and treated with the mixture of two molecules (0.5 mM salicylic acid / 5 mM cysteine or 5 mM iron sulfate / 2.7 mM fosetyl aluminum) or four molecules (60 cuttings for each fungus) .

 [00190] The antibodies and the protocol of incubations and revelation of the results are identical to those presented above.

1. Cuttings inoculated with Phaeomoniella chlamydospora

For each cutting, an impression was made with the basal portion of the shoot and another with the apical portion 14 days after each treatment was performed (Figure 16).

 The signals revealed correspond to the presence of proteins synthesized by the fungi and detected by means of specific antibodies secreted proteins.

First treatment:

Only an impression made with a non-inoculated cuttings has a slight signal (Figure 16A). A strong signal is to be noted for all prunings of untreated inoculated cuttings (Figure 16B). For inoculated and treated cuttings, a signal is detected for all the prints of the branch. However, the intensity of the signal is lower for iron sulfate / fosetyl-aluminum treatment (Figure 16 D) and for treatment involving the four molecules (Figure 16 E).

Second treatment:

A signal was recorded for untreated cuttings and treated cuttings regardless of the treatment.

Third treatment:

 The fingerprints of the basal and apical portions of the shoots of untreated cuttings have a high signal. A signal is detected for all fingerprints of treated cuttings (Figures 16C, D and E). However, the signal is much weaker for fingerprints made from cuttings treated with the iron / fosetyl-aluminum sulfate mixture (Figure 16D) and the mixture of the four molecules with the fingerprints of untreated cuttings (Figure 16). E).

Fourth treatment:

 A signal is still obtained with the cuttings treated with the salicylic acid / cysteine mixture. The signal for fingerprints of cuttings treated with iron / fosetyl-aluminum sulphate mixture is much lower than for untreated cuttings. No signal is no longer detected for 4 out of 5 cuttings treated with the mixture of the four molecules.

2. Cuttings inoculated with Phaeoacremonium aleophilum

 FIG. 17 shows the results obtained with a composition comprising two or four compounds after two or four treatments of cuttings inoculated with Phaeoacremonium aleophilum and makes it possible to compare the intensity of the observed signal with that of untreated inoculated cuttings.

Second treatment:

A signal is recorded for all the impressions made from the basal portion and the apical portion of the branches of the untreated cuttings. Treatments carried out with the association of two molecules do not make it possible to reduce the intensity of the signal. The signal obtained with cuttings fingerprints treated by the combination of the four molecules (Figure 17 E) is weaker than that obtained with untreated cuttings (Figure 17B).

Fourth treatment:

 After four treatments (Figure 17 E), a signal is observed for all cuttings inoculated with Phaeoacremonium aleophilum, whether treated or not. However, the intensity of the signal is much higher for fingerprints made from untreated cuttings (Figure 17B) than with treated cuttings (Figures 17C, D and E). The lowest signal intensity is that obtained with iron / fosetyl-aluminum sulphate treatments (Fig. 17D) and the mixture of the four molecules (Figure 17E).

Four treatments with the salicylic acid / cysteine / iron sulfate / fosetyl-aluminum mixture make it possible to no longer detect the secretion of molecules by Phaeomoniella chlamydospora in planta. This effect is lower for Phaeoacremonium aleophilum.

The signal intensity for fingerprints made from cuttings treated with compositions comprising two or four compounds is much lower than for untreated cuttings.

This example clearly demonstrates that a composition of the invention has an anti-fungal effect and allows in particular to inhibit the secretion of proteins by the fungi in the plant.

List of references

(Ref 1) Daire X., Poinssot B., Bentéjac M., Silué D., Pugin A., 2002 - Stimulation of grape defenses against pathogens. Encouraging results with regard to late blight. Phytoma, Plant Defense, 548: 24-26.

 (Ref 2) White R. F., 1979 - Acetyl salicylic acid (aspirin) resistance to tobacco mosaic virus in tobacco. Virology, 99: 410-412.

 (Ref. 3) Van Loon L. C, 1983 - The induction of pathogenesis related proteins by pathogens and specifies Chemicals. Neth. J. Plant Pathol., 89: 265-273.

 (Ref. 4) Malamy J., Carr J.P., Klessig D.F., Raskin I., 1990 - Salicylic acid: a likely endogenous signal in the resistance response of tobacco to viral infection. Science, 250: 1002-1004.

 (Ref 5) Metals J.P., Signer H., Ryals J., Ward E., Wyss-Benz M.,

Gaudin J., Raschdorf K., Schmid E., Blum W., Inverardi B., 1990 - Increase in salicylic acid on the onset of systemic acquired resistance in cucumber. Science, 250: 1004-1006.

 (Ref 6) Kessmann H., Oostendorp M., Staub T., 1996 - CGA 2455704: A new plant activator. Brighton Crop Prot. Conf., Pests and diseases: 961-966.

 (Ref 7) Amborabé B.E., Fleurât-Lessard P., Chollet J.F., Roblin G., 2002 - Antifungal effects of salicylic acid and other derivatives towards Eutypa lata: structure-activity relationship. Plant Physiol. Biochem., 40: 1051-1060.

(Ref 8) Regnault-Roger C, 2006 - Natural substances of plant origin. Whether they are biopesticides or elicitors (SDN), what are their perspectives in crop protection? Phytoma. Plant Defense, 591: 14-18. (Ref 9) Rocher F., Chollet JF, Jousse C, Bonnemain J.-L, 2006 - Salicylic acid, an ambimobile molecule exhibiting a high ability to accumulate in the phloem. Plant Physiol., 141: 1684-1693.

 (Ref 10) Rocher F., 2004 - Chemical control of plant pathogenic fungi: evaluation of the phloem system of new molecules with fungicidal effect and activators of defense reactions. PhD thesis, University of Poitiers, 146 p.

 (Ref 11) Dong X., 1998 - SA, JA, ethylene, and disease resistance in plants. Current Opin. Plant Biol., 1: 316-323.

 (Ref 12) Van Toor R.F., Jaspers M.V., Stewart A., 2001 - Evaluation of acibenzolar-S-methyl for induction of resistance in Camellia flowers to Ciborinia camelliae infection. New Zealand Plant Protection, 54: 209-212.

 (Ref 13) Ruess W., Mueller K., Krauf-Beiter G., Kunz W., Staub T., 1996 - Plant activator CGA 245704: An innovative approach for disease control in cereals and tobacco. Proceedings of the Brighton Crop Protection Conference. Pests and diseases, 53-60.

 (Ref 14) H. lshii, Tomita Y., Narusaka Y., Nishimura K., Ywamoto S., 1999 - Induced resistance of acibenzolar-S-methyl (CGA 245704) to cucumber and japanese pear diseases. Eur. J. Plant Pathol., 105: 77-85.

 (Ref 15) Leroux P., 2003 - Modes of action of plant protection products on plant pathogenic organisms. R. Biologies, 326: 9-21.

 (Ref 16) Brisset MN, Chartier R., Didelot F., Parisi L., Paulin J.-P., Robert P., Tharaud M., Lemarquand A., Orain G., Toubon J.-F., Brown L., Derridj S., Sauphanor B., 2005 - Inducers of natural plant defenses. Phytoma. Plant Defense, 581: 20-24.

(Ref 17) Alabouvette C, Olivain C, Cordier C, Steinberg C, 2003 - Alternative methods of control of plant diseases. Prophylaxis, natural products, micro-organisms: progress made and to be accomplished. Phytoma. Plant Defense, 566: 41-44. (Ref 18) Conrath U., CMJ Pieterse, Mauch-Mani B., 2002 - Priming in plant-pathogen interaction. Trends Plant Sci., 7 (5): 210-216.

 (Ref 19) A. Spiers, Ward B.G., 2001 - Injection treatment for control of plant diseases. In Patent Cooperation Treaty, PCT Flight WO 01 / 76355A1, New Zealand.

 (Ref 20) Larignon P., Molot B., 2004 - Wood diseases: ongoing experiments and first results. Rhône-Mediterranean wine interviews, 23-26.

 (Ref 21) Williams J.S., S.A. Hall, Hawkesford MJ. , Beale M.H., Cooper R.M., 2002 - Elemental sulphur and thiol accumulation in tomato and defense against a fungal vascular pathogen, Plant Physiol., 128: 150-159.

 (Ref 22) Kahlos K., Tikka V.H., 1994 - Antifungal activity of cysteine, its effect on C-21 oxygenated lanosterol derivatives and other lipids in Inonotus obliquus, in vitro. Appl. Microbiol. Biot., 42: 385-390.

 (Ref 23) Octave S., Amborabé B. E., Luini E., Ferreira T., Lessard Fleurat P., Roblin G., 2005 - Antifungal effects of cysteine towards Eutypa lata, a pathogen of vineyards. Plant Physiol. Bioch., 43: 1006-1013.

 (Ref 24) Daigle DJ. , Cotty J.C., 1991. The influence of cysteine, cysteine analogs and other amino acids on spore germination of Alternaria species. Can. J. Bot., 69: 2353-2356.

 (Ref 25) Granado J., Felix G., Boller T., 1995 - Perception of fungal sterols in plants. Subnanomolar concentrations of ergosterol elicit extracellular alkalinization in tomato cells. Plant Physiol., 107: 485-490.

 (Ref 26) Amborabé B. E., 2000 - Research on Eutypiose: study of Vigne-Eutypa lata relations and antifungal fight with natural molecules. Doctoral Thesis, University of Poitiers, 135 p.

(Ref 27) Amborabe BE, Rossard S., Pérault JM, Roblin G., 2003 - Specifies perception of ergosterol by plant cells. CR Biologies, 326: 363-370. (Ref 28) Luini E., 2003 - Study of signal transduction involved in the response of the plant cell to ergosterol. DEA report, University of Poitiers. 30 p.

 (Ref 29) Rossard S., 2003 - Transport of sterols and specific perception of ergosterol by the plant cell. PhD thesis, University of Poitiers. 123 p.

 (Ref 30) Sekiya J., Schmidt A., Wilson LG., Filner P., 1982 - Emission of hydrogen sulfide by leaf tissue in response to L-cysteine. Plant Physiol., 70: 430-436.

 (Ref 31) Noctor G., Strohm M., Jouanin L, Kunert KJ. , Foyer H.,

Rennenberg H., 1996 - Synthesis of glutathione in transgenic overexpressing γ-glutamylcysteine synthetase. Plant Physiol., 112: 1071-1078.

 (Ref 32) Weinberg E.D., 1966 - Roles of metallic ions in host- parasite interactions. Bacteriol. Rev., 30 (1): 136-151.

 (Ref 33) Brown A.E., Swinburne T.R., 1982 - Iron-chelating agents and lesion development by Botrytis cinerea on leaves of Vicia faba. Physiol. Mol. Plant P., 21: 13-21.

 (Ref 34) Vedie R., The Norman M., 1984 - Modulation of the pathogenic expression of Botrytis fabae Sard. and Botrytis cinerea Pers. by phylloplan bacteria of Vicia faba L, Agronomy, 4 (8): 721-728.

 (Ref. 35) Gouot J. M., 2006 - Field Efficacity of Profiler®, a fluopicolide and fosetyl-AI combianitio fungicide for the control of grape downy mildew (Plasmopara viticola). Pflanzenschutz-Nachrichten Bayer, 59: 293-302.

 (Ref 36) Mazullo A., Calzarano F., Di Marco S., Cesari A., 2000 - Experiments on the control of Esca disease fungi by fosetyl Al. Integrated Control in Viticulture. lOBC / wprs Bulletin, 23 (4): 77-78.

(Ref 37) Leconte F., Bonnemain J. L, of Cormis L, Barchietto T., 1988 - Becoming metabolic, distribution, and forms of transport (systemic xylem and phloem system) of phosethyl-AI in Lycopersicon esculentum. Miii. CR. Acad. Sci. III, 307: 221-227.

 (Ref 38) Leroux P., 2000 - The chemical fight against fungal diseases of the vine: reflection on anti-resistance strategies. Phytoma. Plant Defense, 533: 32-38.

 (Ref 39) Dercks W., Creasy L.L., 1989 - Influence of fosetyl Al on phytoalexin accumulation in Plasmopara viticola-grapevine interaction. Physiol. Mol. Plant P., 34 (3): 203-213.

 (Ref 40) Lacombe J. -P., 2003 - Behavior of the combination of fenamidone and aluminum fosetyl with QoI-resistant late blight. Phytoma. Plant Defense, 560: 45-50.

 (Ref. 41) Molina A., Hunt M.D., Ryals J.A., 1998. Impaired fungicide activity in plants blocked in disease resistance signal transduction. Plant CeII, 10: 1903-1914.

 (Ref 42) Nemestothy G.S., Guest D.I., 1990 - Phytoalexin accumulation, phenylalanine ammonia-lyase activity and ethylene biosynthesis in fosetyl Al treated resistant and susceptible tobacco cultivars infected with Phytophthora nicotianae var. nicotianae. Physiol. Mol. Plant P., 37 (3): 207-219.

 (Ref 43) Serrano M.L., Ferrer M.A., Calderon A.A., Muήoz R., Ros

Barcelo A., Pedreήo M.A., 1994 - Aluminum-mediated fosetyl-AI effects on peroxidase secreted from grapevine cells. About. Exp. Bot., 34 (3): 329-336.

 (Ref 44) Leroux P., 2003 - Modes of action of plant protection products on plant pathogenic organisms. R. Biologies, 326: 9-21.

(Ref. 45) DG Ouimette, Coffey MD, 1990. Symphetic entry and phloem translocation of phosphonate. Pestic. Biochem. Phys., 38 (1): 18-25. (Ref 46) Di Marco S., Mazzullo A., Calzarano F., Cesari A., 2000 - The control of Esca: status and perspectives. Phytopathol. Mediterr., 29: 232-240.

 (Ref 47) Mazullo A., Calzarano F., Di Marco S., Cesari A., 2000 - Experiments on the control of Esca disease fungi by fosetyl Al. Integrated Control in Viticulture. lOBC / wprs Bulletin, 23 (4): 77-78.

 (Ref 48) Di Marco S., Osti F., 2005 - Effect of fosetyl Al foliar applications towards Esca fungi in grapevine. Phytopathol. Mediterr., 44: 114-115.

 (Ref 49) Borie B., Jacquiot L., Jamaux-Despréaux I., Larignon P.,

Peros J. -P., 2002 - Genetic diversity in populations of the fungi Phaeomoniella chlamydospora and Phaeoacremonium aleophilum on grapevine in France. Plant Pathol., 51: 85-96.

 (Ref 50) Bearden J. C, 1978. Quantitation of submicrograms of protein by improved protein binding binding assay. Biochim. Biophys. Acta, 533: 525-529.

 (Ref 51) Leroux P., 1994 - Influence of pH, amino acids and various organic substances on the fungitoxicity of pyrimethanil, glufosinate, captafol, cymoxanil, and fenpiclonil to certain strains of Botrytis cinerea. Agronomy, 14: 541-554

 (Ref 52) Amborabé B.E., Octave S., Roblin G., 2005: Influence of temperature and nutritional requirements for mycelial growth of Eutypa lata, a vineyard pathogenic fungus. CR. Biology, 328: 263-270.

 (Ref 53) Brun L., Geoffrion R., 2003 - Downy mildew, copper and soil: a long common history. Phytoma. Plant Defense, 564: 37-39.

 (Ref 54) Colby S.R., 1967 - Calculating synergistic and antagonistic responses of herbicide combinations. Weeds, 15: 20-22.

Claims

Antifungal composition comprising at least three of the compounds selected from the group consisting of cysteine, salicylic acid, iron (II) sulfate and fosetylaluminum.

The composition of claim 1 wherein the composition comprises cysteine, salicylic acid, iron (II) sulfate and fosetyl aluminum.

3. Composition according to any one of claims 1 to 2, wherein the concentration of cysteine is between 0.01 and 15O mM.

4. Composition according to any one of claims 1 to 2, wherein the concentration of salicylic acid is between 0.01 and 4 mM.

5. Composition according to any one of claims 1 to 2, wherein the concentration of iron (II) sulfate is between 0.01 and 160 mM.

6. Composition according to any one of claims 1 to 3, wherein the concentration of fosetyl-aluminum is between 0.01 and 81 mM.

A method of treating a plant, said method comprising applying the composition of claims 1 to 6.

8. A method of treatment and / or preventive treatment of a plant infected with a pathogen and / or which presents a risk of infection with a pathogen, said method comprising the application of the composition of claims 1 to 6.

The method of treatment of claim 8, wherein the pathogen is a fungus.

10. Treatment process according to any one of claims 7 to 9, wherein the application of the composition is carried out by sprinkling the plant

11. The method of treatment according to any one of claims 7 to 10, wherein the plant is a vine.

The method of claim 11, wherein the fungus is Phaeomoniella chlamydospora and / or Phaeocremonium aleopholilum.