Composition for plants based on chitosan

Abstract

Biostimulant composition for plants based on chitosans The invention relates to a biostimulant composition for plants comprising at least two chitosans selected from: Chitosan I - ultra low molecular weight, viscosity less than or equal to 5 mPa.s., DDA greater than or equal to 70%, Chitosan II - very low molecular weight, viscosity between 6 and 10 mPa.s., DDA greater than or equal to 85%, Chitosan III - low molecular weight, viscosity between 15 and 22 mPa.s., DDA greater than or equal to 85%, Chitosan IV - low medium molecular weight, viscosity between 30 and 45 mPa.s., DDA greater than or equal to 80%, Chitosan V - medium molecular weight, viscosity between 65 and 100 mPa.s., DDA greater than or equal to 80%.

Description

Title of the invention: Chitosan-based composition for plants. Technical field

[0001] The present invention belongs to the agricultural field and more specifically to the field of plant products. The invention will find particular application in stimulating germination rate, vigor, growth, and / or resistance to diseases and abiotic stresses in plants. The invention is applicable to all types of plants, whether cultivated or wild, including shrubs, trees, and other plants. STATE OF THE ART

[0002] Chitin is a natural polymer widely distributed throughout the living world. Chitin can be defined as an organic substance with a structure similar to that of cellulose (a polysaccharide), a constituent of the cuticle of insects and crustaceans, and of the cell wall of certain fungi. Its annual production is estimated at several billion tons.

[0003] Chitin is a high molecular weight, non-toxic, and biodegradable polymer. After cellulose, it is the most abundant polysaccharide in nature. Chitosan is obtained after a chemical modification of chitin. Both chitin and chitosan are polysaccharides belonging to the glycosaminoglycan (GAG) family.

[0004] Chitin consists of a linear chain of acetylglucosamine groups and can perhaps be represented by formula I. [Chem 1]

[0005] Formula I: Chitin

[0006] More specifically, chitin is a highly insoluble N-acetylated polymer of Pl,4-(D)-glucosamine.

[0007] Like cellulose, chitin is a fiber, but it also has exceptional chemical and biological characteristics that can be used in many industrial and medical applications.

[0008] Chitosan is obtained from chitin by removing sufficient acetyl groups (CH3-CO) to allow the molecule to be soluble in most dilute acids. Chitosan is represented by the general structure according to the formula IL

[0009] [Chem.2]

[0010] Formula II: chitosan

[0011] Currently, the production of industrially usable chitin and chitosan is done from waste shells of crustaceans, such as shrimp and / or crabs, or endoskeletons of cephalopods (squid...), but also from fungi which mostly have chitin in their walls.

[0012] The applications of chitosan in agriculture have been studied since the late 20th century. In particular, the elicitor effects of chitosan were studied by NASA in the 1990s. Since 2008, the product has been considered safe for humans and the environment by the EPA (United States Environmental Protection Agency). In 2016, the European Commission recognized chitosan as a basic substance for organic farming.

[0013] Despite these ecological virtues and its enormous potential in agriculture, the use of chitosan in agriculture remains very limited, even negligible.

[0014] Chitosan is often taken as a single molecule, but it turns out that depending on the physico-chemical properties of the molecule, chitosan will have different effects.

[0015] The object of the present invention is to propose chitosan-based plant treatment products that have optimized effectiveness. SUMMARY

[0016] To achieve this objective, according to a first aspect, a composition for the treatment of plants is envisaged comprising at least two chitosans or chitosans' derivatives chosen from: - A first chitosan, named Chitosan I, having an average molecular weight less than or equal to 20,000 Daltons and a degree of deacetylation (DDA) greater than or equal to 70%, - A second chitosan, called Chitosan II, having an average molecular weight less than or equal to 30,000 Daltons and a DDA greater than or equal to 85%, - A third chitosan, called Chitosan III, with an average molecular weight between 50,000 and 100,000 Daltons and a DDA greater than or equal to 85%, - A fourth chitosan, called Chitosan IV, with an average molecular weight between 100,000 and 200,000 Daltons and a DDA greater than or equal to 80%, - A fifth chitosan, called Chitosan V, with an average molecular weight between 200,000 and 350,000 Daltons and a DDA greater than or equal to 80%, Advantageously, a sixth chitosan, called Chitosan VI - Chitosan hydrochloride having a viscosity between 15 and 60 mPa.s., a DDA greater than or equal to 80% and advantageously with a maximum heavy metal content of 40 ppm.

[0017] This selection of chitosans has an optimized effect for the treatment of plants.

[0018] Preferably, the composition comprises three chitosans or derivatives chosen from those mentioned above.

[0019] According to a second aspect, the invention relates to a composition for the treatment of plants comprising a chitosan-metal complex, the chitosan being chosen from chitosan I and / or chitosan II from those mentioned above.

[0020] The presence of the chitosan-metal complex in the composition provides a biofungicidal effect; advantageously, the complex has an effect as a contact biofungicide and a systemic biofungicide.

[0021] According to a third aspect, the invention relates to a method for preparing a composition as described above and in particular according to the first aspect comprising the following steps: - protonation of at least two chitosans by dissolution in water and a mineral acid, - partial crosslinking of at least two protonated chitosans by an organic acid.

[0022] According to a fourth aspect, the invention relates to a composition obtained by the process according to the third aspect.

[0023] According to a fifth aspect, the invention relates to a use of the composition according to the first aspect and / or the second aspect for the treatment or protection of plants and / or as an elicitor and / or biostimulant and / or biofungicide and / or biocontrol. BRIEF DESCRIPTION OF FIGURES

[0024] The aims, objects, features and advantages of the invention will become clearer from the detailed description of an embodiment thereof, which is illustrated by the following accompanying drawings in which:

[0025] [Fig. 1] The [Fig. 1] represents the protonation step of a chitosan by hydrochloric acid.

[0026] [Fig.2] Figure [Fig.2] illustrates the partial cross-linking of four different chitosans: chitosan I, chitosan II, chitosan III and chitosan IV by citric acid.

[0027] [Fig.3] Figure [Fig.3] compares the germination rate of the two experimental groups of wheat from example 2.

[0028] [Fig.4] [Fig.4] illustrates, on days 2 and 3 after placement in the incubator, the rate of germination of the 7 experimental groups of wheat from example 2.

[0029] [Fig.5] Figure [Fig.5] compares the germination rate of the two experimental groups rapeseed from example 3.

[0030] [Fig.6] Figure [Fig.6] compares the germination rate of rapeseed seeds treated according to different conditions described in example 3.

[0031] [Fig.7] Fig.7 compares the time to achieve resistance against pests (4-leaf stage) of seeds treated under different conditions described in example 3.

[0032] [Fig.8] Figure [Fig.8] illustrates the yield in tonnes per hectare of a plot of which a Part is processed according to example 5 and part is not processed.

[0033] [Fig.9] Figure [Fig.9] illustrates the infestation rate of maize seeds from a group of treated seeds and an untreated control group according to example 8.

[0034] [Fig. 10] The [Fig. 10] illustrates the percentage of leaves affected by the disease on day 0 and on day 15 after treatment according to example 10.

[0035] The drawings are given as examples and are not limiting of the invention. DETAILED DESCRIPTION

[0036] Before proceeding to a detailed review of embodiments of the invention, optional features that may be used in combination or alternatively are listed below:

[0037] By way of example, the total weight of chitosans being between 0.1% and 10% by weight of the total weight of the composition, preferably between 0.1 and 6%, for example in the order of 5% for example from 2 to 5%.

[0038] By way of example, the composition includes a first solvent chosen from a mineral acid, more preferably phosphoric acid and / or phosphorous acid and / or hydrochloric acid or a mixture of these.

[0039] By way of example, the composition includes a second solvent chosen from an organic acid, more preferably formic acid and / or citric acid and / or lactic acid and / or acetic acid and / or tartaric acid or a mixture of these.

[0040] By way of example, the composition is in the form of a hydrogel, the at least two chitosans are cross-linked by hydrogen bonds.

[0041] By way of example, the composition comprises a chitosan-metal complex, the chitosan being chosen from chitosan I, chitosan II.

[0042] By way of example, the metal is chosen from among the transition metals.

[0043] By way of example, the metal is chosen from copper, silver, zinc, iron, the manganese or a mixture thereof.

[0044] By way of example, the quantity of metal is less than 10% by weight of the total weight of the composition.

[0045] By way of example, the composition comprises at least one uncomplexed chitosan and at least one chitosan complexed with a metal.

[0046] By way of example, the protonation of the process is carried out under heating, preferably at a temperature between 35 and 40°C.

[0047] By way of example, the crosslinking of the process is carried out under heating, preferably at a temperature between 40 and 50°C.

[0048] By way of example, the process includes a step of complexing at least one chitosan with at least one metal.

[0049] In this context, the expression "biostimulant composition" is used to designate a composition comprising at least one substance that can be described as a "biostimulant" or "plant biostimulant" within the meaning of Regulation (EU) 2019 / 1009, that is to say, as designating a substance that stimulates plant nutrition processes independently of the nutrients it contains, for the sole purpose of improving nutrient use efficiency, tolerance to abiotic stress, qualitative characteristics, and / or the availability of nutrients confined in the soil or rhizosphere. It should be noted, however, that concrete embodiments of the composition according to the invention may be placed on the market under other names, such as, for example, "fertilizer," "fertilizer containing biostimulants," "fertilizing material," "fertilizer combination," etc.The choice of name will depend, in particular, on the legislation and standards to be respected in the country where the product is placed on the market, as well as the specific composition of the product.

[0050] A fertilizing material is intended to ensure or improve plant nutrition as well as the physical, chemical, and biological properties of soils. Fertilizing materials include fertilizers, soil amendments, and mixtures thereof.

[0051] Fertilizers are products that provide plants with the nutrients necessary for their growth. They can be organic or mineral. Nutrients are generally considered to be divided into basic elements, secondary elements, and trace elements. The basic elements—also called macronutrients—are nitrogen (N), phosphorus (P), and potassium (K). The secondary elements include calcium (Ca), sulfur (S), and magnesium (Mg). The trace elements include iron (Fe), manganese (Mn), molybdenum (Mo), copper (Cu), boron (B), zinc (Zn), chlorine (Cl), sodium (Na), cobalt (Co), vanadium (V), and silicon (Si).

[0052] Soil amendments are products whose main purpose is to improve soil quality, particularly in terms of structure and acidity. They often also contain significant amounts of nutrients.

[0053] Among the beneficial effects of biostimulants on plants are: resistance to abiotic stresses, improved plant growth and development, and better nutrient absorption. Unlike fertilizers, their nutrient content is generally not the source of the observed beneficial effects.

[0054] An elicitor is understood to be a substance capable, under certain conditions, of stimulating natural defense mechanisms. These natural defenses are directed, for example, either against bioaggressors (diseases, pests) or against abiotic stresses, such as those caused by frost.

[0055] Pharmaceutical grade is defined as a set of parameters defining the purity of a compound. This grade is defined in the European Pharmacopoeia in force in Europe on the date of this application.

[0056] Food grade and industrial grade are defined in particular according to a maximum heavy metal content of 40 ppm.

[0057] A parameter "approximately equal to / greater than / less than" or "of the order of" a given value means that this parameter is equal to / greater than / less than the given value, to within 10% or even 5% of that value.

[0058] The composition according to the invention comprises at least two different chitosans or chitosan derivatives, preferably three chitosans or chitosan derivatives.

[0059] Preferably, the two chitosans are chosen from the following chitosans or chitosan derivatives: - a first chitosan, called Chitosan I, having an average molecular weight less than or equal to 20,000 Daltons, i.e. 20 kg / mol, being defined as an ultra-low molecular weight, a degree of deacetylation (DDA) greater than or equal to 70%, possibly 85%, and preferably having a viscosity less than or equal to 5 mPa.s, and preferably with a maximum heavy metal content of 40 ppm, possibly with a pharmaceutical grade conforming to the European Pharmacopoeia in force at the date of this patent application, a second chitosan, called Chitosan II, having an average molecular weight less than or equal to 30,000 Daltons, i.e. 30 kg / mol being defined as a very low molecular weight, a DDA greater than or equal to 85%, possibly 90%, and preferably having a viscosity between 6 and 10 mPa.s and preferably with a food grade, more specifically a maximum heavy metal content of 40 ppm and possibly a pharmaceutical grade conforming to the European Pharmacopoeia, a third chitosan, called Chitosan III, having an average molecular weight between 50,000 and 100,000 Daltons (i.e., between 50 and 100 kg / mol), being defined as a low molecular weight, a DDA greater than or equal to 85%, and preferably having a viscosity between 15 and 22 mPa.s, and preferably food grade, more specifically a maximum heavy metal content of 40 ppm, a fourth chitosan, called Chitosan IV, having an average molecular weight between 100,000 and 200,000 Daltons, i.e. 100 to 200 kg / mol being defined as a medium-low molecular weight, a DDA greater than or equal to 80% and preferably having a viscosity between 30 and 45 mPa.s., and preferably food grade, more specifically a maximum heavy metal content of 40 ppm, a fifth chitosan, called Chitosan V, having an average molecular weight between 200,000 and 350,000 Daltons, i.e. 200 to 350 kg / mol, a DDA greater than or equal to 80% and preferably having a viscosity between 65 and 150 mPa.s., and preferably of food grade, more specifically a maximum heavy metal content of 40 ppm, advantageously a sixth chitosan, called Chitosan VI - Chitosan hydrochloride having a viscosity between 15 and 60 mPa.s., a DDA greater than or equal to 80% and advantageously with a maximum heavy metal content of 40 ppm.

[0060] Chitosan derivatives are preferably chitosan hydrochloride, chitosan phosphonate, or chitosan oligosaccharides.

[0061] The chitosans used for the invention are advantageously of crustacean (prawn) origin, advantageously from sustainable fishing, insects, such as, for example, but not limited to: the larvae of Hermetia illucens, the black soldier fly or Tenebrio molitor, the mealworm or fungal, and preferably from the mycelium of an Ascomycete type fungus, and in particular Aspergillus niger, and / or of a Basidiomycete fungus, and in particular Lentinula edodes (shiitake) and / or Agaricus bisporus (button mushroom).

[0062] Chitosans are advantageously of a microbiological quality compatible with its use as a biostimulant.

[0063] The selection of pharmaceutical grade chitosans contributes to optimized preservation of the composition.

[0064] Chitosans are often characterized by two main factors: viscosity or average molecular weight and its degree of deacetylation or its degree of acetylation.

[0065] The degree of deacetylation, or DDA, is expressed as the number of moles of D-glucosamine units relative to the number of moles of total N-acetyl-D-glucosamine and D-glucosamine units present.

[0066] The degree of acetylation DA is the number of N-acetyl-D-glucosamine units relative to the number of moles of total N-acetyl-D-glucosamine and D-glucosamine units present.

[0067] One being the inverse of the other, that is to say that a chitosan having a DDA of 85% has 15% acetyl groups and 85% amine groups on its chains.

[0068] The degree of acetylation DA is, for example, determined by potentiometric titration. Chitosan is dissolved in a hydrochloric acid solution. The excess hydrochloric acid that has not reacted with the amino groups of chitosan is titrated with a standardized sodium hydroxide solution. From this, the number of moles of D-glucosamine units present in the chitosan is deduced, i.e., the degree of deacetylation DDA, and therefore, by subtraction, the degree of acetylation DA.

[0069] Chitosan advantageously has a controlled degree of acetylation. The term "chitosan having a controlled degree of acetylation" means a product whose degree of acetylation, i.e. the proportion of N-acetylglucosamine units, can be adjusted in a controlled manner.

[0070] Advantageously, chitosan has a degree of DDA deacetylation greater than or equal to 70, preferably greater than strictly 70%, more preferably greater than or equal to 80%.

[0071] Viscosity is understood as dynamic viscosity. This viscosity depends, among other things, on the molar mass. Viscosity is measured by capillary viscometry, with a Ubbelohde type capillary viscometer, according to the method of the European Pharmacopoeia monograph EP2.2.17.

[0072] The average molecular weight of chitosan can be estimated from the dynamic viscosity value of a 1% (w / w) chitosan solution in 1% (v / v) acetic acid. The "1% (w / w) chitosan solution" is understood to mean a distilled water solution containing 1% by mass of chitosan relative to the total mass of the chitosan solution in distilled water. Acetic acid is added to this 1% (w / w) chitosan solution to obtain a 1% (w / w) chitosan and 1% (v / v) acetic acid solution. Alternatively, chitosan powder can be added to an acetic acid solution to achieve a concentration of 1%. Acetic acid at 1% (v / v) means a concentration of 1% by volume of acetic acid relative to the volume of the final distilled water solution.

[0073] According to a specific embodiment, the invention uses at least two different chitosans having a degree of deacetylation greater than or equal to 70%, according to a preferred possibility, of the order of 80%, and a viscosity between 5 and 150mPa.s., preferably less than or equal to 145mPa.s.

[0074] According to a preferred embodiment, the composition comprises a percentage by weight of chitosans relative to the weight of the final composition of between 0.1 and 10% w / w. Preferably between 0.1 and 6%, for example around 5%, for example around 2%.

[0075] The composition advantageously comprises water. The amount of water in the composition is configured to make up the quantity to 100% (w / w). The composition is advantageously an aqueous solution of chitosan.

[0076] The composition advantageously comprises a first solvent preferably a mineral acid, more preferably chosen from phosphorous acid and / or phosphoric acid and / or hydrochloric acid or a mixture of these.

[0077] The composition advantageously comprises, as a first solvent, a mineral acid in an amount ranging from 0.1 g / L to 150 g / L depending on the acid chosen. For example, between 0.7% and 2% by weight of the total weight of the composition for hydrochloric acid, between 10% and 15% by weight of the total weight of the composition for phosphorous acid, and between 3% and 5% by weight of the total weight of the composition for phosphoric acid.

[0078] Preferably, the first solvent is chosen to ensure the protonation of chitosans.

[0079] The composition advantageously comprises a second solvent selected from an organic acid, preferably a carboxylic acid with a short carbon chain, preferably less than or equal to 6.

[0080] Advantageously, the organic acid is chosen from acetic acid and / or tartaric acid and / or formic acid and / or citric acid and / or lactic acid or a mixture of these.

[0081] The composition advantageously comprises, as a second solvent, an organic acid in an amount ranging from 1 g / L to 100 g / L depending on the acid chosen. By way of example, the organic acid is preferably present in an amount ranging from 5 to 15% by weight of the total weight of the composition.

[0082] Preferably, the second solvent is chosen to ensure the crosslinking of chitosans.

[0083] The composition is advantageously in the form of a hydrogel. By hydrogel is meant a gel comprising a swelling agent which is water.

[0084] According to one embodiment of the invention, the hydrogel is a network of at least two cross-linked chitosans.

[0085] By the crosslinking of chitosanes, it is understood that chitosan chains made water-soluble by the presence of hydrophilic groups, in particular during the protonation of chitosan by a mineral acid of the first solvent, connect to each other through the carboxylic acid of the organic acid of the second solvent and preferably by a heat treatment.

[0086] According to a preferred embodiment, the selection of the organic acid comprising a carbon chain of a maximum of 6 atoms and advantageously in the case of heat treatment, maintaining the temperature less than or equal to 50°C, preferably 45°C, preferably 40°C, makes it possible to minimize the number of chemical and covalent bonds by promoting crosslinking by hydrogen bonds.

[0087] Advantageously, the partial crosslinking allows that once on the plant, the crosslinking begins to break down and the chitosan molecules are released one by one, and are absorbed by the plant.

[0088] The weaker hydrogen bonds allow for the rapid and stable release of various chitosans immediately upon application of the preparation. Furthermore, the chitosans protonated by the mineral acid (having a positive charge) can rapidly bind to the surface of plants (having a negative charge). This reduces chitosan leaching and increases the duration of treatment efficacy.

[0089] According to a preferred possibility, the at least two chitosanes are partially crosslinked. By way of preferred example, at least 50% of the chitosanes are crosslinked, preferably strictly less than 100%. The percentage of chitosanes crosslinked by a carbon-nitrogen double bond is less than or equal to 10%. The chitosanes in the composition are crosslinked via the carboxylic acid units of the second solvent. Crosslinking is understood to mean the formation of one or more chemical or physical bonds (bridges) between chitosan chains. In this process, partial crosslinking refers to crosslinking in which the crosslinking agent (a carboxylic acid with fewer than 6 carbons) and moderate heat treatment minimize the number of stable bridges (chemical and covalent bonds) by favoring crosslinking via hydrogen bonds. Advantageously, using the aforementioned acids, particularly citric or acetic acid, reduces the formation of imide bonds. Advantageously, crosslinking with citric or acetic acid is used to avoid creating a high level of stable crosslinks via imide bonding. In the invention, the crosslinking is therefore partial.

[0090] The use of chitosans partially cross-linked by organic acid units makes it possible, in particular, to reduce leaching of the composition. However, since the cross-linking is advantageously only partial, it limits the reduction of free amines, allowing the protonated amine units to help the chitosan bind to plants or microorganisms, thus maintaining assimilation of the chitosan by the plant.

[0091] Preferably, the crosslinking of the at least two chitosans of the composition according to the invention is carried out by the second solvent, advantageously chosen from carboxylic acids with a short carbon chain, preferably less than or equal to 6.

[0092] According to one embodiment, the carboxylic acid is mixed with amino acids or a protein hydrolysate containing peptides and free amino acids. The amount added is preferably in the range of 1 to 10%, more preferably 5% by weight of the total weight of the final composition. This option helps to enhance plant resilience in the event of infection.

[0093] According to one embodiment, the composition is stable for a period of 18 months, preferably 24 months. The stability of the composition is preferably verified by the absence of mold. Preferably, the selection of pharmaceutical-grade chitosans contributes to the stability of the composition.

[0094] According to one embodiment, the composition has a pH less than or equal to 4.5, preferably less than or equal to 4, more preferably less than or equal to 3.5.

[0095] According to one aspect, the invention relates to the use of the composition described above for application on seeds in particular by coating the seeds and / or on leaves in particular by spraying and / or on the soil by spraying or watering.

[0096] By way of example, the composition is configured to be applied at a dose of 1 litre of composition per 100 kg of seeds.

[0097] According to one aspect, the invention relates to the use of the composition described above as a biostimulant, and / or as an elicitor and / or as a phytostimulant and / or biofungicide.

[0098] According to one aspect, the present invention relates to a method for manufacturing a composition according to the invention comprising a protonation step of selected chitosans and a partial crosslinking step.

[0099] Advantageously, the protonation step includes the formation of a mixture of chitosans, water and mineral acid.

[0100] Preferably, at least one chitosan, or several chitosans successively or mixed together, is added to advantageously heated water. The first solvent is added to the mixture.

[0101] According to a preferred embodiment, the protonation step comprises heating the water, preferably to a temperature between 35 and 40°C. Then, the protonation step comprises adding at least one chitosan to the heated water, advantageously with stirring. Next, the first solvent is added, preferably with stirring. The first solvent is a mineral acid, or a mixture of one or more mineral acids as described above. In one possibility, each chitosan is added separately to a separate volume of water and then combined. In another possibility, the chitosans are added successively to the heated water. The two possibilities may be combined or used alternatively.

[0102] The resulting solution is designated solution I. Agitation is preferably maintained for 5 to 10 minutes. Depending on one possibility, solution I is degassed. Degassing of solution I is preferably carried out over 20 to 30 minutes. The resulting solution I is advantageously clear.

[0103] Figure 1 illustrates the protonation of chitosan by hydrochloric acid.

[0104] Following the protonation step, the process includes the advantageously partial crosslinking step. The crosslinking step comprises the addition of the second solvent, preferably under stirring and preferably under heating, to the mixture obtained at the end of the protonation step, solution I. The second solvent is advantageously an organic acid or a mixture of organic acids as described above.

[0105] Figure 2 illustrates the partial crosslinking of chitosans according to the invention: four different chitosans are crosslinked: chitosan I, chitosan II, chitosan III and chitosan IV. The crosslinking is partial by hydrogen bonds formed between the carboxylic acids of the organic acid, here citric acid, fixed on the chitosans following their protonation by the mineral acid.

[0106] According to one possibility, the process includes a step of adjusting the pH to a target pH preferably less than 4.5, preferably less than 3.5. Advantageously, the pH is adjusted by the addition of a mineral acid which may or may not be the first solvent described above.

[0107] According to an aspect separable or combinable with the preceding aspect, the invention relates to a composition in which the at least two chitosans of the composition are more particularly selected from: - A first chitosan, named Chitosan I, having an average molecular weight less than or equal to 20,000 Daltons, i.e. 20 kg / mol, being defined as an ultra-low molecular weight, a degree of deacetylation (DDA) greater than or equal to 70%, possibly 85%, and preferably having a viscosity less than or equal to 5 mPa.s. and with a maximum heavy metal content of 40 ppm, possibly with a pharmaceutical grade conforming to the European Pharmacopoeia in force at the date of this patent application. - A second chitosan, called Chitosan II, having an average molecular weight less than or equal to 30,000 Daltons, i.e. 30 kg / mol being defined as a very low molecular weight, a DDA greater than or equal to 85%, possibly 90% and preferably having a viscosity between 6 and 10 mPa.s and a food grade, more specifically a maximum heavy metal content of 40 ppm, and possibly a pharmaceutical grade conforming to the European Pharmacopoeia, and of which at least one chitosan is complexed with a metal.

[0108] The metal being preferably chosen from among the transition metals, more specifically the divalent metal ions, more specifically from group IB of the periodic table. Preferably, the metal is chosen from copper, silver, zinc, iron, manganese or a mixture thereof.

[0109] Chitosan acts as a ligand for a metal cation to form the chitosan-metal complex by chelation.

[0110] This composition has an effect for the treatment of fungal diseases of plants, i.e. a biofungicidal effect.

[0111] This composition also presents a biocontrol role for the prevention of fungal diseases of plants and advantageously for healing.

[0112] Widely used chemical (organic) fungicides present significant risks of toxicity to humans and other living beings. Increasingly stringent regulations are being adopted, and placing this type of product on the market is becoming increasingly difficult.

[0113] In addition, the chemicals that are used cause the development of resistance in pathogens which are harmful in the long term.

[0114] Today, products based on phosphite (or phosphonates), sulfur and copper are used to prevent fungal diseases of plants in order to reduce the use to chemical fungicides. Unfortunately, natural products do not have a sufficient effect on plant diseases, particularly fungal diseases.

[0115] Studies have shown that chitosan has antifungal effects. It is also well established that chitosan can stimulate the natural defense of plants against infectious diseases.

[0116] The antimicrobial effect of chitosan is, however, defined as unstable and sensitive. Metal-chitosan complexes have been developed to optimize the effects of chitosan. However, these complexes are not effective when applied to plants.

[0117] The applicant has observed that the selection of chitosans as described above, particularly for this aspect, allows for a chitosan-metal complex with good solubility and no risk of precipitation or coagulation, thus enabling optimized application and efficacy on plants. The chitosan-metal complex according to the invention has a contact fungicidal effect, the complex itself exhibiting a toxic effect on pathogenic fungi. The complex also has the advantage of ensuring the complexation of the metal and therefore reducing its toxicity to the treated plant.

[0118] The formation of selected chitosan-metal complex allows a synergy of the fungicidal effect while limiting the dormant effect on the culture of chitosan at too high a dose and the potentially toxic effect on the environment of metals at too high a dose also.

[0119] Preferably, the composition comprises a quantity of metal between 50 and 100 g per kilo of composition, preferably the composition is configured to allow compliance with a maximum dose of metal of 250g per hectare treated and more particularly of copper and a maximum dose of chitosan of 800g per hectare treated.

[0120] The quantity of metal in the composition is preferably less than or equal to 10% by weight of the total weight of the final composition.

[0121] Surprisingly, the composition according to the second aspect comprising a chitosan-metal complex has a biostimulating and / or eliciting effect.

[0122] In the composition according to this aspect, at least one of the at least two chitosans in the composition forms a chitosan-metal complex, and advantageously, at least one of the at least two chitosans in the composition does not form a chitosan-metal complex. This arrangement advantageously allows the effects of the chitosan-metal complex and the chitosan according to the invention to be combined.

[0123] According to this aspect, the invention also relates to a method for manufacturing a composition according to this aspect of the invention. The manufacturing method comprises at least one complexation step comprising mixing a solution of chitosan and a metal solution. Advantageously, the complexation step preferably includes a step of preparing a chitosan solution and advantageously a step of preparing a metal solution and advantageously a step of mixing the chitosan solution and the metal solution.

[0124] The mixing of the chitosan solution and the metal solution is preferably carried out at a temperature between 60°C and 70°C and advantageously under stirring, preferably for a minimum duration of 2h, preferably for a duration of 2h to 4h.

[0125] The step of preparing a chitosan solution comprises adding at least one chitosan, advantageously selected from the list of chitosans according to this aspect, to water, followed by the addition of an acid, preferably a weak one. By way of example, the acid is a mineral acid, more specifically selected from phosphoric acid and / or phosphorous acid and / or hydrochloric acid, or a mixture thereof. Preferably, this step of preparing the chitosan solution is carried out under stirring. Preferably, this step, or at least the addition of the acid, is carried out at a temperature between 50°C and 75°C, preferably between 60°C and 70°C, preferably 60°C.

[0126] According to one embodiment, the chitosan solution comprises an amount of chitosan between 1 and 15%, preferably 5% by weight of the total weight of the chitosan solution.

[0127] According to one embodiment, the chitosan solution comprises an amount of acid of between 5% and 20%, preferably between 10% and 15% by weight of the total weight of the chitosan solution. The chitosan solution comprises water in a quantity sufficient to reach 100%.

[0128] The step of preparing a metal solution includes adding a metal to water. Preferably, the metal is that selected as described above and is dissolved in water, preferably with stirring. Preferably, this preparation step is carried out at a temperature between 35°C and 50°C, preferably 40°C. Preferably, the water is heated to said target temperature. According to one possibility, a carboxylic acid is added to the metal solution to facilitate the dissolution of the metal. The carboxylic acid is preferably chosen from acetic acid, lactic acid, or preferably citric acid.

[0129] The chitosan solution and the metal solution are then mixed.

[0130] Optionally, after the complexation step, the process includes the addition of one or more amino acids, or an extract containing proteins and / or peptides and / or free amino acids. The quantity added is preferably in the range of 1 to 10%, more preferably 5% by weight of the total weight of the final composition. This option helps to stimulate plant resilience in case of infection.

[0131] According to a preferred embodiment, the process includes a step of preparing a solution of chitosan uncomplexed with a metal. Preferably, this preparation step includes adding at least one chitosan to water, preferably with the addition of an acid selected from phosphorous acid, acetic acid, citric acid, lactic acid, or a mixture thereof. Preferably, the acid is selected from phosphorous acid and citric acid. Advantageously, this step is carried out at a temperature between 50°C and 70°C, preferably at 60°C. In this embodiment, the uncomplexed chitosan solution is added to the complexed solution obtained at the end of the complexation step. The addition of the uncomplexed solution to the complexed solution is preferably done by drop by drop and preferably at a temperature between 50°C and 70°C, preferably at 60°C. Examples

[0132] Example 1: Preparation of a composition for application to seeds

[0133] Protonation step: 1. Heat 25 mL of water to a temperature between 35 and 40 °C in a compatible container; 2. Add 0.5 grams of IV chitosan and mix for 5 minutes; 3. Add 0.4 mL of 15% hydrochloric acid and mix for 5-10 min; i. If the chitosan is not completely dissolved, repeat step 3 with 0.1 mL of hydrochloric acid; 4. Allow to degas for 20-30 minutes. The solution is clear (solution I); 5. Heat 50 mL of water to a temperature between 35 and 40 °C. Add 0.4 grams of chitosan III and mix for 5 minutes; 6. Add 0.1 gram of chitosan II to the mixture obtained in step 5 and let it mix for 5 minutes; 7. Add 0.4 mL of 15% hydrochloric acid and allow to dissolve (solution he); 8. Add solution I to solution II while stirring;

[0134] Crosslinking step 9. Add 1.0 g of acetic acid or citric acid (any form is acceptable) and stir for 30 minutes at 45°C

[0135] pH adjustment step 10. Adjust the pH of the solution to a pH lower than 3.5; 11. Add enough water to reach the final volume of 100 mL. The concentration of chitosan in the final solution is therefore 1% (w / v).

[0136] Example 2: Instructions for use and dosage

[0137] 100 ml of the composition obtained in example 1 is mixed with 10 grams of seeds. The mixture is stirred until the liquid is homogeneous, then it is left to air dry for 24 hours.

[0138] A first group (Group I) of 25 grams of organic winter soft wheat seed, variety GWENN, is treated by the method indicated above. That is to say, 25 grams of seed are treated with 2.5pl of the composition of Example 1.

[0139] A second group (Group II) called the Control group is treated with distilled water using the same technique.

[0140] 35 grains from each group are sown in an experimental plot in three parallel rows and the total germination rate is counted.

[0141] Figure 3 compares the germination rate of the two experimental groups. Treatment with the preparation described (Group I) increases the wheat germination rate by more than 19% compared to the controls (Group II).

[0142] A second variety of winter soft wheat, organic open-pollinated seeds, known as farm wheat, is treated in the same way: - 100 ml of the composition obtained in example 1 is mixed with 10 grams of seeds. The mixture is stirred until the liquid is homogeneously dispersed and then it is left to air dry for 24 hours.

[0143] Figure 4 illustrates the results of the germination rate for 7 experimental groups: Group 1 Controls Group II Full Composition Group III Mineral acid free Group IV Without organic acid (acetic acid) Group V Chitosan-free II Group VI Without chitosan III Group VII Without chitosan IV

[0144] The results are illustrated in [Fig.4] on days 2 and 3 after incubation, in a moistened paper towel.

[0145] The difference between the germination rate of the treated seeds (group II) and the control seeds (group I) is clearly visible. Furthermore, the treated seeds exhibit a faster growth rate than the untreated control seeds.

[0146] Example 3: Comparison of the biostimulant effect of chitosans (I, II, III) and the acids used in the preparation, separately or in an incomplete manner.

[0147] Rapeseed seeds are treated according to example 2 in two groups: Group I treated with the composition of example 1, Group II control treated with distilled water. Fifty randomly selected grains from each of the three groups were distributed into Petri dishes covered with moistened filter paper. The seeds were then placed in an incubator (25°C) for a period of 7 days. Figure 5 shows the germination rate of the three groups.

[0148] It is observed once again that the composition (Group I) allows the best germination rate compared to Group II (Controls).

[0149] To confirm these results, we treated and sowed the seeds of the same variety with chitosans I, II and III, with and without the acids.

[0150] For the groups Complete preparation, chitosan I, chitosan II, chitosan III, chitosan I + II, chitosan I + III, chitosan II + III, the manufacturing process is that described in example 1.

[0151] For the mineral acid-free group, the process of Example 1 is carried out without adding mineral acid during the protonation step.

[0152] For the group without organic acid, the process of example 1 is carried out without adding organic acid during the crosslinking step.

[0153] Next, 30 seeds for each group are sown in parallel rows.

[0154] Figure 6 shows that the composition obtained by the process of Example 1 is the most effective treatment. The germination rate is improved.

[0155] The growth rate is also compared to [Fig. 7]. The 4-leaf stage is a stage that allows resistance to pests to be achieved; the faster this stage is reached, the better it is for the crop.

[0156] Figure 7 shows that the composition obtained by the process described in Example 1 is the most effective treatment. The time to achieve resistance against pests (4-leaf stage) is reduced by 40%.

[0157] It is observed that the composition according to the invention increases the biostimulant effect of chitosan in a surprising way.

[0158] Example 4: Preparation of a composition for foliar application

[0159] Protonation step: 1. Add 1 gram of chitosan VI to 50 mL of heated water while stirring; 2. Add 0.4 mL of 15% hydrochloric acid and mix, obtaining the solution I; 3. Mix 0.25 grams of chitosan III and 0.25 grams of chitosan IV with 20 mL of heated water while stirring; 4. Add 0.25 mL of 15% hydrochloric acid and stir until completely dissolved, obtaining solution II; 5. Mix 0.25 grams of chitosan II and 0.25 grams of chitosan I with 20 mL of water while stirring. 6. Add 0.25 mL of 15% hydrochloric acid and stir until completely dissolved, obtaining solution III; 7. Allow to cool to room temperature; 8. Add solutions II and III slowly to solution I while stirring; 9. Heat the mixture to 35°C;

[0160] Crosslinking step: 10. Add 2.0 grams of acetic acid or citric acid or formic acid to the mixture obtained in the protonation step; 11. In an optional step, 5 to 10 grams of amino acid extract or peptides of agricultural interest can be added to the mixture. Stir for 60 minutes at a temperature between 35 and 40 °C.

[0161] pH adjustment step - Adjust the pH of the solution to 3.5 by adding a carboxylic acid Add enough water to reach a volume of 100 mL. chitosan concentration in the final preparation and 2% (w / v)

[0162] Example 5: Instructions and dosage

[0163] Dilute 1 mL of the preparation obtained in Example 4 in 25 to 50 mL of water. Shake vigorously and spray on the leaves and branches for complete coverage. Repeat the treatment in 20 to 30 days.

[0164] The resulting composition is used on a natural grassland. Figure 8 illustrates the results obtained on the two plots, the treated and the untreated. The yield in tonnes per hectare is significantly higher for the treated plot.

[0165] Example 6: A process for manufacturing a composition comprising a chitosan-metal complex for seed treatment - Add 2 g of chitosan I to 10 mL of distilled water while stirring; - Add 1 g of citric acid and let it stir for 30 min at 60°C (solution 1); - Add 1 g of chitosan (0.5 g of chitosan II and 0.5 g of chitosan III) to 10 mL of water and 3 grams of acetic acid and wait until completely dissolved; - Heat up to 60°C (solution 2); - Dissolve 16 g of copper sulfate (pentahydrate: CuSO4.5H2O) in 42 mL of distilled water at 40°C; - Add 10 g of zinc sulfate (ZnSO4.H2O) (solution 3); - Add solution 3 slowly and while stirring to solution 1; - Heat to 70°C and let it stir for at least 4 hours; - Add solution 2, drop by drop and let it stir at 60°C for 1 hour; - Add 3 g of amino acid extract powder and let it stir for 1 hour (40°C); - Allow to cool to room temperature (> 20 °C)

[0166] Example 7: process for manufacturing a composition comprising a chitosan-metal complex for foliar use - Add 2.0 grams of chitosan I to 10 mL of distilled water and stir until completely dissolved; - Add 10 grams of phosphorous acid (H3PO3); - Heat to 70°C and stir for 1 hour (solution 1); - Add 0.5 grams of chitosan II and 0.5 grams of chitosan III to 10 mL water. Add 5 grams of phosphorous acid or a carboxylic acid (acetic, citric, etc.) and let it stir for 30 min (solution 2); - Add 20 grams of copper sulfate (pentahydrate: CuSO4.5H2O) to 35 mL of distilled water at 40°C. Add 3 grams of carboxylic acid (acetic or citric...) and stir for 30 min (solution 3); - Add solution 3 to solution 1 slowly at 70°C and let it stir for 4 hours; - Add 5 grams of amino acid extract powder (agricultural biostimulant) and let it dissolve at 70°C; - Add solution 3 slowly (drop by drop) and let it stir for 1 hour; - Allow the mixture to cool to room temperature.

[0167] Example 8: Disease Prevention

[0168] A first group of maize seeds is treated with the composition obtained in Example 6: add 175 pl of the composition obtained in Example 6 and 325 pl of water to 50 g of maize seeds, stir for 2 minutes and let dry for 24 hours and sow.

[0169] A second group of maize seeds is treated with distilled water: add 500pl of water to 50 g of maize seeds, stir for 2 minutes and let dry for 24 hours and sow.

[0170] The two groups of 50 grams of maize seeds were then inoculated with 2 grams of soil infested with Pythium sp. and placed in an incubator at 25°C. Symptoms of the disease (black spots) appeared on the second control group as early as 48 hours after incubation at 25°C. The infestation rate of the seeds in the first group (6%) was 92% lower than that of the control seeds (98%), as illustrated in [Fig. 9].

[0171] Example 9: Treatment of damping-off

[0172] Damping-off is a fungal disease transmitted through the soil, one of the main symptoms of which is rotting of seeds or young shoots during germination.

[0173] Damping-off of pea seedlings is one of the most important diseases of this crop.

[0174] 50 grams of pea seeds are divided into 2 groups. A first group treated The first group received 88 µl of the composition described in Example 6 and 163 µl of distilled water 24 hours before sowing. The second control group received 251 µl of distilled water 24 hours before sowing. The seeds were planted in 3 rows of 20 seeds. More than 73% of the seeds in the control group died due to damping-off, but the seed germination rate of the seeds in the group treated with the composition described in Example 6 was 100%.

[0175] The first group of seeds treated with the composition of the invention is 100% immune to this disease.

[0176] Example 10: Treatment of septoria leaf blotch of cereals

[0177] Every year, several million tons of cereals are lost due to this disease. A 15-hectare plot of einkorn wheat showing symptoms of advanced septoria leaf blotch is treated with the composition of Example 7. 1 liter of the composition is diluted in 100 liters of water and then sprayed per hectare.

[0178] On the day of spraying, the leaves are heavily affected by Septoria leaf spots. 50 plants chosen at random from a 15-hectare plot are counted.

[0179] On day 15 after treatment, 50 plants were randomly selected from the same plot and the number of new leaves affected by the spots was counted. [Fig. 10] shows that the development of the disease was successfully halted and that the plants were completely cured.

[0180] The invention is not limited to the embodiments previously described and extends to all embodiments covered by the invention.

Claims

1. Demands Composition for the treatment of plants comprising at least two different chitosans or chitosan derivatives Characterized in that at least two chitosans or chitosan derivatives are chosen from: • A first chitosan, called Chitosan I: having an average molecular weight less than or equal to 20,000 Daltons and a degree of deacetylation (DDA) greater than or equal to 70%, • A second chitosan, called Chitosan II, having an average molecular weight less than or equal to 30,000 Daltons, a viscosity between 6 and 10 mPa.s and a DDA greater than or equal to 85%, • A third chitosan, called Chitosan III, with an average molecular weight between 50,000 and 100,000 Daltons and a DDA greater than or equal to 85%, • A fourth chitosan, called Chitosan IV, with an average molecular weight between 100,000 and 200,000 Daltons and a DDA greater than or equal to 80%, • A fifth chitosan, called Chitosan V, with an average molecular weight between 200,000 and 350,000 Daltons and a DDA greater than or equal to 80%, • A sixth chitosan, designated Chitosan VI - Chitosan Hydrochloride, having a viscosity between 15 and 60 mPa.s., a DDA greater than or equal to 80%, and advantageously with a maximum heavy metal content of 40 ppm and that the composition comprises a first solvent chosen from a mineral acid, more preferably phosphoric acid and / or phosphorous acid and / or hydrochloric acid or a mixture of these, and a second solvent chosen from an organic acid, more preferably formic acid and / or citric acid and / or lactic acid and / or acetic acid and / or tartaric acid or a mixture of these.

2. Composition according to the preceding claim wherein the total weight of chitosans is between 0.1 and 10% by weight of the total weight of the composition, preferably between 0.1 and 6%, for example in the order of 5%.

3. Composition according to any one of the preceding claims being in hydrogel form, the at least two chitosans are crosslinked by hydrogen bonds.

4. Composition according to any one of the preceding claims comprising a chitosan-metal complex, chitosan being selected from chitosan I, chitosan II.

5. Composition according to the preceding claim wherein the metal is selected from the transition metals.

6. Composition according to any one of the two preceding claims wherein the metal is selected from copper, silver, zinc, iron, manganese or a mixture thereof.

7. Composition according to any one of the three preceding claims wherein the amount of metal is less than 10% by weight of the total weight of the composition.

8. Composition according to any one of the four preceding claims comprising at least one uncomplexed chitosan and at least one chitosan complexed with a metal.

9. A method for preparing a composition according to any one of the preceding claims comprising the following steps: a. protonation of at least two chitosans by dissolution in water and a mineral acid, b. partial crosslinking of the at least two protonated chitosans by an organic acid.

10. A method according to the preceding claim in which protonation is carried out under heating preferably at a temperature between 35 and 40°C.

11. A method according to any one of the two preceding claims wherein the crosslinking is carried out under heating preferably at a temperature between 40 and 50°C.

12. A method according to any one of the three preceding claims comprising a step of complexing at least one chitosan with at least one metal.

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