Field of the invention

The invention relates to formulations for plant care, including plant care products (PCP) and substances useful in agriculture and plant growth and methods of preparation thereof.

Background of the Invention

Plant growth and development is adversely affected by different stressors that are either biotic (pests, diseases, weeds) or abiotic (lack or excess of moisture, low or high temperatures, salinity, heavy metals, other toxic substances, etc.). The resulting loss in crop yield may be substantial. Among the various approaches that have been developed to increase plant resistance to different stressors, chemical agents play the most important role. They act as highly effective pesticides, fertilizers or growth regulators. However, because they accumulate in soil, water and inside living organisms, their widespread use in agriculture has an adverse effect on the environment. There is an increasing need to find solutions that either allow to reduce the concentrations of chemical agents through formulations or to replace them with biological, environmentally friendly solutions (Singh et al., 2020, Journal of Hazardous Materials, 385, 121525; https://doi.org/! 0.1016/j.jhazmat.2019. 121525).

Chitin and its derivative chitosan are promising alternatives to chemical agents (Malerba and C er ana, 2020, Polysaccharides, 1, 21 30; https://doi.org/10.3390/polysaccharidesl010003). Chitin is a long-chain, high molecular weight polysaccharide composed of N-acetyl-D-glucosamine and D-glucosamine. It is the main component of the exoskeleton of arthropods and of the cell wall of mushrooms. Chitosan is derived from chitin by deacetylation. Different factors impact the effectiveness of chitosan-based treatments: molecular weight, chemical modifications, pH, concentration, chelating potential, and the type of microorganism that is targeted. The use of chitosan in various crops is actively studied. Chitosan formulations have been developed to formulate pesticides (Yang et al., 2018, J. Agric. Food Chem., 66, 5, 1 67 1074; https://doi.org/10.1021/acs.jafc.7b04147; Mohamed et al., 2016, Chitosan in the Preservation of Agricultural Commodities, 179-219; https://doi.org/10.1016/B978-0-12- 802735-6.00007-0), to treat seeds prior to sowing (Siddaiah et al, 2018, Sci. Rep., 8, 2485; https://doi.org/10.1038/s41598-017-19016-z') and as soil amendment (Orzali et al. 2017, Chitosan in Agriculture: A New Challenge for Managing Plant Disease, Biological Activities and Application of Marine Polysaccharides, https://doi. org/10.5772/66840). Some plant-care formulations comprising a cross-linked chitosan have been developed for the formulation of plant protecting products by mixing the plant protecting substances with chitosan and inducing cross-linking on the mixture (Vinod et al., 2013, International Journal of Biological Macromolecules, 62(16), 677-683; VinodetaL, 2015, International Journal of Biological Macromolecules, 75 (21), 343-353; Vinod et al, 2016, International Journal of Biological Macromolecules, 64 (31), 6148-6155). However, known cross-linked chitosan formulations of plant protecting products usually rely on tripoly-phosphates (TPP) which leads to particles of reduced adherence to biological tissues in general, and plant tissues in particular. Additionally, the size of these particles results in a low specific surface and limited biological activity. Finally, the manufacturing process for TPP -based particles is an emulsification that depends on many variables and is poorly reproducible and scalable.

The main limiting factor in the use of chitosan in agriculture are its poor solubility (Deepmala et al, 2014, Adv Plants Agric Res., 1(1), 23-30; https://doi. org/10.15406/apar.2014.01.00006) which hampers its use as pesticide carrier, and its physical, chemical and biological fragility, which renders chitosan-based formulations short lived and impractical.

Therefore, there is an increasing need for the development of new formulations of plant care products, in particular those that favorably impact plant growth and development, while preserving the quality and quantity of harvest.

Summary of the invention

The present invention is based on the finding of new use of a chitosan derivative and for the formulation of plant care substances and other agriculturally useful substances. In particular, the present invention is based on the unexpected finding of chemically modified chitosan formulations which exhibit strong adherence to plant tissues, allowing targeted application and preventing runoff and/or leaching of plant care products, in particular pesticides. This addresses a major problem as the actual utilization of biological target uptake is only less than 0.1% after dust drift and rainwater leaching (Zhao et al., 2018, J. Agric. Food Chem., 66, 26, 6504 6512; https://doi.org/10.1021/acs.jafc. 7602004). Further, the formulations of the invention are advantageously frost resistant, enable sustained release of PCPs, and degrade very slowly. Further, it has been found that methods of the invention allow to generate in a single step sprayable solution/suspensions of plant care or protecting products (including plant care or protecting products in solid form which are insoluble in water). Finally, the formulations of the invention provide an advantageous protection against biological (e.g. enzymes, microorganisms) or physical (e.g. UV radiation) degradation of PCPs. The present invention also relates to methods of preparation PCPs having the following advantages: capacity to achieve a formulation in water, methods fast and reliable which are versatile and allow to customize formulation types and obtain formulations which are environmentally friendly.

An aspect of the invention provides a plant-care formulation comprising a soluble crosslinked chitosan and at least one plant protecting product, said plant care formulation comprises from about 0.01% to about 2 % (w/v) of soluble cross-linked chitosan and uses thereof.

Another aspect of the invention provides a method for the preparation of a plant care formulation of the invention.

Another aspect of the invention relates to a plant-care formulation obtainable from a method according to the invention.

Another aspect of the invention relates to a use of a soluble cross-linked chitosan and at least one plant protecting agent for the preparation of a plant-care formulation.

Another aspect of the invention relates to the use of a plant-care formulation according to the invention in agriculture and plant cultivation.

Description of the figures

Figure 1 shows the appearance of leaf disks (permissive Cabernet Sauvignon leaves) 7 days after inoculation with a suspension of P. viticola sporangia following treatment with various formulations of the invention and comparative formulations as described in Example 2. a: water; b: copper oxychloride (0.4%); c: Formulation of the invention Fl; d: Formulation of the invention F2; e: grapevine cane extract (7.5 mg/ml); f: grapevine cane extract (3.75 mg/ml); g: grapevine cane extract (1.875 mg/ml); h: Formulation of the invention F3; h: Formulation of the invention F4; i: Formulation of the invention F5; j: copper sulfate (2.5 mg/ml); k: copper sulfate (1.25 mg/ml); 1: copper sulfate (0.5 mg/ml); m: Formulation of the invention F6; n: Formulation of the invention F7; o: Formulation of the invention F8.

Figure 2 shows the microscopic appearance of initial copper solutions or suspensions and corresponding copper-containing formulations of the invention observed by microscopy as described in Example 3. a: copper sulfate (CuSCU) prior to formulation; b: formulated copper sulfate; c: tribasic copper oxychloride (CU2(OH)3C1) prior to formulation; d: formulated tribasic copper oxychloride; e: copper hydroxide (Cu(OH)2) prior to formulation; f: formulated copper hydroxide.

Figure 3 shows the microscopic appearance of a colloidal sulfur-containing plant care formulation of the invention observed by electron microscopy as described in Example 4.

Detailed description

The term "degree of crosslink" as used herein means the quantity functional groups converted into crosslinking or grafting bonds relative to the total quantity of functional groups The term "degree of crosslink" as used herein means the quantity functional groups converted into crosslinking or grafting bonds relative to the total quantity of functional groups initially present on the chitosan, expressed as a percentage.

The term “alkyl” when used alone or in combination with other terms, comprises a straight or branched chain of C1-C50 alkyl which refers to monovalent alkyl groups having 1 to 50 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, i -propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl, 1 -ethylpropyl, 2-m ethylbutyl, 3- m ethylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-m ethylpentyl, 3 -methylpentyl, 4- methylpentyl, n-heptyl, 2-methylhexyl, 3 -methylhexyl, 4-methylhexyl, 5 -methylhexyl, n- heptyl, n-octyl, n-nonyl, n-decyl, tetrahydrogeranyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-octadecyl, n-nonadecyl, and n-eicosanyl and the like. Preferably, these include C1-C9 alkyl, more preferably Ci-Ce alkyl, especially preferably C1-C4 alkyl, which, by analogy, refers respectively to monovalent alkyl groups having 1 to 9 carbon atoms, monovalent alkyl groups having 1 to 6 carbon atoms and monovalent alkyl groups having 1 to 4 carbon atoms. Particularly, those include Ci-Ce alkyl.

The term "alkenyl” when used alone or in combination with other terms, comprises a straight chain or branched C2-C50 alkenyl. It may have any available number of double bonds in any available positions, and the configuration of the double bond may be the (E) or (Z) configuration. This term is exemplified by groups such as vinyl, allyl, isopropenyl, 1 -propenyl, 2 -m ethyl- 1 -propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-ethyl-l-butenyl, 3- methyl-2-butenyl, 1 -pentenyl, 2-pentenyl, 3 -pentenyl, 4-pentenyl, 4-methyl-3 -pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 1-octenyl, geranyl, 1-decenyl, 1 -tetradecenyl, 1 -octadecenyl, 9-octadecenyl, 1-eicosenyl, and 3, 7, 11, 15- tetram ethyl- 1 -hexadecenyl, and the like. Preferably, these include C2-C8 alkenyl, more preferably C2-C6 alkenyl. Among others, especially preferred are vinyl or ethenyl (- CH=CH2), n-2-propenyl (allyl, -CH2CH=CH2), isopropenyl, 1-propenyl, 2-methyl-l- propenyl, 1-butenyl, 2-butenyl, and 3-methyl-2-butenyl and the like.

The term "alkynyl” when used alone or in combination with other terms, comprises a straight chain or branched C2-C50 alkynyl. It may have any available number of triple bonds in any available positions. This term is exemplified by groups such as alkynyl groups that may have a carbon number of 2-50, and optionally a double bond, such as ethynyl (-C=CH), 1-propynyl, 2-propynyl (propargyl: -CH2C=CH), 2-butynyl, 2- pentene-4-ynyl, and the like. Particularly, these include C2-C8 alkynyl, more preferably C2-C6 alkynyl and the like. Preferably those include C2-C6 alkynyl which refers to groups having 2 to 6 carbon atoms and having at least 1 or 2 sites of alkynyl unsaturation.

The term “aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (c.g, indenyl, naphthyl). Aryl include phenyl, naphthyl, anthryl, phenanthrenyl and the like.

The term “Ci-Ce alkyl aryl” refers to aryl groups having a Ci-Ce alkyl substituent, including methyl phenyl, ethyl phenyl and the like.

The term “aryl Ci-Ce alkyl” refers to Ci-Ce alkyl groups having an aryl substituent, including 3-phenylpropanyl, benzyl and the like.

The term “heteroaryl” refers to a monocyclic heteroaromatic, or a bicyclic or a tricyclic fused-ring heteroaromatic group. Particular examples of heteroaromatic groups include optionally substituted pyridyl, pyrrolyl, pyrimidinyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3- oxadiazolyl, 1,2,4-oxadia-zolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl, [2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, isobenzothienyl, indolyl, isoindolyl, 3H-indolyl, benzimidazolyl, imidazo[l,2-a]pyridyl, benzothiazolyl, benzoxa-zolyl, quinolizinyl, quinazolinyl, pthalazinyl, quinoxalinyl, cinnolinyl, napthyridinyl, pyrido[3,4-b]pyridyl, pyrido[3,2- b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl, tetrazolyl, 5, 6,7,8- tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl, xanthenyl or benzoquinolyl.

The term “Cs-Cs-cycloalkyl” refers to a saturated carbocyclic group of from 3 to 8 carbon atoms having a single ring (e.g., cyclohexyl) or multiple condensed rings (e.g., norbornyl). Cs-Cs-cycloalkyl includes cyclopentyl, cyclohexyl, norbomyl and the like.

The term “heterocycloalkyl” refers to a Cs-Cs-cycloalkyl group according to the definition above, in which up to 3 carbon atoms are replaced by heteroatoms chosen from the group consisting of O, S, NR, R being defined as hydrogen or methyl. Heterocycloalkyl include azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, tetrahydrofuranyl and the like.

Unless otherwise constrained by the definition of the individual substituent, the term “substituted” refers to groups substituted with from 1 to 5 substituents selected from the group consisting of “Ci-Ce alkyl,” “C2-C6 alkenyl,” “C2-C6 alkynyl,” “Cs-Cs-cycloalkyl,” “heterocycloalkyl,” “Ci-Ce alkyl aryl,” “Ci-Ce alkyl heteroaryl,” “aryl Ci-Ce alkyl,” “heteroaryl Ci-Ce alkyl,” “Ci-Ce alkyl cycloalkyl,” “Ci-Ce alkyl heterocycloalkyl,” “amino,” “aminosulfonyl,” “ammonium,” “acyl amino,” “amino carbonyl,” “aryl,” “heteroaryl,” “sulfinyl,” “sulfonyl,” “alkoxy,” “alkoxy carbonyl,” “carbamate,” “sulfanyl,” “halogen,” trihalomethyl, cyano, hydroxy, mercapto, nitro, and the like.

The term “biologically acceptable” refers to a carrier comprised of a material that is not biologically or otherwise undesirable and not especially toxic.

The term “carrier” refers to any component present in a formulation other than the active agent and thus includes diluents, binders, lubricants, disintegrants, fillers, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives and the like.

The term “plant protecting products” (PCPs) or “plant protecting substances” refers to products intended for use in agriculture or in plant growing processes, said products consisting of, or containing substances active as plant safeners or synergists. Such products or substances are useful for protecting plants against all harmful organisms or preventing the action of such organisms (e.g. fungicides, insecticides, bactericides), for influencing the life processes of plants, such as substances influencing their growth, such as fertilizers or preserving plant products (e.g. extending the life of cut flowers), for aiding in recovery after injuries or abiotic stress (e.g. frost damage), for controlling or preventing undesired growth of plants or for destroying undesired plants or parts of plants (e.g. herbicides/weedkillers to kill actively growing weeds). According to an embodiment, PCPs include small molecules, synthetic analogs of DNA and RNA oligonucleotides, low-molecular weight compounds of biological origin: hydrophobic compounds, such as terpenoids and pyrethroids, and others and hydrophilic extracts, such as extracts of polyphenols, polysaccharides, high-molecular weight substances of biological origin: proteins (such as Delta endotoxins from Bacillus thuringiensis) and nucleic acids (such as anti-sense DNA and RNA oligonucleotides), biological entities such as spores, bacteria and other microorganisms such as viruses and multicellular organisms (e.g. nematodes and insects). The term “plant protecting products” or “plant protecting substances” comprises agents also referred to as plant protecting products (PPP) and substances which are also referred to under the generic term “pesticides”. In the context of the present invention, the following class of agents can be formulated according to the invention:

Chemical fungicides: Inhibitors of nucleic acid metabolism: phenylamides (acylalanines, oxazolidinones, butyrolactones), hydroxy-(2-amino-) pyrimidines, heteroaromatics (isoxazoles, isthiazolones), carboxylic acids; Inhibitors of cell division: methyl benzimidazole carbamates (benzimidazoles, thiophanates), N-phenyl carbamates, benzamides, thiazole carboxamide, phenylureas, cyanoacrylates, aryl-phenyl-ketones; Inhibitors of respiration: succinate-dehydrogenase inhibitors (phenyl-benzamides, phenyl-oxo-ethyl thiophene amide, pyridinyl-ethyl-benzamides, phenyl-cyclobutyl- pyridineamide, furan- carboxamides, oxathiin- carboxamides, thiazole- carboxamides, pyrazole-4- carboxamides, N-cyclopropyl-N-benzyl-pyrazole-carboxamides, N- methoxy-(phenyl-ethyl)-pyrazole-carboxamides, pyridine-carboxamides, pyrazinecarboxamides), quinone outside inhibitors (methoxy-acrylates, methoxy-acetamide, methoxy-carbamates, oximino-acetates, oximino-acetamides, oxazolidine-diones, dihydro-dioxazines, imidazolinones, benzyl-carbamates, tetrazolinones), quinone inside inhibitors (cyano-imidazole, sulfamoyl-triazole, picolinamides), uncouplers of oxidative phosphorylation (dinitrophenyl-crotonates, 2,6-dinitro-anilines), tri-phenyl tin compounds, thiophene-carboxamides, triazolo-pyrimidylamine; Inhibitors of amino acid and protein synthesis: anilino-pyrimidines, enopyranuronic acid, hexopyranosyl, glucopyranosyl, tetracycline; Inhibitors of signal transduction: aza-naphthalenes (aryloxyquinoline, quinazolinone), phenylpyrroles, dicarboximides; Inhibitors of lipid transport/membrane biosynthesis and metabolism: piperidinyl-thiazole-isoxazolines, piperazines, pyridines, pyrimidines, imidazoles, triazoles, triazolinthiones, morpholines, piperidines, spiroketal-amines, hydroxyanilides, amino-pyrazolinon, thiocarbamates, allylamines; Inhibitors of cell wall biosynthesis: validamycin, peptidyl pyrimidine nucleoside, carboxylic acid amides (cinnamic acid amides, valinamide carbamates, mandelic acid amides), isobenzo-furanone, pyrrolo-quinolinone, tri azol obenzo-thi azole, cyclopropane-carboxamide, carboxamide, propionamide, trifluoroethyl-carbamate; Host plant defence induction: benzo-thiadiazole, benzisothi azole, thiadi azol e-carb oxami de, ethyl phosphonates, isothiazolylmethyl ether. This class further includes chemical fungicides belonging to the class of cyanoacetamide-oximes, phthalamic acids, benzotriazines, benzene-sulphonamide, pyridazinone, phenyl-acetamide, guanidines, cyano-methylene-thiazolidine, 4-quinolyl-acetates, tetrazolyloximes, glucopyranosyl antibiotics and copper (different salts), sulphur, dithio-carbamates and relatives, phthalimides, chloronitriles, sulfamides, bis-guanidines, triazines, quinones, quinoxalines, maleimide, thiocarbamate. Further possible agents are referred to in Fungicide Resistance Action Committee (FRAC): FRAC Code List 2021: Fungal control agents sorted by cross resistance pattern and mode of action https://www.frac.info/docs/default-source/publications/frac- code-list/frac-code-list- 2021— final.pdf

Biological fungicides: Inhibitors of lipid transport/membrane biosynthesis and metabolism: Bacillus amyloliquefaciens. Melaleuca allernifolia. plant oils (eugenol, geraniol, thymol), amphoteric macrolide from Streptomyces natalensis! Streptomyces chattanoogensi; Host plant defene induction: polysaccharides (chitosan and others), plant extracts (anthraquinones, resveratrol), Bacillus spp., Saccharomyces spp.; Multiple modes of action: plant extracts (lectin, phenols, sesquiterpenes, triterpenoids, coumarins, terpene hydrocarbons, terpene alcohols, terpene phenols) such as grapevine cane extracts, living microbes or extract, metabolites (Trichoderma spp., Clonostachys spp., Coniothyrium spp., Saccharomyces spp., Bacillus spp., Pseudomonas spp., Streptomyces spp.). Further possible agents are referred to in Fungicide Resistance Action Committee (FRAC): FRAC Code List 2021, supra,'

Chemical Insecticides: Agents active on the nervous system: acetylcholinesterase inhibitors (carbamate, organophosphates), GABA-gated chloride channel blockers (cyclodiene organochlorines, phenylpyrazoles), sodium channel modulators (pyrethroids, pyrethrins, DDT, ethoxychlor), nicotinic acetylcholine receptor (nAChR) modulators (neonicotinoids, nicotine, sulfoximines, butenolides, mesoionics, spinosyns), Glutamate- gated chloride channel (GluCl) modulators (avermectins, milbemycins), chordotonal organ TRPV channelmodulators (pyridine azomethine derivatives, pyropenes, nereistoxin analogues, amitraz, oxadiazines, semicarbazones, diamides, flonicamid, meta-diamides Isoxazolines; Juvenile hormone mimics: juvenile hormone analogues (hydroprene, kinoprene, methoprene), fenoxycarb, pyriproxyfen); Non-specific inhibitors: alkyl halides, chloropicrin, fluorides, borates, tartar emetic, methyl isothiocyanate generators (dazomet, metam); Growth regulation: clofentezine, diflovidazin, hexythiazox, benzoylureas, buprofezin, cyromazine, diacylhydrazines; Energy metabolism inhibitors: diafenthiuron, organotin miticides, propargite, tetradifon, pyrroles, dinitrophenols, sulfluramid, hydramethylnon, acequinocyl, fluacrypyrim, bifenazate, fenazaquin, fenpyroximate, pyridaben, pyrimidifen, tebufenpyrad, tolfenpyrad, rotenone, phosphides, cyanides; Lipid synthesis, growth regulation: tetronic and tetramic acid derivatives, beta-ketonitrile derivatives, carboxanilides. Further possible agents are referred to in Insecticide Resistance Action Committee, IRAQ Mode of Action Classification Scheme, Version 9.4, 2020, https://irac- online.org/mode-of-action. This class further includes chemical insecticides belonging to the class of azadirachtin, benzoximate, bromopropylate, chinomethionat, dicofol, lime sulfur, mancozeb, pyridalyl, sulfur;

Antisense oligonucleotides analogs (modified bases);

Biological Insecticides: Microbial disruptors of midgut membranes: Bacillus thuringiensis and the insecticidal proteins they produce (Cry), Bacillus sphaericus, Paenibacillus popillia, Serratia entomophilia, Host-specific occluded pathogenic viruses: Granuloviruses, Nucleopolyhedroviruses, Reoviridae, Parvoviridae, Nudiviruses and GS-omega/kappa HXTXHvla peptide Further possible agents are referred to in Insecticide Resistance Action Committee, 2020, supra. This class further includes biological Insecticides belonging to the class of Burkholderia spp, Wolbachia pipientis, Chenopodium ambrosioides near ambrosioides extract, fatty acid monoesters with glycerol or propanediol, Neem oil, Beauveria bassiana, Metarhizium anisopliae, Paecilomyces fumosoroseus;

Entomopathogenic fungi (Litwin et al., 2020, Rev Environ Sci Biotechnol, 19, 23 42) Insecticidal nematodes ; Earth and minerals: diatomaceous earth, silicates; chemical herbicides: Inhibitors of Photosynthesis: triazines, triazinones, uracils, phenylcarbamates, amides, nitriles, n-phenyl-imides, diphenyl ethers, n-phenyl- oxadiazolones, n-phenyl-triazolinones, pyridiniums, phosphinic acids, cyclopyrimorat, triketones, pyrazoles, phenyl -ethers, n-phenyl heterocycles, diphenyl heterocycles, Isoxazolidinones, amitrole; Inhibitors of cellular metabolism: imidazolinone, sulfonylurea, triazolopyrimidines, triazolinones, pyrimidinyl benzoates, sulfonanilides, cyclohexanediones, aryloxphenoxy-propionates, alpha a-thioacetamides, alphachloroacetamides, alpha-oxyacetamides, oxiranes, isoxazolines, azolyl-carboxamides, benzofuranes, thiocarbamates, benzyl ethers, alkylazines, nitriles, endothall, tetflupyrolimet, glyphosate, asulam; Inhibitors of cell division and growth: dinitroanilines, phosphoroamidates, pyridines, carbamates, dinitrophenols, pyridinecarboxylates, pyridyloxy-carboxylates, benzoates, quinoline-carboxylates, phenoxycarboxylates, aryl-carboxylates, bensulide, bromobutide, cumyluron, difenzoquat, diphenamid, dsma, dymron/daimuron, etobenzanid, flamprop-m, fosamine, naproanilide, napropamide, oxaziclomefone, pelargonic acid, pyributicarb. Further possible agents are referred to in Herbicide Resistance Action Committee, HRAC Mode of Action Classification 2021 Map, https://hracglobal.com/tools/hrac-mode-of-action- classification-2021 -map/.

The chitosan derivative suitable for a use according to the invention is a soluble crosslinked chitosan as described in PCT/EP2021/053204 (WO 2021/160667) or any pharmaceutically acceptable salts thereof. In particular, the cross-linked chitosan is obtainable by a method comprising the following steps: a) providing a chitosan and leaving the said chitosan to swell in a solvent; b) acylating the amino groups of said chitosan with an acrylic compound of Formula (I): wherein Ri is an a halogen or any other leaving group that upon removal, ensures acylation of an amino group such as 3 -hydroxybenzotriazole ester, anhydride (including mixed anhydrides), N-hydroxysuccinimide, pentachlorophenol, 2-nitro-4-sulfophenol esters and other similar leaving groups; R2, R3 and R4 are independently selected from H; optionally substituted alkyl (e.g. Ci-Ce alkyl), optionally substituted alkenyl (e.g. C2-C6 alkenyl), optionally substituted alkynyl (e.g. C3-C6 alkynyl), optionally substituted cycloalkyl (e.g. Cs-Cs-cycloalkyl), optionally substituted cycloalkenyl (e.g. C4-C8 cycloalkenyl), optionally substituted cycloalkynyl (e.g. Cs-Cs cycloalkynyl), optionally substituted heterocycloalkyl; optionally substituted aryl (e.g. optionally substituted phenyl), optionally substituted heteroaryl and optionally substituted aryl Ci-Ce alkyl, in particular benzyl; wherein the term “substituted refers to groups substituted with from 1 to 5 substituents selected from the group consisting of halogen, -COOR', -NR'R", =0, - OR', -COR', -CONR'R", -SR', -SO3R', -SO ₂ NR'R ", -SOR', -SO ₂ R', -NO ₂ , or -CN; or Ri and R2 or Ri and R3, or Ri and R4 together form an optionally substituted 4-24 membered aryl, heteroaryl, cycloalkyl or heterocycloalkyl (e.g. a 6-24 membered aryl, heteroaryl, cycloalkyl or heterocycloalkyl such as an optionally substituted 8-24 membered aryl, heteroaryl, cycloalkyl or heterocycloalkyl); c) reacting the acylation product of step b) in the presence of a base (Aza-Michael reaction); d) purifying the cross-linked chitosan obtained from step c) (e.g. from salt impurities and/or aprotic solvent).

According to a particular embodiment, the solvent used under step a) and/or b) is a protic solvent (such as alcohols or water).

According to a particular embodiment, the free acrylic acids which are formed if a protic solvent (such as alcohols or water) is used under step a) and/or b) to conduct the process are washed off from the reaction mixture before carrying out step c).

According to another particular embodiment, the solvent is an aprotic solvent, in particular under step a) and/or b) and/or c).

According to a particular embodiment, if an aprotic solvent is used under step c) if no further step f) of subsequent acylation is carried out.

According to a particular embodiment, step a) is conducted at room temperature.

According to a particular embodiment, the R3 or R4 but also R2 groups of the acylated product of step b) will react with the primary amino groups of the glucosamine backbone to form a cross-link between glucosamines. The groups reacting will depend on the specific acrylic compound. For example, for acrylic and methacrylic acids, the groups reacting with the primary amino groups of the glucosamine backbone are groups R3 or R4. However, when R3 and R4 groups are replaced by halogen atoms, then R2 groups will react with the primary amino groups of the glucosamine backbone.

According to a particular aspect, the soluble cross-linked chitosan is fully soluble at pH<5.5 in aqueous solution.

Typically, the soluble cross-linked chitosan has a viscosity that is approximately 3-fold inferior to the original chitosan that was used for its synthesis. The working concentrations, for practical liquid handling reasons, range from 0.01% to 2%.

According to a further particular embodiment, this cross-linking leads to the formation of nanoparticles.

According to a further particular embodiment, the soluble cross-linked chitosan forms a nanosuspension, typically particles having a diameter from about 5 nm to about 50 nm (e.g. 20 nm) at pH 7.0 or above. Such a nanosuspension has a lower viscosity than the corresponding solution (at pH<7.0) and can be manipulated as a true solution. In case of any precipitate/sediment formation, it can be easily resuspended.

According to particular aspect, the soluble cross-linked chitosan can be obtained by a method schematized under Scheme 1 as follows: wherein a chitosan (A) wherein m is a integer comprised between 1 and 12’500 and n is a integer comprised between 1 and 12’500, is first provided in a swollen state in an aprotic solvent at room temperature and then the amino groups of said chitosan are acylated with an acrylic compound (I) and the resulting acylation product (Bl) is then reacted in presence of a base to lead to a cross-linked chitosan (B2) which can then be isolated by purification to lead to a purified cross-linked chitosan according to the invention.

According to a particular embodiment, the chitosan can be provided in swollen state, even in dissolved state, in a protic solvent but in this case, side reactions of hydrolysis of the acylating agent can occur and this must be taken into account when calculating reaction loads. Additionally, in this case, it will be necessary to wash the acylated chitosan obtained under step b) to remove the hydrolysis products of the acylating agent before the stage of aza-Michael reaction under step c).

According to a particular embodiment, the chitosan is provided in absence of water and the acylating step is carried out in absence of water. The absence of water advantageously leads to higher yields and avoid the formation of side products.

According to a particularly advantageous aspect, the acylation step is conducted in absence of water. In this case, an aprotic solvent can be used as reaction medium or a supercritical fluid. According to a particular embodiment, the aprotic solvent is a polar aprotic solvent such as for example selected from DMF and DMSO. In a particular embodiment, a supercritical fluid can be a supercritical solvent such as carbon dioxide, nitric oxide (I), freons (chloro(bromo)(fluoro)hydrocarbons) which is used to provide the acylating agent to the reaction medium and then acylation reaction carried out after removal of the supercritical solvent from the reaction medium, for example by lowering the pressure below the critical value.

According to a further particularly advantageous aspect, the acylation step is conducted an anhydrous aprotic medium.

According to a particular aspect, a polar aprotic solvent is selected from dimethylformamide (DMF), dimethylacetamide, acetonitrile (MeCN), N- methylpyrrolidone, dimethyl sulfoxide (DMSO) or a mixture thereof.

According to another particular aspect, di chloromethane, di chloroethane, chloroform, and other chloro(fluoro)hydrocarbons can be also used as a polar aprotic solvent, but when conducting the aza-Michael reaction step c), those solvents should be distilled off right before the provision of the base. It is desirable to perform such distillation at temperatures not exceeding 60°C, which is feasible at atmospheric pressure for most of the mentioned solvents. If a high-boiling solvent was used, distillation must be carried out under reduced pressure. According to another particular aspect, ethers and esters, ketones can also be used as solvents for the reaction steps a) to c) under anhydrous conditions but reactions in such solvents will proceed more slowly. For example, diethyl ether, diisopropyl ether, methyl tert-butyl ether, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, acetone, methyl ethyl ketone and diethyl ketone can be used.

According to a particular aspect, the acylation step b) can be conducted by any method described in the context of acylation of glucosamine with acryloyl chloride (Zhang et al., 2017, Biomacromolecules, 1, 3, 778 786; Bu et al., 2017, Advances, 7, 76, 48166 -

48175) or methods described as useful for the acylation of an amino group such as methods using i) a carbodiimide; ii) an azide; iii) mixed anhydrides; iv) an activated ester and v) others methods described below.

Known acylation methods using carbodiimide with the formation of intermediate enol esters can be used under step b) (WO 2019/60740; Hao-Bin et al, 2018, Carbohydrate Polymers, 196, 359 - 367). If N,N'-dicyclohexylcarbodiimide (DCC) can be used as condensing agent, l-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride and N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide methyl p-toluenesulfonate (CAS Registry Number: 2491-17-0) would be preferred.

Known acylation methods using azides can be used under step b) (Honzl et al, 1961, Coll. Czech. Chem. Commun., 26, N. 9, 2333-2344).

Known acylation methods using mixed anhydrides can be used under step b) (Wieland et al., 1951, Ann. Chem., 572, N3, 190-194; Belleau et al., 1968, J. Amer. Chem. Soc., 90, N 6, 1651-1652; Gorecka et al., 1978, Synthesis, N 6, 474-476; Diago-Meseguer et al., 1980, Synthesis, N 7, 547-551; Leplawy et al., 1960, Tetrahedron, 11, N 1, 39-51). The use of internal anhydrides is also possible for acylation, for example maleic anhydride (Liwschitz et al. 1957, Journal of the Chemical Society, 4399; Kang, et al, 2014, Bioorganic and Medicinal Chemistry Letters, 2, 10, 2364-2367; US 2016/200730; Sanchez et al., 2010, Anna European Journal of Organic Chemistry, 13, 2600 - 2606).

Known acylation methods using activated esters through the formation of activated amides can be used under step b) such as a carbonyldiimidazole method (Paul etal., 1960, J. Amer. Chem. Soc., 82, N 17., 4596-4600), a cyanomethyl ester method (Schwyzer et al., 1955, Helv. Chim. Acta, 38, N 1, 80-83), a thiophenyl ester method (Wieland et al., 1951, supra), a substituted phenyl ester method (Gross et al. , 1983, Mayenhofer, editors. Moscow: Mir., P. 421), an ester method with heteroaromatic compounds together with carbodiimide method (Jakubke et al., 1966, A. Chem. Ber., 99, N 8, 2419-2429; Taschner et al., 1965, Ann. Chem., 690, 177-187), a method of esters with hydroxylamine derivatives together with carbodiimide method (Losse et al., 1964, Ann. Chem., 678, 185- 190; Nefkens et al., 1961, Amer. Chem. Soc., 83, N 5, 1263; Anderson et al., 1963, Ibid, 85, N 19, 3039; Konig et al., 1970, Chem. Ber., 103, N 3, 788-798), interesterification methods (variant of the ester method) (Sakakibara, 1965, Bull. Chem. Soc. Jap., 38, N 1, 1979-1984; Fujino et al, 1968, Ch. Chem. Pharm. Bull, 16, N 5, 929-932; Gudkov etal., 1978, 48, 9, 2146; Devadas et al, 1979, Ind. J. Chem., B16, N 11, 1026-1027).

Other known acylation methods can be can be used under step b) such as ketenimine method (Stevens et al., 1958, J. Amer. Soc., 80, N 15, 4069-4071); Acetylene derivatives method (Arens, 1955, Rec. Trav. Chim., 74, N 6, 759-770; Gais 1978, Aktivierungsmittel fur Peptidsynthesen J. Angew. Chem. Int. Ed, 90(8), 625-626 https://d0i.0rg/l 0.1002/ange.19780900808); method using derivatives of cyanamide (Losse et al, 1960, Ann. Chem., 636, 144-149); Synthesis using isoxazolium salts Woodwart et al., 1961, J. Amer. Chem. Soc., 83, N 4, 1010-1012); Synthesis using imidoyl halides (Bergmann et al, 1936, J. Biol. Chem., 115, N 3, 93-611).

According to a particular embodiment, the acrylic compound of Formula (I) can be an acid, an acid halide, an active ester (e.g. 3 -hydroxybenzotriazole ester, N- hydroxysuccinimide, pentachlorophenol, 2-nitro-4-sulfophenol esters and esters having other similar leaving groups), an anhydride or mixtures thereof.

In particular, said soluble cross-linked chitosan is characterized by a mass spectrum pattern comprising a peak with m/z=252.077 ± 0.01 electrospray positive ions.

According to a particular aspect, a soluble cross-linked chitosan suitable according to the invention can be identified be a method comprising the steps of: providing a chitosan to be characterized in a solvent (e.g. methylene chloride or chloroform); acylating said chitosan as described herein (e.g. as acetyl chloride or acetic anhydride) under vigorous stirring such as for about 0°C to about 20°C, such as about 30 minutes; neutralizing the reaction medium with a base (e.g. diisopropylethylamine as non- nucleophilic base); evaporating the solvents and washing the obtained neutralized product; subjecting the product to a reflux acid hydrolysis (e.g. with preferably 28% hydrochloric acid for about 1 to 3 hours, such as about 2 hours); evaporating the reaction mixture and re-suspending the hydrolysed products in a weak acid such as acetic acid; determining the presence or absence of a product selected from a product according to Formula (Illa), or according to Formula (Illb)

(Hla); (Illb); wherein R2 to R4 are as defined herein, R2’ to R4’ are as defined respectively as R2 to R4 herein and wherein the presence of a product of Formula (Illa) and/or (Illb) is indicative of cross-linked chitosan obtained by a method described above and suitable for formulations according to the invention.

When dissolved, said soluble cross-linked chitosan and formulations thereof according to the invention differ from standard chitosans in that it generates not a viscous gel, but a low-viscosity solution (e.g. for relatively low concentrations (up to 2% [w/v]) Novochizol™ is 3 times less viscous than chitosan) that can be applied to any surface (seed, foliage) as a very fine aerosol.

This property enables to formulate all classes of plant protecting substance or a mixture thereof: small molecules, peptides, proteins, nucleic acids, and supram olecular entities such as inanimate particles, viruses, cells, and multicellular organisms.

According to a particular aspect, the soluble cross-linked chitosan acts as an emulsifier for very hydrophobic plant protecting substances that are otherwise very difficult to formulate.

According to a particular aspect, the soluble cross-linked chitosan allows to prepare plancare formulations with customizable sustained release properties.

According to a particular aspect, is provided a plant care formulation, wherein said formulation is a liquid formulation. According to another particular aspect, is provided a plant care formulation, wherein said formulation is a solid formulation.

According to another particular aspect, is provided a plant-care formulation comprising from about 0.05% to about 1% (w/v) (e.g. from about 0.06% to about 0.15% (w/v)) of a soluble cross-linked chitosan and at least one plant protecting product, wherein said soluble cross-linked chitosan is fully soluble at pH<5.5 in aqueous solution.

According to another particular aspect, is provided a plant care formulation, wherein said formulation contains from about 0.0001% to about 99.99% (w/v) of at least one plant protecting product/sub stance.

According to another particular aspect, is provided a plant care formulation, wherein said formulation may further contain any further biologically acceptable carrier.

According to another particular aspect, is provided a plant care formulation, wherein the mass ratio plant protecting substance: soluble cross-linked chitosan is between 0.00001: 1 to 10’000: 1.

According to another particular aspect, is provided a plant care formulation, wherein mass ratio plant protecting substance: soluble cross-linked chitosan is between 0.5: 1 to 10’000: 1.

According to another particular aspect, is provided a plant care formulation, wherein said at least one plant protecting product is selected from a biological and a chemical fungicide.

According to another further particular aspect, is provided a plant care formulation, wherein said at least one plant protecting product is selected from a plant extract (e.g. grapevine cane extracts) and copper salt or oxide.

According to another further particular aspect, is provided a plant care formulation, wherein said at least one plant protecting product is a semiochemical or a mixture of semi ochemi cals such as pheromones (e.g. gall midge sex pheromones, straight-chained lepidopteran pheromones, mite pheromones and the like) and allochemics (e.g. allomones, kairomones or synomones). According to another further particular aspect, is provided a plant care formulation according to the invention wherein said at least one plant protecting product is grapevine cane extract and the ratio (w/w) grapevine cane extract to soluble cross-linked chitosan is from 10: 1 to 1 : 100.

According to another further particular aspect, is provided a plant care formulation according to the invention wherein said at least one plant protecting product is copper salt or oxide and the ratio (w/w) copper salt or oxide to cross-linked chitosan is from 10: 1 to 1 : 100.

According to another further particular aspect, is provided a plant care formulation according to the invention wherein said at least one plant protecting product is copper salt or oxide and the ratio (w/w) copper salt or oxide to cross-linked chitosan is from 3 : 1 and 10: 1.

According to another further particular aspect, is provided a plant care formulation according to the invention wherein said at least one plant protecting product is a semiochemical or a mixture of semi ochemi cals such as pheromones and the ratio (w/w) pheromone to cross-linked chitosan is from 100: 1 to 1 000 000: 1.

Preparation of a plant-care formulation according to the invention

According to a particular aspect is provided a method for the preparation of a plant-care formulation, said method comprising a step of combining at least one plant protecting substance or a mixture thereof with a soluble cross-linked chitosan (e.g. at 0.01% and 2% (w/v)).

According to a particular embodiment, the method is carried out at a temperature between 0°C and 100°C under atmospheric pressure and between 0°C and 374°C under 21.8MPa. The maximum allowable temperature is 374°C, under 21.8 MPa and the maximum allowable pressure 1’000 MPa. The temperature of the mixture needs to be uniform and therefore should be carried out by adequate heating methods ensuring such a uniform heating of the mixture during mixing step for example by the use of microwave, infra-red or terahertz radiation heating methods.

According to a particular embodiment, the plant care formulation is a liquid formulation. According to a particular embodiment, a method for the preparation of a plant-care formulation, comprises the steps of:

- providing a solution of at least one plant protecting substance in a water-miscible solvent;

- adding a soluble cross-linked chitosan in aqueous solution (e.g. at 0.01% and 2% (w/v)) to the mixture under vigorous mixing such as under ultrasound sonication.

According to a particular embodiment, the soluble cross-linked chitosan is added to the mixture such that mass ratio plant protecting substance: soluble cross-linked chitosan is between 0.00001 :1 to 10’000: 1.

According to a further particular embodiment, the soluble cross-linked chitosan is added to the mixture such that mass ratio plant protecting substance: soluble cross-linked chitosan is between 0.5: 1 to 10’000: 1. In this case, a micro-emulsion of cross-linked chitosan is obtained and the plant protecting substance aggregates (pm) are coated with cross-linked chitosan.

According to another further particular embodiment, the soluble cross-linked chitosan is added to the mixture such that mass ratio plant protecting substance: soluble cross-linked chitosan is between 0.00001 : 1 to 0.5: 1. In this case, the cross-linked chitosan will be impregnated with the plant protecting substance.

According to a particular embodiment, a solution of a plant protecting substance can be prepared such as in methanol, ethanol, acetone, DMF, DMSO, N-Methyl-2 -Pyrrolidone, acetic acid. For plant protecting substances that are fully miscible in a given solvent, a typical solution may comprise 90% (w/v) plant protecting substance and 10% (w/v) solvent, but minimal amounts of solvent (e.g. down to 0.01% w/v.) may be used. In case of very poor solubility, plant protecting substance concentrations as low as 0.001% (w/v) may be used (for example, for derivatives of glycyrrhizinic acid). Plant protecting substances existing in liquid form may be used as such or diluted in a solvent of choice.

According to another particular embodiment, the water-miscible solvent used to introduce the plant protecting substance into the formulation may be removed immediately after formulation by distillation at atmospheric pressure, or at a higher pressure so as to increase the temperature to accelerate solvent elimination process and increase the solubility of the plant protecting substance. Alternatively, the solvent may be removed through dialysis, freezing or barbotage. Unless it is desirable to concentrate the solution, water may be added to the mixture during or after the removal of the solvent to compensate for the loss of volume.

According to another particular embodiment, the method comprises the steps of:

- providing an aqueous solution of a soluble cross-linked chitosan (e.g. at 0.01% and 2% (w/v));

- adding a plant protecting substance in solution to the cross-linked chitosan solution under vigorous mixing such as under ultrasound sonication.

According to another particular embodiment, the liquid mixture between the soluble cross-linked chitosan and the plant protecting substance can be used readily or further subjected to drying or freeze-drying step for storage purposes before use. For example, a number of drying techniques may be used such as spray drying, lyophilization with or without auxiliary substances, drying in a rotary or film evaporator, under atmospheric or reduced pressure. It is also possible to freeze the preparation for storage purposes.

According to another particular embodiment, when the plant protecting substance is insoluble or poorly soluble, is provided a method for the preparation of a plant-care formulation according to the invention wherein the plant protecting substance is combined with a soluble cross-linked chitosan (e.g. at 0.001% and 25% (w/v.)) in solid state wherein one at least one of the two combined agent is in solid state.

In this case, the obtained powder or suspension mixture may then be dissolved in water to obtain a plant-care formulation and used immediately or frozen for storage.

According to further particular embodiment, when the plant protecting substance is insoluble or poorly soluble, is provided method for the preparation of a plant-care formulation, said method comprising the steps of: providing a plant protecting substance in solid form (e.g. as a fine powder of particles having a diameter from about 5 nm to about 200 pm); adding an aqueous solution of a soluble cross-linked chitosan (e.g. at 0.01% and 2% (w/v)) to the plant protecting substance in solid form under vigorous mixing such as under ultrasound sonication or spraying. According to further particular embodiment, when the plant protecting substance is insoluble or poorly soluble, is provided method for the preparation of a plant-care formulation, said method comprising the steps of: providing a soluble cross-linked chitosan (e.g. at 0.01% and 2% (w/v)) or a salt thereof, in dry state; mixing a plant protecting substance in solid form (e.g. as a fine powder of particles having a diameter from about 5 nm to about 200 pm);

- to the cross-linked chitosan in dry form under vigorous mixing such as under ultrasound sonication or spraying.

According to a further particular embodiment, the obtained powder or suspension mixture may then be dissolved/diluted in water with addition of acids with or without additional heating.

According to a further particular embodiment, when a cross-linked chitosan is mixed in dry form with a plant protecting substance both in dry form, the solid particles of the cross-linked chitosan and of the plant protecting substance may either be applied directly to the plant by spraying or the sprayed particles may be dried, frozen or dissolved/resuspended in water or another compatible solvent (e.g. glycerin, propylene glycol, ethanol, N-Methyl-2 -Pyrrolidone, etc.).

According to a particular embodiment, when acid-labile plant protecting substances are used (such as allyl alcohols and aldehydes belonging to the terpenoids class or living cells or viruses), the plant protecting substance solution or suspension may be prepared in a buffer solution to maintain the pH environment of the acid-labile plant protecting substance in a range where the plant protecting substance is not degraded (pH 6.5 - 7.5).

According to another particular embodiment the plant-care formulation of the invention may further include other biologically acceptable ingredients such as salts, oxides, hydroxides, urea, melamine, sugars, polysaccharides, antioxidants, natural and synthetic (polymeric) emulsifiers and synthetic organic chemicals commonly used for plant-care formulations. Those biologically acceptable ingredients may serve different purposes: such as anti-caking agents in case of solid formulations, as anti-oxidants, as antipartitioning agents, as cryoprotectors, as enhancing excipients, as pH buffers, as rheological additives that improve the conditions for product application, as dyes or odorants for the purpose of detecting treated surfaces. According to a particular embodiment, the plant-care formulation according to the invention may be applied to seeds (e.g. by drying an aerosol formulation) to maintain a low degree of humidity (e.g. as little as for example 8%) which is particularly advantageous to in case of seed treatment, as it is important to prevent premature germination.

According to a further particular embodiment, the plant-care formulation obtained by a method according to the invention may then be further mixed with the soluble crosslinked chitosan, typically at a ration from about 0.5: 1 to about 10’000: 1 to create an extra layer on the plant-care formulation particles which may increase adherence to the plant parts. Typically, this extra layer is monolayer, i.e. about 20 nm thick and contributes to delay the release of the plant protecting care/product and to a better adherence to plant tissue.

Examples illustrating the invention will be described hereinafter in a more detailed manner and by reference to the embodiments represented in the Figures.

EXAMPLES

The following abbreviations refer respectively to the definitions below: DMEM (Dulbecco's Modified Eagle Medium).

Example 1: Preparation of plant care formulation of the invention having fungicidal properties

A plant care formulation of the invention comprising a chemical fungicide was prepared according to a method of the invention and tested on Spring wheat, variety Thasos (Strube) (39.8 g/1000 seeds) as compared to commercial formulations as follows. Each experiment comprised 6 groups:

1. Control Group (C): No treatment of seeds;

2. « High DC/D »: Treatment with the commercial fungicide Diamond Super®, KS (https://zemlyakoff.com/katalog-preparatov/zashita-semyan/da jmond-super-ks/ active ingredients: Difenoconazole 30 g/1, Cyproconazol 6.3 g/1) at the recommended dilution (1.5 1/ton);

3. « Low DC/D »: Treatment with the commercial fungicide Diamond Super®, KS (active ingredients: Difenoconazole 30 g/1, Cyproconazol 6.3 g/1) at a 12-fold lower concentration than the recommended dilution (0.125 l/ton); 4. « Low DC/N »: Treatment with a plant-care formulation of the invention (soluble cross-linked chitosan Novochizol™, 0.8%, Succinic acid 0.4%, plant protecting substances: Difenoconazole 2.5 g/1 and Cyproconazol 0.525 g/1) at Diamond Super®, KS’s recommended dilution (1.5 1/ton);

5 « High DC/N »: Treatment with a plant-care formulation of the invention (soluble cross-linked chitosan Novochizol™ 0.8%, Succinic acid 0.4%, plant protecting substances: Difenoconazole 2.5 g/1, Cyproconazol 0.525 g/1) at a 12-fold higher concentration that Dividend Extreme’s recommended dilution (18 l/ton);

6. « Copper N »: Treatment with a plant-care formulation of the invention with Copper as plant protecting substance (soluble cross-linked chitosan Novochizol™ 1%, Cu+27 mg/ml), 0.51/ton.

Seeds were treated under humidification (10 1/t) in closed plastic containers as described below. a) Assessment of the phytosanitary state of the treated seeds

The phytosanitary state of the treated seeds was assessed by the evaluation of the degree of infection, growth and development of sprouted seedlings.

Seeds were treated 5 days prior to laying on a humid substrate (rolls of filter paper). Germination/growth was carried out for a total of 14 days under controlled humidity (7 days at t = +26°C (thermostat-controlled); 7 days under natural light, t = 22°C). At the end of the experiment, the following parameters were determined: degree of infection (determined visually by counting the number of lesions per seedling), the number of normally developed seedlings (visual assessment), the number of seedlings with lesions in the root system (determined visually), the height of the seedlings (determined visually), number of roots per seedling (determined visually), the length of the tap root (determined visually), and the dry biomass of the seedlings. The results are presented under Tables 1 and 2 below where the biological efficacy represents the proportion of seedlings without lesions.

Table 1

Those data support that the plant-care formulations according to the invention exhibited a strong phytosanitary activity against Bipolaris sorokiniana Shoem root rot (degree of infection in the control: 15%, as determined by the number of lesion (dark spots) on the roots). Further, the plant-care formulations according to the invention led to 100% control of bacteriosis (degree of infection in the control (C): 20%). The phyto-expertise can be summarized as follows:

Table 2 b) Assessment of the effect on growth at the initial stages of organogenesis of wheat

Three days after treatment, seeds were laid on a humid substrate (4 layers of filter paper) in Petri dishes (n=100) and put in a germination chamber, under conditions of controlled humidity and under natural light, t = +20-22°C. Germination energy was measured after 1 and 3 days (proportion of seeds that germinate rapidly, without any delays, here after 1 day and after 3 days - 2 measurements) and germination capacity after 7 days which indicates the viability, by measuring the overall proportion of seeds that germinate after a longer period (here 7 days) relative to the number that would germinate under optimal conditions (theoretically 100%). Growth indicators at the initial stages of organogenesis were recorded after 3 days (length of roots of each seedling, total length of seedlings) and after 7 days (number of roots per seedling, length of seedlings, total mass of roots and shoot per seedling, and mass of each root). The results are presented under Table 3 below:

Table 3

Those data support that plant-care formulation of the invention allows to substantially decrease the amounts of difenoconazole and cyproconazol, without affecting efficacy as compared to a commercial preparation.

Example 2: Preparation of plant care formulation of the invention for grapevine treatment against Plasmapora viticola

Crude extracts of grapevine (Vitis vinifera) canes have been shown to have significant antifungal activity, notably against mildew (Schnee et al., 2013, J. Agric. Food Chem., 61, 23, 5459- -5467. https://doi.org/10.1021/jf4010252; Richard et al., 2016, OENO One, 50, 3, https://doi.Org/10.20870/oeno-one.2016.50.3.l 178). However, the antifungal compounds in these extracts (simple and oligomeric stilbenoids) are photolabile. An additional limitation is the leaching of such extracts (Schnee et al., 2013, supra). As a result, grapevine cane extracts cannot be effectively used in the field. Cross-linked chitosan according to the invention, and chitosan nanoparticles in general exhibit strong adherence to plant materials (Maluin and Hussein, 2020, Molecules, 25, 7, 1611, https://doi.org/10.3390/molecules25071617). Formulations of the invention F1-F8 comprising extracts of grapevine canes and similar plant extracts with phytoprotective properties as PCPs are therefore promising formulations. Comparative formulations comprising only the PCPs were used as comparison.

A plant care formulation of the invention was prepared with following ingredients: - 1% w/v soluble cross-linked chitosan (Novochizol™) + 0.5% succinic acid aqueous solution: 1 (soluble cross-linked chitosan in aqueous solution); - 1% (w/v) regular chitosan + 0.5% succinic acid solution, 2 (regular chitosan in suspension in aqueous solution);

- 30 mg/ml grapevine cane extract aqueous solution (Schnee et al., 2013, supra), 3;

- 10 mg/ml copper sulfate aqueous solution, according to a method of the invention, said method comprising the steps of:

The ingredients were slowly added to the soluble chitosan (Novochizol™) suspension under sonication (24.5 kHz) in the following volume ratios: soluble cross-linked chitosan in aqueous solution: 20 ml Novochizol™ solution + one plant protecting substance in a water-miscible solvent: 20 ml regular chitosan solution (final concentrations: 0.5% or 5 mg/ml Novochizol™, 0.5% or 5 mg/ml regular chitosan); soluble cross-linked chitosan in aqueous solution: 30 ml Novochizol™ solution + one plant protecting substance in a water-miscible solvent: 10 ml grapevine cane extract solution (final concentrations: 0.75% or 7.5 mg/ml Novochizol™, 0.75% or 7.5 mg/ml grapevine cane extract); soluble cross-linked chitosan in aqueous solution: 30 ml Novochizol™ solution + one plant protecting substance in a water-miscible solvent: 10 ml copper sulfate solution (0.75% or 7.5 mg/ml Novochizol™, 0.25% or 2.5 mg/ml copper sulfate).

These formulations were diluted in distilled water, to yield specific concentrations of soluble cross-linked chitosan and plant protecting substance as indicated in Table 4 and 5 ml were applied to 10 randomized leaf discs (permissive Cabernet Sauvignon leaves) per dish. Control treatments included 5 ml distilled water and 5 ml copper oxychloride (0.4% in water). At 24 hours post treatment, leaf discs were inoculated with a suspension of P. viticola sporangia. The severity of infection was assessed by measuring the leaf surface carrying spores, after incubating the dishes for 7 days at 22°C, natural day/night cycle. Controls included water (positive control) and 0.4% copper oxychloride (negative control).

Table 4

* Formulations with cross-linked chitosan according to the invention

The results of those experiments demonstrate:

1. the capacity of a formulation of the invention to formulate regular chitosan, retaining or enhancing chitosan’s intrinsic antifungal properties. This is of significant practical importance, as cross-linked chitosan formulations are more stable, versatile and easier to use as plant care products than normal chitosan formulations.

2. the capacity of a formulation of the invention to formulate copper and reduce its toxicity. This finding is likely explained both by the capacity of the cross-linked chitosan to encapsulate active ingredients for slow, sustained release and by intrinsic plant protective effects (eliciting plant defense mechanisms).

3. a synergistic fungicide effect between the cross-linked chitosan with extracts of grapevine canes. This is an unexpected finding, which further supports the advantages of a formulation of the invention comprising extracts of grapevine canes.

Example 3: Reduced-copper plant care formulation of the invention for grapevine treatment against Plasmapora viticola

Copper-based antimicrobial compounds are widely used in both conventional and organic agriculture, and are the most effective contact fungicides against Plasmapora viticola, in grapevine (Koledenkova et al., 2022, Front Microbiol., 11, 13, 889472. https://doi.org/10.3389/fmicb). However, their extensive use over the last 150 years is problematic, as copper is accumulating in the top soils, with deleterious effects on soil biota and toxicity. (Lamichhane et al., 2018, Agron. Sustain. Dev., 38, 28, https://doi.org/! 0.1007/sl3593-018-0503-9 While no effective environmentally- friendly substitutes for copper exist, there is increasing regulatory pressure to reduce the dose of copper applied. The following experiments were conducted to assess the potential to reduce the effective dose of cupric ion Cu ⁺⁺ in formulations of three different copper compounds using formulations of the invention.

Plant care formulations of the invention were prepared as follows:

- concentrate solution of soluble chitosan (Novochizol™) 20X. Soluble crosslinked chitosan (Novochizol™, 3% w/v) was dissolved in succinic acid aqueous solution (1% w/v). This stock was used to prepare all copper-containing 10X concentrate formulations.

- CU ₂ (OH) ₃ C1 10X concentrated formulations. CU2(OH)3C1 (7.246 g, fine powder) were added to 500 ml deionized water and 500 ml of Novochizol 20X concentrate solution were added at a rate of 15 ml/min, under vigorous active mixing (ultrasound homogenization using a 2000W probe sonicator).

- CuSO4 10X concentrated formulations. CU2(OH)3C1 (5.414 g, fine powder) were dissolved in deionized water to 500 ml and 500 ml of 3% w/v Novochizol 20X concentrate solution were added at a rate of 15 ml/min, under vigorous active mixing (ultrasound homogenization using a 2000W probe sonicator).

- CU(OH)2 10X concentrated formulations. CU2(OH)3C1 (5.414 g, fine powder) were dissolved in deionized water to 500 ml and 500 ml of 3% w/v Novochizol 20X concentrate solution were added at a rate of 15 ml/min, under vigorous active mixing (ultrasound homogenization using a 2000W probe sonicator).

These concentrated formulations were used to prepare final treatment solutions as indicated in Tables 5 and 6.

Two-week-old grapevine seedlings of Chasselas variety, grown in pots (3-4 leaf stage, height of 6-7 cm) were treated in a spray cabinet (spray during 14s; 2,5bar = approx.1,2 bar at nozzle; 4 nozzles). Each treatment comprised 6 plants treated with a total of 80-100 ml final treatment solution. Seedlings were left to dry at room temperature for U day (Table 5, all treatments) or 2 days (Table 6, pre-treatment) and then inoculated with a suspension of Plasmapora viticola spores through whole plant spraying as above. Upon inoculation, plants were placed in an environment to induce sporulation (24h at 100% relative humidity and 21 °C, light 16h day/8h night) and then transferred to a growth chamber for disease development (20°C; light regime: 16h day/8h night) for a period of 6 days. Disease incidence was assessed as the proportion (%) of leaves with disease symptoms and disease severity as the proportion (%) of diseased leaf surface. Adherence of formulations (Wash-off, Table 6) were assessed by spraying treated seedlings in a spray cabinet with water, as above, for 20 seconds, ’A day after initial treatment and letting the seedlings dry as above prior to inoculation.

Table 5

** Reference product: Kocid https://www.agrar.bayer.ch/fr-CH/Produkte/Pjlanzenschutzmitt el/Kocide%20Opti

Table 6 https://www.agrar.bayer.ch/fr-CH/Produkte/Pjlanzenschutzmitt el/Kocide%20Opti

The results of these experiments demonstrate:

1. The capacity of using the soluble cross-linked chitosan to formulate a soluble form of copper salt (copper sulfate, Cu SO4) into non-soluble microscopic particles, likely through complex formation between chitosan and cupric ion (Fig. 2, a,b). This characteristic is essential for the use of copper sulfate as a plant protection product, because the active form of copper, the cupric ion, must be removed from solution to be released slowly.

2. The ability of obtain effective plant care formulations at reduced doses of copper, with reductions ranging from 50% (Table 5, CU2(OH)3C1 and CuSCU formulations) to 75 % (Table 5, CuSO double formulations). 3. Resistance of the formulations of the invention to wash-off (Table 6, wash-off) and prolonged efficacy (Table 6, pre-treatment).

Plant-care formulations of the invention provides a sustained release of the plant protecting product, a better adherence to plant tissues, a certain synergy between chitosan’s plant growth and immune defense elicitor effects and the plant protecting product.

Example 4: Novel colloidal sulfur-containing plant care formulation of the invention for wheat treatment against Alternaria triticina and Puccinia triticina

Soluble cross-linked chitosan (20 g) was dissolved in 1 liter tartric acid aqueous solution (7.5% w/v) and added at a rate of 15 ml/min to sodium thiosulfate pentahydrate (413, 5 g) dissolved to 800 ml deionized water, under vigorous active mixing (ultrasound homogenization using a 2000W probe sonicator).

The resulting formulation contained approx. 69 grams of colloidal, elemental sulfur per liter in a 1.1% soluble cross-linked chitosan solution.

The resulting formulation was added to 1.8 liter 3% Novochizol (w/v) in 1.5% succinic acid (w/v), under vigorous active mixing (ultrasound homogenization using a 2000W probe sonicator). The final, double formulation containing approx. 34.5 grams of colloidal, elemental sulfur per liter in a 2% (w/v) soluble cross-linked chitosan solution was diluted to yield various concentrations of working colloidal sulfur-containing plant care formulation.

Four independent field lots (2.5 m ² ) of winter wheat, Hanswin variety at two independent locations were sprayed with 1 liter of plant care formulations at various concentrations of colloidal sulfur at T2 and T3 stages, using 6 Teejet XR 110015 spraying nozzles on a 2- meter spraying bar. The T3 stage coincided with the emergence of Alternaria triticina followed by a superinfection with Puccinia triticina. Disease resistance was assessed as the green area (%) of 10 leaves from each lot. Grain yield was determined by manually harvesting a standardized number of ears of wheat from each lot and weighing the grain. The results are presented under Table 7 below. Table 7

Those results demonstrate that a colloidal sulfur-containing plant care formulation of the invention consists of very fine (20 nm) sulfur particles, a characteristic of interest for a contact fungicide (Fig. 3).

The formulation acts as an effective plant protection product against Alternaria triticina and Puccinia triticina, resulting in a significant increase in harvest.

The absence of any sulfur smell in the preparation and its crystal white color suggests that the mode of action of the sulfur in the plant care formulation does not involve a gas phase and may be novel and more efficient.