Liquid proliposomal composition of plant protection agent and method for its preparation

Abstract

The present invention relates to a liquid proliposomal composition of a plant protection agent and to a method for preparing said composition.

Description

[Technical field]

[0001] The present invention relates to a liquid proliposomal composition of a plant protection agent and to a method for preparing said composition. [Background technology]

[0002] The demand for organic products is increasing due to the growing concern for health and the environment among consumers and the need for chemical-free products. Currently, both market and social trends are forcing manufacturers of plant protection products to look for sustainable and environmentally friendly plant protection products that do not generate toxic waste in the environment and are derived from renewable resources. As part of the legislative work in the European Union, there is an increasing discussion of the need to reduce the concentration of synthetically derived plant protection agents used or to replace them with substances of natural origin. Unfortunately, such a strategy leads to the use of large amounts of active substances that reach the limit of their effectiveness, which not only promotes the phenomenon of resistance of pathogens to certain substances, but also reduces the effectiveness of the substance's action.

[0003] An effective solution to this problem appears to be the encapsulation of pesticides in liposomal carriers. Liposomal carriers consist of a lipid bilayer that allows efficient encapsulation of hydrophobic substances. This bilayer surrounds a water core that may contain hydrophilic substances. Nanocarrier liposomes are one of the main active substance delivery systems today due to their advantages over conventional formulations. They are now widely used as nanocarriers for active substances in pharmaceuticals, cosmetics, and nutraceuticals.

[0004] Compositions comprising liposomes encapsulating an active substance having a size in the range of 100 nm to 1000 nm allow, inter alia, targeted delivery of the active substance with sustained release, improve its stability, reduce its toxicity, increase its activity (leading to a reduction in the amount of insecticide used) and improve penetration through biological barriers, such as, for example, the lipid layer of leaves.

[0005] Moreover, liposomes are made of phospholipids, are 100% biodegradable, have affinity with the leaf surface, and are safe for the environment. Due to their unique vesicular lipid structure and small size (up to a few μm in size, which allows them to snake between leaf cells), liposomes are able to penetrate the leaves, so that the active substances are not washed off the leaf surface by rain, etc. This allows the amount of active substance used to be significantly reduced, and it is gradually released from the liposomes to act for a long period of time on the cells of fungi that attack plants. These properties of liposomal carriers may significantly improve the effectiveness of the active substance and may also reduce the phenomenon of pathogen resistance.

[0006] Despite all the advantages of liposomes and the numerous studies on their application, they are not widely used as carriers for plant protection products. The biggest limitations to the use of liposomes in insecticide compositions include stability problems due to the presence of large amounts of water in the liposomal composition, lack of resistance to negative temperatures (transport and storage of the product during winter), and limitations related to the maximum concentration of active substance.

[0007] Proliposome compositions appear to be a solution to these problems. Proliposomes, i.e. liposome precursors, have low or no water content and therefore can encapsulate both hydrophobic and hydrophilic active substances while maintaining storage stability, minimizing the drawbacks arising from the use of liposomes. Using proliposomes, liposomes can be prepared without loss of active substance and without changing the physicochemical properties of the liposomes formed from them. Additional and equally important advantages of proliposomes are their ease of preparation and use, and the possibility of obtaining concentrates that become the final product after dilution.

[0008] Proliposomal formulations containing plant protection agents as active ingredients are known in the art.

[0009] GB2303791A describes a method for preparing a pesticide solution, which can be effectively used for liposomal microencapsulation of agricultural pesticides (proliposome) by mixing the solution with water. The method includes the steps of a) mixing an organic solvent (capable of dissolving the pesticide) with vegetable lecithin to form a saturated lecithin solution in the solvent at a volume ratio of 1:1 or 1:2; b) allowing the solution to stand and separating the undissolved portion from the solution; c) separating the saturated lecithin solution from the undissolved portion and mixing the saturated solution with the pesticide for further use in the next step; and d) mixing the pesticide with the saturated lecithin solution to form a pesticide solution for agricultural use. As already mentioned, before the solution is used in agriculture, it is further mixed with water to form liposomes. GB2303791A also discloses a pesticide formulation prepared according to the above defined method.

[0010] AU1998053619A1, which belongs to the same owner as GB2303791A, is directed to the development of technology described in GB2303791A, and more specifically to proliposomal formulations of boron-containing insecticides, preferably borate salts, and methods for making such formulations. The manufacturing process for such formulations is the same as GB2303791A.

[0011] WO2013171196A1 relates to liposomal compositions providing control of fungal and microbial infections in food, feed, and agricultural products. The disclosure generally relates to conventional liposomal formulations, but in one aspect of the invention, aqueous and non-aqueous concentrated compositions (stock solutions, pro-liposomal formulations) are disclosed. These concentrates are subsequently mixed with water to form liposomes. The compositions described in this document contain the active ingredient natamycin, but may advantageously further contain herbicides, fungicides, antibacterial agents, insecticides, or nematicides. These compositions may contain natural lipids, semi-synthetic lipids, and synthetic lipids as lipids involved in liposome formation. For example, they contain phospholipids, including but not limited to lecithin, especially vegetable or animal lecithin, with a purity of less than 95%. Lecithin is present in the composition in an amount of 0.02 to 2.0 mg / mL.

[0012] US5004611A describes a proliposomal composition comprising (a) at least one membrane-forming lipid (e.g., lecithin), preferably in an amount of 35-55% by weight, (b) at least one non-aqueous liquid consisting of a water-miscible organic liquid that is a solvent for the lipid (e.g., glycerol, ethanol, propylene glycol, ethanol, isopropanol, ethylene glycol), preferably in an amount of 35-55% by weight, and (c) a bioactive agent, in a lipid weight ratio in the range of 40:1 to 1:20, and in which sufficient active agent is present to produce its bioactive dose. The composition may contain 5-40% water. Upon further addition of water, the mixture spontaneously forms liposomes with a diameter of 0.1-2.5 μm, which contain at least 2 mL of encapsulated aqueous phase per gram of lipid. Additional components, such as fatty acid esters, may also be present in the composition. The composition may be applied by spraying. Although the primary application of the compositions described is as medicine, US Pat. No. 5,004,611A also mentions their possible use in insect control and horticulture.

[0013] The aim of the present invention was to develop a systemic-action proliposomal composition that increases the foliar uptake, allows a reduction in the dose of pesticide used while maintaining effective herbicidal and fungicidal activity, and is characterized by good storage stability and durability.

[0014] Surprisingly, it has been found that all these requirements, and many others, are met by the compositions of the present invention. Summary of the Invention

[0015] The present invention relates to a liquid proliposomal composition of a plant protection agent, said composition comprising: 1% to 50% by weight of at least one active substance; 20% to 75% by weight of at least one lipid; 0.1% to 35% by weight of at least one auxiliary substance, including at least one surfactant in an amount of less than 15% by weight; 20% to 75% by weight of at least one biodegradable, non-flammable, and non-volatile organic solvent; 0 to 12% by weight of water or an aqueous solution of a salt or buffer substance; Contains:

[0016] Preferably, the active substance is a herbicide or a fungicide.

[0017] Preferably, the at least one active agent comprises from 5% to 20% by weight of the composition.

[0018] Preferably, the lipid to active substance ratio is from 25:1 to 2:1.

[0019] Preferably, the lipid comprises 5% to 99.99% phosphatidylcholine.

[0020] In a particularly preferred embodiment of the invention, the lipid is lecithin.

[0021] Preferably, the lipid comprises 20% to 45% by weight of the composition.

[0022] Preferably, the at least one surfactant comprises 3% by weight based on the weight of the composition.

[0023] Preferably, the at least one surfactant is selected from the group consisting of lysophospholipids, mono- and diglycerides, polysorbates, spans, ethoxylated fatty alcohols, alkoxylated alcohols, ethoxylated fatty acid amines, alkanamines, alkyl sulfates, saponins, alkoxylated phosphate esters, butyl block copolymers, and PEO and PPO block copolymers.

[0024] More preferably, the at least one surfactant is selected from the group consisting of polysorbate 20, a mixture of long chain (C12-15) fatty alcohols ethoxylated with 3-5 molecules of ethylene oxide, and octylamine.

[0025] Preferably, the composition contains, in addition to at least one surfactant, at least one other auxiliary substance selected from the group consisting of antifoaming agents, antioxidants, and biodegradable, non-volatile and non-flammable agents that affect the fluidity of the lipid membrane.

[0026] Preferably, the solvent comprises 20% to 30% by weight of the composition.

[0027] Preferably, the solvent is selected from the group consisting of n-butylpyrrolidone, ethylene glycol monobutyl ether, propylene carbonate, N,N-dimethyl lactamide, and 5-dimethylamino-2-methyl-5-oxovaleric acid methyl ester.

[0028] Preferably, the composition comprises 8% by weight water or an aqueous solution of a salt or buffer substance.

[0029] The present invention also relates to a method for the preparation of a composition according to the invention, which method comprises, in order: a) dissolving the lipid in a biodegradable organic solvent and mixing to obtain a mixture; b) adding at least one surfactant to the mixture resulting from step a) while continuing to mix; c) adding at least one active agent to the mixture resulting from step b) while continuing to mix; d) optionally adding water or an aqueous solution of a salt or buffer substance to the mixture obtained from step c); e) mixing the mixture obtained in step d) to form a proliposomal composition; Contains:

[0030] Advantages of the Invention

[0031] Research conducted by the inventors has shown the following. The proliposomal composition according to the invention (composition according to the invention) exhibits stability even after storage under stress conditions. The stability of the aqueous dispersion of the composition according to the invention after preparation is independent of the type of water used: the dispersion is stable when using both soft and hard water. The compositions according to the invention are capable of achieving very high encapsulation efficiencies of the active substance. The compositions according to the invention make it possible to obtain high leaf penetration of the active substances. The fungicide-containing proliposomal composition according to the invention does not cause phytotoxic symptoms. The bactericidal agent-containing proliposomal composition according to the invention exhibits comparable or better bacteriostatic efficacy compared to the reference formulation. The fungicide-containing proliposomal composition according to the invention is effective against a broad range of pathogens, regardless of plant species. The fungicide-containing proliposomal composition according to the invention does not affect or increase the size and quality of the crop yield (mass of 1000 kernels, protein or oil content).

[0032] In vitro studies have shown that the proliposomal composition obtained according to the invention has all the advantages of liposomal compositions, namely reduced toxicity, increased permeability, long residence time of the active substance in the leaves and its extended release, which translates into a long duration of action of this substance, without the drawbacks inherent to liposomes, such as stability and storage problems. Studies have shown that the use of such compositions allows a reduction in the dose of insecticide used per hectare, while maintaining the expected effect, compared to classical compositions of plant protection products. Based on field studies, it has been shown that in the control of certain pathogens, proliposomal compositions containing fungicides allow a reduction in the dose of active substance by up to 60% compared to reference products, while maintaining efficacy. After mixing the proliposomal composition with water to obtain a liposomal composition, it is observed that the active substance is encapsulated in liposomes with an efficiency as high as 98%, which is due to an increase in solubility, which directly affects an increase in bioavailability in comparison to the free substance. At the same time, the compositions of the invention are less toxic than the free active substance.

[0033] Further in vivo studies show that the compositions of the invention show higher leaf penetration compared to conventional products, such as commercially available products of different compositions (see Tables 8 and 9). The increased leaf penetration of a plant protection agent by the use of a proliposomal composition according to the invention, and further by the use of at least one surfactant in said composition, translates directly into an increased activity of this plant protection agent. In addition to the direct impact of the penetration of a substance into the leaf on its bioavailability, the increased penetration has an impact on its protection from external factors, such as rain, when it is on the leaf surface.

[0034] The composition according to the present invention also contributes to the extended release time of active substances thanks to the use of the proliposomal composition according to the present invention. This directly affects the fungicidal and herbicidal activity of the developed formulation. This is important because of the possibility of secondary fungal infection after fungicide application. Furthermore, this extended release time allows the use of lower product concentrations, since the contact time of fungicide with fungus is extended.

[0035] Thanks to the above mentioned advantages, the solution makes it possible to reduce the amount of pesticides used in agriculture in accordance with the new regulations in combination with the Farm2Fork strategy presented by the European Commission. The elaborate composition of the plant protection agent makes it possible to reduce the dose of active substance used and to increase the biological effectiveness of the PPA composition. Furthermore, the use of lecithin as a biodegradable solvent and the main component of the liposomal membrane makes the proliposomes according to the invention an ideal carrier for products based on natural raw materials. [Brief description of the drawings]

[0036] Without limiting the scope of the invention, some embodiments are illustrated in the accompanying drawings.

[0037] FIG. 1 shows a cryo-electron microscope image of a hydrated proliposome composition of the present invention.

[0038] Figure 2 shows the content of difenoconazole in the extracts obtained using different solvents.

[0039] FIG. 3 shows the percentage of difenoconazole on the outside and inside of leaves treated with a dispersion of difenoconazole-containing proliposomes according to the present invention.

[0040] FIG. 4A shows the effect of various herbicides 23 days after foliar application. A) herbicide according to the composition of example 5 in a dose of 25 g / ha of active substance; B) Herbicide Imazamox 40SL at a dose of 25 g / ha of active substance; C) herbicide Imazamox 40SL and used according to the composition of Example 5 at a dose of 25 g / ha of active substance; D) Herbicide Imazamox 40SL and the composition of Example 5, used at a dose of 36 g / ha of active substance. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The object of the present invention is a liquid proliposomal composition for plant protection agents from the group of systemically acting fungicides and herbicides, and a method for the preparation of such a composition.

[0042] Proliposome composition

[0043] The liquid proliposomal composition according to the invention comprises in its composition: at least one active substance, at least one lipid-forming liposome, at least one biodegradable organic solvent, at least one auxiliary substance comprising at least one surfactant, and Optionally, water or an aqueous solution of a salt or buffer substance.

[0044] Such compositions form stable, durable, water-miscible solutions that can be stored and delivered to the site of use. Prior to use, and preferably immediately prior to use, the compositions are mixed with water and, after hydration (dilution with water), liposomal vesicles of less than 1 μm in size are spontaneously formed in which the hydrophobic active agent is encapsulated.

[0045] The active substance according to the present invention is present in the composition in an amount ranging from about 1% to about 50% by weight, preferably from about 5% to about 45% by weight, 40% by weight or 35% by weight, preferably from about 5% to about 30% by weight or 25% by weight, preferably from about 5% to about 20% by weight, more preferably from about 5% to about 10% by weight, based on the weight of the composition. The active substance may be any fungicide or herbicide with systemic action. Examples of fungicides that may constitute the active substance according to the present invention are: difenoconazole, prothioconazole, metconazole, penthiopyrad, fenpropidin, pyraclostrobin, trifloxystrobin, penconazole, and bupirimate. On the other hand, examples of herbicides are fenoxaprop-P-ethyl, florasulam, nicosulfuron, amidosulfuron, iodosulfuron, pyrrolam, clopyralid, pinoxaden, propaquizafop, benfluralin, prosulfocarb, petoxamide, clethodim, picolinafen. Preferably, the active substance is difenoconazole, prothioconazole, clopyralid, or imazamox. The composition may contain at least one of the above-mentioned active substances, and preferably may contain two or more active substances.

[0046] The liposome-forming lipid is present in the composition in an amount ranging from about 20% to about 75% by weight, preferably from about 20% to about 45% by weight, more preferably from about 25% to about 40% by weight, based on the weight of the composition. The lipid content of the composition is preferably less than 70% by weight, preferably less than 65% by weight, more preferably less than 60% by weight, or 55% by weight, or less than 50% by weight, and preferably more than 25% by weight, preferably more than 30% by weight, 35% by weight, or 40% by weight. Such lipids may be natural, semi-synthetic, and synthetic lipids containing 5% to 99.99% phosphatidylcholine, preferably 20% to 99.9% phosphatidylcholine, more preferably 20% phosphatidylcholine. Preferably, the liposome-forming lipid is a phospholipid, more preferably a lecithin, including vegetable lecithin or animal lecithin. Even more preferably, the lecithin used in the present invention is vegetable lecithin, more preferably soybean lecithin.

[0047] In a preferred embodiment, the composition according to the invention is characterized in that the ratio of lecithin to active substance is between 25:1 and 2:1, for example 20:1, 15:1 or 10:1, more preferably it is in the range of 6:1 to 2:1.

[0048] At least one solvent according to the invention is used in the composition in an amount of 20% to 75% by weight, preferably about 20% to about 30% by weight, preferably about 20% to about 25% by weight, based on the weight of the composition. The lipid content of the composition is preferably less than 70% by weight, preferably 65% ​​by weight, more preferably 60% by weight, or 55% by weight, or less than 50% by weight, preferably more than 25% by weight, preferably more than 30% by weight, 35% by weight, 40% by weight. Solvents that can be used according to the invention are any biodegradable, water-miscible, non-flammable, non-volatile organic solvents under storage conditions (ambient temperature). Their amount in the composition must be proportional to the amount of lecithin and active substance used and sufficient to dissolve both substances. Preferred solvents include ethers, glycol ethers, lactams, and particularly preferred are n-butylpyrrolidone, ethylene glycol monobutyl ether, propylene carbonate, N,N-dimethyllactamide, 5-dimethylamino-2-methyl-5-oxovaleric acid methyl ester. More preferably, the solvent is ethylene glycol monobutyl ether. According to the invention, it is possible to use a solvent system comprising two or more solvents.

[0049] The auxiliary substances according to the present invention are present in the composition at about 0.1% to about 35% by weight, preferably 5% to 30% by weight, more preferably 10% to 25% by weight, most preferably 15% by weight, preferably about 20% to about 30% by weight.

[0050] Preferably, the auxiliary substance that can be included in the composition of the present invention is selected from the group including surfactant, antifoaming agent, antioxidant, or agent that affects the fluidity of lipid membrane.The auxiliary substance helps to increase the stability of the system and improve the solubility of both lipid and active substance.Preferably, the composition contains two or more auxiliary substances.For example, glycerol is included in the composition as the agent that affects the fluidity of lipid membrane.

[0051] One of the auxiliary substances is at least one surfactant, which is contained in the composition in an amount of not more than 15%, preferably not more than 14%, even more preferably not more than 13%, for example not more than 12%, particularly preferably not more than 11%, particularly not more than 10%, even more preferably not more than 9%, particularly not more than 8%, even more preferably not more than 7%, particularly not more than 6%, particularly preferably 0.1% to 5% by weight, even more preferably about 3% by weight, based on the weight of the composition. Surfactants are compounds whose molecules are composed of a lipophilic part and a hydrophilic part. The lipophilic part of the surfactant may contain one or more fatty acid residues, aliphatic alcohol residues with different lengths of hydrocarbon chains and degrees of saturation, or other hydrophobic residues with high affinity for lipid membranes, such as aromatic compounds, as well as other branched and cyclic alkyl groups. The hydrophilic part of the surfactant includes hydroxyl groups, carboxyl groups, oxyethylene groups, sugars, carbohydrates, phosphatidylcholine or phosphatidylethanolamine residues, and derivatives thereof. The presence of a surfactant in the composition increases the fluidity of the lipid membrane, which leads to an increased penetration of the hydrated liposomes through the leaves. Furthermore, the presence of a surfactant in the composition allows a more efficient encapsulation of the active substance in the hydrated liposomes. Surfactants that may be used according to the invention include, in particular, lysophospholipids, mono- and diglycerides, polysorbates, spans, ethoxylated fatty alcohols, alkoxylated alcohols, ethoxylated fatty acid amines, alkanamines, alkyl sulfates, saponins, alkoxylated phosphate esters, butyl block copolymers, PEO and PPO block copolymers. Preferably, the surfactant used according to the invention is polysorbate 20, which is a mixture of a long-chain (C12-15) fatty alcohol ethoxylated with 3 to 5 molecules of ethylene oxide (commercially available under the name ROKAnol DB5) and octylamine. The composition according to the invention may contain one surfactant or, preferably, two or more surfactants.

[0052] Preferably, the compositions of the present invention also contain 0 to about 12% by weight, more preferably 5 to 10% by weight, and most preferably about 8% by weight of water or an aqueous solution of a salt (preferably sodium chloride) or a buffer system. The addition of small amounts of water, an aqueous salt solution, or a buffer system increases the solubility of hydrophilic and amphiphilic substances, such as natural lecithin impurities, and reduces the viscosity of the system.

[0053] Process for preparing proliposomal compositions

[0054] Although the compositions of the present invention can be prepared by virtually any method known in the art which involves mixing ingredients to achieve a target composition, preferably, the compositions of the present invention are prepared as follows. a) The lipid is dissolved in a biodegradable organic solvent and mixed to obtain a mixture. b) adding at least one surfactant to the mixture resulting from step a) while continuing to mix, c) adding at least one active to the mixture resulting from step b) while continuing to mix. d) Optionally, water or an aqueous solution of a salt or buffer substance is added to the mixture resulting from step c). e) Mixing the mixture obtained in step d) to form a proliposomal composition.

[0055] All process steps are preferably carried out in a single device or vessel that provides mixing. The above preparation process does not require additional steps, thus reducing production time and costs, and also does not require the use of specialized equipment (e.g., mills, homogenizers, calibrators, etc.).

[0056] Preferably, the process according to the invention is carried out at elevated temperatures, preferably in the range of from 20°C to 70°C.

[0057] According to the present invention, the term "about" as used above and below is to be understood as a deviation of ±5% from the stated value, reflecting imprecision that may occur when carrying out the method for preparing the composition according to the present invention, e.g. affecting during the measurement of the components. EXAMPLES

[0058] Example 1: Composition according to the invention

[0059] 40g of ethylene glycol monobutyl ether and 40g of glycerol were mixed at 60°C. Next, 80g of soybean lecithin (containing 20% ​​phosphatidylcholine) was added, and after dissolution, 4g of polysorbate 20 and 2g of a mixture of long-chain (C12-15) fatty alcohols ethoxylated with 3-5 molecules of ethylene oxide (ROKAnol DB5) were added. While continuing stirring at 60°C, 18g of difenoconazole was added continuously. Finally, 16g of 5% NaCl aqueous solution was added. After mixing, a viscous solution of the substances constituting the composition was obtained.

[0060] Example 2: Composition according to the invention

[0061] 28g of ethylene glycol monobutyl ether and 26g of glycerol were mixed at 60°C. Next, 26g of soybean lecithin (containing 20% ​​phosphatidylcholine) was added, and after dissolution, 2g of polysorbate 20 and 1g of a mixture of long-chain (C12-15) fatty alcohols ethoxylated with 3-5 molecules of ethylene oxide (ROKAnol DB5) were added. While continuing stirring at 60°C, 18g of difenoconazole was added continuously. Finally, 8g of 5% NaCl aqueous solution was added. After mixing, a viscous solution of the substances constituting the composition was obtained.

[0062] Example 3: Composition according to the invention

[0063] 40g of ethylene glycol monobutyl ether and 40g of glycerol were mixed at 60°C. Next, 80g of soy lecithin (containing 20% ​​phosphatidylcholine) was added, and after dissolution, 4g of polysorbate 20 and 2g of a mixture of long-chain (C12-15) fatty alcohols ethoxylated with 3-5 molecules of ethylene oxide (ROKAnol DB5) were added. While continuing stirring at 60°C, 20g of prothioconazole was added continuously. Finally, 16g of 5% NaCl aqueous solution was added. After mixing, a viscous solution of the substances constituting the composition was obtained.

[0064] Example 4: Composition according to the invention

[0065] 42 g of ethylene glycol monobutyl ether and 42 g of glycerol were mixed at 60°C. Next, 84 g of soybean lecithin (containing 20% ​​phosphatidylcholine) was added, and after dissolution, 4 g of polysorbate 20 and 2 g of a mixture of long-chain (C12-15) fatty alcohols ethoxylated with 3-5 molecules of ethylene oxide (ROKAnol DB5) were added. While continuing stirring at 60°C, 10 g of clopyralid was added continuously. Finally, 16 g of 5% NaCl aqueous solution was added. After mixing, a viscous solution of the substances constituting the composition was obtained.

[0066] Example 5: Composition according to the invention

[0067] 50g of ethylene glycol monobutyl ether and 50g of glycerol were mixed at 60°C. Next, 100g of soy lecithin (containing 20% ​​phosphatidylcholine) was added, and after dissolution, 5g of polysorbate 20 and 1.25g of a mixture of long-chain (C12-15) fatty alcohols ethoxylated with 3-5 molecules of ethylene oxide (ROKAnol DB5) were added. While continuing stirring at 60°C, 7g of octylamine and 16g of imazamox were added successively. After mixing, a viscous solution of the substances constituting the composition was obtained.

[0068] Example 6: Composition according to the invention

[0069] 43 g of ethylene glycol monobutyl ether and 43 g of glycerol were mixed at 60°C. Next, 86 g of soybean lecithin (containing 20% ​​phosphatidylcholine) was added, and after dissolution, 4.4 g of polysorbate 20 and 2.2 g of a mixture of long-chain (C12-15) fatty alcohols ethoxylated with 3-5 molecules of ethylene oxide (ROKAnol DB5) were added. While continuing stirring at 60°C, 22 g of metconazole was added continuously. Finally, 16 g of 5% NaCl aqueous solution was added. After mixing, a viscous solution of the substances constituting the composition was obtained.

[0070] Example 7: Composition according to the invention

[0071] 40g of ethylene glycol monobutyl ether and 40g of glycerol were mixed at 60°C. Next, 79g of soybean lecithin (containing 20% ​​phosphatidylcholine) was added, and after dissolution, 4g of polysorbate 20 and 2g of a mixture of long-chain (C12-15) fatty alcohols ethoxylated with 3-5 molecules of ethylene oxide (ROKAnol DB5) were added. While continuing stirring at 60°C, 20g of clopyralid was added continuously. Finally, 16g of 5% NaCl aqueous solution was added. After mixing, a viscous solution of the substances constituting the composition was obtained.

[0072] In this specification, proliposomes refer to liposome precursors that are anhydrous or contain small amounts of water. Liposomes refer to aqueous diluted dispersions formed from proliposomes that spontaneously self-assemble into vesicles composed of phospholipid bilayers and enclose the internal aqueous space. In the following examples, when only the parameters or properties of the aqueous dispersion are mentioned, liposomes are referred to. On the other hand, when summarizing the characteristics, parameters, properties, and effectiveness of proliposomes and their aqueous dispersions (liposomes), we generally refer to proliposomes.

[0073] Example 8: Stability study of the compositions of Examples 1-5

[0074] The stability of the composition according to the invention after dilution with water to obtain liposomes was studied.

[0075] Table 1: Analysis results of proliposomes and their aqueous dispersions [Table 1]

[0076] The composition is stable after dilution with water, both soft (CIPAC Water A) and hard (CIPAC Water D).

[0077] The compositions of Examples 1-5 were also observed for liposome formation. Cryo-electron microscopy images (Figure 1) show the presence of liposomes formed spontaneously upon hydration of the proliposomal composition. Liposomes up to 1 μm in size can be observed. This composition makes it possible to obtain unilamellar or multilamellar liposomes without the need for an additional sizing step. The absence of crystals / precipitates within the field of view proves a high encapsulation efficiency of the active substance (the absence of untrapped active substance).

[0078] Example 9: Stability study of the compositions of Examples 6-7

[0079] The stability of the composition according to the invention after dilution with water to obtain liposomes was studied.

[0080] Table 2: Analysis results of proliposomes and their aqueous dispersions [Table 2]

[0081] The compositions are stable after dilution with water, both soft (CIPAC Water A) and hard (CIPAC Water D), at various dispersion concentrations.

[0082] Example 10: Accelerated aging test of the compositions of Examples 2 and 4

[0083] The proliposomal compositions were subjected to the following accelerated aging test. 7 days at 0°C 14 days at 54°C 56 days at 40°C

[0084] Table 3: Results of the analysis of proliposomes with difenoconazole after aging test (Example 2) [Table 3]

[0085] Table 4: Results of the analysis of proliposomes with clopyralid after aging test (Example 4) [Table 4]

[0086] The composition is stable when stored under stress conditions.

[0087] Example 11: Encapsulation efficiency of difenoconazole for the composition of Example 1

[0088] To determine the encapsulation efficiency of the active substance in hydrated liposomes, the unencapsulated active substance was separated from the liposomes using molecular sieves. The encapsulation efficiency of difenoconazole was measured by HPLC, and the encapsulation efficiency of phospholipids was measured by spectrophotometry. The encapsulation efficiency of the active substance was calculated according to the following formula:

number

[0089] where: Ap: measured concentration of active substance [mg / mL]; Fp: measured concentration of phospholipid [mg / mL]; At: theoretical concentration of active substance [mg / mL]; Ft: theoretical concentration of phospholipid [mg / mL].

[0090] Table 5: Results - Absorbance values, phospholipid and difenoconazole concentrations, and encapsulation efficiency of the composition - Example 1 [Table 5]

[0091] The test results showed a very high encapsulation efficiency (98%) of difenoconazole in liposomes after hydration of the proliposomal composition of Example 1. A high encapsulation efficiency of the active substance leads to a better penetration of the plant protection agent (PPA) into the leaves and therefore to its more effective action.

[0092] Example 12: Leaf penetration study of difenoconazole-containing liposomes in the composition of Example 1

[0093] Approximately 5 mL of buffer A was introduced into each of the six thermostatic Franz chambers (25° C.). An apple leaf was then placed in each of them. 500 μl of difenoconazole-containing liposomes from Example 1 were applied to the leaves in the first three chambers, and in the next three chambers a solution of the commercial formulation Tores 250 EC was applied. Samples (200 μl) were taken from each chamber at 0, 1, 2, 4, 5 and 24 hours, and the difference was replenished with buffer a.

[0094] Table 6: Composition of samples subjected to permeation analysis [Table 6]

[0095] Table 7: Composition of Buffer A [Table 7]

[0096] The active ingredients were then extracted from the leaves in the following four steps. Water (to clean the leaf surface) Ethanol (extraction of active substances adsorbed on the leaf surface) Hexane (extracts active ingredients that penetrate the cuticle layer) Methanol (extraction of active ingredients from the deeper layers of cut leaves)

[0097] At each stage, 5 mL of the indicated solvent was added to the leaves placed in the test tubes, and the entire contents were shaken for 60 seconds.

[0098] result:

[0099] Table 8: Amount of difenoconazole in each phase after extraction [Table 8]

[0100] where: Water + ethanol - amount of active substance remaining on the leaf surface Hexane + Methanol - amount of active substance that penetrated the leaf surface

[0101] The above results are further illustrated in FIG.

[0102] Table 9: Amount of difenoconazole penetrating to the underside of leaves [Table 9]

[0103] FIG. 3 shows the results of measuring the difenoconazole content on the outside and inside of the leaves. Water + ethanol - amount of active substance remaining on the leaf surface Hexane + Methanol - amount of active substance that penetrated the leaf surface

[0104] The results obtained for the leaf penetration of difenoconazole clearly show a much more efficient penetration of the active substance encapsulated in liposomes compared to the conventional form of this substance available on the market.Such results may indicate a better penetration of the composition according to the invention, which may therefore lead to a reduction in the concentration / dosage of plant protection agent used under field conditions.

[0105] Example 13: In vitro bacteriostatic activity study of the composition of Example 1

[0106] Study of fungistatic activity against various plant pathogenic fungal strains on PDA medium under controlled conditions (25 ° C) by the method of poisoning the substrate. Concentrations of active substances in the medium: 200, 20, 5, 2, 1 mg / l.

[0107] Table 10: Bacteriostatic effect [Table 10]

[0108] The results are expressed as % inhibition of filamentous mycelium growth of a given pathogen species under the action of the formulation compared to the control combination (fungal colonies growing on PDA medium containing solvent (water)).

[0109] Results: The proliposomal composition of Example 1 exhibited similar fungistatic effect as the Tores 250 EC standard against the seven pathogenic fungal strains tested on PDA medium.

[0110] Example 15: Study of the phytotoxicity of the fungicide composition of Example 1 against winter oilseed rape under greenhouse conditions in a pot experiment

[0111] Table 12: Phytotoxicity of fungicide compositions against winter oilseed rape (Gemini variety) [Table 12]

[0112] *Scale 0 to 4; 0: no symptoms

[0113] Results: To evaluate the phytotoxicity, three doses were used for each formulation: the lowest and highest recommended doses, doubled taking into account the doses from the labels of other commercial drugs, including difenoconazole, recommended for use in combination with oilseed rape against powdery mildew. The doses of proliposome according to example 1 were calculated based on the active substance content. Seven days after treatment, for combinations No. 1 and 4 (the highest dose of both formulations, 300 g / ha of active substance, corresponds to 1.2 L / ha, 2.4 times the recommended dose for winter oilseed rape - 0.5 L / ha), a slight inhibition of rapeseed plant growth was observed (score 0.5 (0-4)). This means that the fresh mass was slightly reduced by 3-4% in the 4 weeks after treatment. There were no other visible symptoms of the phytotoxic effect of the formulations on rapeseed in the 4-week period after application to all targets at the low dose.

[0114] Example 16: Biological evaluation of the effectiveness of the proliposomal form of the fungicide of Example 1 in controlling Sclerotinia sclerotiorum in winter rapeseed

[0115] Table 13: Phytotoxicity Rating [Table 13]

[0116] Control=0 ** Days after DAA application

[0117] Table 14: Percentage of plants in infested areas and efficacy of fungicides to protect winter oilseed rape shoots against Sclerotinia sclerotiorum (SCLESC) [Table 14]

[0118] *Efficacy calculated using Abbott's formula ** Days after DAA application

[0119] Table 15: Winter oilseed rape yields

[0120] [Table 15]

[0121] *Grain yield recalculated at 9% humidity.

[0122] Table 16: Mass of 1,000 seeds

[0123] [Table 16]

[0124] Conclusion: 1. No phytotoxic effects were observed on winter oilseed rape, cultivar Architect, of the tested fungicides according to the composition of Example 1 at doses of 0.2N, 0.5N and 1N and of the comparator Torres 250 EC at a dose of 1N. 2. The two high doses of the test fungicides according to the composition of Example 1 and the comparator Tores 250 EC significantly inhibited the development of Sclerotinia sclerotiorum in winter oilseed rape plants. 3. The two high doses of the tested fungicide according to the composition of Example 1 inhibited the development of Sclerotinia sclerotiorum at the same level, the effectiveness of their action was significantly higher than that of the comparative agent Tores 250 EC, and the dose of the active substance was halved at the same time. 4. The area of ​​winter oilseed rape plants infested with the fungus Sclerotinia sclerotiorum in experimental combinations using the lowest doses of fungicides tested was slightly lower than in controls, although these differences were not statistically significant. 5. The yield and mass of the thousand winter oilseed rapeseeds obtained reflected the level of control of Sclerotinia sclerotiorum. A significant increase in yield was observed with the two highest doses of fungicide tested according to the composition of Example 1 and the comparator Tores 250 EC. The maximum mass of a thousand seeds was recorded after applying the highest dose of fungicide tested according to the composition of Example 1.

[0125] Example 17: Herbicidal effect 23 days after foliar application of the herbicide Imazamox 40SL and the composition according to Example 5 (weed species: deforestation, goosegrass, lambsquarters, pigweed, charlock, shepherd's purse, self-sown cereals: barley, wheat)

[0126] Pot experiments in soil substrate under greenhouse conditions (temperature during vegetation: 18-28°C) Apply to leaves of 18-day-old indicator plants in appropriate leaf phases 1-3. Evaluation was conducted 7 to 21 days after application.

[0127] Table 17: Herbicidal efficacy of proliposomal herbicides compared to Imazamox 40 SL. Evaluated 7 days after application. [Table 17]

[0128] Table 17a: Evaluation 15 days after application [Table 17a]

[0129] Table 17b: Evaluation after 23 days of application [Table 17b]

[0130] The above effects are also shown in Figures 4A-E. FIG. 4A shows the effect of the herbicide in proliposomal form (example 5) 23 days after foliar application at a dose of 25 g / ha of active substance (control on the left, test sample on the right). · Figure 4B shows the effect of Imazamox 40SL 23 days after foliar application at a dose of 25 g / ha of active substance (control on the left, test samples on the right). FIG. 4C shows the effect of the herbicides Imazamox 40SL and the composition of example 5 23 days after foliar application at a dose of 25 g / ha of active substance (Imazamox 40SL on the left, composition of example 5 on the right). FIG. 4D shows the effect of the herbicides Imazamox 40SL and the composition of example 5 23 days after foliar application at a dose of 36 g / ha of active substance (Imazamox 40SL on the left, composition of example 5 on the right). FIG. 4E shows the effect of the herbicides Imazamox 40SL and the composition of example 5 23 days after foliar application at a dose of 48 g / ha of active substance (Imazamox 40SL on the left, composition of example 5 on the right).

[0131] Results: Preliminary results of the testing of the herbicidal composition of Example 5 show a weaker herbicidal effect on self-sown cereals compared to the Imazamox 40 SL standard at the two lower doses of 36 and 25 g / ha of active substance. 7 days after application, the activity of both compositions was similar in all test species, but differences appeared after 15 days. At the highest dose of 48 g / ha of active ingredient, no significant differences were found in the action of both herbicides.

[0132] Table 20: Doses of phytotoxic formulations (in g active substance / ha) of the herbicidal composition of Example 5 on cultivated plants: soybean (varieties: Aldana, Erica): 36 g (minimum), 48 g (maximum), 96 (2x). Pot experiment, greenhouse conditions. [Table 20]

[0133] Results: One week after applying both formulations at the highest rate (twice the recommended rate), soybean plants of both varieties showed a slight yellowish chlorosis on the leaves, which persisted for approximately 15-16 days after application. No other damage was observed during the growing season.

[0134] Table 22: Herbicidal effect of the proliposomal formulation of Example 5 against shepherd's purse (Capsella bursa-pastoris)

[0135] [Table 22]

[0136] Results: The herbicidal effect of proliposomes with the highest dose of imazamox after 21 days was comparable to that of Imazamox 40 SL (94 and 100%, respectively), whereas for the reference product such results were obtained earlier, after 14 days. At the lower doses of 36 and 25 g / ha of active substance, the effect of proliposomes was about 27% weaker than the standard. The maximum herbicidal effect of Imazamox 40 SL at each concentration was obtained after 14 days, whereas the effect of proliposomes gradually increased over a longer period up to 21 days after application.

[0137] Table 26: Herbicidal efficacy of formulations with composition 5 on spring barley (self-sown) [Table 26]

[0138] Results: After 42 days the herbicidal efficacy of the highest dose of proliposome on barley was good and comparable to that of Imazamox 40 SL (96 and 100%, respectively). The herbicides tested at lower doses showed much lower efficacy. At a dose of 36 g / ha the efficacy of proliposome and Imazamox was 55 and 95%, respectively, and at a dose of 25 g / ha the efficacy was 30 and 86%.

[0139] Example 19: Phytotoxicity of the Herbicide SNS H01 19 (Example 5) on Cultivated Plants - Soybean

[0140] Table 31: Phytotoxicity of proliposomal herbicides on arable plants-soybean, Viola cv. Pot experiment under greenhouse conditions. Developmental stage during application: Initiation-2 whorls. 5 repeats x 9 plants = 45 plants in one combination. [Table 31]

[0141] *Scale 0-9 (0-no damage, 9-plant destruction)

[0142] Results: 5-7 days after application of both formulations at the highest doses (double dose, 96 g active substance / ha and 48 g active substance / ha) a slight yellowish chlorotic discoloration appeared on the leaves of Viola soybean plants. In plants sprayed with the composition according to Example 5 the discoloration disappeared about 10-11 days, whereas in plants sprayed with Imazamox 40 the discoloration remained until about 15-16 days after application and then disappeared. In the remaining subjects no other damage was found during the growing season. Under the influence of Imazamox 40 SL the growth of soybean plants was inhibited from the 12th day (14%) until the end of the observation, 23 days after application. This is also reflected in a mass loss of about 20%.

[0143] Table 32: Effect of proliposomal herbicides on the growth and development of arable plants Soybean, Viola cv. Pot experiment under greenhouse conditions. Developmental stage during application: Initiation-2 whorls. 5 repeats x 9 plants = 45 plants in one combination. [Table 32]

[0144] Example 20: Biological evaluation of the efficacy of difenoconazole-containing proliposomes (Example 1) in controlling powdery mildew (Blumeria graminis (Erysiphe graminis)), brown rust (Puccinia recondita) and wheat leaf stripe septica (Mycosphaerella graminicola (anam. Zymoseptoria tritici)) in winter wheat, cultivar Zyta.

[0145] Table 33: Phytotoxicity [Table 33]

[0146] Control=0 ** Days after DAA application

[0147] Table 34: Mean percentage of leaf surface penetration and fungicide effectiveness for protecting winter wheat from powdery mildew - Blumeria graminis ERYSGR. [Table 34]

[0148] * Potency calculated using Abbott's formula ** Days after DAA application

[0149] Table 35: Mean percentage of leaf surface infestation and efficacy of fungicides in protecting winter wheat against wheat leaf stripe septoria - Zymoseptoria tritici SEPTTR [Table 35]

[0150] * Potency calculated using Abbott's formula ** Days after DAA application

[0151] Table 36: Mean percentage of leaf surface infestation and fungicide efficacy in protecting winter wheat from stem rust - Puccinia recondita PUCCRE

[0152] [Table 36]

[0153] * Potency calculated using Abbott's formula ** Days after DAA application

[0154] Table 37: Preserved green areas for winter wheat [Table 37]

[0155] Table 38: Fresh mass content, grain yield, thousand kernel mass and protein content of winter wheat [Table 38]

[0156] *Grain yield recalculated at 15% humidity.

[0157] result: During the first application, made at the BBCH32-33 arable plant development stage, symptoms of infestation with the fungus Zymoseptoria tritici, the cause of wheat leaf stripe septicemia, were observed on the lower leaves of winter wheat cultivar Zyta at a leaf infestation area level of 10%, while the infestation rate of Blumeria graminis, the cause of powdery mildew, was 3%.

[0158] A second treatment was performed at arable plant development stage BBCH45, 3 weeks after the first application. L-4 leaves were infested with Zymoseptoria tritici and Blumeria graminis. Infestation rates in small control plots averaged 7.38% and 5.5%, respectively. Efficacy of the tested fungicides 3 weeks after the first application ranged from 77 to 100% for powdery mildew, but from 8 to 52% for septicemia stripe on wheat leaves (Table 34, Table 35).

[0159] About 3 weeks after the second application, striped septicemia was observed on the upper leaves of the small control plot with an intensity of about 15% on L-3 leaves and about 5% on L-2 leaves. The efficacy of the tested fungicides was as follows: 45-60% on L-3 leaves and 62-100% on L-2 leaves. The comparison agents inhibited the disease development by 56% on L-3 leaves and 73% on L-2 leaves (Table 35).

[0160] In evaluations carried out on winter wheat, Zyta variety, at developmental stages BBCH 75-77, incidence of stripe septicemia and brown rust on L-2 subflag and L-1 flag leaves was observed at levels of 35.31% and 13% for Zymoseptoria tritici and 5% and 8.63% for Puccinia recondita, respectively. The efficacy of the tested compositions of Example 1 ranged from 25-59% against wheat leaf stripe septicemia and 64-100% against brown rust. Meanwhile, the comparator Daphne 250 EC inhibited leaf stripe septicemia incidence by 40% on L-2 leaves and 51% on L-1 leaves, but inhibited brown rust incidence by 100% on both L-2 and L-1 leaves (Tables 35, 36).

[0161] At plant development stage BBCH75, leaf green area of ​​sub-flag L-2 and flag L-1 was higher in all combinations tested compared to the control (Table 37).

[0162] Grain yield, thousand kernel mass, and protein content were at similar levels for all experimental combinations tested (Table 38).

[0163] Conclusion: 1. The experiments carried out showed that there were no symptoms of phytotoxic effects of different doses of the proliposomal fungicide and the standard Dafne 250 EC on winter wheat of the Zyta variety. 2. Proliposomes tested at doses of 0.8N and 1N showed 100% effectiveness in reducing the incidence of Blumeria graminis on L-4 leaves at the level of the comparator Dafne250 EC(1N). 3. The investigated proliposomal fungicides at doses of 0.8N and 1N applied to Zymoseptoria tritici performed better than the comparator Dafne 250 EC. 4. Proliposomes tested at doses of 0.6N, 0.8N and 1N showed 100% effectiveness in reducing the incidence of Puccinia recondita on L-2 leaves at the level of the comparator Dafne250 EC(1N). 5. Increases in green leaf area (GLA) were observed following application of all doses of the proliposomal agent and the comparator Daphne 250 EC tested compared to the control. 6. The use of Proliposomes and Dafne250 EC did not affect the yield size and quality (mass of 1000 kernels, protein content) of winter wheat Zyta cultivar, regardless of the dose used.

[0164] Example 21: Biological evaluation of the efficacy of difenoconazole-containing proliposomes (Example 1) in controlling powdery mildew (Blumeria graminis (Erysiphe graminis)), wheat leaf stripe septica (Mycosphaerella graminicola (anam. Zymoseptoria tritici)) in winter wheat, Arkadia cultivar.

[0165] Table 39: Phytotoxicity [Table 39]

[0166] Control=0 ** Days after DAA application

[0167] Table 40: Mean percentage of leaf surface infestation and fungicide efficacy in protecting winter wheat against powdery mildew (Blumeria graminis ERYSGR)

[0168] [Table 40]

[0169] "*Efficacy calculated using Abbott's formula" ** Days after DAA application

[0170] Table 41: Mean percentage of leaf surface infestation and efficacy of fungicides in protecting winter wheat against wheat leaf stripe septoria - Zymoseptoria tritici SEPTTR

[0171] [Table 41]

[0172] * Potency calculated using Abbott's formula ** Days after DAA application

[0173] Table 42: Winter wheat conservation green space

[0174] [Table 42]

[0175] ** Days after DAA application

[0176] Table 43: Fresh mass content, grain yield, thousand kernel mass and protein content of winter wheat. [Table 43]

[0177] *Grain yield recalculated at 15% humidity.

[0178] result: During the first application, made at the BBCH32-33 arable plant development stage, symptoms of infestation with the fungus Zymoseptoria tritici, the cause of wheat leaf stripe septicemia, were observed on the lower leaves of winter wheat cultivar Arkadia at a leaf infestation area level of 15%, while infestation with Blumeria graminis, the cause of powdery mildew, was at a level of 4%.

[0179] A second treatment was performed 3 weeks after the first application on arable plant developmental stages BBCH49-53. The infestation rate of Zymoseptoria tritici on the L-4 leaf sub-control was about 6.38%, and the infestation rate of Blumeria graminis on the L-4 leaf sub-control was about 7.81%. The efficacy of the tested fungicides 3 weeks after the first application was about 56-94% on L-4 for powdery mildew, but at the level of 10-57% for septicemia stripe on wheat leaves (Table 40, Table 41).

[0180] Approximately 2 weeks after the second application, powdery mildew was observed on the upper leaves in the small control field, L-2, with an intensity of about 5%. The efficacy of the tested fungicides was 54-100% on the L-2 leaves, depending on the dose used. The comparison agent suppressed the disease on the L-2 leaves by 100%. Meanwhile, in the small control field, striped peeling of the leaves occurred with an intensity of about 21% on the L-2 leaves and about 5% on the L-1 leaves. The efficacy of the tested fungicides was 36-57% on the L-2 leaves, while it was 40-62% on the L-1 leaves, depending on the dose used. The comparison agent inhibited the disease development by 32% on the L-2 leaves and 50% on the L-1 leaves (Table 40, Table 41).

[0181] In evaluations performed on winter wheat, Arkadia variety, developmental stages BBCH75-77, the incidence of leaf stripe septicemia in L-1 flag leaves was 21.38%. The efficacy of the test composition of Example 1 ranged from 49-68% against L-1 (Table 41).

[0182] At plant development stage BBCH75-77, the green area of ​​L-1 flag leaves was higher in all combinations tested compared to the control (Table 42).

[0183] Conclusion: 1. The experiments carried out showed no phytotoxic effects of different doses of proliposomal fungicide and standard Dafne 250 EC on winter wheat of Arkadia variety. 2. The efficacy of the proliposomal formulation against Blumeria graminis was comparable to that of the standard formulation at a dose of 1N. 3. Two applications of the tested proliposomes at doses of 0.8N and 1N reduced the incidence of Zymoseptoria tritici at the level of the standard agent Dafne250EC. 4. A significant increase in green leaf area (GLA) was observed following application of all doses of the proliposomal agent and the comparator Daphne 250 EC tested compared to the control. 5. A significant increase in mean yield and 1000 seed mass was observed in the small fields treated with proliposomes compared to the small control fields, regardless of the dose used.

[0184] Example 22: Biological evaluation of the efficiency of difenoconazole-containing proliposomes (Example 1) in controlling powdery mildew (Blumeria graminis (Erysiphe graminis)), brown rust (Puccinia recondita), yellow leaf spot (Pyrenophora tritici-repentis), wheat leaf stripe septica (Mycosphaerella graminicola (anam. Zymoseptoria tritici)) in winter wheat, Tobak variety.

[0185] Table 44: Phytotoxicity [Table 44]

[0186] Control=0 * Days after DAA application

[0187] Table 45: Mean percentage of leaf surface infestation and fungicide efficacy in protecting winter wheat against powdery mildew (Blumeria graminis ERYSGR).

[0188] [Table 45] *Efficacy calculated using Abbott's formula ** Days after DAA application

[0189] Table 46a: Mean percentage of leaf surface infestation and efficacy of fungicides in protecting winter wheat against wheat leaf stripe septoria - Zymoseptoria tritici SEPTTR [Table 46a]

[0190] *Efficacy calculated using Abbott's formula ** Days after DAA application

[0191] Table 46b: Mean percentage of leaf surface infestation and efficacy of fungicides in protecting winter wheat against wheat leaf stripe septoria - Zymoseptoria tritici SEPTTR [Table 46b]

[0192] *Efficacy calculated using Abbott's formula ** Days after DAA application

[0193] Table 47: Mean percentage of leaf surface infestation and fungicide efficacy in protecting winter wheat from stem rust - Puccinia recondita PUCCRE [Table 47]

[0194] *Efficacy calculated using Abbott's formula ** Days after DAA application

[0195] Table 48: Mean percentage of leaf surface penetration and fungicide efficacy against leaf spot disease of winter wheat - Pyrenophora tritici-repentis PYRNTR [Table 48]

[0196] *Efficacy calculated using Abbott's formula ** Days after DAA application

[0197] Table 49: Winter wheat conservation green space [Table 49]

[0198] ** Days after DAA application

[0199] Table 50: Fresh mass content, grain yield, thousand kernel mass and protein content of winter wheat. [Table 50]

[0200] *Grain yield recalculated at 15% humidity.

[0201] result: During the first application, made at the BBCH32-33 arable plant development stage, symptoms of infestation with the fungus Zymoseptoria tritici, the cause of wheat leaf stripe septicemia, were observed on the lower leaves of winter wheat cultivar Tobak at a leaf infestation area level of 3%, while infestation with Blumeria graminis, the cause of powdery mildew, was at a level of 2%.

[0202] Three weeks after the first application, a second treatment was performed at arable plant development stages BBCH 45-47, when plants in the sub-control were infested with Zymoseptoria tritici at the order of 8.44% of the L-4 leaves, and plants in the sub-control were infested with Blumeria graminis at the order of 14% of the L-4 leaves and 5.13% of the L-3 leaves. The efficacy of the tested fungicides three weeks after the first application was in the order of 57-89% on L-4 for powdery mildew and 85-93% on L-4, but at the level of 7-28% for septicemia of wheat leaves (Table 45, Table 46a).

[0203] Approximately two weeks after the second application, powdery mildew occurred on the upper leaves in the small control plots at approximately 22% on L-3 leaves and 7% on L-2 leaves. The efficacy of the tested fungicides was 47-79% on L-3 leaves, whereas it was 62-88% on L-2 leaves, depending on the dose used. The comparison agent suppressed the disease at 65% on L-3 leaves and 78% on L-2 leaves. Meanwhile, in the small control plots, striped peeling of the leaves occurred at an intensity of approximately 13% on L-2 leaves and 7% on L-1 leaves. The efficacy of the tested fungicides was 26-46% on L-3 leaves, whereas it was 15-61% on L-2 leaves, depending on the dose used. The comparison agent inhibited the disease occurrence at 39% on L-3 leaves and 50% on L-2 leaves (Table 45, Table 46a).

[0204] Evaluations of winter wheat, Tobak variety, at growing seasons BBCH 75-77 showed incidences of leaf stripe septicemia, brown rust and yellow leaf spot. Stripe septicemia occurred at levels of 29.69% and 9.19% on L-2 sub-flag leaves and L-1 flag leaves, respectively. The efficacy of the tested proliposomal agents ranged from 23-50% on L-2 and 51-70% on L-1 (Table 46b). In the sub-control plots, Puccinia recondita and Pyrenophora tritici-repentis appeared on flag leaves at levels of 10% and 9%, respectively. The efficacy of the tested proliposomal fungicides ranged from 53-82% against brown rust and 26-54% against yellow leaf spot, depending on the dose used. On the other hand, the comparative agent Dafne250EC suppressed the occurrence of brown rust by 79% and the occurrence of yellow leaf spot by 33% (Tables 47 and 48).

[0205] At plant development stage BBCH75, leaf green area of ​​sub-flag L-2 and flag L-1 was greater in all combinations tested compared to the control (Table 49).

[0206] Grain yield, thousand kernel mass, and protein content were at similar levels for all experimental combinations tested (Table 50).

[0207] Conclusion: 1. The experiments carried out showed that there were no symptoms of phytotoxic effects of different doses of proliposomal fungicide and standard Dafne 250 EC on winter wheat of Tobak variety. 2. The tested proliposomal fungicides at doses of 0.8N and 1N applied to Blumeria graminis performed better than the comparator Dafne 250 EC. 3. Two applications of the tested proliposomes at a dose of 1N reduced the incidence of Zymoseptoria tritici more than the standard agent Dafne250EC. Proliposomes tested at a dose of 4.1N inhibited the development of Puccinia recondita at comparable drug levels. 5. The investigated proliposomal fungicides at doses of 0.8N and 1N applied to Pyrenophora tritici-repentis performed better than the comparator Daphne 250 EC. 6. The use of Proliposomes and Dafne250 EC did not affect, on average, the yield size and quality (mass of 1000 kernels, protein content) of winter wheat Zyta cultivar.

[0208] Example 23: Biological evaluation of the efficacy of difenoconazole-containing proliposomes (Example 1) in controlling powdery mildew (Blumeria graminis (Erysiphe graminis)), wheat leaf stripe septica (Mycosphaerella graminicola (anam. Zymoseptoria tritici)) in winter wheat, cultivar Opoka.

[0209] Table 51: Phytotoxicity [Table 51]

[0210] Control=0 * Days after DAA application

[0211] Table 52a: Mean percentage of leaf surface infestation and efficacy of fungicides in protecting winter wheat against wheat leaf stripe septoria - Zymoseptoria tritici SEPTTR [Table 52a]

[0212] *Efficacy calculated using Abbott's formula ** Days after DAA application

[0213] Table 52b: Mean percentage of leaf surface infestation and efficacy of fungicides in protecting winter wheat against wheat leaf stripe septoria - Zymoseptoria tritici SEPTTR [Table 52b]

[0214] *Efficacy calculated using Abbott's formula ** Days after DAA application

[0215] Table 53: Winter wheat conservation green space [Table 53]

[0216] ** Days after DAA application

[0217] Table 54: Fresh mass content, grain yield, thousand kernel mass and protein content of winter wheat. [Table 54]

[0218] *Grain yield recalculated at 15% humidity.

[0219] result: During the first application, carried out at the developmental stage of cultivated plants BBCH32-33, symptoms of Zymoseptoria tritici infestation causing wheat leaf stripe septicemia were observed on the lower leaves of winter wheat of the Opoka variety at a leaf infestation area level of 2%.

[0220] Three weeks after the first treatment, a second treatment was applied at the BBCH45 arable plant development stage. At that time, the average infestation of plants by Zymoseptoria tritici on L-4 leaves in the small control field was about 8%. Three weeks after the first application, the efficacy of the tested fungicides ranged from 17 to 28% in the case of septicemia stripe on wheat leaves (Table 52a).

[0221] Approximately two weeks after the second application, striped septicemia was observed on the upper leaves of the small control plots with an intensity of about 14% on L-3 leaves and about 8% on L-2 leaves. The efficacy of the tested fungicides ranged from 33 to 57% on L-3 leaves, whereas it ranged from 58 to 86% on L-2 leaves. The comparator agents inhibited disease development by 55% on L-3 leaves and 88% on L-2 leaves (Table 52a).

[0222] In evaluations performed on winter wheat, Opoka variety, at developmental stage BBCH75, incidence of leaf stripe septicemia in L-2 subflag leaves and L-1 flag leaves was observed at levels of 25.63% and 9.88%, respectively. The efficacy of the tested proliposomal formulations against wheat leaf stripe septicemia ranged from 40-74%. The comparator formulations inhibited disease incidence by 55% in L-2 leaves and 59% in L-1 leaves (Table 52b).

[0223] At plant development stage BBCH75, leaf green area of ​​sub-flag L-2 and flag L-1 was higher in all combinations tested compared to the control (Table 53).

[0224] Conclusion: 1. The experiments carried out showed no phytotoxic effects of different doses of proliposomal fungicide and standard Dafne 250 EC on winter wheat variety Opoka. 2. The tested proliposomal forms of fungicides, applied twice at a dose of 1N, reduced the incidence of Zymoseptoria tritici more than the standard agent Dafne250EC. 3. Increases in green leaf area (GLA) were observed following application of all doses of the proliposomal agent and the comparator Daphne 250 EC tested compared to the control. 4. Increased yield and 1000 seed mass were observed in the small fields treated with proliposomes compared to the small control fields, irrespective of the dose used.

[0225] Example 24: Biological evaluation of the effectiveness of the action of difenoconazole-containing proliposomes (Example 1) in controlling blackleg (Leptosphaeria maculans) in winter oilseed rape, cultivar Architect.

[0226] Table 55: Phytotoxicity [Table 55]

[0227] Table 56: Mean percentage of surface infestation and fungicide efficacy in protecting winter oilseeds against blackleg - Leptosphaeria maculans LEPTMA

[0228] [Table 56]

[0229] * Potency calculated using Abbott's formula ** Days after DAA application

[0230] Table 57: Grain yield, mass per 1000 seeds, and oil content. [Table 57]

[0231] *Grain yield recalculated at 9% humidity.

[0232] result: Infestation of winter oilseed rape, cv. Architect, by Leptosphaeria maculans assessed in phase 85 of BBCH was at average levels, reaching 27% in small controls. Infestation by the pathogen was at a level sufficient to assess the efficacy of the fungicides tested.

[0233] Conclusion: 1. No phytotoxic effects were observed on winter oilseed rape, cultivar Architect, at a dose of 1N for either the tested proliposomal fungicides or the comparator Dafne 250 EC. 2. Proliposomal fungicides and the comparator Dafne 250 EC significantly inhibited the development of blackleg in winter oilseed rape plants, regardless of the dose used. An increase in the effectiveness of proliposomes with increasing applied dose was observed. 3. The investigated proliposomal fungicides at a dose of 1N applied to Leptosphaeria maculans performed better than the comparator Dafne 250 EC. 4. A significant increase in yield and a moderate increase in 1000 seed mass were observed in the small fields treated with proliposomes compared to the small control fields, regardless of the dose used.

[0234] Example 25: Biological evaluation of the effectiveness of the action of difenoconazole-containing proliposomes (Example 1) in controlling Sclerotinia sclerotiorum in winter oilseed rape, cultivar Architect.

[0235] Table 58: Phytotoxicity [Table 58]

[0236] Control=0 ** Days after DAA application

[0237] Table 59: Mean percentage of surface infestation and fungicide efficacy in protecting winter oilseeds against Sclerotinia sclerotiorum (SCLESC). [Table 59]

[0238] * Potency calculated using Abbott's formula ** Days after DAA application

[0239] Table 60: Grain yield, mass per 1000 seeds, and oil content. [Table 60]

[0240] *Grain yield recalculated at 9% humidity.

[0241] result: The first symptoms of infection by Sclerotinia sclerotiorum were observed at BBCH 75. Infestation of winter oilseed rape, Architect variety, by Sclerotinia sclerotiorum evaluated at BBCH 85 was moderate, reaching 28% in small controls. The infestation by the pathogen was at a level sufficient to evaluate the efficacy of the fungicides tested.

[0242] Conclusion: 1. No phytotoxic effects were observed on winter oilseed rape, cultivar Architect, at a dose of 1N for any of the tested proliposomal fungicides and the comparator Dafne250 EC. 2. Proliposomal fungicides and the comparator Dafne 250 EC significantly inhibited the emergence of Sclerotinia sclerotiorum in winter oilseed rape plants, regardless of the dose used. An increase in the effectiveness of proliposomes with increasing applied dose was observed. 3. The investigated proliposomal fungicides used on Sclerotinia sclerotiorum at doses of 0.8N and 1N performed better than the comparator Dafne250 EC. The results obtained demonstrate the possibility of a two-fold dose reduction maintaining the efficacy of the proliposomal composition at a dose of 0.5N, comparable to the comparator Dafne250EC at a dose of 1N. 4. Increased yield and 1000 seed mass were observed in the small fields treated with proliposomes compared to the small control fields, irrespective of the dose used.

[0243] Example 26: Biological evaluation of the efficacy of difenoconazole-containing proliposomes (Example 1) in controlling Sclerotinia sclerotiorum in winter oilseed rape, variety Visby.

[0244] Table 61: Phytotoxicity [Table 61]

[0245] Control=0 ** Days after DAA application

[0246] Table 62: Mean percentage of surface infestation and fungicide efficacy in protecting winter oilseeds against Sclerotinia sclerotiorum (SCLESC). [Table 62]

[0247] * Potency calculated using Abbott's formula ** Days after DAA application

[0248] Table 63: Grain yield, mass per 1000 seeds, and oil content. [Table 63] *Grain yield recalculated at 9% humidity.

[0249] result: The first symptoms of infection by Sclerotinia sclerotiorum were observed at BBCH 75. Infestation of winter oilseed rape, cv. Visby, evaluated at BBCH 85 was moderate, reaching 28% in small controls. The infestation by the pathogen was at a level sufficient to evaluate the effectiveness of the fungicides tested.

[0250] Conclusion: 1. No phytotoxic effects were observed on winter oilseed rape (variety Visby) at a dose of 1N for either the tested proliposomal fungicides or the comparator Dafne 250 EC. 2. Proliposomal fungicides and the comparator Dafne 250 EC significantly inhibited the emergence of Sclerotinia sclerotiorum in winter oilseed rape plants, regardless of the dose used. An increase in the effectiveness of proliposomes with increasing applied dose was observed. 3. The investigated proliposomal fungicides used against Sclerotinia sclerotiorum performed better than the comparator Dafne 250 EC at all tested doses. The results show the possibility of a 60% dose reduction in the proliposomal formulation while maintaining the same efficacy as the comparator Dafne 250 EC. 4. Increased yield and 1000 seed mass were observed in the small fields treated with proliposomes compared to the small control fields, irrespective of the dose used.

[0251] Example 27: Biological evaluation of the effectiveness of the action of difenoconazole-containing proliposomes (Example 1) in controlling Sclerotinia sclerotiorum in winter oilseed rape, Alibaba variety.

[0252] Table 64: Phytotoxicity [Table 64]

[0253] Control=0 ** Days after DAA application

[0254] Table 65: Mean percentage of surface infestation and fungicide efficacy in protecting winter oilseeds against Sclerotinia sclerotiorum (SCLESC). [Table 65]

[0255] * Potency calculated using Abbott's formula ** Days after DAA application

[0256] Table 66: Grain yield, mass per 1000 seeds, and oil content. [Table 66]

[0257] *Grain yield recalculated at 9% humidity.

[0258] result: The first symptoms of infection by Sclerotinia sclerotiorum were observed at BBCH 75. Infestation of winter oilseed rape, Alibaba variety, by Sclerotinia sclerotiorum assessed at BBCH 85 was moderate, reaching 29% in small control plots. The infestation by the pathogen was at a level sufficient to evaluate the efficacy of the fungicides tested.

[0259] Conclusion: 1. No phytotoxic effects were observed on winter oilseed rape, Alibaba cultivar, at a dose of 1N for either the tested proliposomal fungicides or the comparator Dafne250EC. 2. The investigated proliposomes and the comparator Dafne250 EC significantly inhibited the development of Sclerotinia sclerotiorum in winter oilseed rape plants, regardless of the dose used. An increase in the effectiveness of proliposomes with increasing applied dose was observed. 3. The investigated proliposomal fungicides used at doses of 0.5N, 0.8N and 1N against Sclerotinia sclerotiorum performed better than the comparator Dafne 250EC. The results obtained demonstrate the possibility of a two-fold dose reduction in the proliposomal composition compared to the comparator Dafne 250EC while maintaining comparable efficacy. 4. The use of Proliposomes and Dafne250EC did not affect, on average, the yield size and quality (mass of 1000 kernels, oil content) of winter oilseed rape, Alibaba variety.

Claims

1. A liquid proliposomal composition of a plant protection agent, comprising: At least one plant protection agent, at least one phospholipid, and at least one organic solvent; At least one plant protection agent is present in the range of 1% to 50% by weight; at least one phospholipid is lecithin and is present in the range of 20% to 75% by weight; the at least one organic solvent is selected from the group consisting of ethers, glycol ethers, n-butylpyrrolidone, ethylene glycol monobutyl ether, propylene carbonate, N,N-dimethyllactamide, and 5-dimethylamino-2-methyl-5-oxovaleric acid methyl ester and is present in the range of 20% to 75% by weight; The composition comprises: 0.1% to 35% by weight of at least one agriculturally acceptable auxiliary material, comprising at least one surfactant selected from the group consisting of polysorbate 20, a mixture of long chain (C12-15) fatty alcohols ethoxylated with 3 to 5 molecules of ethylene oxide, and octylamine, in an amount of less than 15% by weight; 0-12% by weight of water or an aqueous solution of an agrochemically acceptable salt or buffer substance; The composition further comprises:

2. 2. The composition according to claim 1, characterized in that the at least one plant protection agent is a herbicide or a fungicide.

3. 3. The composition according to claim 1, wherein the at least one plant protection agent constitutes from 5% to 20% by weight of the composition.

4. 3. The composition according to claim 1, characterized in that the ratio of said at least one phospholipid to said at least one plant protection agent is between 25:1 and 2:

1.

5. 3. The composition according to claim 1, wherein said at least one phospholipid comprises from 5% to 99.99% phosphatidylcholine.

6. 6. The composition according to claim 5, wherein said at least one phospholipid constitutes from 20% to 45% by weight of said composition.

7. 3. The composition according to claim 1, wherein said at least one surfactant constitutes 3% by weight based on the weight of said composition.

8. 3. The composition according to claim 1 or 2, characterized in that in addition to said at least one surfactant, it contains at least one other auxiliary substance selected from the group consisting of antifoaming agents, antioxidants and biodegradable, non-volatile and non-flammable agents that influence the fluidity of the lipid membrane.

9. 3. The composition according to claim 1 or 2, characterized in that said at least one organic solvent constitutes from 20% to 30% by weight of said composition.

10. A composition according to claim 1 or 2, characterized in that it contains 5 to 10% by weight of water or an aqueous solution of an agrochemically acceptable salt or buffer substance.

11. A method for preparing a composition according to claim 1 or 2, comprising, in order: a) dissolving and mixing at least one phospholipid in an amount of 20% to 75% by weight of at least one organic solvent in an amount of 20% to 75% by weight of the final composition according to claim 1 or 2 to obtain a mixture; b) while continuing to mix, adding to the mixture resulting from step a) at least one surfactant selected from the group consisting of polysorbate 20, a mixture of long chain (C12-15) fatty alcohols ethoxylated with 3-5 molecules of ethylene oxide, and octylamine in an amount of less than 15% by weight of the final composition according to claim 1 or 2; c) adding, while continuing to mix, to the mixture obtained from step b) at least one plant protection agent in an amount of 1% to 50% by weight of the final composition according to claim 1 or 2; d) optionally adding water or an aqueous solution of an agrochemically acceptable salt or buffer substance to the mixture obtained from step c) in an amount of 0% to 12% by weight of the final composition according to claim 1 or 2; e) optionally adding at least one agrochemically acceptable auxiliary substance to the mixture resulting from step c) or d) in such an amount that it constitutes from 0.1% to 35% by weight of the final composition according to claim 1 or 2, including the surfactant added in step b); f) mixing the mixture obtained in step e) to form the final proliposomal composition according to claim 1 or 2; The method includes:

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