Composite microspheres and fungicidal formulations

Abstract

A composite microsphere enhancing the efficacy of fungicides, comprising chitosan with an average molecular weight ranging from 190,000 to 310,000 g / mol, and a cross-linking agent, in the absence of a fungicidal active ingredient; and a fungicidal formulation comprising a fungicidal active ingredient and said composite microspheres

Description

[0001] COMPOSITE MICROSPHERES AND FUNGICIDAL FORMULATIONS

[0002] Cross-reference to related applications

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 621,881, filed on January 17, 2024, which is incorporated by reference herein in its entirety.

[0004] Field of the Invention

[0005] The present invention relates to the field of smart materials designed to enhance the efficacy of chemical products used in agricultural processes. Specifically, the present invention pertains to a microstructured polymeric compound with tailored chemical composition, charge distribution, and advantageous properties for use in the dosage and controlled release of agrochemicals for sustainable agricultural processes. In particular, it relates to fungicidal formulations enhanced with composite microspheres. The present invention also includes scalable, high-conversion, verified, and validated methods for their preparation.

[0006] State of the Art

[0007] In general terms, an agrochemical refers to any substance or mixture of natural or synthetic substances used to prevent, eliminate, and / or control pests, diseases, or weeds in agricultural activities. These substances are commonly known as pesticides or crop protection agents - also referred to as phytosanitary products - which include insecticides, herbicides, fungicides, acaricides, among others. This categorization of agrochemicals also encompasses substances aimed at providing elements that promote plant growth, such as fertilizers and plant growth regulators.

[0008] Fungicides are used to prevent or control harmful fungi and molds that affect both plants and animals.

[0009] In addition to pests, fungal diseases also affect crops worldwide. Fungi are responsible for approximately 70% of diseases in major agricultural crops [Agrios, 2005], such as wheat, potato, cotton, tomato, peanut, grapevine, and cotton [Dhekney, 2005], The severity of the situation becomes evident when considering that annual crop losses in the agricultural industry due to fungal diseases, both in the field and post-harvest, exceed $200 billion. In contrast, over $600 million are spent annually on fungicides in the United States alone [Fernandez-Perez, 2000], Globally, approximately one-quarter of food crops are damaged by fungal toxins such as aflatoxins, ergot toxins, Fusarium toxins, patulin, and tenuazonic acid [Razzaghi-Abyaneh, 2014].

[0010] The use of agrochemicals can result in undesired effects on human health, such as the development of chronic diseases. Acute problems due to accidental exposure to high doses can cause acute intoxications, including symptoms such as dizziness, vomiting, respiratory difficulties, and even death. Risks in sensitive populations, such as pregnant women and children, are particularly concerning, as they are especially susceptible to the neurotoxic and endocrine-disrupting effects of some agrochemicals, potentially impacting physical and cognitive development [Islam, 2024],

[0011] Indiscriminate use of agrochemicals has disrupted natural balances, causing harmful effects on various ecosystems, such as soil contamination that reduces its natural fertility, alters the microbiota, and leads to salinization or acidification processes. Excessive application of agrochemicals can leach into rivers, lakes, and groundwater, causing water contamination and phenomena such as eutrophication, where an excess of nutrients in the water promotes the proliferation of toxic algae, affecting water quality, aquatic life, and biodiversity.

[0012] Additionally, the repeated and excessive use of agrochemicals has led to the development of resistance in pests, weeds, and pathogens. A report on the environmental and health impacts of pesticides and fertilizers, prepared in close collaboration and consultation with the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), highlights the need for transformative actions and better management of pesticides and fertilizers as the global demand and use of these products increase.

[0013] (wedocs.unep.org / xmlui / bitstream / handle / 20.500.11822 / 38409 / pesticides.pdf)

[0014] There is a pressing need to adopt sustainable procedures that mitigate the negative impacts of excessive agrochemical use. In recent years, traditional agriculture has been complemented by alternatives aimed at reducing these adverse effects, such as precision agriculture. Precision agriculture seeks to optimize farming practices by leveraging advanced technologies that collect, analyze, and interpret data on crops, soil, climate, and other factors to make more informed decisions. Its primary goal is to maximize the efficacy of resources such as water, fertilizers, and pesticides while minimizing environmental impacts and enhancing productivity.

[0015] Nanotechnology has provided developments and technologies aimed at addressing these issues by maximizing resource optimization. Nanotechnology is widely applied in modern agriculture to realize the concept of precision agriculture [Duhan, 2017], Nanotechnology includes nanoparticles with one or more dimensions in the range of 100 nm or less. Nanomaterials find applications in plant protection, nutrition, and agricultural management practices due to their small size, high surface-to-volume ratio, and unique optical properties [Auffan, 2009],

[0016] Biopolymeric systems offer additional benefits, such as reduced toxicity, increased stability / shelf life, and improved solubility of pesticides. These systems are also cost- effective, readily available, biocompatible, biodegradable, and environmentally safe (i.e., they gradually release associated active compounds without harming the environment) and have a low carbon footprint. Biopolymeric nanoformulations support plant growth while enhancing soil aeration and microbial activity, benefiting the environment.

[0017] The development of controlled-release formulations (CRFs) is highly desirable to meet international environmental and biodiversity laws. The use of polymers for sustained release began in the early 1970s, owing to the greater efficacy of their encapsulated components compared to commercial formulations. To enhance the value of traditional pesticides, many natural molecules, such as carrageenan, galactomannans, chitosan, alginate, pectin, cellulose, gum arabic, guar gum, cashew gum, chitin, agar, tamarind seed polysaccharides, and starch, are frequently processed into nanomaterials for sustained and targeted delivery to specific action sites of various agrochemicals [Salgueiro, 2013; Albuquerque, 2016; Sharma, 2019; Slade, 2019; Rashidipour, 2019; Elabasy, 2020],

[0018] In the state of the art, one of the most widely used polymers is chitosan, which is a polysaccharide composed of repeated units of D-glucosamine and N-acetyl-D- glucosamine. Chitosan is the primary industrial derivative of chitin and can be obtained through the N-deacetylation of chitin. It is poorly soluble in water, and when the degree of deacetylation reaches at least 50%, it dissolves as a polycation in diluted aqueous acids [Robert, 1992]. The degree of deacetylation and the molecular weight of chitosan strongly determine its physicochemical and biological properties, such as solubility, hydrophobicity, crystallinity, and cellular response. In recent decades, chitosan has garnered increasing interest in the agricultural sector. For example, chitosan dissolved in an acidic solution activates its antimicrobial properties and its ability to stimulate plant defense mechanisms, giving it the potential to become a new class of plant stress protectants [Bautista-Banos, 2006; Sahariah, 2017],

[0019] Another way to use chitosan in various applications, particularly in agricultural applications, is by forming three-dimensional structures through the crosslinking of chitosan molecules, creating stable structures that allow for the nanoencapsulation of active components and are characterized by more controlled release of the encapsulated component. The slow release, protection against degradation, and low solubility of the encapsulated insecticide are the most important characteristics of polymeric nanoformulations, making them the first choice for nanoencapsulation. This valuable information has paved the way for the development and practical application of polymeric nanoinsecticides with immense potential.

[0020] Additionally, different types of nanoformulations, both polymer-based and non- polymer-based, have been proposed, such as nanospheres, nanocapsules, nanogels, micelles, nanofibers, nanometals, and nanoemulsions, for the encapsulation of insecticides. Among these, nanocapsules are the most widely used for the controlled release of insecticides. Very recently, a novel concept of hybrid nanoformulation (nanoemulsion encapsulation or liposome coating) has also been suggested for the controlled release of certain insecticides. However, the efficacy of this novel approach needs to be tested on a broader range of insecticides [Das, 2014].

[0021] In the document by Grenha from 2012 [Grenha, 2012], the preparation methods of chitosan-based nanostructured systems are described, primarily through emulsification, different types of coacervation, or even slight modifications of both. More specifically, the methods include emulsion droplet coalescence [Tokumitsu et al., 1999], solvent diffusion in emulsions [El-Shabouri, 2002], reverse micelle methods [Mitra et al., 2001], ionic gelation, polyelectrolytic complexation [Calvo et al., 1997; Sarmento et al., 2006], and desolvation [Tian & Groves, 1999], All these methods involve bottom-up manufacturing processes, which consist of the assembly of molecules in solution to form defined structures [Chan & Kwok, 2011], In this case, composite nanomicrocapsules.

[0022] The release systems resulting from bottom-up technologies often exhibit size polydispersity [Wang et al., 2011], which in certain cases limits the utility of nanoparticles. Indeed, it is assumed that in a polydisperse system, larger nanoparticles might have a greater capacity to load active ingredients, while smaller nanoparticles are expected to be more efficient in delivering agrochemicals to tissues or cells [Fan et al., 2012],

[0023] Chitosan microparticles and nanoparticles have been fabricated through chemical crosslinking with glutaraldehyde, glyoxal, and ethylene glycol diglycidyl ether. Although these are highly effective crosslinking agents, they are not preferred due to their physiological toxicity. Chitosan, being polycationic in acidic media (pKa 6.5), can interact with negatively charged species, such as the crosslinker TPP (sodium tripolyphosphate) and sodium sulfate. This characteristic can be leveraged to prepare crosslinked chitosan nanoparticles. The interaction of chitosan with TPP leads to the formation of biocompatible crosslinked chitosan nanoparticles, which can be efficiently used in the delivery of proteins, vaccines, and many other types of compounds. The crosslinking density, crystallinity, and hydrophilicity of crosslinked chitosan allow for the modulation of encapsulated drug release and expand its range of potential applications in drug delivery [Bhumkar, 2006],

[0024] Chitosan has a high degree of protonation in its amine functions and demonstrates the capacity to form hydrogels in the presence of specific polyanions. This process results from inter- and intramolecular crosslinking mediated by anionic molecules [Janes et al., 2001; Terbojevich & Muzzarelli, 2009] and is referred to as ionic gelation or polyelectrolytic complexation. It has therefore been used to produce chitosan nanoparticles. This method involves an ionic interaction between the positively charged amino groups of chitosan and the polyanion sodium tripolyphosphate (TPP), which acts as the crosslinker for chitosan. It is important to note that the term "ionic gelation" is preferred when chitosan gelation is induced by small anionic molecules, such as phosphate, citrate, or sulfate. On the other hand, it is considered "polyelectrolytic connplexation" when macromolecular anions are used instead of small molecules [Bhattarai et al., 2010].

[0025] Mancozeb, a protective contact fungicide belonging to the dithiocarbamate group, is widely used in agricultural crops to control fungal diseases (FAO, 2020). In a 2022 publication, Kumar et al. demonstrated the fungicidal activity of Mancozeb encapsulated in chitosan nanocapsules. In this study, chitosan and gum arabic nanoparticles crosslinked with TPP were evaluated, loaded with three concentrations of Mancozeb during the synthesis process, allowing the active ingredient to be encapsulated. The same study describes that the encapsulated formulations exhibit controlled release of Mancozeb over a prolonged period of at least 10 hours compared to the commercial formulation.

[0026] The Mancozeb-loaded nanoparticles showed at least 85.2% inhibition against the fungus Alternaria alternata and 100% inhibition against the fungus Stemphylium lycopersici when applied at concentrations of 1.0 and 1.5 ppm, respectively, compared to the commercial Mancozeb control. The same authors presented chitosan and carrageenan nanocapsules containing Mancozeb and demonstrated antifungal effects against the fungus Sclerotinia sclerotiorum and S. lycopersici at concentrations of 1.0 and 1.5 ppm [Kumar, 2022],

[0027] Finally, in a study by Butstraen et al. [Butstraen, 2014], chitosan and gum arabic microcapsules crosslinked with TPP and loaded with commercial Miglyol 812 N are described. The microcapsules were obtained using the coacervation method, and different chitosan-to-gum arabic mass ratios of 1:4 and 1:5 were evaluated, with TPP as the crosslinking agent and the presence of Miglyol during the synthesis. The physicochemical parameters of the microparticles, their encapsulation capacity, and the zeta potential of the particles were analyzed. This document presents research aimed at identifying optimal conditions for encapsulating Miglyol but does not describe or suggest any correlation between TPP concentration and the Z potential of the particles formed. This document suggests an optimal chitosan-to-gum arabic mass ratio of 1:4, a range far from the preferred embodiments of the present invention. None of the prior art mentioned discloses or provides any indication that could lead an expert to a composite microsphere like the one in the present invention, which does not encapsulate agrochemical actives but is instead added to a fungicidal formulation to enhance its efficacy, its efficiency, and thereby reduce the concentration of fungicide used while maintaining effective activity.

[0028] Additionally, none of the prior art suggests that the Z potential of such a composite microsphere could be regulated by the concentration of the crosslinking agent during the synthesis process. Furthermore, none of the prior art has proposed the use of gum, a preferred embodiment of the present invention, to improve the redispersion stability of composite chitosan microspheres when combined with the fungicide, preventing clogging of spray nozzles and applicators. The state of the art proposes the use of gums as dispersants during the manufacturing process of natural polymer microparticles, such as chitosan, to regulate dispersion during the synthesis process of the microparticles. However, the use of gums to improve the stability of the redispersion of such particles when added to a fungicidal solution has not been reported.

[0029] The present invention provides a composite microsphere comprising chitosan and a crosslinking agent, preferably tripolyphosphate (TPP), and in another preferred embodiment, a dispersant such as gum. The composite microsphere of the invention can be synthesized with a regulated Z potential by adjusting the concentration of the crosslinking agent, allowing the zeta potential to be tuned in an aqueous solution. This feature of the present invention enables the design and selection of the desired composite microsphere, allowing it to be combined with different agrochemical compounds. The composite microsphere of the present invention, formulated with a fungicidal active ingredient for its application, enhances the effect of said fungicide.

[0030] The present invention provides composite microspheres that can be used as adjuvants in fungicidal formulations. Particularly, when combined with Mancozeb, they enhance fungicidal activity against fungi. Collectively, these properties make the composite microsphere an innovative and promising tool for improving the efficiency of agricultural inputs, reducing environmental impact, and promoting sustainability in modern agriculture. The present invention differs from similar inventions in the state of the art, among other reasons, in that it can be combined with the fungicidal active ingredient prior to its application and does not require encapsulation of the active ingredient during the particle synthesis process.

[0031] Therefore, the present invention addresses the problem posed by the state of the art by reducing the use of agrochemicals with a solution that is safe, biocompatible, biodegradable, and environmentally friendly.

[0032] Brief Description of the Invention

[0033] The composite microsphere enhancing the efficacy of fungicides, preferably in agriculture, which is the object of the present invention, comprises chitosan with an average molecular weight in the range from 190,000 to 310,000 g / mol, and a crosslinking agent, wherein said microsphere is synthesized in the absence of a fungicidal active ingredient. This composite microsphere of the invention has an adjustable surface electrical charge (Z potential) in an aqueous medium modulated by the concentration of said crosslinking agent, and the chitosan-to-crosslinker mass ratio is 1:X, where X varies in the range from 0.08 to 1.5; more preferably in the range from 0.4 to 1.2; even more preferably 0.8. Said crosslinking agent is selected from the group consisting of sodium tripolyphosphate (TPP), sodium hexametaphosphate, and their derivatives, preferably sodium tripolyphosphate (TPP).

[0034] In a preferred embodiment, the present invention further comprises a dispersant that improves the redispersion of said microsphere when combined with an aqueous solution of a fungicidal active ingredient, wherein said dispersant is selected from the group consisting of gum arabic, xanthan gum, guar gum, carrageenan, pectin, alginates, dextrins, cyclodextrins, maltodextrins, tragacanth gum, mesquite gum, polyetilenglycol and mixtures thereof; preferably gum arabic. The chitosan-to-dispersant mass ratio is Y:l, where Y is in the range from 5 to 20. That is, the gum is incorporated into the microsphere of the invention at up to 20% of the chitosan mass.

[0035] Another main object of the present invention is a fungicidal formulation comprising a fungicidal active ingredient and the composite microspheres of the invention that enhance the fungicidal efficacy on crops, characterized in that said composite microsphere is added at a concentration between 2 and 200% w / w relative to said fungicidal active ingredient; preferably between 2 and 100%.

[0036] In preferred embodiments of the present invention, said fungicidal active ingredient is selected from the group consisting of pre-emergent, contact, systemic, residual fungicides, prothioconazole, trifloxystrobin, cyproconazole, carbendazim, difenoconazole, isopyrazam, chlorothalonil, epoxiconazole, fluoxastrobin, mancozeb, zineb, thiram, tebuconazole, propiconazole, azoxystrobin, pyraclostrobin, thiabendazole, metalaxyl, boscalid, captan, folpet, dithiocarbamates, triazoles, strobilurins, benzimidazoles, anilides, organochlorines, phthalimides, sedaxane, their derivatives, and combinations thereof. Preferably, said fungicidal active ingredient is mancozeb.

[0037] In a preferred embodiment of the fungicidal formulation of the invention, said composite microsphere comprises chitosan with an average molecular weight in the range from 190,000 to 310,000 g / mol, a dispersant, and a crosslinking agent, and the chitosan-to-crosslinker mass ratio is 1:X, where X varies in the range from 0.08 to 1.2 and defines the Z potential in the aqueous solution of said microspheres. Preferably, the microspheres comprise chitosan, gum arabic, and tripolyphosphate (TPP), in the absence of agrochemical active ingredients.

[0038] The composite microspheres of the invention have a Z potential in the range of +30 mV to -30 mV in an aqueous solution, adjustable by the concentration of the crosslinking agent. When TPP is present at low concentrations, such as 8% of the chitosan mass, the potential is around +30. As the TPP concentration increases, the Z potential decreases, reaching zero when TPP constitutes approximately 40% of the chitosan mass. When the TPP concentration continues to increase, the Z potential becomes more negative, reaching values around -30 at TPP concentrations exceeding 80% of the chitosan mass.

[0039] In a preferred embodiment, the fungicidal formulation of the invention further comprises a co-adjuvant selected from the group consisting of an adhesive, a surfactant, a stabilizer, an antifoaming agent, a pH buffer, a sequestrant, their derivatives, and combinations thereof. In a preferred embodiment, the fungicidal formulation of the invention comprises the fungicidal active ingredient at a concentration equal to or less than the recommended field application rate. This means that the fungicidal formulation of the invention enables a reduction in the amount of fungicides used in agriculture.

[0040] A particular feature of the fungicidal formulation of the invention is that the concentration of the composite microspheres is in the range of 5 to 200% w / w relative to the fungicidal active ingredient, preferably from 5 to 100% w / w. Furthermore, the fungicidal active ingredient and the composite microspheres are mixed in the field prior to application. Preferably, they are mixed at least 2 hours before application, more preferably 4 hours before, and even more preferably 24 hours before.

[0041] In one preferred embodiment, the fungicidal formulation of the present invention is an aqueous composition comprising a fungicidal active ingredient and composite microspheres packaged in containers, as it has been mixed at the manufacturing plant and jointly packaged for sale and distribution.

[0042] In an alternative preferred embodiment, the fungicidal formulation of the present invention is an aqueous composition containing the fungicidal active ingredient and, separately, the composite microsphere powder of the invention to be mixed prior to use.

[0043] Another object of the present invention is a method for obtaining the fungicidal formulation of the invention, comprising the following steps: a) preparing an aqueous solution of the fungicidal active ingredient at the desired concentration, b) adding the dry composite microspheres at a concentration of up to 100% relative to the fungicidal active ingredient, c) mixing at least 2 hours before application. Preferably, a co-adjuvant is also added.

[0044] Another object of the present invention is a method for preventing and controlling fungal infections, which comprises applying the fungicidal formulation of the present invention to a crop. Said crop is selected from the group consisting of cereals, oilseeds, forest species, fruit trees, ornamental plants, vegetables, wheat, barley, rye, triticale, oats, maize, sunflower, rice, soybean, pea, broad bean, common bean, peanut (groundnut), rapeseed, kala, cotton, potato, sugar beet, sugarcane, chard (Beta vulgaris), peppers (Capsicum spp.), garlic and onions (Allium spp.), celery (Apium graveolens), eggplant (Solanum melongena), squash (Cucurbita moschata), chayote (Sechium edule), cabbage (Brassica oleracea), spinach (Spinacia oleracea), common bean (Phaseolus vulgaris), lettuce (Lactuca sativa), maize (Zea mays), peanut (Arachis hypogaea), tomato (Solanum lycopersicum), cucumber (Cucumis sativus), okra (Hibiscus esculentus), radish (Raphanus sativus), beetroot (Beta vulgaris), carrot (Daucus carota), avocado (Persea americana), sugar apple (Annona squamosa), star apple (Chrysophyllum cainito), canistel (Pouteria campechiana), cherry (Malpighia punicifolia), custard apple (Annona reticulata), hog plum (Spondias dulcis), coconut (Cocos nucifera), papaya (Carica papaya), soursop (Annona muricata), guava (Psidium guajava), pomegranate (Punica granatum), lime (Citrus aurantifolia), lemon (Citrus limonum), mamey sapote (Calocarpum mammosum), Santo Domingo apricot (Mammea americana), mamoncillo (Melicoccus bijugatus), mandarin (Citrus reticulata), mango (Mangifera indica), passion fruit (Passiflora laurifolia), watermelon (Citrullus vulgaris), bitter orange (Citrus aurantium), sweet orange (Citrus sinensis), pineapple (Ananas comosus), banana (Musa paradisiaca), plantain (Musa balbisiana), tamarind (Tamarindus indica), grapefruit (Citrus paradisi), native grapefruit (Citrus grandis), grape (Vitis vinifera), common bean (Phaseolus vulgaris), rice (Oryza sativa), coffee (Coffea arabica), sugarcane (Saccharum officinarum), cotton (Gossypium hirsutum), gherkin (Melothria guadalupensis), sweet potato (Ipomoea batatas), potato (Solanum tuberosum), native potato (Solanum phureja), cape gooseberry (Physalis peruviana), cassava (Manihot esculenta), soybean (Glycine max), strawberries (Fragaria spp.), mulberry (Morus spp.), brambles (Rubus spp.), palms of the family Aracaceae, oil palm (Elaeis guineensis), apple, cacao (Theobroma cacao), tamarillo (Solanum betaceum), and lulo (Solanum quitoense). The fungicidal formulation is applied at an active fungicide concentration lower than that indicated on the market label, and it enhances fungicidal action by at least 15% compared to the action of the fungicide in the absence of composite microspheres. On the other hand, the present invention addresses infections caused by fungi selected from the group consisting of Magnaporthe oryzae, Botrytis cinerea, Puccinia spp., Fusarium spp. (including but not limited to Fusarium graminearum, Fusarium oxysporum), Blumeria graminis, Mycosphaerella spp., Mycosphaerella fijiensis, Mycosphaerella graminicola, Colletotrichum spp., Ustilago maydis, Melampsora lini, Phakopsora pachyrhizi, Stemphylium lycopersici, and Rhizoctonia solani.

[0045] Specifically, the present invention comprises, in a preferred embodiment, a fungicidal formulation that enhances the efficacy of a fungicidal active ingredient by at least 15%, characterized by comprising said fungicidal active ingredient and composite microspheres; wherein said microspheres comprise chitosan with an average molecular weight ranging from 190,000 to 310,000 g / mol, and a cross-linking agent; wherein the mass ratio of chitosan to cross-linking agent is 1:X, where X is variable within a range of 0.5 to 1.5; and wherein the concentration of said composite microspheres is between 2% and 200% w / w relative to said fungicidal active ingredient in an aqueous phase, preferably 100% w / w, and more preferably 20%.

[0046] In a preferred embodiment of the invention, said composite microspheres further comprise a dispersing agent, where said dispersing agent is selected from the group consisting of gum arabic, xanthan gum, guar gum, carrageenan, pectin, alginates, dextrins, cyclodextrins, maltodextrins, tragacanth gum, mesquite gum, and mixtures thereof, more preferably gum arabic. The dispersing agent has a mass ratio of chitosan to dispersing agent of Y:l, where Y ranges from 5 to 20.

[0047] In another embodiment, the fungicidal formulation of the present invention includes composite microspheres with an adjustable surface charge or zeta potential, modulated by the concentration of the cross-linking agent, where X ranges from 0.08 to 0.8, and where said cross-linking agent is TPP.

[0048] In a preferred embodiment of the fungicidal formulation of the present invention, the fungicidal active ingredient is contained in a container, and said composite microspheres are a powder added to said container.

[0049] Another object of the present invention is a process for obtaining said composite microspheres, comprising the following steps: mixing acetic acid, chitosan, and water while stirring; adding said dispersing agent and stirring; adding the cross-linking agent solution dropwise; allowing the mixture to rest; and drying the obtained microspheres. Preferably, said process comprises the following steps: a. dissolving chitosan in 1% v / v acetic acid under constant stirring at 20°C until no lumps are observed; b. adding the dispersing agent and stirring until completely dissolved, achieving a chitosan-to-dispersing agent mass ratio of Y:l, where Y may range from 5 to 20; c. adding a cross-linking agent solution dropwise to the mixture, achieving a chitosan- to-cross-linking agent mass ratio of 1:X, where X may vary from 0.08 to 1.2, in the absence of a herbicidal active ingredient; d. adjusting the pH to approximately 6.5; e. drying the composite microspheres.

[0050] In a preferred embodiment, step e) involves freeze-drying the microspheres with a lyophilizer operating at 220 V, -45°C, and 10 Pa pressure, followed by grinding with a mortar and sieving the composite microspheres through a 100-mesh filter. Alternatively, step e) may involve spray drying at an inlet temperature of 200°C.

[0051] Finally, a preferred embodiment of the process for obtaining the fungicidal formulation of the invention comprises the following steps: a) preparing an aqueous solution of the fungicidal active ingredient, b) adding dry composite microspheres to the aqueous solution of the fungicidal active ingredient, c) leaving the mixture in contact for at least 2 hours, d) stirring and applying to the crop.

[0052] Description of the Figures

[0053] Figure 1. Scanning Electron Microscopy (SEM) images of the composite microsphere of the invention with 10% dispersing agent and cross-linking agent concentrations ranging from 8% to 150%, all expressed as mass percentages relative to the concentration of chitosan. Microspheres are labeled as MCS 0.08-0.1, MCS 0.3-0.1, MCS 0.5-0.1, MCS 0.8- 0.1, MCS 1.2-0.1, and MCS 1.5-0.1. Figure 2. Transmission Electron Microscopy (TEM) images of MCS 0.08-0.1, MCS 0.3-0.1, MCS 0.8-0.1, and MCS 1.2-0.1.

[0054] Figure 3A. SEM images of composite microspheres:

[0055] • Top Left Panel: SEM image of microspheres formulated with Mancozeb. Magnification: 1600x. Composite microspheres MCS 0.8-0.15 added to a Mancozeb suspension and mixed for 24 hours.

[0056] • Top Center Panel: SEM image of microspheres formulated with Mancozeb. Magnification: 6000x. Composite microspheres MCS 0.8-0.15 added to a Mancozeb suspension and mixed for 24 hours.

[0057] • Top Right Panel: SEM image of microspheres formulated with Mancozeb. Magnification: 20,000x. Composite microspheres MCS 0.8-0.15 added to a Mancozeb suspension and mixed for 24 hours.

[0058] • Bottom Left Panel: SEM image of microspheres. Magnification: 3050x. The image shows agglomerated microspheres after spray drying.

[0059] • Bottom Center Panel: SEM image of microspheres. Magnification: 2760x. The image shows concentrated agglomerated microspheres after spray drying.

[0060] • Bottom Right Panel: SEM image of microspheres. Magnification: 18,000x. The image shows individual microspheres after freeze-drying.

[0061] These images clearly show the reconfiguration of microspheres when combined with an aqueous solution of Mancozeb (MZ) for a sufficient time.

[0062] Figure 3B. SEM images of composite microspheres:

[0063] • Left Panel: SEM image of Mancozeb suspension in water. Magnification: 1600x.

[0064] • Right Panel: SEM image of microspheres formulated with Mancozeb. Magnification: 6000x. Composite microspheres MCS 0.8-0.15 added to a Mancozeb suspension and mixed for 2 hours.

[0065] Figure 4. Fungicidal activity assay of MZ combined with MCS 0.8-0.1 against B. cinerea. Bar heights represent group means, and error bars represent standard errors (n = 3). Figure 5. Fungicidal activity assay of MZ combined with MCS 0.08-0.1 and MCS 0.8-0.1 at different pre-incubation times (2, 4, and 24 hours) before application against B. cinerea. Bar heights represent group means, and error bars represent standard errors (n = 3).

[0066] Figure 6. Antifungal activity assay of MZ combined with MCS containing different concentrations of TPP against B. cinerea. Bar heights represent group means, and error bars represent standard errors (n = 6). Two pre-incubation times (2 and 24 hours) were evaluated with each particle containing different TPP concentrations (MCS 0.08-0.1, MCS 0.3-0.1, MCS 0.5-0.1, MCS 0.8-0.1, MCS 1.2-0.1).

[0067] Figure 7. Photograph showing sprayer filters clogged by the microspheres of the invention without gum.

[0068] Figure 8. SEM images of MCS 0.8 without gum (left) and with gum arabic (right).

[0069] Detailed Description of the Invention

[0070] For the purposes of the present invention, "agrochemical" refers to any natural or synthetic substance or mixture of substances used to prevent, eliminate, and / or control any pest, disease, or weed in agricultural activities and / or to preserve or enhance soil fertility and / or the quality and / or yield of crops.

[0071] For the purposes of this document, agrochemicals include fertilizers, plant growth regulators, biostimulants, pesticides, herbicides, fungicides, nematicides, insecticides, rodenticides, and plant protection products.

[0072] For the purposes of the present invention, the terms "adjuvant," "co-adjuvant," "agricultural adjuvant," or "agricultural co-adjuvant" refer to natural or synthetic products that improve or facilitate the action of an agrochemical (plant protection product, fertilizer, or biostimulant) by modifying certain characteristics of the solution and whose properties enhance the activity of the agrochemical. Forthe purposes of this document, "fungicidal active ingredient" is defined as a chemical or biological product, whether natural, modified, or synthetic, with the direct or derived ability to eliminate, prevent, or control harmful fungi affecting plants.

[0073] For the purposes of the present invention, the term "MCS" refers to the composite microspheres of the invention made with chitosan:gum:TPP.

[0074] For the purposes of the present invention, the term "MCS0.08" refers to the composite microspheres of the invention made with chitosan:gum:TPP in a mass ratio of 1:0.1:0.08.

[0075] For the purposes of the present invention, the term "MCS0.3" refers to the composite microspheres of the invention made with chitosan:gum:TPP in a mass ratio of 1:0.1:0.3.

[0076] For the purposes of the present invention, the term "MCS0.5" refers to the composite microspheres of the invention made with chitosan:gum:TPP in a mass ratio of 1:0.1:0.5.

[0077] For the purposes of the present invention, the term "MCS0.8" refers to the composite microspheres of the invention made with chitosan:gum:TPP in a mass ratio of 1:0.1:0.8.

[0078] For the purposes of the present invention, the term "MCS1.2" refers to the composite microspheres of the invention made with chitosan:gum:TPP in a mass ratio of l:0.1:1.2.

[0079] In this document, "e.a. ha-1" refers to the amount of acid equivalent of a compound, such as Glyphosate, applied per hectare of land. It is a unit commonly used to specify dosages in agricultural applications.

[0080] Cross-linking agents are molecules that may contain at least two reactive functional groups, enabling the formation of covalent bonds between polymer chains.

[0081] One of the major challenges associated with agricultural activity is the excessive use of agrochemicals due to their adverse effects on human health, the environment, and the emergence of resistant crops. The present invention provides a solution to this issue identified in the state of the art. The invention introduces composite reticular microspheres based on chitosan, preferably spherical in shape and with a size ranging from 100 to 200 nanometers, which can aggregate to form structures in the micrometer range. When combined at low concentrations with a fungicide, these microspheres enhance its efficacy, enabling the application of reduced doses while maintaining the same fungicidal effect.

[0082] The composite microspheres of the present invention are characterized by their composition of chitosan and a cross-linking agent, and preferably a dispersing agent. These microspheres exhibit a surface charge that can be positive, neutral, or negative, with the surface charge being adjustable through the concentration of the cross-linking agent in the microsphere.

[0083] The chitosan used in the present invention preferably has a degree of deacetylation of at least 90%.

[0084] In preferred embodiments of the invention, the cross-linking agent comprises tripolyphosphate (TPP) in a chitosan / cross-linking agent / dispersing agent weight ratio of 100 / 8 / 15 and 100 / 80 / 10.

[0085] The present invention provides hierarchical composite microspheres fabricated from chitosan with precisely defined and known chemical composition, size, shape, and charge distribution. These microspheres serve as co-formulators to reduce herbicide doses in an agrochemical formulation. The microspheres exhibit a well-defined microstructure and an adjustable surface charge, which can be positive, neutral, or negative. The amount of cross-linking agent, preferably sodium tripolyphosphate, is strategically defined according to the expected charge distribution of the particle, varying from 8% to 150% w / w relative to chitosan.

[0086] The dispersing agent, typically a natural polymer, imparts rapid dispersibility to the microspheres and enables their use across a broad range of salinity, pH, and temperature. This dispersing agent is selected from the group consisting of gum arabic (and its derivatives), xanthan gum (and its derivatives), guar gum (and its derivatives), poly(ethylene glycol)-poly(propylene glycol) triblock copolymers, polyacrylamide derivatives, microcrystalline cellulose (and its derivatives), and carboxymethyl cellulose (and its derivatives). The dispersing agent is present at a concentration of up to 20% w / w relative to chitosan.

[0087] The present invention proposes a range of dispersant concentrations from 0% to 20% w / w relative to the mass of chitosan, ensuring that in the preparation of the herbicidal formulation of this invention, its composite microspheres are easily redispersible in an aqueous agrochemical solution. This maintains suspension stability, thereby preventing the clogging of sprayer nozzles in the field. Consequently, one of the functions of the dispersant in the present invention, within agronomic practice, is to prevent nozzle clogging during agrochemical application processes using the fungicidal formulation of the invention.

[0088] The composite microspheres of the present invention differ from those identified in the state of the art in their adjustable physicochemical properties, particularly their zeta potential or charge distribution. The inventors have successfully adjusted or defined the zeta potential of the microspheres in the present invention based on the amount of cross-linking agent incorporated during their synthesis. Particles were developed with a cross-linking agent content varying in a range from 0.02 to 1.5 per unit of chitosan, achieving a zeta potential adjustable within the range from +30 mV to -30 mV. This allows the synthesis of particles with optimal physicochemical characteristics for compatibility with a broad spectrum of agrochemicals.

[0089] The control of the zeta potential facilitates the design of microspheres that selectively interact with specific surfaces or ions, catering to the needs of crops and / or soil. This provides advantages for the targeted delivery of active ingredients to specific action sites (plant, pathogen, soil), optimizing the use of agricultural inputs.

[0090] Notably, the use concentration of the composite microspheres in the invention can range from 2% to 200% w / w relative to the weight of the fungicidal active ingredient, with excellent results observed even in ranges as low as from 5% to 10%.

[0091] The ability to adjust the zeta potential enables the customization of microspheres for different pH levels, salinity conditions, and soil types. This adaptability allows these microspheres to be used across a wide variety of crops and agricultural systems. By regulating the zeta potential through the concentration of the cross-linking agent, the microspheres can control surface charge, influencing the release of active compounds (such as agrochemicals, fertilizers, pesticides, herbicides, fungicides, micronutrients, among others) in aqueous solution. From an application standpoint, the invention allows for the sustained and efficient delivery of agrochemicals and fertilizers, both chemical and biological, as well as plant protection products. This reduces losses due to leaching or evaporation while increasing availability at biological targets (plants, fungi, insects). Additionally, the preferred components (chitosan, gum arabic, and TPP) are biodegradable and non-toxic, making the microspheres environmentally friendly. Consequently, the invention reduces environmental impact compared to synthetic matrices, promoting sustainable agriculture and protecting soil biodiversity. Moreover, chitosan has antimicrobial properties that can protect plants against pathogens and fungal diseases.

[0092] Unlike similar particles identified in the prior art, which encapsulate the agrochemical active ingredient within the particle during synthesis, the composite microspheres of the present invention are combined with the active ingredient after synthesis, preferably just before field application. Key advantages include compatibility with a wide variety of agrochemicals and a boosting effect on the agrochemical with which they are formulated. This enhanced efficiency allows for lower application doses than those commercially recommended on product labels.

[0093] The ability to dry the composite microspheres and combine them at the time of application results in a stable product under regular environmental conditions, with an extended shelf life. Being biodegradable, the composite microspheres do not accumulate in the environment, thus avoiding ecological damage. No cytotoxicity indices have been detected in human cells.

[0094] The inventors of the present invention have formulated these composite microspheres with fungicidal active ingredients and found that their addition enhances the fungicidal effect. Specifically, they demonstrated that the microspheres alone have minimal fungicidal activity at concentrations of 25 pg / mL, achieving less than 20% fungicidal activity against B. cinerea. Furthermore, they demonstrated that fungicidal formulations composed of 25 pg / mL of Mancozeb combined with the composite microspheres of the invention, with different TPP compositions, in a mass ratio of 1:1 (25 pg / mL Mancozeb and 25 pg / mL microspheres), enhance the fungicidal activity of Mancozeb by 14X to 16X compared to free Mancozeb. An unexpected synergistic effect was observed due to the combination of the fungicidal active ingredient with the microspheres containing different TPP levels (MCS0.08, MCS0.3, MCS0.5, MCS0.8, MCS1.2).

[0095] The inventors demonstrated that the enhancement of the fungicidal effect by the chitosan-based particles requires pre-incubation of the composite microspheres with the fungicide for at least about 2 hours, preferably 24 hours, before application.

[0096] Laboratory trials have provided surprising evidence of the agricultural application of the fungicidal formulation of the present invention. These trials, involving a fungicidal active ingredient like Mancozeb combined with the composite microspheres described herein, exhibited enhanced fungicidal activity. The results show that the chitosan:gum:TPP microspheres of the present invention are non-toxic and can amplify the fungicidal activity by up to 1400%. This remarkable fungicidal effect occurs when the composite microspheres are mixed with the fungicidal active ingredient for over 2 hours, preferably around 24 hours, prior to applying the agrochemical formulation.

[0097] In the present document, the formula for calculating the enhancement of a fungicidal active ingredient with the composite microspheres of the present invention is as follows:

[0098] ((Effect of the invention - Effect of fungicide alone) / Effect of fungicide alone) x 100

[0099] In this case, the effect can be measured by fungicidal activity. However, it can also be measured by other variables, such as yield, when the yield depends on the control exerted by the agrochemical in question.

[0100] In the present invention, the composite microspheres are obtained through an ionic gelation method, where the required amounts of chitosan and the dispersant (in embodiments that include it) are mixed, followed by the dropwise addition of TPP at the desired concentration.

[0101] Unlike inventions described in the prior art, where the fungicide is incorporated during the particle synthesis process, the fungicidal formulation of the present invention is obtained simply by contacting the microspheres with a fungicidal active ingredient solution for at least 2 hours, preferably 24 hours, before application.

[0102] The composite microspheres of the present invention differ from those known in the prior art due to their versatility, allowing them to be combined with various agrochemicals. They can also be handled independently, combining them at the time of use and favoring particle stability.

[0103] The present invention provides a process for producing these composite microspheres through physical gelation. In a preferred embodiment, the synthesis is strategically designed to involve three components: chitosan, a cross-linking agent, and a dispersant.

[0104] The production process has been successfully scaled up to a reaction volume of 20 liters. Throughout the process, the pH is maintained at 4.5 using acetic acid, while agitation and temperature are kept constant at 130 rpm and 20°C, respectively. At the end of the process, the pH is adjusted to 6.5, and the microspheres are allowed to decant naturally. Subsequently, they are dried using spray drying and sieved through a 100-mesh screen.

[0105] The present invention also provides a production process for obtaining the composite microsphere, comprising the following steps: a. Dissolve chitosan in 1% v / v acetic acid under constant stirring at a temperature of 20°C until no lumps are observed; b. Add gum arable a nd stir until completely dissolved, achieving a chitosan-to-gum arabic mass ratio of Y:l, where Y may range from 5 to 20; c. Add a TPP solution dropwise to the mixture until the desired TPP concentration is reached, achieving a chitosan-to-TPP mass ratio of 1:X, where X may range from 0.03 to 1.2, in the absence of a herbicidal active ingredient; d. Adjust the pH to approximately 6.5; e. Dry the particles.

[0106] In a preferred embodiment of the invention:

[0107] • Step e) involves freeze-drying with a lyophilizer operating at 220 V, -45°C, and 10 Pa pressure, followed by grinding with a mortar and sieving through a 100-mesh screen;

[0108] • Alternatively, step e) involves spray drying at an inlet temperature of 200°C. The present invention provides a method for preventing and controlling fungal infections, comprising applying the fungicidal formulation of the invention to a crop, where said crop is selected from the group consisting of trees, cereals, oilseeds, vegetables, fruits, ornamentals, Swiss chard (Beta vulgaris), peppers (Capsicum spp.), garlic and onions (Allium spp.), celery (Apium graveolens), eggplant (Solanum melongena), pumpkin (Cucurbita moschata), chayote (Sechium edule), cabbage (Brassica oleracea), spinach (Spinacia oleracea), common bean (Phaseolus vulgaris), lettuce (Lactuca sativa), maize (Zea mays), peanut (Arachis hypogaea), tomato (Solanum lycopersicum), cucumber (Cucumis sativus), okra (Hibiscus esculentus), radish (Raphanus sativus), beet (Beta vulgaris), carrot (Daucus carota), avocado (Persea americana), sugar apple (Annona squamosa), star apple (Chrysophyllum cainito), canistel (Pouteria campechiana), cherry (Malpighia punicifolia), custard apple (Annona reticulata), plum (Spondias dulcis), coconut (Cocos nucifera), papaya (Carica papaya), soursop (Annona muricata), guava (Psidium guajava), pomegranate (Punica granatum), lime (Citrus aurantifolium), lemon (Citrus limonum), apple (Malus domestica), sapote (Calocarpum mammosum), mamey (Mammea americana), Spanish lime (Melicoccus bijugatus), mandarin (Citrus reticulata), mango (Mangifera indica), passion fruit (Passiflora laurifolia), watermelon (Citrullus vulgaris), sour orange (Citrus aurantium), sweet orange (Citrus sinensis), pear (Pyrus communis), pineapple (Ananas comosus), banana (Musa paradisiaca), plantain (Musa balbisiana), tamarind (Tamarindus indica), grapefruit (Citrus paradisi), local grapefruit (Citrus grandis), grape (Vitis vinifera), common bean (Phaseolus vulgaris), maize (Zea mays), rice (Oryza sativa), coffee (Coffea arabica), sugarcane (Saccharum officinarum), cotton (Gossypium hirsutum), gherkin (Melothria guadalupensis), sweet potato (Ipomoea batatas), potato (Solanum tuberosum), yellow potato (Solanum phureja), cape gooseberry (Physalis peruviana), cassava (Manihot esculenta), soybean (Glycine max), strawberries (Fragaria spp.), mulberries (Morus spp.), blackberries (Rubus spp.), palms of the Arecaceae family, oil palm (Elaeis guineensis), cocoa (Theobroma cacao), tree tomato (Solanum betaceum), naranjilla (Solanum quitoense), potato (Solanum tuberosum), soybean (Glycine max), chickpeas (Cicer arietinum), among others. The infections are caused by fungi selected from the group consisting of Magnaporthe oryzae, Botrytis cinerea, Puccinia spp., Fusarium spp. (including, but not limited to, Fusarium graminearum, Fusarium oxysporum), Blumeria graminis, Mycosphaerella spp., Mycosphaerella fijiensis, Mycosphaerella graminicola, Colletotrichum spp., Ustilago maydis, Melampsora lini, Phakopsora pachyrhizi, and Rhizoctonia solani, among others.

[0109] The present invention is further described through the following examples, which should not be construed as limiting the scope of the invention. The invention is not restricted to the illustrative examples presented below.

[0110] Examples

[0111] Example 1: Synthesis and Production of Chitosan:Gum Arabic:Tripolyphosphate (TPP) Microspheres at Laboratory Scale

[0112] Materials Used:

[0113] Chitosan (degree of deacetylation: 95%, Mw 275,000), Gum Arabic (Mw 70,000), TPP, 1% acetic acid, 1 M sodium hydroxide, distilled water, 600 mL and 250 ml_ beakers, Dragon Lab mechanical stirrer, peristaltic pump, pH meter, 100 and 450 mesh sieves.

[0114] Procedure:

[0115] For a 600 mL reactor:

[0116] 1. Prepare 200 mL of a 1% w / v acetic acid solution (10 mL / L) and add 4 g of chitosan. Stir continuously at 1300 RPM using a Dragon Lab mechanical stirrer until the solution is fully dissolved, achieving a uniform, clear, and lump-free solution, for 10 minutes (maintain temperature between 19-24°C).

[0117] 2. Add 0.4 g of gum arabic (10% or 15% chitosan ratio). Stir for 10 minutes until completely dissolved.

[0118] 3. Prepare an aqueous TPP solution with a concentration of 1.6% w / v.

[0119] 4. Using a peristaltic pump, add TPP dropwise into the beaker at a rate of 0.55 L / h, until 200 mL of TPP is added (23-25 minutes). Maintain stirring at 1300 RPM for an additional 30 minutes after the dropwise addition is completed. 5. Measure and adjust the pH to 6.5 using 1 M sodium hydroxide.

[0120] 6. Centrifuge for 10 minutes at 1200 RPM and discard the supernatant.

[0121] 7. Freeze the particles for at least 24 hours.

[0122] 8. Dry the particles using a PeetLab BK-FD10PT lyophilizer at 220 V, -45°C, and 10 Pa pressure.

[0123] 9. Grind and sieve the particles first through a 100-mesh sieve and then through a 450-mesh sieve.

[0124] For a 20 L reactor:

[0125] The same procedure was followed, with adjustments to material quantities to match the 20 L reactor's dimensions. The drying step was modified as follows:

[0126] Briefly, in a 20 L borosilicate glass reactor (Figmay brand) with a 4-flat-paddle mechanical stirrer and no heating, 5 L of 1% acetic acid (10 mL / L) and 100 g of chitosan (degree of deacetylation: 95%, Mw 275,000) were added. The mixture was stirred at 130 RPM for 10 minutes. The reactor temperature was maintained at 21 ± 2°C. Complete dissolution was achieved, resulting in a uniform, clear solution without visible lumps.

[0127] Then, 10 or 15 g of gum arabic (as dispersing agent, depending on the case) were added, and the solution was stirred for 10 minutes.

[0128] In a second 10 L reactor, a TPP solution was prepared at a concentration of 16 g / L by adding 5 L of distilled water and 80 g of TPP, stirring until completely dissolved for 5 minutes at room temperature.

[0129] In the 20 L reactor containing the chitosan solution, stirring was maintained at 130 RPM, and two peristaltic pumps were used to add TPP dropwise at a rate of 2 L / h. TPP was added to achieve the desired final concentration, and stirring continued for 30 minutes. The pH was then adjusted to 6.5 using 1 M NaOH.

[0130] The reactor contents were discharged into a 20 L container and left to settle overnight (12 hours) at room temperature. The supernatant was discarded. The particles were dried using a JISL LSD-48 benchtop spray dryer with a drying capacity of 1 L / h. Drying was conducted at 200°C with a feed rate of 60%. During the drying process, the particles were kept in suspension by stirring. Example 2: Production of Chitosan Microspheres with Variable Proportions of Gum Arabic and Tripolyphosphate

[0131] Microspheres were produced following the procedures described in Example 1, adjusting the amounts of gum arabic relative to the grams of chitosan and the volumes of TPP added dropwise relative to the amount of chitosan. This allowed the production of microspheres with different mass ratios of Chitosan:Gum Arabic:TPP.

[0132] The following microspheres were prepared with the mass ratios shown in Table 1.

[0133] Table 1. Particles with Mass Ratios of Gum Arabic and TPP per Unit of Chitosan

[0134] Example 3: Production of Composite Chitosan Microspheres Using Sodium Hexametaphosphate (HMP) as Cross-Linking Agent and Xanthan Gum and Polyethylene Glycol as Dispersing Agents

[0135] In a 20 L borosilicate glass reactor (brand Figmay), with a 4-flat-paddle mechanical stirrer and no heating, 5 L of a 15% citric acid solution (10 mL / L) and 100 g of chitosan (degree of deacetylation 92%, Mw 210,000) were added. The mixture was stirred at 150 RPM for 10 minutes, maintaining the reactor temperature at 21 ± 2°C. A complete dissolution was achieved, resulting in a uniform, clear solution without visible lumps. Subsequently, 5 g of xanthan gum and 10 g of polyethylene glycol were added as dispersing agents, and the solution was stirred for another 10 minutes.

[0136] In a second 10 L reactor, a sodium hexametaphosphate (HMP) solution with a concentration of 25 g / L was prepared by adding 5 L of distilled water and 125 g of HMP. The mixture was stirred until fully dissolved for 10 minutes at room temperature.

[0137] The chitosan solution in the 20 L reactor was stirred at 150 RPM, and HMP was added dropwise using a peristaltic pump at a rate of 0.5 L / h (10 hours). Stirring continued for an additional 60 minutes after all the HMP had been added. The reactor contents were then discharged into a 20 L container and left to rest overnight (12 hours) at room temperature. The supernatant was transferred to another container, ensuring no material loss during the process, and then centrifuged for 15 minutes at 1500 RPM using a "Rolco MOD.CM-2036" laboratory centrifuge.

[0138] The samples were frozen for 24 hours at -24°C. Using a benchtop multi-collector laboratory lyophilizer, the samples were freeze-dried for 72 hours. Finally, the samples were ground and sieved using a 450-mesh sieve.

[0139] Example 4: Determination of the Size and Morphology of Chitosan:Gum ArabicTripolyphosphate Microspheres

[0140] A thorough physicochemical characterization was performed on the composite microspheres obtained according to the procedure in Example 1 and with the chemical composition described in Example 2 to define their structure and properties.

[0141] Three techniques were used to study and determine the size and morphology of the composite microspheres:

[0142] 1. Dynamic Light Scattering (DLS): o Three measurements of 60 seconds each were performed, with 10- second intervals between measurements, at 25°C using a Malvern Zetasizer NanoS90 device. o The Brownian motion of the composite microspheres in suspension was analyzed. The light scattered by the composite microspheres provided information on the diffusion coefficient, which was used to determine the hydrodynamic radius and size distribution of the material.

[0143] Table 2. Physicochemical Parameters of Composite Microspheres (MCS) for Different TPP / CS Ratios, All Samples Containing 10% Gum Arabic . Transmission Electron Microscopy (TEM): o A JEOL JEM 2100 TEM with 200 kV and a B6La filament was used. o Photographs were taken at magnifications of 50,000x or higherto analyze the morphology and size of the composite microspheres.

[0144] 3. Scanning Electron Microscopy (SEM): o A ZEISS Crossbeam 350 Field Emission Scanning Electron Microscope (FESEM) was used. o Samples were prepared with Cr or Au coating. o Images were captured at magnifications of 30x, 500x, 1500x, 6000x, 20,000x, and 40,000x to analyze the general appearance, size, and morphology of the composite microspheres.

[0145] The characterization of the composite microsphere sizes using DLS allowed us to conclude that there are populations with different size distributions (Table 2). In many cases, up to three distinct hydrodynamic diameters were observed: approximately 200- 300 nm, 600-1000 nm, and 2000-5000 nm.

[0146] When using more precise techniques to study the morphology and size of the composite microspheres, such as SEM and TEM, spherical particles approximately 20-30 nm in diameter were observed. These particles interact with one another, forming larger aggregates, some amorphous and others spherical (Figures 1 and 2). These aggregates range from a few hundred nanometers to several micrometers, confirming the DLS results regarding the size distribution of the composite microspheres obtained during the synthesis process.

[0147] Example 5: Determination of the Charge Distribution of Chitosa Gum Arabic:Tripolyphosphate Microspheres

[0148] To determine the zeta potential, three measurements of 100 seconds each were conducted with 10-second intervals between measurements, at 25°C, using a Horiba SZ- 100 device. The average of the measurements was calculated and reported. The results for the different composite microspheres (MCS) with 10% Gum Arabic and variable TPP concentrations are shown in Table 2.

[0149] From the results of the material characterization, it was observed that composite microspheres with gum and a TPP / CS ratio from 0.08 to 0.3 exhibited positive zeta potential values, while those with a TPP / CS ratio from 0.5 to 1.5 exhibited negative values (Table 2). The inflection point appeared to correspond to a TPP / CS ratio of 0.4, where the surface charge of the composite microspheres was near neutrality, with the zeta potential value around 0, considering the measurement error (±5 to ±10 mV). These results demonstrate that increasing TPP concentrations raises the particle's zeta potential, allowing the composite microspheres to be designed with the desired zeta potential by adjusting TPP levels.

[0150] Example 6: Characterization of the Morphology of Composite Microspheres Combined with Mancozeb

[0151] The morphology of composite microspheres combined with Mancozeb was characterized using electron microscopy techniques. The top panels of Figure 3A show composite microspheres MCS0.8-0.15 formulated with Mancozeb at different magnifications after 24 hours of contact. These images reveal a change in the morphology of the system, forming lamellar structures resembling flower petals. The bottom panels of Figure 3A display images of composite microspheres MCS0.8-0.15 obtained using different drying methods (lyophilization or spray drying). Figure 3B shows SEM images of Mancozeb alone (left) and composite microspheres MCS0.8-0.15 formulated with Mancozeb after 2 hours of contact (right). In the latter, the microstructure of Mancozeb evolves in the presence of the particles, showing a lamellar microstructure (flower petal-like) and areas with an intermediate microstructure (worm-like).

[0152] Example 7: Fungicidal Activity Assay of Mancozeb Combined with Composite Microspheres Against Botrytis cinerea

[0153] In vitro assays were conducted to evaluate the fungicidal activity of Mancozeb, alone or combined with composite microspheres (MCS), against Botrytis cinerea spores, a fungus of agronomic interest that significantly affects strawberry and grape crops.

[0154] The 8. cinerea spores used in all assays were obtained from a culture grown on potato dextrose agar (15 g / L agar, 20 g / L dextrose, 4 g / L potato extract). The spores were harvested, suspended in water, and quantified using a Neubauer chamber.

[0155] The spores, in a suspension of 2x1052 \times 10A52xl05 spores / mL, were incubated in multiwell plates (30 mm x 100 mm x 6 mm, Marienfield Superior) containing either water as a control or the following treatments:

[0156] 1. 25 pg / mL Mancozeb (MZ),

[0157] 2. 25 pg / mL composite microspheres MCS0.8-0.1,

[0158] 3. A combination of 25 pg / mL MZ + 25 pg / mL MCS0.8-0.1.

[0159] Incubation was carried out in darkness at 23°C for 24 hours (Table 3). Sucrose was added at 2% w / v to all incubation solutions. For the MZ + MCS0.8-0.1 combination (Treatment 4), both compounds were pre-incubated for 24 hours prior to application on the spores.

[0160] Table 3. Treatments to Evaluate the Fungicidal Efficacy of Mancozeb Formulations Combined with MCS0.8-0.1 Against B. cinerea Spores

[0161] At the time of quantification and analysis, spores subjected to each treatment and control were taken from 3 independent replicates (3 wells per treatment, n = 3) and observed under an Eclipse E200 light microscope (Nikon). For each treatment, the percentage (%) of spore germination was quantified. Spores were considered germinated when the germ tube length exceeded half the conidial length [Plascencia- Jatomea et al., 2003], Three independent photographs were taken from each replicate, and the germination percentages quantified in each replicate were averaged. At least 100 spores were counted for each replicate corresponding to each treatment. A value of 100% fungicidal activity in a treatment indicates that after 24 hours of incubation, spore germination was 0%.

[0162] Treatments with 25 pg / ml MZ or 25 pg / ml MCS 0.8-0.1 showed residual fungicidal activity on B. cinerea spores, very similar to what was observed in the water control incubation (Figure 4). The combination of 25 pg / ml MCS 0.8-0.1 + 25 pg / ml MZ showed a 73% increase in MZ efficacy compared to the action of MZ alone. The addition of composite microspheres enhances the fungicidal effect of Mancozeb.

[0163] This result is extraordinary and demonstrates remarkable synergy between a fungicidal active ingredient such as Mancozeb and the composite microspheres of the invention. Considering enhancement as the percentage improvement in the fungicidal action of an active ingredient due to the addition of the microspheres of the invention, in this case, we observe a 1460% enhancement. Since the fungicidal effect of Mancozeb alone is 5%, and the effect of the combination is 78%, the enhancement of the fungicidal effect of Mancozeb is calculated as the increase divided by the effect of the fungicide alone, multiplied by 100. That is:

[0164] ((Effect of the invention - Effect of fungicide alone) / Effect of fungicide alone) x 100

[0165] In this example:

[0166] ((78-5) / 5)xl00=1460% enhancement.

[0167] Absolutely disruptive.

[0168] Example 8: Fungicidal Activity Assay of Mancozeb Combined with Composite Microspheres with Different Pre-lncubation Times

[0169] To evaluate how the pre-incubation or contact time between the fungicide and the composite microspheres (MCS) influences fungicidal activity, an experimental design was carried out to compare the fungicidal activity of the formulations after 2, 4, and 24 hours on B. cinerea. The 8. cinerea spores used in all the assays were obtained from a culture grown on potato dextrose agar (15 g / L agar, 20 g / L dextrose, 4 g / L potato extract). The spores were harvested, suspended in water, and quantified using a Neubauer chamber.

[0170] The spores, in a suspension of 2xl05spores / mL, were incubated in multiwell plates measuring 30 mm x 100 mm x 6 mm (Marienfield Superior) containing water as a control or the treatments of 25 pg / mL Mancozeb (MZ), 25 pg / mL composite microspheres (MCS), or the combination of 25 pg / mL MZ + 25 pg / mL MCS (in all cases) in darkness at 23°C for 24 hours (Table 4). Sucrose was added to each incubation solution at a concentration of 2% w / v.

[0171] In this experimental scheme, different pre-incubation times of the formulation (MZ + MCS) were tested: 2, 4, and 24 hours before application on the spores. Two composite microspheres with different TPP concentrations, MCS0.08-0.1 and MCS0.8-0.1, were tested.

[0172] Table 4. Treatments to Evaluate the Fungicidal Efficacy of Mancozeb Formulations Combined with MCS at Different Pre-lncubation Times on B. cinerea Spores.

[0173] 10

[0174] At the time of quantification and analysis, spores subjected to each treatment and control were taken from 3 independent replicates (3 wells per treatment, n = 3) and observed under an Eclipse E200 light microscope (Nikon). For each treatment, the percentage (%) of spore germination was quantified. Spores were considered germinated when the germ tube length exceeded half the conidial length [Plascencia- Jatomea et al., 2003], Three independent photographs were taken from each replicate, and the germination percentages quantified in each replicate were averaged. At least 100 spores were counted for each replicate corresponding to each treatment. A value of 100% fungicidal activity in a treatment indicates that after 24 hours of incubation, spore germination was 0%.

[0175] In Figure 5, it can be observed that with short pre-incubation times (2 hours and 4 hours), both MCS0.08-0.1 and MCS0.8-0.1 composite microspheres combined with Mancozeb showed fungicidal activity not exceeding 20% efficacy. In contrast, with 24- hour pre-incubation prior to application, efficacy increased to around 75% and 95% with MCS0.08-0.1 and MCS0.8-0.1 particles, respectively. This assay demonstrates that the enhanced efficacy of Mancozeb is significantly increased when the particles are preincubated with Mancozeb for at least 2, moste preferably 24 hours. Example 9: Fungicidal Activity Assay of Mancozeb Combined with Composite Microspheres of Different TPP Concentrations on B. cinerea Spores

[0176] To evaluate whether the TPP content influences the fungicidal effect enhancement of the particles on Mancozeb, formulations of Mancozeb combined with different composite microspheres (MCSO.08-0.1, MCSO.3-0.1, MCSO.5-0.1, MCSO.8-0.1, and MCS1.2-0.1) were tested. The B. cinerea spores used in all the assays were obtained from a culture grown on potato dextrose agar (15 g / L agar, 20 g / L dextrose, 4 g / L potato extract). The spores were harvested, suspended in water, and quantified using a Neubauer chamber.

[0177] The spores, in a suspension of 2x1052 \times 10A52xl05 spores / mL, were incubated in multiwell plates measuring 30 mm x 100 mm x 6 mm (Marienfield Superior) containing water as a control or the treatments of 25 pg / mL Mancozeb (MZ), 25 pg / mL composite microspheres (MCS), or the combination of 25 pg / mL MZ + 25 pg / mL MCS (in all cases) in darkness at 23°C for 24 hours (Table 5). Sucrose was added to each incubation solution at a concentration of 2% w / v.

[0178] In this experimental scheme, different pre-incubation times of the formulation (MZ + MCS) were tested: 2 hours and 24 hours prior to application on the spores. Particles with different TPP concentrations (MCSO.08-0.1, MCSO.3-0.1, MCSO.5-0.1, MCSO.8-0.1, and MCS1.2-0.1) were tested.

[0179] Table 5. Treatments to Evaluate the Fungicidal Efficacy of Mancozeb Formulations Combined with MCS at Different TPP Concentrations on B. cinerea Spores.

[0180]

[0181] At the time of quantification and analysis, spores subjected to each treatment and control were taken from 3 independent replicates (3 wells per treatment, n = 3) and observed under an Eclipse E200 light microscope (Nikon). For each treatment, the percentage (%) of spore germination was quantified. Spores were considered germinated when the germ tube length exceeded half the conidial length [Plascencia- Jatomea et al., 2003], Three independent photographs were taken from each replicate, and the germination percentages quantified in each replicate were averaged. At least 100 spores were counted for each replicate corresponding to each treatment. A value of 100% fungicidal activity in a treatment indicates that after 24 hours of incubation, spore germination was 0%.

[0182] In Figure 6, it can be observed that free MZ (without MCS) applied at a concentration of 25 pg / mL exhibits antifungal activity not exceeding 6%. In accordance with the results from the assay in Example 8, the pre-incubation of Mancozeb with the particles for 24 hours achieves fungicidal activity ranging from 80% to 93% for particles tested with different TPP concentrations.

[0183] This assay confirms that pre-incubation time before applying the Mancozeb and MCS formulations is critical. Specifically, for Mancozeb, the TPP concentration was not a determining factor in fungicidal effect. A synergistic effect is observed in formulations with particles, increasing fungicidal activity by 14X to 16X compared to free Mancozeb. This means that the enhancement effect of the composite microspheres of the invention ranges from 1233% to 1450% compared to Mancozeb alone when the formulation is pre-mixed approximately 24 hours in advance. In contrast, when the formulation is premixed only 2 hours before application, the enhancement is between 230% and 300%.

[0184] An antifungal enhancement effect is observed for particles with different TPP concentrations, particularly in assays 5 and 7. After 2 hours of contact between MZ and the composite microspheres of the invention, the enhancement is around 300%, reaching nearly 20% fungicidal power, compared to 6% for MZ alone.

[0185] Example 10: Stability Assay of Composite Microspheres During Synthesis and Redispersion of Microspheres Before Application by Adding Gum

[0186] The composite microsphere formulation of the invention, defined in Table 1 of Example 2, was applied, i.e., MCS0.8-0 microspheres with a mass ratio of chitosan:gum:TPP of 1:0:0.8, meaning in the absence of gum, by spraying with a portable backpack sprayer. It was observed that the filter of the sprayer nozzles became clogged by the agglomerated microspheres, as shown in Figure 7. This is due to the low redispersion capacity of the microspheres of the invention in the absence of gum.

[0187] When the assay was repeated with the microspheres from assay 4 of Table 1 in Example 2 (MCSO.8-0.1), containing a gum concentration of 10% relative to the chitosan mass, the sprayer nozzles did not clog, and it was not necessary to clean their filters.

[0188] In Figure 8, it can be seen that a gum concentration between 5% and 20% improves the formation of the microspheres of the invention.

[0189] Example 11: Case Study MZ + MCS0.8 Field Trial for the Control of Foliar Diseases (Alternaria and Phytophthora infestans) in Potato Cultivation

[0190] The objective is to evaluate the yield response and health performance of a potato crop using carrier technology as a biological fungicide for the control of foliar diseases (Alternaria and Phytophthora infestans).

[0191] The synergistic effect between MZ and MCS0.8-G (without gum arabic) was evaluated on the growth of phytopathogenic fungi in potatoes and the yield of the harvest.

[0192] A randomized complete block design with four replicates was used. Each plot consisted of 4 rows 5 meters long, spaced 85 cm apart. "Seed potatoes" of the prefoundation category of the Innovator variety were used. Five cuts were planted per linear meter. Four treatments were evaluated, as indicated in Table 6.

[0193] The treatments were sprayed using a constant-pressure backpack sprayer with a CO2 source.

[0194] Table 6. Treatments to Evaluate Yield Response and Health Performance of a Potato Crop Treated with MZ and MCS0.8.

[0195] Agronomic (vigor) and sanitary data were collected during the crop cycle. The severity, incidence, and control efficacy of the products for the evaluated diseases were estimated. At the end of the growth period, production was weighed, and commercial yield was calculated by subtracting the yield of tubers smaller than 50 mm, rotted tubers, and defective tubers from the total yield.

[0196] The crop developed without sanitary problems, with good plant growth. There was no water deficit during the crop cycle, and the plants remained vigorous, following their growth cycle. No climatic, sanitary, or nutritional events negatively impacted normal development, and full surface coverage was achieved in the trial.

[0197] The yield of the crop was quantified, distinguishing between total yield (gross production) and commercial yield (production meeting commercial standards and, therefore, suitable for marketing).

[0198] Table 7. Total and Commercial Yield of Potato Crop for Each Treatment Evaluated.

[0199]

[0200] By using 60% less MZ than the labeled dose or full dose, that is, 50 g instead of 125 g of MZ per 20 liters of formulation, and mixing it with 5% by weight of composite microspheres of the invention without dispersant (without gum), potato production increased by 20%. This is another common way to measure fungicide efficacy: the difference in yield, as fungi drastically reduce crop yield.

[0201] Example 12: Study of the Interaction of Composite Microspheres with Mancozeb

[0202] Additionally, studies were conducted to evaluate the interaction capacity of composite microspheres with MZ in aqueous solution to determine the percentage of association between the two materials.

[0203] Nuclear Magnetic Resonance (NMR):

[0204] Using the technique described in "Probing interactions by means of pulsed field gradient nuclear magnetic resonance spectroscopy," S. Cozzolino, et al. Magn. Reson. Chem. 2008, 46, S16-S23, DOI: 10.1002 / mrc.2345, diffusion coefficients were calculated via the NMR technique. From these coefficients, the percentage of association (physicochemical) of various analytes with the composite microspheres (MCS) can be determined.

[0205] The analyte and the particle each have their own free-state diffusion coefficients, reflecting their molecular weight and shape. However, when a complex is formed and the analyte and the particle are strongly bound, they should share the same diffusion coefficient as they diffuse as a single molecular entity. In the case of weak or negligible association, the diffusion coefficients of the analyte and the particle remain unchanged. For any other case, assuming rapid exchange on the NMR timescale, the observed (measured) diffusion coefficient is a weighted average of the free and bound diffusion coefficients and can therefore be used to calculate the bound fraction.

[0206] Diffusion coefficients were determined using a PFGSTE26 sequence on a Spinsolve80 Ultra from Magritek GmbH, with membrane strips placed in a 5 mm tube and operated at 26°C.

[0207] For the study, samples of MCSO.8-0.1 (with gum) and MCS0.8-0 (without gum) from Example 2, as well as MZ, were used to study the percentage of association between them. Components were added to a laboratory test tube with distilled water and manually stirred for 1 minute. Suspensions were prepared from the individual components and mixtures of each microparticle with glyphosate in a mass ratio of 1:5 glyphosate: microspheres.

[0208] MZ is a paramagnetic compound, so the signal obtained when studied by NMR shows only the solvent and not the diffusion coefficient of MZ. When mixed with the particles, it is possible to determine the percentage of association of microspheres with MZ, but not vice versa.

[0209] Table 8. Association Percentages of Composite Microspheres (with and without gum) with MZ. Percentages were obtained from diffusion coefficients determined by the NMR technique. This table demonstrates that the presence of gum does not alter the association capacity between Mancozeb and the composite microspheres of the invention. Additionally, it can be determined that the maximum amount of microspheres that can have a positive effect when interacting with Mancozeb is 2:1 in a microsphere-to- Mancozeb mass ratio (approximately 40% of microspheres were associated with Mancozeb, 40% x 5:1 = 2:1 w / w).

[0210] Example 13: Control of Yellow Spot and Yellow Rust in Wheat, Crypton + MCS0.8-0.1

[0211] Field trials were conducted for the control of Yellow Rust fungus in wheat crops in Miramar (La Totora Station), Argentina. The final yield of the treated plots was measured.

[0212] Crypton is a commercial fungicide composed of Bixafen, Prothioconazole, and Trifloxystrobin.

[0213] Optimizer is a commercial adjuvant used in combination with Crypton to optimize product wetting during application. It is a mixture of methyl esters of fatty acids from vegetable oils.

[0214] Table 9. Field Trials

[0215] It was evidenced that with a 29% reduction in the Crypton dose and the use of the composite microspheres of the invention, an equivalent yield was achieved.

[0216] The foliar application was carried out at the flag leaf stage (Zadoks 39). At harvest, the yield was determined in kg / ha, corrected for moisture (14%). The use of the lower Crypton dose (500 cc / ha) in combination with the microsphere allowed achieving yields similar to those obtained with the higher Crypton dose alone, enabling a 29% reduction in the fungicide dose.

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Claims

Claims1. A composite microsphere with fungicidal efficacy-enhancing properties, comprising chitosan with an average molecular weight in the range from 190,000 to 310,000 g / mol, and a crosslinking agent, in the absence of a fungicidal active ingredient.

2. The composite microsphere of claim 1, wherein it comprises an adjustable surface electrical charge (Z potential) in an aqueous medium, adjustable by the concentration of said crosslinking agent, and wherein the mass ratio of chitosan to crosslinking agent is 1:X, where X varies in the range from 0.08 to 1.5.

3. The composite microsphere of claim 1, wherein said crosslinking agent is selected from the group consisting of sodium tripolyphosphate (TPP), sodium hexametaphosphate, and their derivatives.

4. The composite microsphere of claim 1, wherein said crosslinking agent is sodium tripolyphosphate (TPP).

5. The composite microsphere of claim 1, wherein it further comprises a dispersant that improves the redispersion of said microsphere when combined with an aqueous fungicide solution, wherein said dispersant is selected from the group consisting of gum arabic, xanthan gum, guar gum, carrageenan, pectin, alginates, dextrins, cyclodextrins, maltodextrins, tragacanth gum, mesquite gum, polyetilenglycol and mixtures thereof.

6. The composite microsphere of claim 5, wherein it comprises a mass ratio of chitosan to dispersant of Y:l, where Y is in the range from 5 to 20.

7. The composite microsphere of claim 5, wherein said dispersant is gum arabic.

8. A fungicidal formulation comprising a fungicidal active ingredient and composite microspheres of claim 1 that enhance the fungicidal effectiveness on crops, wherein said composite microsphere is added at a concentration of 2 to 100% w / w relative to said fungicidal active ingredient.

9. The fungicidal formulation of claim 8, wherein said fungicidal active ingredient is selected from the group consisting of pre-emergent, contact, systemic, residual fungicides, prothioconazole, trifloxystrobin, cyproconazole, carbendazim, difenoconazole, isopyrazam, chlorothalonil, epoxiconazole, fluoxastrobin, mancozeb, zineb, thiram, tebuconazole, propiconazole, azoxystrobin, pyraclostrobin, thiabendazole, metalaxyl, boscalid, captan, folpet, dithiocarbamates, triazoles, strobilurins, benzimidazoles, anilides, organochlorines, phthalimides, sedaxane, their derivatives, and combinations thereof.

10. The fungicidal formulation of claim 8, wherein said fungicidal active ingredient is mancozeb.

11. The fungicidal formulation of claim 8, wherein said microsphere comprises chitosan with an average molecular weight in the range from 190,000 to 310,000 g / mol, a dispersant, and a crosslinking agent, and wherein the mass ratio of chitosan to crosslinking agent is 1:X, where X varies in the range from 0.08 to 1.2 and defines the Z potential of said microspheres in an aqueous solution.

12. The fungicidal formulation of claim 8, wherein said composite microspheres comprise chitosan, gum arabic, and sodium tripolyphosphate, in the absence of agrochemical active ingredients.

13. The fungicidal formulation of claim 8, wherein said composite microsphere has a Z potential in an aqueous solution in the range from +30 mV to -30 mV, adjustable by the concentration of the crosslinking agent.

14. The fungicidal formulation of claim 8, further comprising a co-adjuvant selected from the group consisting of an adhesive, a surfactant, a stabilizer, an antifoaming agent, a pH buffer, a sequestrant, their derivatives, and combinations thereof.

15. The fungicidal formulation of claim 8, wherein said fungicidal active ingredient is at a concentration equal to or lower than the recommended field application rate.

16. The fungicidal formulation of claim 8, wherein the concentration of said composite microspheres is in the range from 5 to 200% w / w relative to the fungicidal active ingredient.

17. The fungicidal formulation of claim 8, wherein the fungicidal active ingredient and composite microspheres are mixed in the field prior to application.

18. The fungicidal formulation of claim 8, wherein the fungicidal active ingredient and composite microspheres are mixed at least 2 hours before application.

19. The fungicidal formulation of claim 8, wherein it comprises an aqueous composition of fungicidal active ingredient and composite microspheres packaged in containers after being mixed at a manufacturing facility and jointly packaged for sale and distribution.

20. The fungicidal formulation of claim 8, wherein it comprises, on one side, an aqueous composition of the fungicidal active ingredient and, separately, a powder of composite microspheres to be mixed prior to use.

21. A method for obtaining the fungicidal formulation of claim 8, comprising the following steps: a) preparing an aqueous solution of the fungicidal active ingredient at the desired concentration, b) adding dry composite microspheres at a concentration of up to 100% relative to the fungicidal active ingredient, c) mixing at least 2 hours prior to application.

22. The method of claim 21, wherein a co-adjuvant is further added.

23. A method for preventing and controlling fungal infections, comprising applying the fungicidal formulation of claim 8 to a crop.

24. The method for preventing and controlling fungal infections of claim 23, wherein said crop is selected from the group consisting of cereals, oilseeds, forest crops, fruit trees, ornamental plants, vegetables, wheat, barley, rye, triticale, oats, maize, sunflower, rice, soybean, pea, faba bean, common bean, peanut (Arachis hypogaea), rapeseed, kala, cotton, potato, sugar beet, sugarcane, chard (Beta vulgaris), peppers (Capsicum spp.), garlic and onions (Allium spp.), celery (Apium graveolens), eggplant (Solanum melongena), pumpkin (Cucurbita moschata),chayote (Sechium edule), cabbage (Brassica oleracea), spinach (Spinacia oleracea), green beans (Phaseolus vulgaris), lettuce (Lactuca sativa), maize (Zea mays), peanut (Arachis hypogaea), tomato (Solanum lycopersicum), cucumber (Cucumis sativus), okra (Hibiscus esculentus), radish (Raphanus sativus), beetroot (Beta vulgaris), carrot (Daucus carota), avocado (Persea americana), sugar apple (Annona squamosa), star apple (Chrysophyllum cainito), canistel (Pouteria campechiana), cherry (Malpighia punicifolia), custard apple (Annona reticulata), hog plum (Spondias dulcis), coconut (Cocos nucifera), papaya (Carica papaya), soursop (Annona muricata), guava (Psidium guajava), pomegranate (Punica granatum), lime (Citrus aurantifolia), lemon (Citrus limon), mamey sapote (Calocarpum mammosum), mamey apple (Mammea americana), mamoncillo (Melicoccus bijugatus), mandarin (Citrus reticulata), mango (Mangifera indica), passion fruit (Passiflora laurifolia), watermelon (Citrullus vulgaris), bitter orange (Citrus aurantium), sweet orange (Citrus sinensis), pineapple (Ananas comosus), banana (Musa paradisiaca), plantain (Musa balbisiana), tamarind (Tamarindus indica), grapefruit (Citrus paradisi), native grapefruit (Citrus grandis), grape (Vitis vinifera), common bean (Phaseolus vulgaris), rice (Oryza sativa), coffee (Coffea arabica), sugarcane (Saccharum officinarum), cotton (Gossypium hirsutum), gherkin (Melothria guadalupensis), sweet potato (Ipomoea batatas), potato (Solanum tuberosum), native potato (Solanum phureja), goldenberry (Physalis peruviana), cassava (Manihot esculenta), soybean (Glycine max), strawberry (Fragaria spp.), mulberry (Morus spp.), blackberry (Rubus spp.), palms of the Aracaceae family, oil palm (Elaeis guineensis), apple tree, cacao (Theobroma cacao), tree tomato (Solanum betaceum), and lulo (Solanum quitoense).

25. The method for preventing and controlling fungal infections of claim 23, wherein said fungicidal formulation is applied at a concentration of fungicidal active ingredient lower than that indicated on the commercial label.

26. The method for preventing and controlling fungal infections of claim 23, wherein the fungicidal efficacy is enhanced by at least 15% compared to the fungicide's efficacy in the absence of composite microspheres.

27. The method for preventing and controlling fungal infections of claim 23, wherein said infections include fungi selected from the group consisting of Magnaporthe oryzae, Botrytis cinerea, Puccinia spp., Fusarium spp., Fusarium graminearum, Fusarium oxysporum, Blumeria graminis, Mycosphaerella spp., Mycosphaerella fijiensis, Mycosphaerella graminicola, Colletotrichum spp., Ustilago maydis, Melampsora lini, Phakopsora pachyrhizi, Stemphylium lycopersici, and Rhizoctonia solan! .

28. A fungicidal formulation that enhances the effectiveness of a fungicidal active ingredient by at least 15%, comprising said fungicidal active ingredient and composite microspheres, wherein said microspheres comprise chitosan with an average molecular weight in the range from 190,000 to 310,000 g / mol, and a crosslinking agent, wherein the mass ratio of chitosan to crosslinking agent is 1:X, where X varies in the range from 0.5 to 1.5, and wherein the concentration of said composite microspheres is from 2 to 200% w / w relative to said fungicide, in an aqueous phase.

29. The fungicidal formulation of claim 28, wherein said composite microsphere further comprises a dispersing agent, wherein said dispersing agent is selected from the group consisting of gum arabic, xanthan gum, guar gum, carrageenan, pectin, alginates, dextrins, cyclodextrins, maltodextrins, tragacanth gum, mesquite gum, and mixtures thereof.

30. The fungicidal formulation of claim 29, wherein said dispersing agent is gum arabic.

31. The fungicidal formulation of claim 29, wherein the dispersing agent comprises a mass ratio of chitosan to dispersing agent of Y:l, where Y is in the range from 5 to 20.

32. The fungicidal formulation of claim 28, wherein said composite microsphere comprises an adjustable surface electrical charge or Z potential modulated by the concentration of the crosslinking agent, with X ranging from 0.08 to 0.8, and wherein said crosslinking agent is TPP.

33. The fungicidal formulation of claim 28, wherein the fungicidal active ingredient is in an aqueous solution stored in a container, and the composite microsphere, in powder form, is configured for addition to said container.

34. A process for obtaining the composite microsphere of claim 1, characterized by comprising the following steps: mixing acetic acid, chitosan, water, and stirring; adding said dispersing agent and stirring; adding the cross-linking agent solution dropwise; allowing to rest; and drying the obtained microspheres.

35. The process for obtaining the composite microsphere of claim 34, characterized by comprising the following steps: a. dissolving chitosan in 1% v / v acetic acid with constant stirring at a temperature of 20°C until no lumps are observed; b. adding dispersing agent and stirring until completely dissolved to achieve a chitosan-to-dispersing agent mass ratio of Y:l, where Y can vary in the range from 5 to 20; c. adding a cross-linking agent solution dropwise to the mixture, at a chitosan- to-cross-linking agent mass ratio of 1:X, where X can vary in the range from 0.08 to 1.2, in the absence of a herbicidal active ingredient; d. adjusting the pH to approximately 6.5; e. drying the composite microspheres.

36. The process of claim 35, characterized in that said step e) involves freeze-drying using a 220 V lyophilizer at -45°C and 10 Pa pressure, followed by grinding with a mortar and sieving through a 100-mesh filter to obtain the composite microspheres.

37. The process of claim 35, characterized in that said step e) involves spray drying at an inlet temperature of 200°C.

38. A process for obtaining the fungicidal formulation according to claim 28, characterized by comprising the following steps: a) preparing an aqueous solution of fungicide;b) adding dry composite microspheres to the aqueous fungicide solution; c) allowing them to remain in contact for at least 2 hours; d) stirring and applying to the crop.

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