GREEN ANTIFUNGAL AGROCHEMICAL FORMULATION BASED ON NANOMATERIALS OF SILICON AND ZINC

Abstract

The present invention relates to a stable agrochemical formulation, comprising a silicate salt, zinc oxide nanoparticles, a vegetable oil, a surfactant or a mixture of surfactants, a dispersant or a mixture of dispersants and, a stabilizer or mixture of stabilizers useful in the prevention or control of a plant disease produced by a microorganism, such as fungi or oomycetes. Additionally, the present invention relates to a method for preparing such agrochemical formulations. The agrochemical formulations and the method of the invention facilitate the preparation of agrochemical formulations containing silicate salts and zinc oxide nanoparticles, which furthermore decrease the environmental impact produced by the dispersion of pesticidal, bactericidal, fungicidal and oomyceticidal compounds.

Claims

1. An agrochemical formulation comprising: a silicate salt; a vegetable oil; a surfactant or a mixture of surfactants; a dispersant or a mixture of dispersants; zinc oxide nanoparticles; and a stabilizer or mixture of stabilizers.

2. The agrochemical formulation according to claim 1, wherein the stabilizer or mixture of stabilizers is a thickener.

3. The agrochemical formulation according to claim 1, wherein the formulation is in the form of an emulsion.

4. The agrochemical formulation according to claim 1, wherein the silicate salt is selected from the group consisting of sodium silicate, potassium silicate, manganese silicate, magnesium silicate or calcium silicate.

5. The agrochemical formulation of claim 1, wherein the silicate salt is in a concentration between 0.01 and 250 g/L.

6. The agrochemical formulation according to claim 1, wherein the surfactant is selected from the group consisting of ethoxylated linear alcohols, ethoxylated alkylphenols, fatty acid esters, amine derivatives, amide derivatives, alkylpolyglucosides, ethylene glycol/propylene glycol copolymers, polyols, ethoxylated polyols, thiols (mercaptans), or a mixture thereof.

7. The agrochemical formulation of claim 1, wherein the surfactant is in a concentration between 0.01 and 10 g/L.

8. The agrochemical formulation according to claim 1, wherein the vegetable oil is selected from the group consisting of soybean oil, corn oil, cottonseed oil, olive oil, palm oil, peanut oil, canola oil, safflower oil, sesame oil, sunflower oil, hazelnut oil, almond oil, walnut oil, macadamia oil, pecan oil, and pistachio oil.

9. The agrochemical formulation of claim 1, wherein the vegetable oil is in a concentration between 0.01 and 50 g/L.

10. The agrochemical formulation of claim 1, wherein the dispersant is selected from the group consisting of glycerol, ammonium citrate or sodium hexametaphosphate.

11. The agrochemical formulation of claim 1, wherein the dispersant is in a concentration between 0.1 and 500 g/L.

12. The agrochemical formulation according to claim 1, wherein the stabilizer is selected from the group consisting of polyvinyl alcohol, methylcellulose, xanthan gum, hydroxymethylcellulose, gum arabic, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, gellan gum, guar gum, locust bean gum, tragacanth gum, succinoglucan gum, gelatin, carrageenan, starch, sago, tapioca, pectin, collagen and agar.

13. The agrochemical formulation of claim 1, wherein the stabilizer is in a concentration between 0.01 and 5 g/L.

14. The agrochemical formulation of claim 1, wherein the zinc oxide nanoparticles are between 1 and 100 nm in size.

15. The agrochemical formulation of claim 1, wherein the zinc oxide nanoparticles are in a concentration between 0.1 and 300 g/L.

16. A method for preparing the agrochemical formulation of claim 1 comprising the steps of: a) mixing an aqueous solution of the silicate salt with a vegetable oil; b) adding a surfactant or a mixture of surfactants; c) adding a stabilizer or a mixture of stabilizers; d) preparing a nanofluid comprising zinc oxide nanoparticles and a dispersant; e) mixing the product resulting from step a) with the nanofluid from step d).

17. A use of the agrochemical formulation according to claim 1 for the prevention or control of a plant disease caused by a microorganism.

18. The use of claim 17, wherein the plant is selected from the group consisting of plants producing fruits or infructescences, ornamental plants, medicinal plants, legumes, grains and tubers.

19. The use of claim 17, wherein the plant is selected from the group consisting of chard (Beta vulgaris), chili peppers (Capsicum spp.), garlic and onions (Allium spp.), celery (Celery gravolens), eggplant (Solanum meolongena), pumpkin (Curcurbita moschata), chayote (Sechium edule), cabbage (Brassica oleracea), spinach (Spinaca oleracea), 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 auericana), anon (Annona squamosa), caimito (Chrysophyllum cainito), canistel (Pouteria campechiana), cherry (Malpighia punicifolia), custard apple (Annona reticulata), plum (Spondias dulcis), coconut (Coco nucifera), papaya (Carica papaya), soursop (Annona muricata), guava (Psidium guajaba), pomegranate (Punica granatum), lime (Citrus aurantifolium), lemon (Citrus limonum), red mamey (Calocarpum mammosum), mamey santo domingo (Mammea americano), mamoncillo (Melicocea bijuga), mandarin (Citrus reticulata), mango (Mangifera indica), passion fruit (Passiflora laurifolia), water melon (Citrullus vulgaris), sour orange (Citrus aurantium), sweet orange (Citrus sinensis), pineapple (Ananas comosus), banana (Musa paradisiaca), plantain (Musa balsisiana), tamarind (Tamarindus indica), grapefruit (Citrus paradisi), Creole grapefruit (Citrus grandin), grape (Vitis vinifera), bean (Phaseolus vulgaris), maize (Zea mays), rice (Oryza sativa), coffee (Coffea arabica), sugar cane (Saccharum officinarum), cotton (Gossypium hirsutum), gherkin (Melothria guadalupensis), sweet potato (Ipomoes batatas), potato (Solanum tuberosum), creole potato (Solanum phureja), cape gooseberry (Physalis peruviana), cassava (Manihot esculenta), soybean (Glycine max), strawberry (Fragaria spp.), blackberry (Morus spp.), bramble (Rubus spp.), palms of the family Aracaceae, oil palm (Elaeis guineensis), cocoa (Theobroma cacao), tree tomato (Solanum betaceum), lulo (Solanum quitoense).

20. The use according to claim 17, wherein the microorganism is a oomycete selected from a group comprising Phytophthora spp, Phytophthora infestans, Phytophthora ramorum, Phytophthora sojae, Phytophthora capsici, Phytophthora cinnamomi, Phytophthora palmivora and Phytophthora parasitica, Hyaloperonospora arabidopsidis, Plasmopara viticola, Pythium ultimum, Albugo candida, and Peronospora farinosa.

21. The use according to claim 17, wherein the microorganism is a fungus selected from a group comprising Magnaporthe oryzae, Botrytis cinerea, Puccinia 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.

22. (canceled)

23. (canceled)

24. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0019] FIG. 1 Powder X-ray diffractogram of (A) ZnO nanoparticles synthesized via Sol-Gel (Example 1), (B) dry-synthesized ZnO nanoparticles (Example 2) and (C) commercial USP ZnO nanoparticles.

[0020] FIG. 2 FT-IR spectra of ZnO nanoparticles: (A) Sol-Gel synthesis (Example 1) and (B) dry synthesis (Example 2).

[0021] FIG. 3 UV-VIS spectra of zinc oxide nanoparticles (A) dry synthesis of solvent H.sub.2O, (B) Sol-Gel synthesis of solvent H.sub.2O, (C) Sol-Gel synthesis of solvent PEP-H.sub.2O and (D) Sol-Gel synthesis of solvent PEP-CH.sub.3H.sub.8O.

[0022] FIG. 4 Scanning electron microscopy (SEM): (A) Dry-synthesized zinc oxide nanoparticles; (B) Sol-Gel-synthesized zinc oxide nanoparticles; (C). Commercial USP zinc oxide nanoparticles.

[0023] FIG. 5 Scanning electron microscopy (SEM) for agrochemical formulations of Example 7: (A) Agrochemical formulation D; (B) Agrochemical formulation F and (C). Agrochemical formulation H.

[0024] FIG. 6 UV-VIS spectra of agrochemical formulations of Example 7 (for 10 days) (A) Agrochemical formulation D and (B) Agrochemical formulation H.

[0025] FIG. 7 Stability curve of the agrochemical formulations of Example 7 (A) Agrochemical formulation D, (B) Agrochemical formulation F and (C) Agrochemical formulation H.

[0026] FIG. 8 Antifungal activity of the agrochemical formulations of Example 7 against B. cinerea based on time: (A) Agrochemical formulation D1, (B) Agrochemical formulation F1, (C) Agrochemical formulation H1, (D) Agrochemical formulation I, (E) Agrochemical formulation J.

[0027] FIG. 9 Antifungal activity of the agrochemical formulations of Example 7 against M. fijiensis based on time: (A) Agrochemical formulation D1, (B) Agrochemical formulation F1, (C) Agrochemical formulation H1, (D) Agrochemical formulation I, (E) Agrochemical formulation J.

[0028] FIG. 10 Antifungal activity of the agrochemical formulations of Example 7 against P. palmivora based on time: (A) Agrochemical formulation D, (B) Agrochemical formulation F, (C) Agrochemical formulation H, (D) Agrochemical formulation I, (E) Agrochemical formulation J.

[0029] FIG. 11 Antifungal activity of the agrochemical formulations of Example 7 against P. infestans based on time: (A) Agrochemical formulation D, (B) Agrochemical formulation F, (C) Agrochemical formulation H, (D) Agrochemical formulation I, (E) Agrochemical formulation J.

[0030] FIG. 12 Antifungal activity based on time with the commercial fungicides: Siganex (C1), Mancozed 80% WP (C2) and Forum 500 WP (C3) against; (A) B. cinerea, (B). M. fijiensis, (C) P. palmivora and (D) P. infestans.

DETAILED DESCRIPTION

[0031] The present invention corresponds to an agrochemical formulation comprising a silicate salt and zinc oxide nanoparticles in a liquid matrix, containing vegetable oil, a surfactant or a mixture of surfactants, a dispersant or a mixture of dispersants and a stabilizer or mixture of stabilizers, as well as methods for their preparation. In particular, this invention provides an agrochemical formulation comprising a silicate salt and zinc oxide nanoparticles that are stable over time.

[0032] For the purposes of the present application, the terms “silicate” or “silicate salt” refer to a salt comprising the (SiO.sub.3).sup.2− anion and any cation, including but not limited to sodium, potassium, manganese, magnesium and calcium. In one embodiment of the invention, the silicate or silicate salt is selected from potassium silicate, sodium silicate, manganese silicate, magnesium silicate and calcium silicate.

[0033] The silicate salt is contained in the agrochemical formulation of the invention in a concentration between 0.01 and 250 g/L. In another embodiment of the invention, the concentration is selected between 0.05 and 240 g/L, 0.1 and 230 g/L, 0.5 and 220 g/L, 0.8 and 210 g/L, 1.0 and 200 g/L, 1.5 and 190 g/L, 2.0 and 180 g/L, 2.5 and 170 g/L, 3.0 and 160 g/L, 3.5 and 150 g/L, 4.0 and 140 g/L, 4.5 and 130 g/L, 5.0 and 120 g/L, 5.5 and 110 g/L, 6.0 and 100 g/L, 6.5 and 95 g/L, 7.0 and 90 g/L, 8.5 and 85 g/L, 9.0 and 80 g/L, 10 and 75 g/L, 15 and 70 g/L, 20 and 65 g/L, 25 and 60 g/L, 30 and 55 g/L, 35 and 50 g/L, and 40 and 45 g/L.

[0034] Additionally, the term “vegetable oil” refers to a viscous, liquid mixture at room temperature extracted from a plant and made of non-polar compounds, mainly triglycerides. Possible vegetable oils include, but are not limited to soybean oil, corn oil, cottonseed oil, olive oil, palm oil, peanut oil, canola oil, safflower oil, sesame oil, sunflower oil, hazelnut oil, almond oil, walnut oil, macadamia oil, pecan oil and pistachio oil. In one embodiment of the invention, the vegetable oil is selected from the group consisting of soybean oil, corn oil, cottonseed oil, olive oil, palm oil, peanut oil, canola oil, safflower oil, sesame oil and sunflower oil. Vegetable oil is found at a concentration between 0.001 to 5.0% v/v, 0.01 to 3.0% v/v and 0.1 to 1.0% v/v.

[0035] Additionally, the term “surfactant” used in the present application refers to a compound which comprises polar and non-polar groups and decreases the surface tension of a liquid or the interfacial tension of two immiscible substances. Synonyms thereof are “surfactant” and “surface-active agent”. The surfactant comprising the agrochemical formulation of the invention is selected from ionic and non-ionic surfactants. In one embodiment of the invention, the surfactant is non-ionic. For example, the non-ionic surfactant is selected from, but not limited to, the group comprising ethoxylated linear alcohols, ethoxylated alkylphenols, fatty acid esters, amine derivatives, amide derivatives, alkylpolyglucosides, ethylene glycol/propylene glycol copolymers, polyols, ethoxylated polyols, thiols (mercaptans) and derivatives thereof or a mixture thereof. Particularly, the non-ionic surfactant is selected, but not limited to, the group comprising polysorbate, poloxamer, octylglycoside, polyglycerol, polyricinoleate, Triton X-100, cetyl alcohol and Cirrasol. Especially, it may be polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 65 (Tween 65), polysorbate 80 (Tween 80), Cirrasol or a mixture thereof. The surfactant or mixture of surfactants is at a concentration between 0.0001 to 1% v/v, 0.001 to 0.1% v/v, and 0.001 to 0.01% v/v.

[0036] As used in the present application, the term “thickener” refers to a compound that increases the viscosity of a liquid. A synonym for this term is “viscosifier”. The thickener or viscosifier comprising the formulation of the present invention is an organic, polysaccharide, protein, thickener or viscosifier, a derivative thereof or a mixture thereof. For example, polyvinyl alcohol, methylcellulose, xanthan gum, hydroxymethylcellulose, gum arabic, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, gellan gum, guar gum, locust bean gum, tragacanth gum, succinoglucan gum, gelatin, carrageenan, starch, sago, tapioca, pectin, collagen and agar. In particular, the thickener or viscosifier is a polysaccharide, such as methylcellulose, xanthan gum, hydroxymethylcellulose, gum arabic, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, gellan gum, guar gum, locust bean gum, tragacanth gum, succinoglucan gum, gelatin, carrageenan, starch, agar. The thickener is at a concentration between 0.0001 to 10.0 g/L, 0.001 to 8.0 g/L, 0.01 to 6.0 g/L, 0, yl to 4.0 g/L.

[0037] As used herein, the term “nanofluid” refers to a fluid containing uniformly and stably suspended nanoparticles.

[0038] As used in the present invention, the term “stable” refers to the formulation containing silicates and zinc oxide nanoparticles remaining homogeneous, without separation of its components or phases for a storage time exceeding 30 days.

[0039] In one embodiment of the invention, the agrochemical formulation is in the form of an emulsion, consisting of a two-phase system, in which one of the non-miscible liquids is uniformly dispersed in the other. In the present invention, the two phases are formed with the silicate and the vegetable oil, together with the surfactant. This emulsion also comprises zinc oxide nanoparticles. As used in the present application, the term “nanoparticles” refers to a particle with a size between 1 and 999 nm.

[0040] In one embodiment of the present invention, the agrochemical formulation comprises a silicate, a vegetable oil, a surfactant or a mixture of surfactants, a dispersant or a mixture of dispersants, a stabilizer or mixture of stabilizers and zinc oxide nanoparticles. The zinc oxide nanoparticles are in a concentration between 0.1 and 300 g/L. In one embodiment of the invention, the zinc oxide nanoparticles have a size of 1 to 100 nm, 10 to 80 nm, 25 to 80 nm and 25 to 50 nm.

[0041] In a further embodiment of the invention, the agrochemical formulation comprises a silicate salt, a vegetable oil, a surfactant or a mixture of surfactants, a stabilizer or mixture of stabilizers, zinc oxide nanoparticles, further comprising a dispersant or a mixture of dispersants. As used in the present application, the term “dispersant” refers to a substance that allows the zinc oxide nanoparticles to remain dispersed in solution preventing their aggregation. In one embodiment of the invention, the dispersant is selected from a mixture of ammonium citrate and glycerol (in a 1:1.5 ratio) and sodium hexametaphosphate. The dispersant is in a concentration between 0.1 to 500 g/L, 0.5 to 400 g/L, 1.0 to 300 g/L, 5.0 to 200 g/L, 10.0 g/L to 100 g/L, and between 0.1 to 40 g/L.

[0042] The zinc oxide nanoparticles incorporated in the agrochemical formulations of the present invention may be synthesized by any technique known to a person averagely versed in the matter, such as the colloidal method, photochemical and radiochemical reduction, microwave irradiation, use of dendrimers, solvothermal synthesis, sol-gel synthesis, dry synthesis, among others. In one embodiment of the invention, zinc nanoparticles are synthesized by modifications of the Sol-Gel methodology developed by “Synthesis and characterization of zinc oxide nanoparticles: application to textiles as UV-absorbers”, Becheri A., et al., J. Nanopart. Res, 10: 679-689 and by the methodology developed by “Low temperature synthesis of ZnO nanoparticles using mechanochemical route: a green chemistry approach” Azam A et al., IJTAS, 2009, 1(2): 12-14.

[0043] The preparation of the foregoing agrochemical formulations is carried out through the following methods, corresponding to invention modalities. The formulation comprising a silicate salt, a vegetable oil and a surfactant or a mixture of surfactants, a dispersant or a mixture of dispersants, zinc oxide nanoparticles, and a stabilizer or mixture of stabilizers is made according to a method comprising the steps of: [0044] a) mixing an aqueous solution of the silicate salt with a vegetable oil; [0045] b) adding a surfactant or a mixture of surfactants; [0046] c) adding a stabilizer or a mixture of stabilizers; [0047] d) preparing a nanofluid comprising zinc oxide nanoparticles and a dispersant; [0048] e) mixing the product resulting from step a) with the nanofluid from step d).

[0049] The formulations of the invention are directed to use for the prevention or control of a plant disease produced by a microorganism. For example, the plants may be vegetables, plants producing fruits or infructescences, ornamental plants, medicinal plants, legumes, grains and tubers. In particular, crops of chard (Beta vulgaris), peppers (Capsicum spp.), garlic and onions (Allium spp.), celery (Celery gravolens), eggplant (Solanum meolongena), pumpkin (Curcurbita moschata), chayote (Sechium edule), cabbage (Brassica oleracea), spinach (Spinaca oleracea), 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 auericana), anon (Annona squamosa), caimito (Chrysophyllum cainito), canistel (Pouteria campechiana), cherry (Malpighia punicifolia), custard apple (Annona reticulata), plum (Spondias dulcis), coconut (Coco nucifera), papaya (Carica papaya), soursop (Annona muricata), guava (Psidium guajaba), pomegranate (Punica granatum), lime (Citrus aurantifolium), lemon (Citrus limonum), red mamey (Calocarpum mammosum), mamey santo domingo (Mammea americano), mamoncillo (Melicocea bijuga), mandarin (Citrus reticulata), mango (Mangifera indica), passion fruit (Passiflora laurifolia), water melon (Citrullus vulgaris), sour orange (Citrus aurantium), sweet orange (Citrus sinensis), pineapple (Ananas comosus), banana (Musa paradisiaca), plantain (Musa balsisiana), tamarind (Tamarindus indica), grapefruit (Citrus paradisi), Creole grapefruit (Citrus grandin), grape (Vitis vinifera), bean (Phaseolus vulgaris), maize (Zea mays), rice (Oryza sativa), coffee (Coffea arabica), sugar cane (Saccharum officinarum), cotton (Gossypium hirsutum), gherkin (Melothria guadalupensis), sweet potato (Ipomoes batatas), potato (Solanum tuberosum), creole potato (Solanum phureja), cape gooseberry (Physalis peruviana), cassava (Manihot esculenta), soybean (Glycine max), strawberry (Fragaria spp.), blackberry (Morus spp.), bramble (Rubus spp.), palms of the Aracaceae family, oil palm (Elaeis guineensis), cocoa (Theobroma cacao), tree tomato (Solanum betaceum), lulo (Solanum quitoense).

[0050] In particular, the microorganism is a bacterium, fungus or oomycete. For example, the bacterium is Pseudomonas syringae, Ralstonia solanacearum, Agrobacterium tumefaciens, Xanthomonas spp. (including but not limited to Xanthomonas oryzae pv. oryzae, Xanthomonas campestris, Xanthomonas axonopodis, Erwinia amylovora, Xylella fastidiosa, Dickeya dadantii, Dickeya solani, Pectobacterium carotovorum, Pectobacterium atrosepticum, Clavibacter michiganensis, Clavibacter sepedonicus, Pseudomonas savastanoi. In particular, the fungi are Magnaporthe oryzae, Botrytis cinerea, Puccinia spp. (including but not limited to Fusarium graminearum, Fusarium oxysporum), Blumeria graminis, Mycosphaerella spp. (including but not limited to Mycosphaerella fijiensis and Mycosphaerella graminicola), Colletotrichum spp., Ustilago maydis, Melampsora lini, Phakopsora pachyrhizi and Rhizoctonia solani. As for the oomycete, this may be Phytophthora spp. (including but not limited to Phytophthora infestans, Phytophthora ramorum, Phytophthora sojae, Phytophthora capsici, Phytophthora cinnamomi, Phytophthora palmivora and Phytophthora parasitica, Hyaloperonospora arabidopsidis, Plasmopara viticola, Pythium ultimum, Albugo candida and Peronospora farinosa).

EXAMPLES

Example 1: Sol-Gel Synthesis of ZnO Nanoparticles

[0051] An amount of 5.5 g of ZnCl.sub.2 was dissolved in 200 mL of water at 90° C. in an oil bath. Then, 16 mL of 5 M of NaOH was added dropwise to the ZnCl.sub.2 solution with gentle stirring over a period of 10 minutes at 90° C. The particles were separated from the supernatant dispersion by sedimentation and, then, the supernatant solution was discarded. The suspension was washed five times with distilled water to reduce the NaCl concentration to less than 1 μM by measuring the removal of NaCl from the suspension through AgNO.sub.3 solution. The obtained purified particles were peptized with 2-propanol in an ultrasonic bath for 10 min to disintegrate the micro-agglomerates and obtain ZnO nanounits. Then, the particles were separated by centrifugation at 6000 rpm for 15 min. The washing procedure was carried out three times. Finally, the particles were subjected to 250° C. for 5 hours to obtain 1.5 g of zinc oxide nanoparticles.

Example 2: Dry Synthesis of ZnO Nanoparticles

[0052] An amount of 21.9 g of zinc acetate and 18 g of tartaric acid were mixed by grinding in an agate mortar for 30 min at room temperature to produce zinc tartrate. The zinc tartrate was subjected to 450° C. for one hour to obtain 7 g of zinc oxide nanoparticles.

Example 3: X-Ray Powder Diffraction Characterization of ZnO Nanoparticles

[0053] As seen in FIGS. 1A and 1B, the zinc oxide nanoparticles synthesized according to Example 1 and 2 present a crystal structure corresponding to the hexagonal wurtzite phase and with similar lattice parameters (a=3.251 y c=5.205 Å). No additional phase is observed, assuring the purity of the obtained compound. Particularly the diffractograms obtained from the synthesized nanoparticles present the same peaks of the commercial USP nanoparticles (FIG. 1C).

Example 4: Characterization by FT-IR Spectroscopy of ZnO Nanoparticles

[0054] The FT-IR spectra of the zinc oxide nanoparticles synthesized according to Example 1 and 2 (FIGS. 2A and 2B) show the Zn—O absorption band near 430 cm.sup.−1. The peaks at 3450 and 2350 cm.sup.−1 indicate the presence of —OH and C═O traces, probably due to the moisture and CO.sub.2 present in the atmosphere. The same spectrum was obtained from the nanoparticles produced by the following dry synthesis (ST SC), sol-gel synthesis (ST SOL-GEL), sol-gel synthesis with 2-propanol pectization (ST SOL-GEL PECT), sol-gel synthesis with 2-propanol pectization and oven drying at 120° C. (ST SOL-GEL PECT SC).

[0055] It is interpreted in the FT-IR spectrum the presence of zinc oxide with the intense band in the wavelength of 400 cm.sup.−1 in the ZnO NPs by dry synthesis of analytical precursors (ZnO NPs SS Analit), using the same synthesis and changing the precursor tartaric acid by the commercial one (ZnO NPs SS AT) presents similar spectrum and the compound ZnO USP also showed the same spectrum.

Example 5: UV-Vis Spectroscopy Characterization of ZnO Nanoparticles

[0056] The UV-Vis spectra of zinc oxide nanoparticles synthesized according to Example 1 and 2 using different solvents are shown in FIGS. 3A, 3B, 3C and 3D. The absorbance is presented from 372 and 362 nm for aqueous and organic. The highest absorbance of 1.72 for the sol-gel synthesis with pectization dissolved in deionized water (ST SOL-GEL PECT-H2O), the intermediate magnitudes 0.36-0.56 corresponding to: sol-gel synthesis dissolved in distilled water (ST SOL-GEL-H2O), propanol dissolved in 2-propanol (ST SOL-GEL PECT-C3H8O, and the lower magnitude 0.228 corresponding to dry synthesis dissolved in deionized water (ST SC-H2O), the latter value is also reported by the authors [4, 6], typical of ZnO NPs.

[0057] By UV-visible spectroscopy the zinc oxide is reported with wavelength in the range of 370-390 nm with an absorbance percentage between 0.22% to 1.80% allowing to identify, evaluate qualitatively the stability and purity of the compound.

Example 6: Scanning Electron Microscopy (SEM) Characterization of ZnO Nanoparticles

[0058] As seen in FIG. 4, the zinc oxide nanoparticles in Example 2 (FIG. 4A) are spherical and have diameters between 25 and 47.2 nm or are irregular and form agglomerates. As for the zinc oxide nanoparticles synthesized in Example 1 (FIG. 4B), they form agglomerates of defined petal-shaped sheets 29.83 nm wide and 72.05 nm long. Finally, with regard to the commercial USP zinc oxide nanoparticles (FIG. 4C), these are in the form of defined rods of 205 to 500 nm in length and 99 to 177 nm in base, and agglomerates of several shapes.

Example 7: Preparation of Agrochemical Formulations

[0059] Different formulations (formulations A to H) were tested according to the concentrations shown in Tables 1 to 5. For this purpose, two initial compositions were made. The first one was prepared by initially mixing an aqueous solution of K.sub.2SO.sub.3 40% w/v with soybean oil, then Tween 80 and Cirrasol were added and finally Xanthan gum was added. The second composition was prepared by initially mixing the ZnO nanoparticles with ammonium citrate and glycerol in a ball mill, then mixing by ultrasound for 10 minutes. Finally, both compositions were mixed. As shown in Tables 1 to 5, not all formulations comprise all components. In case one of them is absent, the foregoing procedure was simply continued by skipping the step related to such component. Alternatively, after preparing a concentrated formulation according to the foregoing method, it was diluted to achieve other concentrations. For example, the formulations of Tables 2 to 5 were also prepared by dilution in water from the concentrated formulation of Table 1. Accordingly, the formulation of Table 2 shows a dilution factor equal to 40 with regard to the formulation of Table 1, the formulation of Table 3 shows a dilution factor equal to 200 with regard to the formulation of Table 1, the formulation of Table 4 shows a dilution factor equal to 400 with regard to the formulation of Table 1 and the formulation of Table 5 shows a dilution factor equal to 800 with regard to the formulation of Table 1.

TABLE-US-00001 TABLE 1 Ingredients and Concentrations of Concentrated Formulations A through H (NA: Not Applicable) Zinc Sodium Potassium oxide Hexameta silicate Soy oil Tween 80 Cirrasol Xanthan NPs Glycerol Ammonium phosphate Formulation (g/L) (% v/v) (& v/v) (% v/v) gum (g/L) (g/L) (% v/v) citrate (g/L) (g/L) A 172 3.4 0.15 0.35 NA NA NA NA NA B NA 3.4 0.15 0.35 NA NA NA NA NA C NA NA NA NA 200 NA NA NA 40 D NA NA NA NA 200 25.5 25.5 200 NA E 20.416 NA NA NA 200 NA NA NA 40 F 20.416 NA NA NA 200 25.5 25.5 200 NA G 20.416 3.4 0.15 0.35 200 NA NA NA 40 H 20.416 3.4 0.15 0.35 200 25.5 25.5 200 NA

TABLE-US-00002 TABLE 2 Ingredients and concentrations of formulations A to H with dilution factor 40 with regard to the concentrations in Table 1 (NA: Not applicable). Zinc Sodium Potassium oxide Hexameta silicate Soy oil Tween 80 Cirrasol Xanthan NPs Glycerol Ammonium phosphate Formulation (g/L) (% v/v) (& v/v) (% v/v) gum (g/L) (g/L) (% v/v) citrate (g/L) (g/L) A 4.3   0.085 0.00375 0.00875 0.005 NA NA NA NA B NA 0.085 0.00375 0.00875 0.005 NA NA NA NA C NA NA NA NA 1.53125 5 NA NA 1 D NA NA NA NA 1.53125 5 0.6375 5 NA E 0.5104 NA NA NA 1.53125 5 NA NA 1 F 0.5104 NA NA NA 1.53125 5 0.6375 5 NA G 0.5104 0.085 0.00375 0.00875 1.53125 5 NA NA 1 H 0.5104 0.085 0.00375 0.00875 1.53125 5 0.6375 5 NA

TABLE-US-00003 TABLE 3 Ingredients and concentrations of formulations A to H with dilution factor 200 with regard to the concentrations in Table 1 (NA: Not applicable). Zinc Sodium Potassium oxide Hexameta silicate Soy oil Tween 80 Cirrasol Xanthan NPs Glycerol Ammonium phosphate Formulation (g/L) (% v/v) (& v/v) (% v/v) gum (g/L) (g/L) (% v/v) citrate (g/L) (g/L) A 0.86   0.017 0.00075 0.00175 0.001 NA NA NA NA B NA 0.017 0.00075 0.00175 0.001 NA NA NA NA C NA NA NA NA 0.30625 1 NA NA 0.2 D NA NA NA NA 0.30625 1 0.1275 1 NA E 0.10208 NA NA NA 0.30625 1 NA NA 0.2 F 0.10208 NA NA NA 0.30625 1 0.1275 1 NA G 0.10208 0.017 0.00075 0.00175 0.30625 1 NA NA 0.2 H 0.10208 0.017 0.00075 0.00175 0.30625 1 0.1275 1 NA

TABLE-US-00004 TABLE 4 Ingredients and concentrations of formulations A to H with dilution factor 400 with regard to the concentrations in Table 1 (NA: Not applicable). Zinc Sodium Potassium oxide Hexameta silicate Soy oil Tween 80 Cirrasol Xanthan NPs Glycerol Ammonium phosphate Formulation (g/L) (% v/v) (& v/v) (% v/v) gum (g/L) (g/L) (% v/v) citrate (g/L) (g/L) A 0.43   0.0085 0.000375 0.000875 0.0005 NA NA NA NA B NA 0.0085 0.000375 NA 0.0005 NA NA NA NA C NA NA NA NA 0.153125 0.5 NA NA 0.1 D NA NA NA NA 0.153125 0.5 0.06375 0.5 NA E 0.05104 NA NA NA 0.153125 0.5 NA NA 0.1 F 0.05104 NA NA NA 0.153125 0.5 0.06375 0.5 NA G 0.05104 0.0085 0.000375 0.000875 0.153125 0.5 NA NA 0.1 H 0.05104 0.0085 0.000375 0.000875 0.153125 0.5 0.06375 0.5 NA

TABLE-US-00005 TABLE 5 Ingredients and concentrations of formulations A to H with dilution factor 800 with regard to the concentrations in Table 1 (NA: Not applicable). Zinc Sodium Potassium oxide Hexameta silicate Soy oil Tween 80 Cirrasol Xanthan NPs Glycerol Ammonium phosphate Formulation (g/L) (% v/v) (& v/v) (% v/v) gum (g/L) (g/L) (% v/v) citrate (g/L) (g/L) A 0.215  0.00425  0.0001875 0.0004375 0.00025 NA NA NA NA B NA 0.00425  0.0001875 0.0004375 0.00025 NA NA NA NA C NA NA7 NA NA 0.0765625 0.25 NA NA 0.05 D NA NA NA NA 0.0765625 0.25 0.375 0.25 NA E 0.02552 NA NA NA 0.0765625 0.25 NA NA 0.05 F 0.02552 NA NA NA 0.0765625 0.25 0.375 0.25 NA G 0.02552 0.0001875 0.0001875 0.0004375 0.0765625 0.25 NA NA 0.05 H 0.02552 0.0001875 0.0001875 0.0004375 0.0765625 0.25 0.375 0.25 NA

Example 8: Stability Evaluation of the Invention Formulations

[0060] The stability of the formulations D, F and H was determined through EDS, SEM (FIG. 5), UV-Vis spectrometry (FIGS. 6A and 6B) and Turbiscan techniques, the latter two to evaluate the stability of the formulations in a 10-day period of time. Considering the EDS results, the presence of zinc, oxygen and carbon in formulation D is evident. In addition, this formulation presents zinc oxide nanoparticles with particle size between 42 to 60 nm, spherical shapes, with abundant agglomeration of irregular shapes and totally coated (FIG. 5A). As for formulation F, it is characterized by the presence of zinc, oxygen, carbon, silicon and potassium. In addition, this formulation presents zinc oxide nanoparticles with a particle size between 22 to 47.4 nm, spherical shapes, with spherical agglomeration with diameter sizes from 542.0 nm to 638.2 nm, within a linear network covering them, given by the emulsifying compounds of the formulation (FIG. 5B). Finally, in formulation H the presence of zinc, oxygen, silicon and potassium is evidenced. The zinc oxide nanoparticles of formulation H have a particle size between 24.0 to 68.0 nm, spherical shapes with varied agglomeration, spherical with spherical deformations and polygons, within linear networks that surround and cover them, given by the emulsifying compounds of the formulation (FIG. 5C). All the formulations evaluated show high stability over long periods of time as evidenced in FIG. 7.

Example 9: Antifungal Activity Evaluation of the Formulations in Example 7

[0061] The antifungal activity of formulations D, F, H, I and J of Example 7 and ½ and ¼ dilutions thereof were tested on four microorganisms: Botrytis cinerea ATCC 36634, Mycosphaerella fijiensis ATCC 36055, Phytophthora palmivora ATCC 46634 and Phytophthora infestans ATCC 48716. In order to compare the effect of the formulations of the present invention, commercial fungicides and oomyceticides were also tested: Misilk 360, Nitrofil FT, Siganex, Mancozeb 80% WP and Forum 500 WP. Additionally, in order to determine the effect of the size of the ZnO nanoparticles, the foregoing formulations were prepared using commercial zinc oxide USP, characterized by a particle size larger than the nanoparticles used in the formulations of the invention. The formulations containing commercial USP zinc oxide are designated with an asterisk (*).

[0062] The fungi and oomycetes were grown on 2% potato dextrose agar (PDA) at a temperature of 24±1° C. for 7 to 30 days of incubation, depending on the strain. Subsequently, new PDA medium containing the formulations of the invention was inoculated. The formulations were used at the concentration shown in Tables 3, 4 and 5 and sterile distilled water was used as a control. Then, using a Vernier caliper, the radial growth of the pathogen was measured based on time. All assays were performed three times. The formula used to calculate the percentage of inhibition is as follows:

[00001]$\mathrm{Inhibition}\mathrm{Percentage}=\frac{\mathrm{Control}\mathrm{diameter}-\mathrm{Treatment}\mathrm{diameter}}{\mathrm{Control}\mathrm{diameter}\times 100}$

[0063] Table 6 presents the results obtained for B. cinerea ATCC 36634 after 7 days of growth for the formulations evaluated (FIGS. 8A, 8B, 8C, 8D and 8E). According to the results obtained at day 7 of the assay, formulations F and H were those that exhibited the highest in vitro antifungal activity on the Botrytis cinerea strain with growth inhibition percentages between 80 to 99% with regard to the growth control without antifungals. The antifungal activity of the highest evaluated concentration of formulations F and H was comparable to that exhibited by the commercial fungicides Siganex and Mancozeb (FIG. 12A).

[0064] The results of growth inhibition of M. fijiensis ATCC 36055 for the evaluated formulations (FIGS. 9A, 9B, 9C, 9D and 9E) at day 40 are shown in Table 7. The growth of this fungus was stabilized even up to day 55. For this reason, day 40 was considered as the maximum point of fungus radial growth. Considering the results, formulations D, H and H* exhibited the highest in vitro antifungal activity on M. fijiensis strain, inhibiting 100% of the fungal growth, as well as the commercial fungicides evaluated as inhibition control (FIG. 12B).

[0065] Similarly, Table 8 presents the results obtained for the strain P. palmivora ATCC 46634 for the formulations evaluated (FIGS. 10A, 10B, 10C, 10D and 10E) at day 17 of the study. According to the results, compounds F, H, H* and Misilk 360 (FIG. 12C) exhibited the highest P. palmivora activity even at the lowest concentrations evaluated (dilution factor 800), exhibiting growth inhibition percentages higher than 94% with regard to the control. The maximum concentration of the evaluated formulations inhibited more than 99% of the oomycete, with the exception of the compound Nitrofil FT.

[0066] The results of growth inhibition of P. infestans ATCC 46634 for the evaluated formulations (FIGS. 11A, 11B, 11C, 11D and 11E) at day 20 are shown in Table 9. According to the results obtained, with the exception of compound (J), the formulations of the invention may inhibit the growth of P. infestans by 98 to 100%.

[0067] In conclusion, it may be considered that the highest concentration of the formulations F, F*, H and H* are the best formulations with antifungal or antioomycete activity in vitro on the different phytopathogens evaluated, being comparable to the commercial fungicides Siganex, Mancozeb 80% WP and Forum 500 WP.

TABLE-US-00006 TABLE 6 Growth inhibition of B. cinerea ATCC 36634 against the invention formulations. The formulations marked with asterisk (*) were prepared using commercial zinc oxide nanoparticles (USP). Dilution Factor: 200 Dilution Factor: 400 Dilution Factor: 800 Colony Colony Colony Diameter Growth Diameter Growth Diameter Growth (mm) ± Inhibition (mm) ± Inhibition (mm) ± Inhibition Standard Percentage Standard Percentage Standard Percentage Formulation Deviation (%) Deviation (%) Deviation (%) White 86.00 ± 0.00 — 86.00 ± 0.00 — 86.00 ± 0.00 — A — — — — — — B — — — — — — C — — — — — — C* — — — — — — D 19.14 ± 0.31 85.39 30.17 ± 0.59 71.30 85.20 ± 0.72  1.02 D* 27.69 ± 0.90 74.47 63.67 ± 0.77 28.52 85.00 ± 1.00  1.28 E — — — — — — E* — — — — — — F  8.33 ± 0.42 99.20  8.60 ± 0.36 98.85 10.07 ± 0.31 96.97 F* 11.57 ± 0.51 95.06 18.53 ± 0.50 86.17 22.23 ± 0.40 81.44 G — — — — — — G* — — — — — — H  9.37 ± 0.32 97.87 12.23 ± 0.64 94.21 15.27 ± 0.64 90.33 H*  9.77 ± 0.68 97.36 12.60 ± 0.40 97.74 23.07 ± 1.10 80.37 Misilk 360 17.63 ± 0.32 87.32 19.93 ± 1.01 84.38 23.13 ± 0.81 80.29 Nitrofil FT 85.80 ± 0.35  0.26 85.67 ± 0.58  0.42 86.00 ± 0.00  0.00 Siganex  7.70 ± 0.00 100.00   7.70 ± 0.00 100.00  — — Mancozeb 80% WP  7.70 ± 0.00 100.00   7.70 ± 0.00 100.00  — — Forum 500 WP 11.07 ± 0.30 95.70 11.90 ± 0.20 94.64 — —

TABLE-US-00007 TABLE 7 Growth inhibition of M. fijiensis ATCC 36055 against the invention formulations. The formulations marked with asterisk (*) were prepared using commercial (USP) zinc oxide nanoparticles. Dilution Factor: 200 Dilution Factor: 400 Dilution Factor: 800 Colony Colony Colony Diameter Growth Diameter Growth Diameter Growth (mm) ± Inhibition (mm) ± Inhibition (mm) ± Inhibition Standard Percentage Standard Percentage Standard Percentage Formulation Deviation (%) Deviation (%) Deviation (%) White 38.07 ± 0.00  — 38.07 ± 0.00 — 38.07 ± 0.00 — A — — — — — — B — — — — — — C — — — — — — C* — — — — — — D 7.70 ± 0.00 100.00  9.47 ± 0.31 94.18 10.73 ± 0.25 90.01 D* 10.07 ± 0.50   92.21 12.43 ± 0.71 84.41 14.27 ± 0.64 78.38 E — — — — — — E* — — — — — — F 7.80 ± 0.17  99.67 13.17 ± 0.72 82.00 16.43 ± 0.78 71.24 F* 8.23 ± 0.06  98.24 10.23 ± 0.25 91.66 11.40 ± 0.36 87.82 G — — — — — — G* — — — — — — H 7.70 ± 0.00 100.00 10.80 ± 0.72 89.79 14.93 ± 0.61 76.18 H* 7.70 ± 0.00 100.00 12.30 ± 0.46 84.85 15.83 ± 0.76 73.22 Misilk 360 8.53 ± 0.42  97.26  9.17 ± 0.29 95.17 10.70 ± 0.44 90.12 Nitrofil FT 11.77 ± 0.21   86.61 14.63 ± 0.60 77.17 17.83 ± 0.35 66.63 Siganex 7.70 ± 0.00 100.00  7.70 ± 0.00 100.00  — — Mancozeb 80% WP 7.70 ± 0.00 100.00  7.70 ± 0.00 100.00  — — Forum 500 WP 7.70 ± 0.00 100.00  7.70 ± 0.00 100.00  — —

TABLE-US-00008 TABLE 8 Growth inhibition of P. palmivora ATCC 46634 against the invention formulations. Formulations marked with an asterisk (*) were prepared using commercial zinc oxide nanoparticles (USP). Dilution Factor: 200 Dilution Factor: 400 Dilution Factor: 800 Colony Colony Colony Diameter Growth Diameter Growth Diameter Growth (mm) ± Inhibition (mm) ± Inhibition (mm) ± Inhibition Standard Percentage Standard Percentage Standard Percentage Formulation Deviation (%) Deviation (%) Deviation (%) White 86.00 ± 0.00  — 86.00 ± 0.00  — 86.00 ± 0.00 — A — — — — — — B — — — — — — C — — — — — — C* — — — — — — D 8.33 ± 0.15 99.19 18.37 ± 0.78  86.38 23.07 ± 0.31 80.37 D* 8.10 ± 0.17 99.49 62.67 ± 1.60  29.80 79.00 ± 1.40  8.94 E — — — — — — E* — — — — — — F 8.13 ± 0.15 99.45 9.60 ± 0.53 97.57 11.80 ± 0.72 94.76 F* 8.33 ± 0.21 99.19 9.93 ± 0.12 97.15 20.53 ± 1.86 83.61 G — — — — — — G* — — — — — — H 8.00 ± 0.00 99.62 8.00 ± 0.00 99.62  8.00 ± 0.00 99.62 H* 8.00 ± 0.00 99.62 8.30 ± 0.26 99.23 11.27 ± 0.64 95.44 Misilk 360 8.23 ± 0.21 99.32 8.63 ± 0.15 98.81  8.60 ± 0.30 98.85 Nitrofil FT 86.00 ± 0.00   0.00 86.00 ± 0.00   0.00 86.00 ± 0.00  0.00 Siganex 7.70 ± 0.00 100.00  7.70 ± 0.00 100.00  — — Mancozeb 80% WP 7.70 ± 0.00 100.00  7.70 ± 0.00 100.00  — — Forum 500 WP 7.70 ± 0.00 100.00  7.70 ± 0.00 100.00  — —

TABLE-US-00009 TABLE 9 Growth inhibition of P. infestans ATCC 48716 against the invention formulations. The formulation marked with asterisk (*) were prepared using commercial zinc oxide nanoparticles (USP). Dilution Factor: 200 Dilution Factor: 400 Dilution Factor: 800 Colony Colony Colony Diameter Growth Diameter Growth Diameter Growth (mm) ± Inhibition (mm) ± Inhibition (mm) ± Inhibition Standard Percentage Standard Percentage Standard Percentage Formulation Deviation (%) Deviation (%) Deviation (%) White 86.00 ± 0.00  — 86.00 ± 0.00  — 86.00 ± 0.00  — A — — — — — — B — — — — — — C — — — — — — C* — — — — — — D 7.70 ± 0.00 100.00 7.70 ± 0.00 100.00 9.53 ± 0.50 97.66 D* 7.70 ± 0.00 100.00 8.23 ± 0.25  99.32 17.53 ± 0.50  87.44 E — — — — — — E* — — — — — — F 7.70 ± 0.00 100.00 8.17 ± 0.15  99.40 8.90 ± 0.36 98.47 F* 7.70 ± 0.00 100.00 7.70 ± 0.00 100.00 7.70 ± 0.00 100.00  G — — — — — — G* — — — — — — H 8.70 ± 0.36  98.72 9.10 ± 0.26  98.21 9.37 ± 0.15 97.87 H* 7.70 ± 0.00 100.00 7.70 ± 0.00 100.00 7.70 ± 0.00 100.00  Misilk 360 7.70 ± 0.00 100.00 7.70 ± 0.00 100.00 8.57 ± 0.40 98.89 Nitrofil FT 80.33 ± 1.53   7.24 83.33 ± 1.53   3.41 85.67 ± 0.58   0.43 Siganex 7.70 ± 0.00 100.00 7.70 ± 0.00 100.00 — — Mancozeb 80% WP 7.70 ± 0.00 100.00 7.70 ± 0.00 100.00 — — Forum 500 WP 7.70 ± 0.00 100.00 7.70 ± 0.00 100.00 — —