Obtaining coacervates and their use as a biological control tool in agriculture

Abstract

Obtaining coacervates and their use as a biological control tool in agriculture. Method for obtaining coacervates of a polymer and spores of at least one fungus used for the biological control of agricultural pests, for use as a tool in the biological control of plant pests and diseases. The method comprises the following steps: - dissolution of chitosan in a weak acid buffer solution, - Addition of Pochonia chlamydosporia spores to the solution from the previous step, and shake to obtain a homogeneous dispersion, - add the homogeneous dispersion from the previous step to an aqueous solution at basic pH.

Description

OBTAINING COACERVATES AND THEIR USE AS A CONTROL TOOL BIOLOGICAL IN AGRICULTURE FIELD OF INVENTION The present invention falls within the field of agricultural biotechnology and, in particular, relates to obtaining coacervates of a polymer for practical application against pests and diseases in plants. STATE OF THE ART Polymers, biopolymers, and more specifically chitosan, have been used as antimicrobials primarily against bacteria and fungi, applied in solutions, films, and other formulations. It has been shown to be effective in controlling plant diseases and pests. It also induces defense mechanisms in plants against pre- and post-harvest plant diseases (Lopez-Moya et al., 2017. Induction of auxin biosynthesis and WOX5 repression mediate changes in root development in Arabidopsis exposed to chitosan. Scientific reports, 7 (1), 16813). Pochonia chlamydosporia is able to reduce root colonization by plant pathogenic fungi and the damage they cause. In turn, Pochonia chlamydosporia parasitizes the eggs of cyst-forming and root-knot nematodes. The fungus uses appressoria and extracellular enzymes (mainly proteases, chitin deacetylases, and chitosanases) to do this. Furthermore, it promotes plant growth by producing growth regulators. Encapsulation consists of forming a protective layer around an active ingredient or a cell. Among the processes that use chitosan encapsulation are ionotropic gelation, precipitation, atomization or spray-drip and spray-ing, simple coacervation, complex coacervation, chemical crosslinking, thermal crosslinking, and emulsification (Dhawan et al., 2004 Evaluation of mucoadhesive properties of chitosan microspheres prepared by different methods. AAPS Pharmscitech, 5 (4), 122-128). Simple coacervation is based on the decrease in the solubility of the polymer, which forms a layer or particle by dripping an acidic solution of the polymer onto a basic solution. This results in the precipitation of the polymer and the formation of a polymer capsule (Park and Chang, 2000. Microencapsulation of microbial cells. Biotechnology Advances. 18 (4), 303-319). Some fungi have the ability to remain viable encapsulated in different polymers. In the study by Liu C. and Liu S, 2008. Formulation and characterization of the microencapsulated entomopathogenic fungus Metarhizium anisopliae (MA126) . Journal of Microencapsulation, 26 (5) 377-384, a coating technique for the microencapsulation of conidia of the entomopathogenic fungus Metarhizium anisopliae (MA126) is explored. In the aforementioned study, calcium alginate is used to prepare microcapsules along with the conidia of M. anisopliae. Once the microcapsules are formed, they use, among other compounds (dextrin, sodium salt of alginic acid from brown algae (HPMC)), chitosan as a coating method for the capsule to obtain greater stability. This document does not disclose that the matrix where the conidia are encapsulated is entirely made of chitosan.Additionally, they indicate that the most effective compound for the formulation of these capsules is HPMC and not chitosan. The present invention discloses a method of coacervation or obtaining coacervates in which the main component is a biopolymer, preferably chitosan, which is used as a matrix to encapsulate spores (conidia and chlamydospores) of Pochonia chlamydosporia, a fungus used for the biological control of agricultural pests, preferably a nematophagous fungus used as a biological control agent of phytopathogenic nematodes and promoter of crop development and defenses. In the present invention, a coacervate is understood to be the formation of a protective layer or the creation of a particle with at least one polymer and one or more cells or spores. In this report, the terms "cells" and "spores" are synonymous and are used interchangeably. Coacervation is primarily used for the controlled release of compounds. This process is based on decreasing the solubility of the polymer forming the layer by adding another compound to the solution, changing the pH, or altering the temperature, thereby causing the polymer to precipitate. The present invention discloses a method for obtaining coacervates comprising the encapsulation of spores (conidia and chlamydospores) of a fungus used for biological control in agriculture in a polymer, preferably chitosan, for subsequent application in crops. In this document, the terms "polymer" and "biopolymer" are synonymous and used interchangeably. A biopolymer is one that was first observed in nature, although it may later be obtained by artificial methods. In this document, the expressions "as a biological control agent in agriculture" and "for the biological control of agricultural pests" are synonymous and are used interchangeably. A first object of the invention relates to a method of obtaining coacervates from: - a linear cationic polymer comprising at least one monomer that is an amino monosaccharide and - spores of at least one fungus used as a biological control agent in agriculture which includes the following stages: - dissolution of said linear cationic polymer in a weak acid buffer solution, - addition of spores of at least one fungus used as a biological control agent in agriculture to the solution from the previous step, and stirring to obtain a homogeneous dispersion - add the homogeneous dispersion from the previous step to an aqueous solution at basic pH. solid particles are obtained from a coacervate formed by the polymer and the spores. In one particular embodiment the amino monosaccharide has at least 5 carbons, preferably 5 or 6 carbons. In another particular embodiment, the monomer is selected from glucosamine, N-acetyl-glucosamine, galactosamine, sialic acid, N-acetylgalactosamine, and N-acetylmannosamine. In another particular embodiment, the linear cationic polymer comprises at least 2 different monomers, preferably 2 different monomers. In another particular embodiment, the two different monomers are glucosamine and N-acetylglucosamine. In another particular embodiment, the linear cationic polymer is chitosan. In this document, a linear cationic polymer is one that has no side chains beyond the atoms or functional groups that are part of the monomer(s) of the polymer, or the side chains are each equal to or less than 50 atoms, preferably equal to or less than 40 atoms, more preferably equal to or less than 30 atoms, even more preferably equal to or less than 25 atoms, or 20 atoms, or 15 atoms, or 10 atoms, or 5 atoms. In another particular embodiment, at least 90% of the chitosan particles have a particle size of less than 1 mm, preferably less than 0.8 mm, and more preferably less than 0.5 mm. It is desirable that the ash and heavy metal content be as low as possible; in a preferred embodiment, the ash content is less than 0.5% and the heavy metal concentration is less than 100 ppm. In another embodiment, chitosan exhibits a degree of deacetylation of between 60% and 100%, preferably between 70% and 100%, more preferably between 80% and 100%, and even more preferably between 90% and 100%. A higher degree of deacetylation results in fewer acetyl groups from chitin and more free amino groups from chitosan, which imparts its cationic character, thus providing greater stability to the coacervate. This is because the cationic ions bind with the anionic ions of the aqueous solution at basic pH, providing a larger and therefore more stable bond network in the formulation or coacervate. Fungal biological control agents in agriculture are capable of degrading the polymer coacervate, preferably chitosan, obtaining from it the necessary substrate to develop and grow, colonizing the soil and roots of crops treated with the coacervates of the invention. Furthermore, a higher concentration of biopolymer, preferably chitosan, can be added, since in coacervate form it releases more slowly, thus reducing potential phytotoxic reactions to crops at high doses, as occurs when it is added in liquid form (Lopez-Moya et al., 2017. Induction of auxin biosynthesis and WOX5 repression mediate changes in root development in Arabidopsis exposed to chitosan. Scientific reports, 7 (1), 16813). The buffer solution in which the linear cationic polymer is dissolved can be a weak acid solution, such as hydrochloric acid, acetic acid, citric acid, or other weak acid solutions that a person skilled in the art would be able to identify. Preferably, the weak acid buffer solution comprises glacial acetic acid and anhydrous sodium acetate in water, since when the linear cationic polymer is dissolved in this solution, it becomes more viscous / dense than when dissolved in buffer solutions of other weak acids, which result in a practically liquid solution. The increased viscosity / density facilitates the formation of the coacervates. The pH of the buffer solution is between 3 and 6.5, preferably between 3.5 and 6, more preferably between 4 and 6, even more preferably around 5. The concentration of the biopolymer in the buffer solution is between 0.5% and 20% by weight / volume (w / v), preferably between 1% and 15% w / v, more preferably between 2% and 10% w / v, even more preferably between 2.5% and 5% w / v. When the polymer is chitosan, the maximum concentration that can be used is around 5 mg / ml of buffer solution, preferably around 4 mg / ml of buffer solution, more preferably around 3 mg / ml of buffer solution. In another particular embodiment, the spores can be conidia and / or chlamydospores. In another particular embodiment, the fungus used as a biological control agent in agriculture is selected from entomopathogenic fungi, nematophagous fungi, and combinations of the above. In another particular embodiment, the fungus used for the biological control of agricultural pests is a fungus that is resistant to the concentration of chitosan used in the object of the invention and is capable of degrading it. In a particular embodiment, the spores of the nematophagous fungus Pochonia chlamydosporia are selected from among spores of the strains Pc123, IMI SD 187, Pc10, Pc104, Pc133, Pc147, Pc190, Pc280, Pc392, Pc400, Pc69, Pc399, Pc4624, Pc75, Pc21, PcCat, Pc132 and combinations thereof, preferably Pc123. As well as from other biological control agent fungi. Spores of the nematophagous fungus Pochonia chlamydosporia can be obtained from any source, preferably isolated from Heterodera avenae eggs in Seville (ATCC No. MYA-4875; CECT No. 20929). Spore collection (conidia and / or chlamydospores) can be carried out using any standard protocol in the field, for example, as described in the scientific article: Güerri-Agulló, B., López-Follana, R., Asensio, L., Barranco, P., & Lopez-Llorca, LV (2011). Use of a solid formulation of Beauveria bassiana for biocontrol of the red palm weevil (Rhynchophorus ferrugineus) (Coleoptera: Dr. Ophthoridae) under field conditions in SE Spain. Florida Entomologist, 737-747. The mixture to obtain a homogeneous dispersion can be stirred for a period of time between 1 min and 10 hours. In a particular embodiment, the spore concentration in the homogeneous dispersion is between 1*10⁴ spores / ml and 1*10⁸ spores / ml, preferably 1*10⁵ spores / ml and 1*10⁷ spores / ml, more preferably 5*10⁵ spores / ml and 5*10⁶ spores / ml. The addition of the homogeneous dispersion to the aqueous solution at basic pH is done by dripping, falling by gravity, from the first onto the second, for example, by using an injector between 0.5 mm and 6 mm in diameter, preferably between 1 mm and 5 mm in diameter, more preferably between 2 and 4 mm in diameter, even more preferably around 3 mm in diameter. More than 90% of the coacervates obtained by the method described above have a volume per coacervate between 0.0001 mm³ and 0.05 mm³, preferably between 0.001 mm³ and 0.01 mm³, and more preferably between 0.014 mm³ and 0.005 mm³. The diameter of the injector used is directly related to the diameter of the coacervates obtained. If the injector producing the droplet has a larger outlet diameter, larger coacervates will be obtained. It is important that the aqueous solution be at a basic pH because this is what causes the coacervate or matrix to form when it reacts with chitosan. The reaction of a strong base with chitosan causes a decrease in the number of free amino groups of the latter, which causes them to interact ionicly with the base, resulting in greater gelation efficiency and a higher degree of crosslinking than simply adding fungal spores and a polymer to an aqueous solution. In a particular embodiment, the basic pH aqueous solution is selected from a sodium hydroxide solution, potassium hydroxide solution, or a basic sodium tripolyphosphate solution, preferably sodium hydroxide. In this document, basic pH means a pH greater than 7, preferably greater than 8, more preferably greater than 9, even more preferably greater than 10, up to 14, the maximum pH. The aqueous solution at basic pH, preferably of sodium hydroxide, can be between 0.05% w / v and 20% w / v, preferably between 0.5% and 10% w / v, more preferably between 1% and 8% w / v, even more preferably between 1.5% and 5% w / v. In one particular embodiment, chitosan is dissolved in a buffer solution, and then conidia and chlamydospores of Pochonia chlamydosporia are added. The mixture is stirred for at least one hour to ensure homogeneous spore dispersion. The mixture is then dripped into a 2% w / v sodium hydroxide (NaOH) solution, generating solid particles of a coacervate composed of the polymer and the fungal spores. In a preferred embodiment, the method of the invention comprises dripping a homogeneous dispersion of chitosan with spores (conidia and chlamydospores) of the nematophagous fungus Pochonia chlamydosporia (strain 123), into a basic sodium hydroxide solution of about 2% w / v. The advantage of the method of the invention, specifically the combination of spores of a fungus used for the biological control of agricultural pests, preferably *Pochonia chlamydosporia*, with a biopolymer, preferably chitosan in coacervates, is that if this fungus colonizes the plant roots, it prevents other phytopathogenic fungi from doing so. Furthermore, the chitosan added to the soil promotes the growth of this fungus and also causes the plant to activate its defense systems, which complements the action of the biological control agent. Moreover, chitosan promotes crop growth at low doses. Adding chitosan in coacervate form results in the slow release of this biopolymer, allowing for the application of higher concentrations, up to 5 mg / ml, preferably around 2 mg / ml, which would not be harmful to the plants, unlike when added in liquid form.Furthermore, *Pochonia chlamydosporia* is able to degrade this substrate and feed on it, giving it an advantage over other fungi in growing and colonizing the soil and / or roots of crops. It also causes the release of the compound from the coacervate to produce toxicity in other microorganisms sensitive to chitosan. Additionally, the method of the invention comprises washing the obtained coacervates until the pH decreases below 10, preferably around 8.5. Any suitable solution for lowering the pH can be used, preferably autoclaved distilled water. Once the pH has been lowered, the coacervates must be dried under anexic conditions for at least 6 h, preferably 24 h. The coacervates can be dried, for example, in a laminar flow hood. They can then be stored in sterile containers with a humidity level below 30%. An additional object of the invention relates to coacervates obtained according to the method of the invention described above. In one particular embodiment, the coacervates are composed of chitosan and spores of the nematophagous fungus Pochonia chlamydosporia. Another object of the invention relates to a formulation comprising the coacervates described above and one or more inert substances used in agriculture. For example, emulsifiers, solvents, conidium carrier compounds, aerosol propellants, fragrances, and colorants. These may be present in a proportion, for example, of between 0 and 10% by volume relative to the total volume of the composition. All these substances are conventional and known in the prior art for this type of composition (Shinoda and Saito, Journal of Colloid and Interface Science, 1969). In a further embodiment, the inert substances are selected from the group consisting of: sodium tripolyphosphate, monopotassium phosphate, monoammonium phosphate, clay, talc, potassium nitrate, ammonium sulfate, amino acids, peptides, proteins, carboxylic acids, such as dicarboxylic acids, citric acid, ascorbic acid, glutamates, acetates, humates, potassium carbonate, potassium sorbate, potassium malate, citrulline malate, urea, potassium chloride, silica gel, oxalate, zeolite, seaweed extracts, alginates, diatomaceous earth, xanthan gum, gum arabic, methylcellulose, agarose, acrylamide, gels, lignin, pectin, lignosulfonates, molybdates, diammonium phosphate, mica, kaolin, fructose, sucrose, chitosan, calcium hydroxide, quartz sand, bicarbonate sodium, whey (e.g., lactoseura) or mixtures thereof. The inert substance or substances serve an important function, such as improving the formulation in aspects like solubility, stability, and providing anti-caking, anti-wetting, and / or coloring effects. Some of these properties are also essential for marketing the product in good condition and suitable for use. In the event that any of the inert substances mentioned above could have biological activity on their own, that is, perform an additional function beyond those indicated in the previous paragraph, it will be understood that the concentration of said inert substances in the formulation must be less than that necessary to show biological activity. Another additional object of the invention relates to the use of the coacervates described above and / or produced by the method described above in agriculture. In one particular embodiment, the use in agriculture includes the control of pests and / or diseases in agricultural crops. In one particular embodiment, chitosan in coacervate form causes a slow release of this biopolymer and higher concentrations can be applied, for example, up to 5 mg / ml, preferably around 2 mg / ml, which would not be harmful to plants. In another particular embodiment, pests and / or diseases are caused by a pathogen selected from nematodes, insects, and combinations of the above. Another additional object of the invention relates to the use of the coacervates described above and / or produced by the method described above as biofertilizers, biostimulants and / or inducers of defense mechanisms in agricultural crops. Another additional object of the invention relates to the coacervates described above or produced by the method described above for use in veterinary medicine, preferably for use in the treatment of intestinal nematodes in animals. BRIEF DESCRIPTION OF THE FIGURES Figure 1: Germination of Pochonia chlamydosporia from chitosan coacervates (n=10) with a degree of deacetylation of 90.1%, at 2% NaOH in an incubation period of 19 days on water agar. Figure 2: Development of Pochonia chlamydosporia from a chitosan coacervate with a degree of deacetylation of 90.1%, at 2% NaOH in an incubation period of 19 days in water agar growth medium. Figure 3: Chitosan coacervates and Pochonia chlamydosporia spores obtained with 2% NaOH in fresh. Figure 4: Chitosan coacervates and Pochonia chlamydosporia spores obtained with 2% dry NaOH. Figure 5: Chitosan coacervate and spores of the fungus Pochonia chlamydosporia transformed with GFP (Green Fluorescent Protein), obtained with 2% NaOH, viewed under a confocal microscope. The image shows the chitosan coacervate matrix (dark green autofluorescence) and within it, the hyphae of the fungus Pochonia chlamydosporia GFP growing (green fluorescence). EXAMPLES Fungus A species of nematophagous fungus (Pochonia chlamydosporia) was used. The fungus Pochonia chlamydosporia strain 123 (Pc123), Pc123 was isolated from Heterodera avenae eggs, in Seville (ATCC No. MYA-4875; CECT No. 20929). The fungal cultures were maintained in darkness at 4°C on cornmeal agar (CMA; BBL Sparks, MD). For the use of the fungus Pc123 to produce chitosan coacervates, solid formulations of the fungus were prepared from rice substrate (Orgyza sativa) according to Güerri-Agulló, B., López-Follana, R., Asensio, L., Barranco, P., & Lopez-Llorca, LV (2011). Use of a solid formulation of Beauveria bassiana for biocontrol of the red palm weevil (Rhynchophorus ferrugineus) (Coleoptera: Drugy Ophthoridae) under field conditions in SE Spain. Florida Entomologist, 737-747. 250 ml flasks were used, to which 75 g of rice were added as a substrate. A solution of conidia and chlamydospores was obtained from these flasks using sterile distilled water. Once the inoculum was extracted, it was counted in a Neubauer chamber. Alternatively, the procedure for obtaining fungal spores described in patent P201931071 can be followed. Polymer The polymer used to form the coacervates was a biopolymer, specifically chitosan, more specifically chitosan T8 (Chitosan 90 / 60 / A1) with a particle size less than 0.5 mm, with a loss on drying of 5.2%, with an ash content of 0.1%, dynamic viscosity 20 °C (± 5%) (1% sol. In 1% acetic acid sol.) of 69.0 mPa.s (millipascals), information provided by the commercial manufacturer of the biopolymer, a degree of deacetylation of 90.1% and a concentration of heavy metals less than 50 ppm. Preparation of coacervates To dissolve chitosan, the following protocol developed by the Faculty of Pharmacy of the Complutense University of Madrid is followed. To prepare the buffer solution in which the chitosan powder will be dissolved, we first prepare two stock solutions of glacial acetic acid (3M; pH 2.09) and anhydrous sodium acetate (2M; pH 8.62), adding 25.7 ml of glacial acetic acid and 24.61 g of anhydrous sodium acetate to a final volume of 150 ml adjusted with distilled water respectively. From these solutions, we prepared another solution by adding 10 mL of glacial acetic acid and 10 mL of anhydrous sodium acetate from the solutions prepared according to the previous paragraph, and diluting to 100 mL with distilled water, obtaining a 0.3 M and 0.2 M solution of each compound. The pH of the final solution was adjusted to 5 using the stock of 2 M anhydrous sodium acetate. When the solutions were mixed, the pH was around 4.2. Since glacial acetic acid is more acidic, it was necessary to add sodium acetate to raise the pH to around 5, as it is more basic. The resulting buffer solution was autoclaved. Once the buffer solution is prepared, 100 ml of it is taken and chitosan 3% (3 g in 100 ml) is added and the mixture is stirred for 24 hours, in a magnetic stirrer. A 2% w / v NaOH solution is prepared by adding 20 g of NaOH (1M) to 1 L of distilled water and autoclaving it to ensure sterility. Prepare sterile material, distilled water (7 L), a 1 L beaker, a stainless steel strainer (pore size of 2 mm) and filter paper. After 24 hours of agitation, the conidia are counted as previously described in a Neubauer chamber, and the necessary volume is added to obtain a final concentration of 1 × 10⁶ conidia / ml. The final conidial suspension, mixed with chitosan, is agitated for one hour to obtain a homogeneous mixture. This mixture is then dripped onto the 2% NaOH solution using a 20 ml syringe. (Approximately 20 drops of 50 microliters each are obtained per ml, generating solid coacervates with a volume of 0.0025 mm³ per coacervate). The volume of the drops was calculated as follows: The chitosan solution was drawn up with a syringe, and the number of drops per milliliter of solution was measured. This was repeated three times, and the average was calculated. The result was that each drop contained 50 microliters of solution, and from this, the volume was calculated in cubic millimeters. Once the coacervate formation process is complete, the coacervates are removed from the 2% NaOH solution and washed with sterile distilled water until the pH of the wash water is reduced to approximately 8.5. The initial pH is usually 12. When the coacervates reach the indicated pH, they are placed on sterile filter paper for drying in a vertical laminar flow hood (TELSTAR BV-100) for at least 24 hours. After this time, they are collected and stored at room temperature in sterile containers. Germination of the coacervates of the invention Once dry, a chitosan coacervate is placed in a 90 mm Petri dish with European Type Bacteriological Agar (WA; PanReac). The plate is observed daily until the fungus germinates from the coacervate with Pochonia chlamydosporia spores. (Figures 1-4). In the agar-water medium there are no nutrients available for the development of the fungus, this experiment demonstrates that Pochonia degrades the chitosan of the coacervate and uses it for its growth, the time is the same as it takes for the fungus to grow in a normal culture medium (Figure 2). Figure 3 shows freshly made coacervates washed with distilled water at neutral pH. Figure 4 shows dried coacervates after 24 hours of air drying. They are dry, not dehydrated, because adding water does not return them to their initial (fresh) state before drying. Drying coacervates has the advantage of increasing their shelf life under optimal conditions for application. Figure 5 shows coacervates obtained with spores of the same fungus but expressing the GFP protein, which allows visualization of fungal hyphal growth within the coacervate using confocal microscopy and fluorescence. As can be seen, the fungus develops correctly within the coacervate, and its inclusion within the chitosan coacervate does not affect its biological function of pest control.

Claims

1. A method for obtaining coacervates from: - a linear cationic polymer comprising at least one monomer that is an amino monosaccharide and - spores of at least one fungus used as a biological control agent in agriculture, comprising the following steps: - dissolving said linear cationic polymer in a weak acid buffer solution, - adding spores of at least one fungus used as a biological control agent in agriculture to the solution from the previous step, and stirring to obtain a homogeneous dispersion, - adding the homogeneous dispersion from the previous step to an aqueous solution at basic pH, obtaining solid particles of a coacervate formed by the polymer and the spores.

2. The method according to the preceding claim, wherein the linear cationic polymer comprises at least two different monomers.

3. The method according to any one of the preceding claims, wherein the linear cationic polymer is chitosan. 4.Method according to the preceding claim, wherein the chitosan has a degree of deacetylation of between 60% and 100%.

5. Method according to any one of the preceding claims, wherein when the polymer is chitosan, the maximum concentration of said polymer in the buffer solution is 5 mg / ml.

6. Method according to any one of the preceding claims, wherein the buffer solution is a solution of glacial acetic acid and anhydrous sodium acetate in water at a pH between 3 and 6.

5.

7. Method according to any one of the preceding claims, wherein the concentration of the biopolymer in the buffer solution is between 0.5% and 20% w / v.

8. Method according to any one of the preceding claims, wherein the fungus used as a biological control agent in agriculture is a fungus that is resistant to the concentration of chitosan used in the invention and is capable of degrading it. 9.Method according to any one of the preceding claims, wherein the fungus used as a biological control agent in agriculture is selected from entomopathogenic fungi, nematophagous fungi and combinations thereof.

10. Method according to the preceding claim, wherein the nematophagous fungus is selected from one or more of the following strains of Pochonia chlamydosporia: strain Pc123, IMI SD 187, Pc10, Pc104, Pc133, Pc147, Pc190, Pc280, Pc392, Pc400, c69, Pc399, Pc4624, Pc75, Pc21, PcCat, Pc132 and combinations thereof, preferably Pc123.

11. A method according to any one of the preceding claims, wherein the spore concentration in the homogeneous dispersion is between 1*10⁴ spores / ml and 1*10⁸ spores / ml.

12. A method according to any one of the preceding claims, wherein the addition of the homogeneous dispersion to the basic pH aqueous solution is carried out by dripping the former onto the latter. 13.A method according to any one of the preceding claims, wherein the basic pH aqueous solution is selected from a sodium hydroxide solution, potassium hydroxide solution, or a basic sodium tripolyphosphate solution, preferably sodium hydroxide.

14. A method according to the preceding claim, wherein the sodium hydroxide solution is between 0.05% and 20% w / v.

15. A method according to any one of the preceding claims, wherein the coacervate solid particles have a volume per coacervate of between 0.0001 mm³ and 0.05 mm³.

16. A method according to any one of the preceding claims, comprising washing the coacervate solid particles until the pH decreases below 10.

17. A method according to the preceding claim, comprising drying the coacervate solid particles under anexic conditions for at least 6 hours. 18.Coacervates of a polymer and spores of a fungus used as a biological control agent in agriculture obtained according to any one of claims 1 to 17.

19. Coacervates according to the preceding claim, wherein the polymer is chitosan and the fungus used for the biological control of agricultural pests is a nematophagous fungus, preferably Pochonia chlamydosporia. 20.A formulation comprising coacervates according to any one of claims 18 to 19 or obtained according to the method described in any one of claims 1 to 17, and one or more inert substances used in agriculture selected from: sodium tripolyphosphate, monopotassium phosphate, monoammonium phosphate, clay, talc, potassium nitrate, ammonium sulfate, amino acids, peptides, proteins, carboxylic acids such as dicarboxylic acids, citric acid, ascorbic acid, glutamates, acetates, humates, potassium carbonate, potassium sorbate, potassium malate, citrulline malate, urea, potassium chloride, silica gel, oxalate, zeolite, seaweed extracts, alginates, diatomaceous earth, xanthan gum, gum arabic, methylcellulose, agarose, acrylamide, gels, lignin, pectin, lignosulfonates, molybdates, phosphate diammonium, mica, kaolin, fructose, sucrose, chitosan, calcium hydroxide, quartz sand, sodium bicarbonate, whey and mixtures thereof. 21.Use of the formulation described in claim 20, or the coacervates according to any one of claims 18 to 19, or obtained according to the method described in any one of claims 1 to 17, in agriculture.

22. Use according to the preceding claim, wherein agriculture comprises the control of pests and / or diseases in agricultural crops.

23. Use according to claim 21, as biofertilizers, biostimulants, and / or inducers of defense mechanisms in agricultural crops.

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