BR102020002179A2 - BIOPESTICIDES NANOFORMULATIONS BASED ON SECONDARY METABOLITES AND MICRONUTRIENTS, THEIR PROCESS FOR OBTAINING AND THEIR USE IN PATHOGEN AND PEST CONTROL - Google Patents

Abstract

biopesticidal nanoformulations based on secondary metabolites and micronutrients, their production process and their use in the control of pathogens and pests. the present invention is situated in the field of nanotechnology. these are nanoformulations based on carbon-dots and copper and carbon-dots and essential oils of citronella, cloves and sweet orange. such nanoformulations have antifungal and larvicidal activity, therefore, useful for the development of biopesticides to be used in the control of pathogens and pests.

Description

BIOPESTICIDES NANOFORMULATIONS BASED ON SECONDARY METABOLITES AND MICRONUTRIENTS, THEIR PROCESS FOR OBTAINING AND THEIR USE IN PATHOGEN AND PEST CONTROL FIELD OF THE INVENTION

[001] The present invention is located in the field of nanotechnology. These are nanoformulations based on Carbon-dots and copper and Carbon-dots and essential oils of citronella, cloves and sweet orange. Such nanoformulations have antifungal and larvicidal activity, therefore, useful for the development of biopesticides to be used in the control of pathogens and pests.

TECHNICAL STATUS

[002] Plant diseases are caused by pathogens, which lead to significant losses in agricultural production, and the control of these pathogens is extremely important for the agro-industrial market.

[003] An example of this is coffee rust, the main disease of the coffee tree, which is annually responsible for significant financial losses, as it directly interferes in the production of Arabica coffee (Coffea arabica L.) (TALHINHAS, Pedro, et al. Molecular Plant Pathology, 18: 1039-1051, 2017).

[004] In addition to economic issues related to agribusiness, pest control is also a public health issue. The control of diseases transmitted by insects such as dengue, yellow fever, chikungunya and Zika is a problem mainly in countries with tropical and subtropical climate, since there are no effective vaccines and specific medications for these viruses, making it necessary to control of the mosquito vector of transmission through insecticides that act in a repellent and/or larvicide manner (YOON, Jong, et al. Journal of Parasitology Research, 2015: 1-6, 2015).

[005] Given this problem, one of the solutions for pest control is the use of pesticides, a general name for chemical compounds developed to control pests, which can be plants, fungi or animals. There are several types of pesticides, for each specific pest, including: herbicides, insecticides, bactericides, fungicides, biopesticides, animal repellents and others. The main use of pesticides is in agriculture, with the purpose of protecting crops against plant pathogens (bacteria, fungi and viruses), insects and weeds (RAMADASS, Manjula; THIAGARAJAN, Padma. IOP Conf. Series: Mater. Sc. Eng, 263: 1-12, 2017).

[006] Over the last few decades, the Green Revolution has promoted a significant increase in food and feed production based on the indiscriminate use of agrochemical inputs (MOSSA, Abdel-Tawab H. Journal of Envir. Sc. Tech., 9: 354 -378, 2016). Unrestrained applications of conventional pesticides have contributed to increased resistance to pests and diseases, the resurgence of previously controlled pests, genetic anomalies in plants and animals, and the presence of toxic residues in food. Furthermore, the dependence on synthetic pesticides and their indiscriminate application are also responsible for several harmful effects on the environment.

[007] The extensive use of pesticides for sanitary use in agriculture is associated with toxicity to the environment, contributing to the presence of toxic material on the surface of the soil and groundwater and, in addition, can be toxic to the health of plants and animals (DUHAN, Joginder et al. Biotechnology Reports, 15: 11–23, 2017).

[008] In the case of arthropod insect control, such as Aedes aegypti L., for example, insecticides based on the compound DEET (N,N-diethyl metatoluamide) are often used, which is associated with some severe adverse effects, such as sensory disturbances and alteration in motor ability, memory and learning ability (YOON, Jong, et al. Journal of Parasitology Research, 1-6, 2015). Furthermore, DEET in high concentrations can cause encephalopathy and other side effects in children (LEE, Mi Young. BioMed Research International, 1-9, 2018).

[009] One of the alternatives to traditional chemical pesticides is the use of biopesticides. Biopesticides are compounds that contain biocontrol agents such as natural organisms (such as bacteria, fungi, plants or animals) or substances derived from living beings, including their genes and/or metabolites, for pest control.

[010] Such compounds can be classified into: microbial pesticides, plant-incorporated protectants (PIPs) and biochemical pesticides (SPORLEDER, Marc; LACEY, Lawrence. Academic Press, 463–497, 2013).

[011] These agents are highlighted as they are environmentally safe, target-specific, biodegradable, and suitable for integrated pest management (IPM) programs (MOSSA, Abdel-Tawab H. Journal of Envir. Sc. Tech., 9: 354-378, 2016).

[012] Biochemical pesticides refer to chemicals that are wholly or partially derived from biological sources, such as, for example, plants, algae, crops, animals and other organic wastes, such as by-products from the food chain. They are natural substances, such as metabolites, hormones, plant extracts and natural insect growth regulators, that control pests through non-toxic mechanisms (SPORLEDER, Marc; LACEY, Lawrence. Academic Press, 463–497, 2013).

[013] Unlike traditional chemical pesticides, those biochemicals do not need to directly eliminate pests, but can control their multiplication and prevent the development of the biological cycle, in addition to being more specific (VISHWAKARMA, Kanchan et al. Academic Press, 473–500 , 2018).

[014] Plant extracts comprise several types of metabolites (alkaloids, terpenoids and phenolic compounds) and secondary chemicals, a complex mixture that the plant has developed to protect itself from different pests. These chemical compounds are bioactive agents against pathogens and other pests, and have a mode of action through control (being repellent or interfering in the biological cycle) and/or lethal effect (eliminating pests) (PINHEIRO, Patrícia Fontes et al. Ciênc. agrotec. Lavras, 37: 138–144, 2013). Thus, these metabolites are used as biopesticides for the protection of agricultural crops (SPORLEDER, Marc; LACEY, Lawrence. Academic Press, 463–497, 2013).

[015] Essential oils (EOs) are also bioactive, biodegradable, non-toxic molecules with potential use in pest management, as they are ecological sources that have activity against agents such as larvae and adult mosquitoes. EOs are a mixture of volatile, lipophilic and low molecular weight compounds, whose most common constituents are terpenoids and phenylpropanoids (PAVELA, Roman, et al. Ind. Crops Prod., 129: 631–40, 2019).

[016] In contrast to synthetic pesticide molecules, EOs are particularly attractive because most of them are recognized as safe by the Food and Drug Administration (FDA) and the EPA (Environmental Protection Agency). EOs are not only easy to handle at work, but the presence of different bioactive compounds reduces the chance of resistance development. Currently, more than 3000 constituents have been identified and some of them have shown insecticidal effects against various types of pests (MOSSA, Abdel-Tawab H. Journal of Envir. Sc. Tech., 9: 354-378, 2016).

[017] All these characteristics are indicative that biopesticides based on EOs could be applied to control a wide variety of pests, including pests caused by insects, since recent reports have shown that some chemical constituents of these EOs can specifically interfere with the system octopaminergic nervous system of these animals, without presenting risk to mammals (JANKOWSKA, Milena, et al. Molecules, 23: 1-20, 2018).

[018] Although the biological activities of EOs are promising, some limitations need to be overcome for their use on a large scale, such as volatility, low water solubility and easy oxidation by exposure to light and heat. (MOSSA, AbdelTawab H. Journal of Envir. Sc. Tech., 9: 354-378, 2016).

[019] In the State of the Art works that report the application of EOs as natural pesticides are found.

[020] Pereira et al. (2012) describe the use of citronella EO (extracted from Cymbopogon nardus) as a potential fungicide in coffee cultivars. In this study, citronella EO partially controlled coffee rust (Hemileia vastatrix) and brown spot (Cercospora coffeicola) with efficacy of 47.2% and 29.7%, respectively. In addition to activating the plant's defense system, promoted by the increase in the activities of the enzymes peroxidase and chitinase, as well as the accumulation of lignin in the coffee leaves after 14 days of spraying the OE (PEREIRA, Ricardo Borges et al. Ciênc . agrotec, 36: 383-390, 2012).

[021] Mahakittikun et al. (2014) showed the use of clove EO (extracted from Eugenia caryophyllus) as a potential acaricide, bactericide and fungicide. In this study, an herbal extract (containing 2% clove EO) had its biological activity tested against house dust mites (Dermatophagoides pteronyssinus), bacteria (Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa) and fungi (Candida albicans and Aspergillus fumigatus) . The clove EO solution showed expressive acaricide activity, with a 99% mortality rate for the mite. In addition, the solution also showed inhibitory activity on fungal and bacterial growth (MAHAKITTIKUN, Vanna et al. Asian Pacific jour. all. immun., 32: 46–52, 2014).

[022] Metoui et al. (2015) reported the use of sweet orange EO (extracted from Citrus aurantium) as a potential fungicide. Antifungal activity tests were carried out for the fungi Fusarium oxysporum, Fusarium solani, Botrytis cinerea, Bipolaris sorokiniana and Fusarium avenaceum, in which a growth inhibition rate of 80% was observed for all tested fungi, showing significant antifungal activity for the concentration of 4 µL∙ml-1 of the EO (METOUI, Nadia et al. Natural Product Research, 29: 2238-2241, 2015).

[023] Thus, the works cited indicate that the EOs of citronella, clove and sweet orange have antimicrobial activity with potential for application as a biopesticide to control insect-pests and diseases caused by fungi and bacteria. However, problems such as volatility, low solubility in aqueous media, stability of EOs and delivery of bioactive molecules to the target site are not addressed by the works described. Thus, the present invention surpasses the studies described because it is a nanoformulation in which the EOs are associated with carbon-dots nanoparticles. This association contributes to the reduction of EO volatilization rates, as well as to the improvement in dispersion in aqueous media and targeted delivery of bioactive molecules.

[024] In addition to EOs, some micronutrients, such as copper, can be used as bioactive agents in the control of pathogens and pests (RENNÓ, Marcelo, et al. SPCB, 8: 220-226, 2013).

[025] Copper is an important chemical element in the health of plants and animals, and copper deficiency in agricultural crops is supplemented by the use of fertilizers and nanofertilizers (VISHWAKARMA, Kanchan et al. Academic Press, 473–500, 2018) .

[026] Copper is in the composition of several fungicides used in the world agrochemical market (ELGUINDI, Jutta, et al. Appl. Microb. Biotech., 91: 237–249, 2011).

[027] Regarding the concentration of copper to be used in pest control, it is noteworthy that the formulations should be established below 100 ppm. However, current agrochemicals go beyond this parameter and, therefore, present toxicity (CAMPOY, Sonia; ADRIO, José L. Biochemical Pharmacology, 133: 86–96, 2017).

[028] Copper is accepted and widely used in organic agriculture, as a composition of Bordeaux mixture (copper sulphate with quicklime diluted in water) (BRAZIL. Ministry of Agriculture, Livestock and Supply. Normative Instruction No. 46, of 6 October de ; CIDADE JUNIOR, HA; SOURCE, NN da; CAMARGO, RFR Basic information on organic agriculture. Curitiba: SENAR-PR, 2007, 128p. HENZ, GP; ALCÂNTRA, FA; RESENDE, FV Organic production of vegetables: the producer asks , Embrapa answers. Brasília: Embrapa technological information, 2007, 308p; IT'S EASY TO DO! CALDA BORDALESA 1%, Vitória: Alternative Technologies Project – FASE, 1986, 4p).

[029] In this sense, nanotechnology is a possible alternative to solve the problem of high concentrations of copper used in the formulation of traditional chemical pesticides. The development of nanoscale formulations with bioactive agents, the so-called nanobiopesticides, makes it possible to make the metal available in a targeted way, reducing its concentrations in the medium (HAYLES, John et al. Academic Press, 193–225, 2017).

[030] For this, it becomes necessary to choose a system for carrying to the target through the selection of nanomaterials. With the definition of the nanoparticles that will be used in the construction of the nanoformulations, the physical and chemical characteristics of these nanomaterials will lead to the successful application of the final product (PODDAR, Kingshuk et al. Woodhead Publishing, 281–303, 2018).

[031] In this context, the present invention proposes a nanoformulation based on the association of copper to Carbon-dots nanoparticles.

[032] Carbon quantum dots, Carbon-dots, is a class of spheroidal nanoparticles, with dimensions smaller than 10 nm, composed of a highly ordered polyaromatic domain (core) covered by an amorphous carbon border (CHOI, Yuri et al. Chem. Asian J., 13: 586-598, 2018; KWON, Woosung et al. Scientific Reports, 5: 12604-12614, 2015).

[033] Carbon-dots have characteristics that can be exploited in various applications, such as spectroscopic properties, low toxicity, biocompatibility and good chemical and physical robustness. Carbon-dots have emerged as a promising nanomaterial that may meet the need for the development of green pesticides (LUO, Pengju et al. RSC Advances, 4:10791-10807, 2014).

[034] Another important aspect is related to the perspective of occupational safety, as agricultural workers are chronically exposed to pesticides during their work routine. However, as it is a non-toxic material, human exposure to Carbon-dots does not cause health problems for workers.

[035] The results presented by Zheng et al. (2015) showed that citric acid carbon-dots did not show toxicity (acute and subacute, LD50% ~ 400 mg∙kg-1) or genotoxicity in mice. In this study, Carbon-dots were administered intravenously and, after a few hours, were excreted through the urinary tract without any evidence of bioaccumulation (ZHENG, Xiao, et al. RSC Advances, 5: 91398–406, 2015).

[036] Therefore, nanoformulations may have unique useful characteristics compared to their bulk material analogues, due to the small size and huge surface area/volume ratio, in addition to increasing the solubility and dispersion of bioactive agents and even synthetic chemicals in water. Such formulations can increase the bioavailability of active compounds, reduce the toxicity inherent to certain agents and deliver specifically and gradually to target sites (ATHANASSIOU, Christos, et al. J Pest Sci, 91: 1-15, 2018).

[037] The international publication WO2010/068275A1 reports on a silica-based nanoformulation that can be used to treat citrus canker caused by the bacteria Xanthomonas axonopodis, inhibit mold and mildew growth and add nutrients to the soil. It is a nanostructure based on copper-loaded silica gel (CuSiNG), which uses silica nanoparticles that have an exposed hydroxyl group on their surface. However, this characteristic limits the type of molecule that will be conjugated on the surface of the nanomaterial, reducing its carrying applicability. As it is a nanogel, the mode of application and the type of treatment are limited (WU, Si-Han, et al. Chem. Soc. Rev., 42: 3862-3875, 2013).

[038] The present invention overcomes such limitations, since, depending on the starting material for the synthesis of Carbon-dots, nanoparticles have different functional groups that enable interaction with different molecules, in addition to being a smaller carrier (diameter <10 nm), which improves dispersion and incorporation of the material. These characteristics extend the field of application of nanoformulations based on Carbon-dots.

[039] Already the international publication WO2017/172634A1 reports on the composition of nanoparticles and method for targeted delivery of a bioactive agent in plants. These are coronatin-coated nanoparticles produced from a planetary ball mill comprising a nano-matrix core, a release coating layer and at least one bioactive agent, the release coating layer being based on polycaprolactone (PCL) and polyethylene glycol (PEG). The composition of these nanoparticles was developed to provide the bioactive agent through plant stomata.

[040] The nanoparticles obtained in the patent document are polydispersed, with an average size ranging between 5 and 1000 nm. Considering that the particle size distribution influences the formulation properties, such as dispersion in aqueous media and bioavailability, obtaining particles with a wide size distribution range can limit the application of these materials (PODDAR, Kingshuk et al. Woodhead Publishing, 281–303, 2018).

[041] In addition, it is inferred that because it consists of coronatine, PCL and PEG, the ability to use this technology is reduced, since such polymeric nanomaterials are limited in terms of carrying molecules, in addition to being quickly removed from the living organism without reaching the intended active sites (Kumari, Avnesh et al. Coll. Surf. B: Biointerfaces, 75: 1-18, 2010). Therefore, the nanoformulations based on Carbon-dots of the present invention surpass the invention of the aforementioned patent document by presenting a greater capacity for carrying bioactives in different target sites in the treated object.

[042] That said, the State of the Art does not foresee or suggest the development of nanoformulations based on Carbon-dots containing micronutrients and EOs as biostimulants and biopesticides for applications in agriculture and in the control of the mosquito vector for transmission of arboviruses, as presented herein invention.

[043] In addition, the present invention is characterized as being safe nanoformulations, little degrading in relation to its bioactive agents, with biopesticide feasibility and provided with target-oriented release of bioactive agents. Furthermore, the proposed nanoformulations enable the increase of the solubility of active compounds that are poorly soluble in aqueous media.

BRIEF DESCRIPTION OF THE FIGURES

[044] The invention can be better understood based on Figures 1 to 8, the description of which follows below:

[045] Figure 1 presents the “process flow chemistry” scheme.

[046] Figure 2 shows, on the left, the leaf discs of coffee leaf accommodated in a Petri dish, on the right, fluorescence microscopy image of Hemileia vastatrix spores.

[047] Figure 3 shows the graph that shows the inhibition of germination of Hemileia vastatrix spores as a function of different treatments.

[048] Figure 4 shows the larvicide bioassay with L3 larvae of Aedes aegypti. The 12-well plates used to accommodate larvae during the assay for periods of 24 to 72 hours are shown.

[049] Figure 5 shows the graph that shows the mortality rate of L3 larvae of Aedes aegypti as a function of different treatments.

[050] Figure 6 shows the graph that shows the inhibition of the mycelial diameter of the fungus Lasiodiplodia theobromae as a function of different treatments, evaluated after 48 hours of testing.

[051] Figure 7 shows the images of Petri dishes containing the fungus Lasiodiplodia theobromae and its inhibition to different treatments for a period later than 26 hours, in which "A" is observed the control treatment BDA (Potato-Dextrose- Agar), in "B" the treatment with Carbon-dots is observed, in "C" is observed the treatment with citronella essential oil (OEC), in "D" the treatment with Cdots-OEC nanoformulation is observed and in " E” the treatment with the C-dots-Cu nanoformulation is observed.

[052] Figure 8 shows the images of Petri dishes containing the fungus Lasiodiplodia theobromae and its respective inhibition to different treatments for a period later than 120 hours.

DETAILED DESCRIPTION OF THE INVENTION

[053] The present invention relates to biopesticidal nanoformulations based on Carbon-dots and copper and Carbon-dots and OEs of citronella, clove and sweet orange, obtained from the binding of copper and essential oils on the surface of constituted Carbon-dots with carboxyl, hydroxyl and amine groups.

[054] The incorporation of EOs and copper in Carbon-dots nanoparticles gives biopesticidal nanoformulations characteristics such as: target-oriented release of bioactive agents, reduced degradation of these agents, greater efficiency in action, with reduced dose used in control of a given pathogen, and for EOs, reduced volatilization rates and increased solubility in aqueous media.

[055] In addition, nanoformulations are made with non-bioaccumulative nanomaterials, their use reduces the adverse impact of trophic transfer of conventional nanoparticles in the food chain, and non-toxic, when used for crop protection and control mosquito vectors of transmission arboviruses reduce the environmental impact in relation to soil and water contamination, compared to conventional agrochemicals and do not harm the human body.

[056] One of the modalities of the present invention refers to the process of obtaining nanoformulations based on Carbon-dots, through a continuous flow reaction, as shown in Figure 1.

• (A) Formação de nanopartículas de Carbon-dots;
• (B) Purificação das partículas;
• (C) Modificação da superfície das nanopartículas com compostos bioativos.

[057] In general, this modality can be understood through the process of obtaining the Carbon-dots and surface modifications that consist of the following steps:

• (A) Formation of Carbon-dots nanoparticles;
• (B) Particle purification;
• (C) Surface modification of nanoparticles with bioactive compounds.

[058] Step (A) consists of the formation of carbon-dots nanoparticles. The nanoparticles are obtained from different carbon sources, and citric acid, glucose, amino acids and agricultural residues such as fruit peels, bagasse and animal excrement can be used, preferably citric acid, due to the partial acid neutralization reaction -base in which a basic solution, a source of a hydroxyl functional group, is used to form the acid salt and contributes to the formation of several nucleation points through the electrostatic repulsion induced by ions (OH-) in solution. The bases used can be calcium hydroxide, sodium hydroxide, potassium hydroxide, ammonium hydroxide, aniline, metalamine, urea and thiourea, preferably ammonium hydroxide and urea.

[059] An acidic solution, carbon source, and a basic solution, functional group source, are prepared and placed in the flow system.

[060] With the use of a dosing pump and a valve, these solutions are dosed in a mixer, which then presents another mixing vessel in the region where it allows to receive the recycle and dopants (micro and macronutrients or stimulants). The reagents, after passing through the mixers, are pumped into a tubular piston flow reactor (PFR) which is equipped with a valve that restricts the output of the material, maintaining a constant pressure inside, preventing the vaporization of the reagents, as this reactor is heated range from 100 to 250 °C, preferably at 150 °C.

[061] The PFR reactor must have a reduced diameter only a few millimeters and also be constructed of material with little roughness to reduce pressure loss during material flow. Another important factor in the process is to maintain a turbulent hydrodynamic flow regime, all of which must be calculated by the equation of motion Bernoulli's Equation, as this reactor must be accommodated inside a greenhouse for heating the mixture.

[062] This reactor must be heated to a temperature between 100 and 250 ºC, depending on the concentration of the material used, the pressure inside the reactor is controlled by the outlet valve preventing the solution from boiling, the pressure will vary in reaction for reaction depending on the density of the salts involved. This pressure is high, so the material of construction of the reactor must withstand a pressure of up to 5 bar. The material flow that leaves this first reactor, the tubular, discharges into a continuous stirred tank reactor (CSTR) which is also heated and has agitation, the exit flow of this reactor goes through a separation system, which works by density difference hydrocyclone or decanter that allows the removal of the finished product by overflow and returns the underflow stream to reflux.

[063] If necessary, a backflow of the material until the optimal operating conditions are established. The product formed has a density lower than the solution of precursor salts, therefore, the product can be separated by this principle, removing the supernatant and recirculating the bottom material, inserting it before the reactor, adding the initial current of the reactants. Until the stabilization process, in which the material is refluxed, lasts between 0.5 and 2 hours, preferably 1 hour, from that period onwards, the density of the material starts to change and to form the product.

[064] This process has a yield of 95% (713 g after 2 hours of cycle. This amount of material is enough to be applied in 24 hectares.

[065] This product can be stored liquid or go through a drying process, in which the material expands its surface area, increasing its volume by approximately 05 times.

[066] Step (B) consists of the purification of Carbon-dots nanoparticles. After cooling to room temperature, the solution obtained in step A is subjected to centrifugation at 10,000 rpm and then filtered to remove salt and unreacted residues.

[067] Step (C) consists of modifying the surface of the nanoparticles obtained in step (B). The modification process is carried out by incorporating EOs such as citronella, clove, sweet orange, eucalyptus, mint and copaiba, preferably using citronella, clove and sweet orange EOs or even with the insertion of bioactive elements such as copper , phosphorus, magnesium and aluminium, with copper being preferably used.

[068] In surface modification processes, citronella, clove and sweet orange EOs are loaded onto Carbon-dots particles through non-covalent interactions between the COOH, OH and NH2 groups on the surface of the nanoparticles and the functional group on the EOs, with mass fractions between 0 and 100% of each essential oil, preferably 3%. To modify the nanoparticles in a solution of 50 mg/L of Carbon-dots, 3% essential oil is slowly added under constant agitation. After the addition of essential oils, the container is capped and remains under agitation for 60 minutes at 40oC. The biopesticides solutions produced are kept at 10 oC and protected from light. Storage is an important parameter to maintain the larvicidal, fungicidal, bactericidal and insecticidal activities of the formulations for 12 months.

[069] Regarding the incorporation of copper, the insertion into the nanoparticle is performed by adding Cu and Cu2+, preferably Cu2+. Copper cations are loaded onto Carbon-dot particles through coordinated bonds between functional groups on the surface of nanoparticles and copper atoms arising from copper(II) complexes. For this procedure, a solution of 50 mg/L of Carbon-dots and 30-110 mg/L of copper ions are pumped to a mixer at room temperature. Then the solution is pumped into a tubular reactor (PFR) at 100 °C in order to ensure coordination of the ions on the surface of the particles. The biopesticides solutions produced are kept at 10 °C and protected from light. Storage is an important parameter to maintain the larvicidal, fungicidal, bactericidal and insecticidal activities of the formulations for 12 months.

[070] The technology for the production of Carbon-dots presents scaling characteristics that allow a feasible transition to industrial scale without negative impacts on the quality of the formed Carbon-dots. Tests carried out with a scale-up of 100 times the production of the C-dot produced on a bench, verified the possible production and maintenance of the same properties and qualities. Under a high demand for low environmental impact production, as required in the agricultural inputs industry, this technology is disruptive and cohesive with today's new production demands.

[071] Versatility is also one of the main features of the production platform developed, one that allows for variation in its composition and properties in order to meet the demands of the production chain.

[072] Another modality of the present invention refers to the use of nanoformulations based on Carbon-dots and copper and Carbon-dots and essential oils of citronella, clove and sweet orange in the production of biopesticides for the control of pathogens and pests. These nanoformulations increase the solubility and dispersion in aqueous media, increase the bioavailability and reduce the toxicity of the bioactive agents used in the preparation. These characteristics make the nanoformulations described in this invention efficient for the treatment of various pathogens and insects, in addition to being of low production cost, low toxicity and sustainable. By our process, 1.0 Kg of Carbon-dots and 1.0 L yield about 33 thousand liters of nanobiopesticides.

[073] Thus, nanoformulations based on Carbon-dots can be explored as biostimulant systems and intelligent biopesticides, since this technology can simultaneously provide several nutrients to plants, improve the efficiency of plant absorption, reduce the adverse impact of applications pesticides and carry out ecologically safe control of pathogens and pests. Furthermore, nanoformulations based on Carbon-dots presented in this technology do not require quarantine periods as they are non-toxic to humans, animals and fish.

[074] The biopesticidal nanoformulations of the present invention demonstrate significant efficiency in the control of Hemileia vastatrix (coffee rust fungus), Aedes aegypti and Lasiodiplodia theobromae larvae. Therefore, its use in various crops such as coffee, cocoa, soybeans, wheat and tomatoes, has wide applicability.

[075] In addition, the nanoformulation of Carbon-dots with citronella essential oil promotes the inhibition of the growth of the fungus Lasiodiplodia theobromae with 100% efficiency, thus making its use viable in various crops such as avocado, citrus, coconut, Argentine eucalyptus, jackfruit, cassava, ornamental ficus, melon, fig, oiticica mango, guava, papaya, rose, sapodilla, cocoa and vine.

EXAMPLES

[076] The examples below are represented in order to illustrate the best execution of the invention. It is noteworthy that the present invention is not limited to the examples cited, and can be used in all applications described or any other equivalent variations.

EXAMPLE 1: Evaluation of nanoformulations in the control of Hemileia vastatrix

[077] The evaluation of antifungal activity for Hemileia vastatrix (coffee rust fungus) is carried out on discs of coffee leaves with a surface area of 1 cm2, conditioned in Petri dishes with a layer of 2% w/v of gel. agarose.

[078] On each leaf disc are deposited a drop of treatment solutions prepared from the stock solutions of the formulations with copper and essential oils of citronella, clove and sweet orange, containing 30 µL of solution with spores of the fungus Hemileia vastatrix with a concentration of 100 urediniospores per disc. The Petri dishes shown on the left in Figure 2 are kept at 26°C in a dark environment.

[079] After 24 hours, the relative percentages of spore germination are assessed by observation under a fluorescence microscope, as shown on the right in Figure 2. Each test is performed in triplicate and the final results are obtained by arithmetic mean.

[080] These results are tabulated in data sheets for the application of statistical tests. First, the Shapiro-Wilk normality test is applied, which indicates that the collected data do not have a normal distribution. As it is free distribution data, it is necessary to use a non-parametric statistical test. Therefore, the Kruskal-Wallis test of independent samples is applied to evaluate the results. The Kruskal-Wallis test shows that there is a statistically significant difference in the spore germination inhibition rates between the different treatments applied, x2(10) = 30.330, p = 0.001.

[081] To show the differences in the rates of inhibition of spore germination between the treatments applied in the bioassay, a boxplot graph is made. As seen in Figure 3, there is a higher germination inhibition rate of Hemileia vastatrix when using the nanoformulations of Carbon-dots-OEs (Nanoformulations 1, 2 and 3) and Carbondots-Cu (Nanoformulation 4) when compared to negative and positive (water and copper oxychloride, respectively) after 24 hours. Thus indicating that the use of nanoformulations inhibits the development of the fungus in coffee leaf discs.

EXAMPLE 2: Evaluation of nanoformulations in the control of Aedes aegypti larvae

[082] The larvicidal activity of the nanoformulations is tested in cell culture plates – 12 wells, in which 10 stage 3 (L3) Aedes aegypti L. larvae are placed in each well. The experiment being carried out in quadruplicate, 40 larvae are destined for each treatment. The control solutions and formulations are added to these plates and then kept under the same creation conditions (dark environment at 27 ºC). Evaluations are carried out at 24, 48 and 72 hours after the start of the test, where the relative percentages of mortality of the larvae, which are accounted for by observation with the naked eye.

[083] These results are tabulated in data sheets for the application of statistical tests. First, the Shapiro-Wilk normality test was applied, which indicated that the collected data do not have a normal distribution. As it is data with free distribution and repeated measures, it is necessary to use a paired non-parametric statistical test. Therefore, the Friedman test of related samples is applied in the evaluation of the results. The Friedman test showed that there is a statistically significant difference in larval mortality rates between the different treatments applied, x2(9) = 25.991, p = 0.002.

[084] To show the differences in mortality rates of Aedes aegypti larvae between treatments applied in the bioassay, a boxplot graph is made. As seen in Figure 5, where the treatments are: Control 1 - distilled water, Control 2 - C-dots, Control 3 - citronella essential oil (OEC), Control 4 - clove essential oil (OECr), Control 5 - sweet orange essential oil (OELD), Control 6 - copper (Cu), Nanoformulation 1 - C-dots-OEC, Nanoformulation 2 - Cdots-OECr, Nanoformulation 3 - C-dots-OELD and Nanoformulation 4 - C-dots-Cu , there is a higher mortality rate of L3 larvae when using the nanoformulations of Carbon-dots-OEC (Nanoformulation 1), Carbon-dots-OECr (Nanoformulation 2), and Carbon-dots-Cu (Nanoformulation 4), when compared to Control 1 and Control 2 . The Carbon-dots-OECr nanoformulation is the one with the highest larvicidal activity after 24 hours.

EXAMPLE 3: Evaluation of nanoformulations in the control of Lasiodiplodia theobromae

[085] The antifungal activity of nanoformulations for the fungus Lasiodiplodia theobromae is tested by adding 10 mL of treatment solution prepared from the stock solutions of copper and essential oils of citronella, clove and sweet orange in 90 mL PDA culture medium (Potato- Dextrose-Agar) melt and then pour into 70 cm Petri dishes. For the control, distilled and sterilized water is added to the culture medium.

[086] After 24 hours, a mycelial disc (inoculum of the fungus) 5 mm in diameter is transferred to the center of the plate. Measurements of 2 perpendicular diameters are taken on the back of the plate every 24 hours to observe the mycelial growth rate. The test is performed with 4 repetitions, 1 plate per repetition.

[087] After 48 hours, the diameter of the mycelial disc is measured by observation with the naked eye. These results are tabulated in data sheets for the application of statistical tests. First, the Shapiro-Wilk normality test is applied, which indicates that the collected data do not have a normal distribution. As it is free distribution data, it is necessary to use a non-parametric statistical test.

[088] Thus, the Kruskal-Wallis test of independent samples is applied in evaluating the results. The Kruskal-Wallis test shows that there is a statistically significant difference in the inhibition rates of the fungus mycelial diameter between the different treatments applied, x2(5) = 28.456, p < 0.001.

[089] To show the differences in the inhibition rates of the fungus mycelial diameter between the treatments applied in the bioassay, a boxplot graph is made. As seen in Figure 6, there is a higher inhibition rate of the diameter of the mycelial disc of Lasiodiplodia theobromae when using the nanoformulations of Carbon-dotsOEC (Nanoformulation 1) and Carbon-dots-Cu (Nanoformulation 2) when compared to controls 1, 2 , 3 and 4 (Potato Dextrose-Agar, C-dots, OEC and copper, respectively) after 48 hours. Thus indicating that the use of nanoformulations inhibits the development of the fungus in Petri dishes.

[090] As shown in Figure 7, in which "A" shows the control treatment with Potato-DextroseAgar, in "B" the treatment with C-dots is observed, in "C" the treatment with oil citronella essential (OEC), in "D" the treatment with C-dots-OEC nanoformulation is observed and in "E" the treatment with the C-dots-Cu nanoformulation is observed, the inhibition of fungus growth is observed Lasiodiplodia theobromae after 26 hours, using both Carbon-dots-OEC and Carbon-dots-Cu nanoformulations. The nanoformulation of Carbon-dots with citronella essential oil is able to inhibit the growth of the fungus with 100% efficiency having elapsed a period of 120 hours from the inoculum, shown in Figure 8.

Claims (16)

BIOPESTICIDE NANOFORMULATIONS characterized by comprising carbon nanoparticles with antifungal and larvicidal activity. BIOPESTICIDE NANOFORMULATIONS, according to claim 1, characterized in that carbon nanoparticles are doped with secondary metabolites and micronutrients. BIOPESTICIDE NANOFORMULATIONS, according to claim 1 or 2, characterized in that the carbon nanoparticles are doped with citronella, clove, sweet orange, eucalyptus, mint and copaiba essential oil, preferably using citronella, clove and orange essential oils candy. BIOPESTICIDE NANOFORMULATIONS, according to claims 1 to 3, characterized in that carbon nanoparticles have their surface modified with bioactive elements, such as copper, phosphorus, magnesium and aluminum, preferably copper. PROCESS FOR OBTAINING BIOPESTICIDES NANOFORMULATIONS, according to one of claims 1 to 4, characterized in that it consists of the steps:

• A. Formation of Carbon-dots nanoparticles;
• B. Particle purification;
• C. Surface modification of nanoparticles with bioactive compounds.

PROCESS FOR OBTAINING BIOPESTICIDE NANOFORMULATIONS, according to claim 5, characterized in that step A is a partial acid-base neutralization reaction. PROCESS FOR OBTAINING BIOPESTICIDES NANOFORMULATIONS, according to one of claims 5 to 6, characterized in that the formation of Carbondots nanoparticles in step A occur from carbon-rich matrices, such as glutamines, organic acids, citric acid, tartaric acid, acid oleioc, malic acid, fumaric acid, isocitric acid, corn starch, preferably citric and tartaric acid. PROCESS FOR OBTAINING BIOPESTICIDES NANOFORMULATIONS, according to one of claims 5 to 7, characterized in that the basic solution used in step A is composed of calcium hydroxide, sodium hydroxide, potassium hydroxide, ammonium hydroxide, aniline, metalamine, urea and thiourea, preferably ammonium hydroxide and urea. PROCESS FOR OBTAINING BIOPESTICIDES NANOFORMULATIONS, according to one of claims 5 to 8, characterized in that it uses water as a solvent. PROCESS FOR OBTAINING BIOPESTICIDE NANOFORMULATIONS, according to one of claims 5 to 9, characterized in that the formation of Carbondots nanoparticles occur in a continuous flow system in which acidic and basic solutions are injected into a mixing vessel by means of a metering pump, being later redirected to a second mixing vessel for receiving recycling and dopants (micro and macronutrients or stimulants), being further pumped to a tubular piston flow reactor, which is equipped with a valve that restricts the output of the material, maintaining the constant pressure inside and the temperature between 100 and 250 ºC, preferably at 150 ºC, in which the process is kept in turbulent hydrodynamic flow regime; being a posteriori the flow of material leaving the tubular reactor discharged into a continuous stirred tank reactor, under the same heating, in which the output flow of this reactor passes through a separation system, in which it is possible to remove the supernatant and recirculate the background material, inserted before the reactor, which is adding the initial current of the reactants; resuming the process by refluxing until product formation occurs. PROCESS FOR OBTAINING BIOPESTICIDES NANOFORMULATIONS, according to claim 5, characterized in that step C occurs the modification of the surface of the nanoparticles, through the surface engineering technique, being carried out by incorporating essential oils such as citronella, clove, sweet orange, eucalyptus, mint and copaiba, preferably using citronella, clove and sweet orange essential oils with mass fractions between 0 and 100% of each essential oil, preferably 3%. PROCESS FOR OBTAINING BIOPESTICIDES NANOFORMULATIONS, according to claim 5, characterized in that in step C the modification of the surface of the nanoparticles is carried out by inserting bioactive elements, through the surface engineering technique, such as copper, phosphorus, magnesium and aluminum, preferably the use of copper, in the form of Cu and Cu2+, and preferably Cu2+. USE OF BIOPESTICIDE NANOFORMULATIONS, as defined in one of claims 1 to 4, characterized by being used in the control of pathogens and pests. USE OF NANOFORMULATIONS BIOPESTICIDES, as defined in one of claims 1 to 4, characterized by being used as biopesticides to control the fungus Hemileia vastatrix. USE OF NANOFORMULATIONS BIOPESTICIDES, as defined in one of claims 1 to 4, characterized in that it is used as biopesticides for the control of Aedes aegypti larvae. USE OF NANOFORMULATIONS BIOPESTICIDES, as defined in one of claims 1 to 4, characterized in that it is used as biopesticides to control the fungus Lasiodiplodia theobromae.

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