Method for producing an agricultural husk extract and use thereof for promoting plant growth under normal conditions or under abiotic stress

Abstract

The present invention relates to the improvement made to a method for producing an active acid extract from agricultural husks, particularly from rice or oat husks, and the use thereof for promoting plant growth and development under normal conditions or under abiotic stress, particularly saline stress. The invention comprises the use of effective doses of said active acid extract, either alone or formulated with glycine betaine. The improvement made to the method lies in the implementation of the adsorption or retention phase, the purification phase and the desorption phase. The resulting active acid extract is used in both monocotyledon and dicotyledon plants.

Description

[0001] Method for obtaining an extract from agricultural husks and its use to promote plant growth under normal conditions or under abiotic stress.

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to improvements to a method of extracting an active acid extract from agricultural husks, particularly from rice or oat husks, and its use to promote the growth and development of plants under normal conditions or under abiotic stress, particularly saline stress, and which comprise the application of effective doses of said active acid extract, alone or formulated with glycine betaine.

[0004] BACKGROUND OF THE INVENTION

[0005] The demand for food has risen dramatically due to the rapid growth of the world's population in the last 20 years (Savary et al., Food Secur. 2020, 12, 695-717), so improving crop yield and quality is a global issue, highly priority and that must be addressed, although to do so a series of obstacles must be overcome, many of them related to abiotic stress in plants and aggravated by climate change. Among these, soil salinization is the greatest environmental challenge threatening global agriculture (Hazell et al., Philos. Trans. R. Soc. B Biol. Sci. 2007, 363, 495-515) since it is estimated that approximately 20% of irrigated agricultural lands worldwide are adversely affected by soil salinization (Qadir et al., Nat. Resour. Forum 2014, 38, 282-295).Salinization problems are aggravated by environmental degradation, poor irrigation practices, and climate change (Park et al., Mol. Cells 2016, 39, 447–459; Ziska et al., Proc. R. Soc. B Boil. Sci. 2012, 279, 4097–4105). Given this scenario, it is critical to develop new tools that help increase crop yield and quality by mitigating the effects of growing threats such as different types of abiotic stress, mainly derived from climate change, soil salinization, and poor agricultural practices.

[0006] Stress is defined as a physiological reaction of an organism, in which various response mechanisms are activated to cope with a situation perceived as threatening or demanding. Any unfavorable substance or condition that affects or blocks plant metabolism, growth, or development is considered a stress factor. In plants, stress signals evolved from energy quantification, promoting ion and water homeostasis, as well as cellular stability under stress conditions. In plants, SnRK regulators are responsible for halting different processes, such as cell division and, consequently, normal plant development. In contrast, the TOR regulator activates different functions and represses abscisic acid (ABA) synthesis (Zhang et al., Develop. Cell 2020, 55, 529–543).

[0007] Different environmental factors, such as temperature, wind, soil salinity, water availability (excess or deficiency) and nutrients, ultraviolet radiation, chemical pollution, among others, cause different types of stress in plants, such as oxidative stress, osmotic stress, and ionic stress (toxicity). Of the different stressors, salinity is one of the most acute, causing all three types of stress (Munns, Plant, Cell and Environ., 2002, 25, 239-250; Yang and Guo, New Phytologist, 2018, 217, 523-539).

[0008] Oxidative stress reflects an imbalance between reactive oxygen species (ROS) and a biological system's ability to readily detoxify reactive intermediates or repair the resulting damage. (Malik, et al (eds.), Plant Biotechnology: Principles and Applications, 2017). However, it is important to consider that ROS are necessary at basal levels for proper plant physiological activity, acting as messenger molecules in multiple processes such as cell proliferation, stress acclimation, signal transduction, differentiation and development, metabolic regulation, defense against pathogens, and physiological cell death.The problem arises when their production exceeds the plant's detoxification capacity, so the accumulation of ROS causes cytotoxicity, damaging vital components such as DNA and RNA, the lipid layer of the cell membrane by causing its peroxidation, as well as the oxidation of different proteins, amino acids and photosynthetic pigments (Mittler, Trends in Plant Sci., 2017, 22, 1; Hasanuzzaman et al., Cells, 2021, 10, 2537). To regulate the concentration of ROS, plants have a battery of control mechanisms classified as enzymatic and non-enzymatic responses. As part of the enzymatic response, there are a number of enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and guaiacol peroxidase (GP), which catalyze different reactions to deactivate reactive oxygen species.For its part, the non-enzymatic response includes a series of molecules, such as ascorbic acid, reduced glutathione, α-tocopherol, carotenoids, flavonoids and proline that act as cosubstrates of some of the enzymes previously mentioned, or as scavengers of some of the reactive species, such as singlet oxygen, hydroxyl radical and / or hydrogen peroxide, protecting the cell membrane from lipid peroxidation or other components of oxidation and damage (Das and Roychoudhury, Front. Environ. Sci., 2014, 2, 53).

[0009] Osmotic stress, on the other hand, is related to the flow of water through a semipermeable membrane, such as the cell membrane. When the concentration of water-soluble compounds outside the cell is greater than inside, a condition called hypertonic stress is created, which causes water to escape into the hypertonic medium (osmosis), thus dehydrating the cell and reducing its size. This phenomenon, known as plasmolysis, can cause an irreversible separation of the plasma membrane from the plant cell wall, preventing the cell from returning to its normal state. If dehydration is not severe, there may be incipient plasmolysis, in which case the cell can return to its natural state.To reduce the effects of osmotic stress, plants have developed an increase in the synthesis of a series of osmolytes that seek to balance the concentration of soluble molecules outside the cell, thereby preventing the flow of water by osmosis. The osmolytes produced by the cell are of different chemical nature, such as sugars (sorbitol, mannitol, sucrose, glucose, fructose, galactose, mannose, etc.), amino acids (proline), quaternary amines (glycine betaine) and polyamines (putrescine, spermine, spermidine) (Zhu, Cell, 2016, 167, 313-234).

[0010] Ionic stress is mainly related to the accumulation of sodium cation, which causes an imbalance in the homeostasis of other important ions, such as K + and the Ca 2+, in addition to hindering proper glycation of RuBisCo, decreasing its catalytic efficiency in CO2 fixation and therefore photosynthesis; therefore, sodium is considered to be toxic. To avoid the toxicity caused by sodium accumulation, plants have developed a series of responses, one of which is regulated by the SOS system that activates proton pumps (ATPases) and sodium transporters that expel sodium from the cell or encapsulate it in the vacuole. This same system, complemented by other genes, such as AKT1 , promotes the entry of potassium and prevents its exit in an attempt to restore homeostasis (Assaha et al., Front. Physiol., 2017, 8, 509).

[0011] The ability of plants to resist or tolerate abiotic stress is considered to be related to the efficiency, at the first level, of their signal sensors, perception and transduction, among which are osmosensors (eg AtHK1), phospholipases (eg PLD), secondary messengers (eg Ca 2+ , PtdOH, ROS), MAP kinases, Ca sensors 2+(eg SOS3), calcium-dependent protein kinases (eg CPDKs). At the second level is the efficiency in transcriptional control, in which different factors participate (eg CBF / DREB, ABF, HBF, HSF, bZIP, MYC / MIB). Finally, the third level is in the stress response mechanisms with the activation of different genes that promote detoxification (eg SOD, PX), osmoprotection (proline, glycine betaine, polypeptide sugars), chaperone functions (eg Hsp, SP1, LEA, COR) and the movement of water and ions (eg aquaporins, ion transporters). These mechanisms seek to reestablish cellular homeostasis and the structural and functional protection of proteins and membranes (Wang et al., Planta, 2003, 218, 1-14).In this context, different products, such as phytohormones and / or biochemical or microbial biostimulants, which particularly improve the third level of response, may favor the tolerance or resistance of plants to abiotic stress. For example, Esan et al (EJFOOD, 2020, 2(6), 1-4) reported the direct correlation between the exogenous application of IAA and the increase in photosynthetic pigments, such as chlorophylls and carotenoids. One of the first systems to be damaged or stopped in situations of abiotic stress, particularly salinity stress, is the photosynthesis system.This is why increasing the content of photosynthetic pigments and improving or maintaining the photosynthetic system has a double effect in stressful situations: 1) pigments, particularly carotenoids, act by deactivating reactive oxygen species (ROS); therefore, they are elements that mitigate oxidative stress; 2) an intact or improved photosynthetic system maintains or increases the production of sugars, polyamines and polypropylene derived from central metabolism; molecules that are considered osmolytes, understood as elements that reduce osmotic stress.

[0012] A group of molecules of botanical origin, classified as phenolic compounds, are possibly the most abundant secondary metabolites in plants and are often found in the cell walls and vacuoles of epidermal and subepidermal cells (Lattanzio et al (editors), Phytochemistry: Advances in Research, 2006, 661 , 23–67). These phenolic compounds can be constitutive or inducible. Constitutive phenolic compounds are synthesized during normal growth and development, whereas inducible ones are synthesized in response to biotic and abiotic stresses. The functions of endogenous phenolic compounds in plants are very diverse.They act as antioxidants, scavenging reactive oxygen species; they accumulate in the epidermis to act as shields against harmful UV radiation; they defend plants against herbivores, pathogens, and weeds; they improve nutrient mobilization and absorption; and they are involved in seed germination and dormancy. Phenols, especially anthocyanins, act as pigments that attract pollinators and seed dispersal agents, etc. (Jain et al., Int. J. Pharm. Sci. Res., 2019, 10, 494-504; Takshak and Agrawal, J. Photochem. Photobiol. B Biol., 2019, 193, 51-88; Bhattacharya et al., Mol. Plant Pathol., 2010, 11, 705-719; Sharma et al., Molecules 2019, 24, 2452). One of the recent reviews clearly shows the response and role of endogenous phenols in plant tolerance to abiotic stress factors such as heavy metals, drought, salinity, ultraviolet light and heat among others (Sharma et al., Molecules 2019, 24, 2452).It has been suggested that these effects are achieved because phenolic compounds regulate the activity of phytohormones, modulate the activity of certain enzymes and increase the synthesis of some important metabolites, managing to influence metabolism, photosynthesis, nutrient absorption and other physiological processes in plants to stimulate their growth and development (Yakhin et al., Front. Plant Sci. 2017, 7, 2049). Phytohormones such as auxins, cytokinins and gibberellins are known to promote growth, while others, such as abscisic acid, inhibit it; having found that phenolic compounds and extracts increase the levels of the former and decrease those of the latter (Abdalla, Int. J. Plant Physiol. Biochem., 2013, 5, 42-49).However, although several studies show the influence of phenolic compounds and extracts on the activity of phytohormones, a thorough understanding of their modes of action and the underlying mechanisms at the molecular level is still lacking. Similarly, phenolic compounds and extracts modulate the activity of antioxidant enzymes, such as catalase, peroxidase, and superoxide dismutase, by stimulating the production of antioxidant compounds such as ascorbic acid and flavonoids, which inactivate reactive oxygen species whose accumulation during periods of stress can cause damage to cellular structures. In Abdalla's study (Int. J. Plant Physiol. Biochem., 2013, 5, 42-49), Moringa oleifera extract decreased the activity of peroxidase, superoxidase dismutase and catalase enzymes, as well as lipid peroxidation, while increasing the levels of endogenous phenolic compounds and ascorbic acid in treated plants.In addition to promoting the synthesis of these types of compounds, phenolic extracts and compounds have been shown to also promote the formation of chlorophyll, carotenoids, proteins, sugars, and amino acids, among many others. While endogenous phenols and ascorbic acid allow plants to counteract the accumulation of reactive oxygen species, an increase in chlorophyll and carotenoids helps plants improve their photosynthetic rates and transpiration (stomatal conductance) (Abdalla, Int. J. Plant Physiol. Biochem., 2013, 5, 42-49).

[0013] Although the exogenous application of different extracts and phenolic compounds has shown that it can improve different agronomic variables (germination, emergence, growth, vigor, yield, quality, etc.) of different crops (Kisihko et al., Molecules, 2021, 26(21), 6343), to date there is no report that refers to a phenolic extract or compound having a strong and effective effect against abiotic stress, particularly against saline stress. Nor, about a phenolic extract or compound, combined with other molecules, such as glycine betaine, having a synergistic, strong and effective effect against abiotic stress, particularly against saline stress.

[0014] Numerous studies have established the important roles of glycine betaine (GB) in plant responses to abiotic stresses, including salinity, drought, and low temperature (Munns and Tester, Annu. Rev. Plant. Biol., 2008, 59, 651–81; Annunziata et al., Front. Plant Sci., 2019, 10, 230; Bai et al., Biotechnol. Lett., 2022, 44, 367–386). Certain glycophyte plants can synthesize and accumulate GB to adapt to salt stress (Annunziata et al., Front. Plant Sci., 2019, 10, 230). However, the biosynthesis of this compound in plant tissues is energy intensive (Mákelá et al., Plant Science, 1996, 121, 221–230). Therefore, exogenous application of GB may be crucial for improving plant tolerance to abiotic stresses, including salinity.Exogenous application of GB improves plant stress tolerance by activating the ROS scavenging system, which reduces membrane peroxidation and helps maintain membrane stability under saline conditions (Banu et al., Biosci. Biotechnol. Biochem., 2010, 74, 2043-2049; Gupta and Huang, Int. J. Genom., 2014, 18). Similarly, GB, being an osmolyte, reduces osmotic water loss (Ashraf and Foolad, Environ. Exp. Bot., 2007, 59, 206-216; Bai et al., Biotechnol. Lett., 2022, 44, 367-386). Furthermore, GB preserves the thermodynamic stability of proteins, preventing their aggregation and helping maintain their normal functional activities. Studies conducted over the past 25 years have shown that GB protects the normal structure and activity of the photosynthetic reaction center (PSII) and enhances RuBisCo activity under drought stress (Papageorgiou and Murata, Photosynth. Res., 1995, 44(3), 243-52; Huang et al., Plant Physiol., 2000, 122, 747-756). GB has also shown that it is capable of promoting the overexpression of genes, such as ATPase and sodium transporters (ZmMHA2, ZmMHA4, ZmYP1 and ZmNHXI), involved in the SOS response to salt stress, causing the expulsion of the sodium cation out of the cell or its encapsulation in the vacuole, although this overexpression occurs only under salt stress conditions (Zhu et al., Front. Plant Sci., 2022, 13, 978304.) However, to date there is no report that GB has a synergistic effect against abiotic stress, particularly against salt stress, when applied jointly with a phenolic extract or compound.

[0015] It was discovered that a composition containing polyphenols (PF), obtained from the acid hydrolysis of agricultural husks, particularly rice husks, improves plant growth under both normal and abiotic stress conditions, particularly saline stress. In the prior art related to the invention, as described in US Patent 5,352,264, polyphenols (PF) are obtained from agricultural husks according to the process described in the patent, with some modifications. Basically, said invention describes "the extraction of polyhydroxycarboxylic acids, carbohydrates and alcohols from plant residues, mainly rice and oat husks." As described in US Patent 5,352,264, polyhydroxycarboxylic acids come from the partial oxidation of pentoses obtained during the hydrolysis of hemicellulose contained in agricultural husks.However, processes with dilute acids, such as the one described in US patent 5,352,264, also promote the removal of lignin, as described in different scientific articles (Bhutto et al., Energy, 2017, 122, 724-745; Kim, et al., Carbohydrate Polymers, 2014, 99, 563-567). Precisely, the improvements or modifications to the method that includes the introduction of adsorption / desorption processes for the effective recovery of polyphenols (PF) derived from lignin and found in the acid solution of the hydrolysis of agricultural husks.The selection of adsorbent materials with a high specific adsorption capacity but which also facilitate the release of adsorbates during the desorption stage, the selection of the desorption or elution solvent, as well as the establishment of the process parameters, are not obvious to a person skilled in the art since the particular characteristics of the adsorbates and the rest of the components of the solution must be considered in order to achieve high selectivity in the adsorption operation and facilitate the desorption or elution operation.

[0016] As regards other differences and the object of the invention, it can be pointed out that in a second aspect, the present invention is directed to providing methods for improving plant growth and comprising applying effective amounts of PF recovered from an acid extract of agricultural husks, particularly rice husks; in a third aspect, the present invention is directed to providing a method for improving abiotic stress tolerance of plants, particularly salt stress, comprising applying an effective amount of PF recovered from an active acid extract of agricultural husks, particularly rice husks;and finally, in a fourth aspect, the present invention is directed to providing a method for improving plant tolerance to abiotic stress, particularly saline stress, comprising applying an effective amount of PF recovered from an active acid extract of agricultural husks, particularly rice husks, and glycine betaine, both acting synergistically and applied in a ratio (w / w) of 1:1 to 1:3; wherein the polyphenol (PF) content is expressed as gallic acid equivalents in 100 g of sample, according to the Folin Ciocalteu method;

[0017] BRIEF DESCRIPTION OF THE FIGURES

[0018] The characteristic details of the invention are described in the following paragraphs together with the accompanying figures, which are intended to define the invention, but without limiting its scope.

[0019] Figure 1. Expression level of the genes evaluated in the roots of tomato seedlings (Solatium lycopersicum). The graphs represent the average expression level (2-AACt fold change) of the genes evaluated at different times after the application of PF extracted from rice husks (white bars) versus control plants (untreated, black bars). The lines in the bars represent the standard error of two biological replicates and two technical replicates. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention relates to improvements to a method for extracting an active acid extract from agricultural husks, particularly rice husks, where the active acid extract is obtained through the following phases: a) Pentose extraction phase:

[0021] Sieve agricultural husks, preferably rice or oat, individually or in combination, until obtaining a particle size between 24 and 90 mesh, to subsequently promote acid hydrolysis by adding an acid solution, preferably nitric acid and preferably at a concentration of 1 to 25% (w / v), in a liquid / solid ratio of 3:1 to 5:1. Subsequently, steam is injected, preferably at a pressure of 7.0 to 9.0 kg / cm 2and stir the liquid-solid mixture until a temperature between 75 and 85 ° C is obtained. Once the temperature is reached, an additional amount of acid, preferably nitric acid, is added until a concentration of 1.7 to 41% (w / v) is reached, maintaining the same temperature until the end of the hydrolysis, which in total lasts 8 to 20 hours, with stirring provided intermittently by the injection of steam through diffusers to maintain the temperature, to finally obtain a hydrolyzed solution containing pentoses. b) Oxidation phase: Add an additional amount of acid, preferably nitric acid, until a concentration of 2.2 to 47% (w / v) is reached, to the hydrolyzed solution containing the pentoses, and maintain the temperature in a range of 70 to 85 ° C, for 4 to 10 hours, by injecting steam, preferably at a pressure of 7.0 to 9.0 kg / cm 2. Subsequently, the mixture is allowed to cool to room temperature to obtain a mixture of acids comprising monocarboxylic and dicarboxylic acids, such as oxalic acid and 2,3,4-trihydroxypentanedioic acid, and other non-oxidized or partially oxidized molecules, such as alcohols and aldehydes, and other compounds such as carbon dioxide. The resulting mixture of acids is then allowed to settle to facilitate phase separation by decantation, and finally, the supernatant free of large particles, such as agricultural husk residues, is recovered. c) Clarification and concentration phase:

[0022] Filter the supernatant free of large particles to obtain a primary filtrate. The primary filtrate is then concentrated by vacuum evaporation at an absolute pressure of 145 to 200 mm Hg and a maximum boiling point of 45 to 60 °C to obtain a clarified and concentrated product with a 1.5:1 to 3.5:1 ratio. d) Adsorption or retention phase:

[0023] Circulate the filtered or clarified and concentrated product through columns packed with 1 to 4 kg, preferably 1.5 to 3 kg of granular activated carbon or a quantity of high specific adsorption capacity resin that can be packed in a volume equivalent to the effective volume of the columns given by an internal diameter of 10.16 cm and an effective height of 85 cm. The selected resins with a high specific adsorption capacity are included in the group of macroporous polystyrene resins functionalized with tertiary amines, such as MN-102 from Purolite®, or with other hydrophilic molecules, or resins from the amberlite family.

[0024] Apply an optimal influent flow rate between 1.0 to 6.0, preferably between 1.5 to 4.5 ELV h-1 (packed bed volumes per hour) for the development and inclusion of an adsorption process so that phenolic compounds and flavonoids contained in low concentrations in the clarified and concentrated product and circulated as influent in the columns, are adsorbed and retained in the adsorbent material; while other secondary compounds, such as aliphatic polyhydroxycarboxylic acids, carbohydrates, alcohols and aldehydes, are eliminated in the effluent. e) Purification phase:

[0025] Wash in counterflow with 5 to 50 L, preferably with 15 to 30 L of acidified water (pH < 4), the columns containing the phenolic compounds and flavonoids adsorbed on the activated carbon or resins with a high specific adsorption capacity, to eliminate other residual compounds, such as aliphatic polyhydroxycarboxylic acids, carbohydrates, alcohols and aldehydes, absorbed between the pores of the activated carbon or macroporous resins that could act as contaminants during the desorption or elution process. Additionally, and to facilitate the subsequent desorption or elution process, the columns are drained of the wash water and dried by circulating air in counterflow for 5 to 30 minutes, preferably for 10 to 20 minutes. f) Desorption phase:

[0026] Circulate in counterflow 10 to 40 L, preferably 15 to 25 L of 96% (v / v) ethanol or an alkaline aqueous solution of potassium hydroxide 0.05 to 0.2 M, preferably 0.1 to 0.2 M, through the columns packed with the adsorbed polyphenolic compounds, maintaining the pH in a range of 7.0 to 10, preferably between 7.5 to 9.0, to favor the solubility of the adsorbed compounds and facilitate the desorption or elution process to obtain a concentrated active acid extract with polyphenols in a concentration range between 3 to 10 g equivalent of gallic acid / 100 mL.

[0027] In one of the preferred embodiments of the present invention, the resulting active acid extract with polyphenols in concentrations between 3 to 10 g equivalents of gallic acid / 100 mL is formulated together with glycine betaine until reaching a final concentration between 0.5 to 2.5% g equivalents of gallic acid / 100 g of sample, preferably between 1.0 to 2.0%, while GB is in a concentration between 1 to 5%, preferably between a concentration between 2 to 3%, keeping a polyphenols (PF) / glycine betaine (GB) ratio between 1:1 to 1:3. For the application of the formulation for plant treatment, said formulation must be diluted in water until reaching final concentrations of effective field doses of 0.0014 to 0.0105% (w / v) of polyphenols (PF) and 0.0030 to 0.0225% of glycine betaine (GB), applying between 400 to 1000 L / Ha of the diluted solution.The “effective amount or dose” may vary, depending on the concentration of the PF and GB in the formulations, the variety of plant or plant species to be treated, the severity of the stress, the desired result and / or the stage of phonological development of the plants, among the most relevant factors, therefore, it is not always possible to specify exactly an “effective amount”, although in each particular case, this can be established by a common expert in the art.

[0028] The active acid extract alone or in formulation with GB, is used to promote the growth and development of plants under normal conditions or under abiotic stress, particularly under salinity stress, and which comprise the application of effective doses in solution to monocotyledonous plants, such as a cereal or grass, preferably corn or to an amahlidae, preferably onion; and dicotyledons of the Solanaceae family, preferably tomato, chili, pepper and potato or of the Rosaceae family, preferably apple, or of the Malvaceae family, preferably cotton, or of the Cucurbitaceae family, preferably watermelon, melon or cucumber, or of the Asparagaceae family, preferably asparagus (table 1 and table 2).The active acid extract individually or in formulation with GB, promotes the overexpression of genes involved in the response to oxidative stress, such as APX, ascorbate peroxidase; GST1, glutathione S-transferase-1; ion transport, such as ATPse4, ATPase4; auxin transport, such as PIN1, PINFORMED; and response to auxins, such as IAA5, Inducible lndole-3-acetic acid 5; under normal conditions or under abiotic stress, particularly under salinity stress in monocotyledonous plants, such as a cereal or grass, preferably corn or an amahlidae, preferably onion; and dicotyledons of the Solanaceae family, preferably tomato, chili, pepper and potato or of the Rosaceae family, preferably apple, or of the Malvaceae family, preferably cotton, or of the Cucurbitaceae family, preferably watermelon, melon or cucumber, or of the Asparagaceae family, preferably asparagus (Figure 1).In the case of cereals, such as corn, the formulation with the active acid extract is applied during the vegetative stage of growth, starting in V2 and ending in VT, at a concentration of 1 to 3 L / Ha, divided into 2 to 3 applications. In the case of apple crops, between 1 to 5 L per hectare is applied, preferably 2 to 4 L per hectare, divided into 3 applications, starting after the sprouting period and at intervals of 20 to 30 days. For vegetable crops, such as asparagus, chili, tomato and onion, the same concentration is applied divided into 3 applications, starting after the sprouting period and at intervals of 15 to 20 days, while in cotton crops, in a single application, of the same concentration after the sprouting period.

[0029] Optionally, the formulation with the active acid extract is applied with a herbicide, such as glyphosate, halosulfuron, or others; a fungicide, such as strobirulins, tetraconazole, or others; a nematicide, such as strobirulins, tetraconazole, or others; and nitrogen fertilizers, such as ammonium nitrate, potassium nitrate, urea, or micronutrients.

[0030] Optionally, the formulation with the active acid extract is applied directly to the soil by any convenient means, including sprinkling, drip irrigation systems, strip irrigation systems, or others that are obvious to anyone skilled in the art. The following examples are intended to illustrate the present invention and to teach one skilled in the art how to use the formulations and methods of the present invention, but are not limiting in any way.

[0031] EXAMPLE 1. EFFECT AT THE GENETIC LEVEL, IN TOMATO SEEDLINGS, OF THE APPLICATION OF THE FORMULATION WITH THE ACTIVE ACID EXTRACT CONTAINING POLYPHENOLS (PF).

[0032] In one of the preferred embodiments, the application of the formulation with the active acid extract to tomato seedlings promotes changes in the expression of key genes in metabolic pathways related to growth and tolerance to abiotic stress, as illustrated in Figure 1.

[0033] The experiment was conducted using a randomized complete block design with three replicates. The experimental unit consisted of 20 plants for each treatment, resulting in a total of 120 plants for the entire study.

[0034] To evaluate the effect of PF on gene expression in tomato seedlings (30 days after germination), two treatments were applied as follows: 1) Absolute control (water), 2) PF in aqueous solution (0.0028% gallic acid equivalents). Three applications of one liter per plant were made with an interval of 7 days between each, the first application being at the time of transplantation. After the third application of the treatments, root samples were taken at 0.5 h and 4 h after application. Finally, expression levels were determined using the 2-AACt method (Nolan et al., Nat. Protoc., 2006, 1 (3), 1559-82) comparing the expression levels of treated plants versus control plants.

[0035] An overexpression of up to 100% of the IAA5 gene and up to 200% of the PIN1 gene was observed, the first related to the endogenous production of auxins, such as indole acetic acid (IAA) and the second involved in its polar transport. In addition to promoting better root development, an increase in chlorophyll and carotenoid content was observed, coinciding with the known correlation between the exogenous application of IAA and the increase in photosynthetic pigments, such as chlorophyll and carotenoids. An overexpression of up to 400% of the GST1 gene and up to 350% of the APX gene was also observed, both involved in the response to oxidative stress. Another of the most relevant discoveries of the present invention is that PF promote an overexpression of up to 1500% of genes from the ATPase family, key elements of the SOS response of plants to ionic stress caused by sodium. EXAMPLE 2.APPLICATION OF THE FORMULATION WITH THE ACTIVE ACID EXTRACT CONTAINING POLYPHENOLS (PF) AND GLYCINE BETAINE (GB) AND ITS EFFECT AT THE PHYSIOLOGICAL AND DEVELOPMENT LEVEL OF TOMATO PLANTS, GROWN IN A GREENHOUSE, UNDER NORMAL CONDITIONS OR IN SALINE SOILS.

[0036] Table 1. Effect of the treatments evaluated on tomato plants under three soil salinity conditions.

[0037] The experiments were conducted at the Polytechnic University of Valencia (Valencia, Spain). The experiment was established under a multi-strand design consisting of three salinity conditions (non-saline (NS), saline (S), and very saline (VS) soils), using two doses of a formulation containing 1 to 2% (gallic acid equivalents / 100 mL of sample) of PF and 2 to 4% of GB (w / v), (D1 = 0.4 mL / L and D2 = 0.8 mL / L), and two evaluation periods (30 and 60 days after transplanting). Control plants (DO, without the formulation) were grown in parallel, maintaining the same experimental design. Each experimental unit consisted of 6 plants for each treatment, making four applications of one liter per plant with an interval of 15 days between each one, the first application being at the time of transplanting. After the fourth application of the treatments, the roots and aerial parts were collected separately for their respective quantification.

[0038] The results of the variables evaluated are summarized in the following table, showing significant differences between the witness (control) and the treatments (*), mainly at 60 days after transplantation.

[0039] Indicates significant difference (p<0.05) in relation to the control group

[0040] Fresh weight analysis of treated plants showed a favorable response to plant growth, especially under high salinity conditions (S and VS), where mean values ​​(root, stem, and leaves) were considerably higher than in control plants. Furthermore, treatment application significantly increased the average levels of photosynthetic pigments (chlorophylls and carotenoids) after the second application.

[0041] Of particular note is the fact that lower levels of Na were found in treated plants. +, both in roots and leaves, at 30 and 60 days, compared to tomato plants grown in the absence of treatments.

[0042] The higher content of sugars (TSS) and other osmolytes, such as glycine betaine (GB) in treated plants is also notable, particularly under salinity conditions (S and VS). As a consequence of a greater accumulation of pigments, particularly carotenoids, the greater accumulation of sugars and GB, and relevantly, the lower accumulation of Na in tissues (root and leaves). + , there is a clear mitigation of oxidative, osmotic and saline stress respectively, demonstrated through lower values ​​of practically all the different stress indicators (PRO, MDA, H2O2, SOD, CAT and GR) in the treated plants, in relation to the corresponding controls.

[0043] EXAMPLE 3. APPLICATION OF THE FORMULATION WITH THE ACTIVE ACID EXTRACT CONTAINING POLYPHENOLS (PF) AND GLYCINE BETAINE (GB) AND ITS EFFECT AT THE PHYSIOLOGICAL AND DEVELOPMENT LEVEL OF TOMATO PLANTS, GROWN IN A GREENHOUSE, UNDER NORMAL CONDITIONS OR UNDER IRRIGATION WITH BRACKISH WATER

[0044] The experiments were conducted in the greenhouses of the Polytechnic University of Valencia (Valencia, Spain). The experiment was established under a multi-strand design consisting of four conditions, two salinity conditions (non-saline soils (NS) and saline soils (SS)) and two irrigation water conditions (non-saline (WN) and saline (WS)), using two doses of a formulation containing between 1 and 2% (gallic acid equivalents / 100 mL of sample) of PF and 2 to 4% of GB (w / v), (D1 = 0.4 mL / L and D2 = 0.8 mL / L), and two evaluation periods (30 and 60 days after transplant). Control plants (DO, untreated) were grown in parallel, maintaining the same experimental design. The experimental unit consisted of 120 plants in total for the test. Four applications of one liter per plant were made with an interval of 15 days between each one, the first application being at the time of transplant.After the fourth application of the treatments, the roots and aerial parts were collected separately.

[0045] The results of the variables evaluated 60 days after transplantation are summarized in the following table, showing significant differences between the control and the treatments (*).

[0046] Table 2. Effect of the treatments evaluated on tomato plants subjected to increasing salinity by different combinations of saline or non-saline soil and irrigation water.

[0047] A first aspect to highlight is the fact that the formulation stimulates plant development under normal conditions, according to the values ​​of the root fresh weight, stem fresh weight, and leaf fresh weight variables, respectively. As salinity conditions increased, reaching the most extreme condition of saline soil and brackish water irrigation (SSWN). <NSWS<SSWS), el desarrollo de las plantas fue disminuyendo significativamente, aunque en las plantas tratadas, dicha disminución fue marcadamente menos drástica que en los grupos control. Por ejemplo, en la condición de mayor salinidad (SSWS), los pesos frescos de raíz, tallo y hoja del grupo tratado fueron 55, 50 y 67% más altos que los del grupo control, respectivamente.Likewise, this effect was reflected in a less marked reduction in other growth parameters compared to the control group, such as plant height, stem diameter, and number of leaves (data not shown). A second aspect worth highlighting is the effect of the formulation on the increase in photosynthetic pigments, such as chlorophylls and carotenoids, under normal growing conditions. As salinity conditions increased, reaching the most extreme condition of saline soil and brackish water irrigation (SSWS), the production of these pigments declined sharply, affecting the photosynthetic process, which explains the drastic decrease in plant development. However, although the production of these pigments was also affected in treated plants grown under saline conditions, the decrease was markedly less drastic than in the corresponding control groups.For example, under the highest salinity condition (SSWS), the chlorophyll a, chlorophyll b and carotenoid contents of the treated group were 167, 166 and 100% higher than those of the control group, respectively.

[0048] A third aspect to highlight is the effect of the formulation on the sodium ion content (Na + ). This content increased significantly in the roots and leaves of tomato plants in the control groups in proportion to the increase in salinity (SSWN <NSWS<SSWS). Si bien en las plantas tratadas también hubo un incremento en el contenido del ion Na + , this increase was 15 to 25% lower than the increases observed in the control groups, under the various conditions evaluated. Also relevant, under the different salinity conditions, the contents of the K cations + and Ca 2+At both the leaf and root levels, the concentrations increased by 45 to 140% in treated plants compared to the control groups. This clearly demonstrates an improvement in cellular homeostasis, which is achieved by the SOS response mechanism of plants to salinity stress.

[0049] A fourth aspect to highlight is the increase in the content of osmolytes, such as total soluble sugars (TSS) and glycine betaine (GB), in the treated plants, both under normal conditions and under conditions of different degrees of salinity (SSWN). <NSWS<SSWS), con valores entre 35 a 140% superiores a los grupos control correspondientes. Claramente, la mejora o mantenimiento de los sistemas de fotosíntesis, que es donde se da la fijación de CO2 y la síntesis de carbohidratos, afecta también positivamente la producción de azúcares.

[0050] Finally, a fifth aspect to highlight is that as a consequence of a greater accumulation of pigments, particularly carotenoids, the greater accumulation of sugars and GB, and relevantly, the lower accumulation in tissues (root and leaves) of Na + , there is a clear mitigation of oxidative, osmotic and saline stress respectively, demonstrated through lower values ​​of practically all the different stress indicators (PRO, MDA, H2O2, SOD, CAT and GR) in the treated plants, in relation to the corresponding controls.

[0051] EXAMPLE 4. EFFECT OF A POLYPHENOL FORMULA (PF) WITH GLYCINE BETAINE (GB) AND GLYCINE BETAINE ALONE IN THE MANAGEMENT OF SALINITY STRESS ON PRODUCTION PARAMETERS OF CORN (Zea mays L.).

[0052] The trial was carried out in the agricultural area located in Hacienda Humboldt (Julimes, Mexico) on an area of ​​20 hectares, whose soil presented salinity problems (9.1 1 dS m-1 of electrical conductivity (EC) determined from an extract ratio 1 : 1). Three treatments were carried out with 3 repetitions each, which included T0: control treatment, using only water; T1: Treatment with a formulation containing between 1 to 2% (gallic acid equivalents / 100 mL of sample) of PF and 2 to 4% of GB (w / v), 3 L / Ha; and T2: Treatment with a formulation containing 1 to 2% (gallic acid equivalents / 100 mL of sample) of PF. 3 L / ha of each formulation was used, which was applied directly to the furrow using equipment with a boom and a Streamjet TP0001 -SS nozzle at 2 bars of pressure and with a flow rate of 1,152 L / ha. Applications were made at stages V1, V4 and V6 (16, 32 and 44 days after sowing) of crop development.

[0053] For each of these treatments, the ears of corn were harvested from a 30-meter row. Fresh weight parameters were measured from 20 randomly selected ears of corn, and the kernels contained in the 20 ears were dried and weighed.

[0054] The results showed the following: for the fresh weight of the 20 ears, the highest mean was T1 with 55.733 g, followed by T2 with 47.650 g; and the lowest T0 with 39.566 g, all with statistically significant differences (p<0.05). For the dry weight per grain, the highest mean was T1 with 0.71 g, followed by T2 with 0.62 g; and the lowest T0 with 0.52 g, all with statistically significant differences (p<0.05).

[0055] Based on the results, it was determined that both PF alone and PF co-formulated with GB have an effect on grain yield in corn, observing a synergistic effect between the components of the formulation.

[0056] EXAMPLE 5. EFFECT OF BIOSTIMULATION OF THE ROOT SYSTEM OF A PRODUCT BASED ON A PF AND GB CONCENTRATE ON THE INCREASE IN YIELD DUE TO THE EFFECT OF BIOSTIMULATION ON THE ROOT SYSTEM OF BASIL (Ocimum basilicum) UNDER GREENHOUSE CONDITIONS.

[0057] The trial was conducted in the greenhouses of a Green Mex farm located in Axochiapan (Morelos, Mexico). To evaluate the yield of export-quality foliage through biostimulation under poor water quality conditions (EC 15.2 dS*nr) 1 ) , was carried out in a 1,000 m greenhouse building 2, which was divided into 6 plots to establish a total of 2 treatments with 3 repetitions each, these being: T0, for the control; and T1 , for the treatment with a formulation containing between 1 to 2% (gallic acid equivalents / 100 mL of sample) of PF and 2 to 4% of GB (w / v), carrying out every 15 days 3 applications of 3 L / ha each.

[0058] The treatment was applied during the last irrigation of the day, injecting the product in the last third of the irrigation period to ensure complete application. The irrigation system consists of irrigation tapes, emitters every 20 cm, and a flow rate of 1.1 L / hour, placed every 60 cm.

[0059] From a total of 7 cuts, the number of boxes harvested per treatment was recorded (sum of the 3 replicates). The plots treated with the formulation generated better yields, with a mean difference of 65 boxes per 500 m². 2, i.e. an increase of 8.3% in the number of export-quality boxes of basil leaves.

[0060] EXAMPLE 6. BIOSTIMULATION FOR THE MANAGEMENT OF SALT STRESS IN THE YIELD OF CHILI AND JALAPEÑO CROP THROUGH ROOT APPLICATION OF A PF AND GB FORMULA.

[0061] The trial was carried out at the Invernaderos Yucatán agricultural farm, located in the town of Jiménez (Chihuahua, Mexico) on an area of ​​3.72 hectares, divided into two irrigation sectors of 1.56 (sector 1) and 2.15 hectares (sector 2). The soil used had salinity problems (11.19 dS * m-1 of electrical conductivity (EC) determined from a 1:1 ratio extract. For each of the treatments, 5 blocks of 6 plants were marked, each one randomly, for evaluation. 3 applications of a formulation containing between 1 to 2% (gallic acid equivalents / 100 mL of sample) of PF and 2 to 4% of GB (w / v) were made; each application used 3 L / Ha of the formulation, applied with a bar equipment with 16 nozzles for SJ7 liquid fertilizers and an expenditure of 1,230 L / ha., injected through the irrigation system, seeking to position the assets between 5 to 30 cm, sector where the highest proportion of roots is found.To evaluate the biostimulant effect of the formulation on the yield and quality of the jalapeño pepper crop, Orizaba variety, stem thickness (mm) was measured; the leaf area index, AIF (cm. 2 ); plant height (mm); and yield (number of fruits / plant).

[0062] At the end of the season, there was a 25.7% increase in the average number of fruits per plant; a 7.14% increase in average stem thickness; a 3% increase in average leaf area index; and a 3.9% increase in average plant height. However, significant differences were only observed between the control and treatment groups in the variable number of fruits.

[0063] EXAMPLE 7. EFFECT OF APPLYING A ROOT BIOSTIMULANT FORMULATED WITH POLYPHENOLS (PF) AND GLYCINE BETAINE (GB) ON THE INCREASE IN YIELD IN MEXICAN LEMON (Citrus aurantifolia) UNDER CONDITIONS OF HIGH LEVELS OF BICARBONATE. The experiments were carried out under field conditions, in the agricultural farm of the company Green Mex located in the town of Buena Vista de Tomatlán (Michoacán, Mexico).

[0064] To evaluate the effect on stress produced by high bicarbonate levels (183 mg / L), two treatments were established (control treatment and treatment with a formulation containing between 1 to 2% (gallic acid equivalents / 100 mL of sample) of PF and 2 to 4% of GB (w / v); on 2 rows, where 15 homogeneous trees were selected for each treatment, from which 20 random fruit samples were obtained (5 fruits for each of the 4 sides of the tree). Productivity was measured in terms of average fruit weight (g / fruit) and average juice content (mL / fruit).

[0065] The treatment containing PF and GB was applied via irrigation, injecting the corresponding dose of 3 L / Ha directly onto the row line, which meant an expenditure of 3.6 mL of the formulation per tree, and for the row it meant injecting 76 mL (1.35 mL / linear meter) in the last third of the irrigation period. The first application was made at full bloom, the second with fruit between 5 and 10 mm, the third with fruit between 14 and 20 mm, and the last with fruit > 30 mm.

[0066] The effect of the application corresponded to a 5.11% increase in fresh weight per fruit and a 9.6% increase in juice content per fruit. EXAMPLE 8. SALT STRESS MANAGEMENT THROUGH A FORMULA CONTAINING PF AND GB AND ITS EFFECT ON THE YIELD OF THE ONION CROP, VARIETY BLANCA MONTEJO

[0067] The trial was conducted at the Agrolosa agricultural farm in Lázaro Cárdenas, Chihuahua, Mexico, covering 5 hectares, divided into two irrigated areas. The soil used had an EC of 13.8 dS*nr. 1 determined from a 1:1 ratio extract, which is considered saline. Three applications were made every 15 days, 3 L / h each, applied using a bar equipment with nozzles for SJ7 liquid fertilizers and an expenditure of 1,536 L / ha, of a formulation containing between 1 to 2% (gallic acid equivalents / 100 mL of sample) of PF and 2 to 4% of GB (w / v). For both the control group (TO) and the treated group (T1), 15 plants were marked and harvested from 3 blocks randomly arranged in each sector.

[0068] At the end of the season, an increase of 1.33% in onion yield was obtained (TO: 94,644 and T1: 95,904 Kg / ha). Although no significant differences were observed in yield, there was a significant improvement in bulb size, corresponding to an average value of 7.41 inches for T0 and 8.17 inches for T1 (10.26% increase), which translates into a price differential for the quality and classification that corresponds to these sizes. EXAMPLE 9. EFFECT OF ROOT SYSTEM BIOSTIMULATION OF A PRODUCT BASED ON A POLYPHENOLS (PF) AND GLYCINE BETAINE (GB) FORMULA ON THE INCREASE IN ASPARAGUS YIELD (Asparagus officinalis).

[0069] The trial was carried out in the agricultural area in the town of Lea, Peru. The crop has a plant density of 19,000 units per hectare, irrigated by means of a tape with well water with a high concentration of salts (EC 21.3 dS*nr). 1 ).

[0070] To evaluate the yield of tuhones, the quantity of 5 kg boxes with quality A and B and size XL was measured during a cutting cycle that lasted 6 days of 1 hectare per sector. Two sectors of 1 Ha each were available, corresponding to T0 the control sector and T1 for the sector treated via foliar with a formulation containing between 1 to 2% (gallic acid equivalents / 100 mL of sample) of PF and 2 to 4% of GB (w / v), at a dose of 3 L / Ha and a wetting of 650 L / Ha. Applications were made with the first, second and third shoot with the same doses of 3 L / Ha each.

[0071] From a total of 6 harvests, the number of boxes harvested per treatment was counted, yielding 631 boxes, equivalent to 3,155 kg, for the control sector, and 714 boxes, equivalent to 3,570 kg, for the treated sector; that is, a 13% increase in yield was obtained in favor of the treated sector. According to the examples provided, the formulation of polyphenols (PF) and glycine betaine (GB) clearly provides a novel and unique method for mitigating abiotic stress, particularly one of the most complex, such as salinity stress, by comprehensively and effectively reducing oxidative stress, osmotic stress, and ionic stress, triggered by excess salts.

Claims

CLAIMS Having sufficiently described the invention, I consider the following clauses to be novel and therefore claim exclusive ownership.

1. Improvements to a method for extracting an active acid extract from agricultural husks, particularly rice husks, where the active acid extract is obtained through the following phases: a) Pentose extraction phase, b) Oxidation phase, c) Clarification and concentration phase, d) Adsorption or retention phase, e) Purification phase and f) Desorption phase; Characterized in that in the d) adsorption and concentration phase, the filtered or clarified and concentrated product resulting from the classification and concentration phase is circulated through columns packed with 1 to 4 kg, preferably 1.5 to 3 kg of granular activated carbon or a quantity of resin with a high specific adsorption capacity that can be packed in a volume equivalent to the effective volume of the columns given by an internal diameter of 10.16 cm and an effective height of 85 cm. The selected resins with a high specific adsorption capacity are included within the group of macroporous polystyrene resins functionalized with amines. tertiary, such as Purolite® MN-102, or with other hydrophilic molecules, or resins from the amberlite family; applying an optimal influent flow rate between 1.0 to 6.0, preferably between 1.5 to 4.5 VLE h-1 (packed bed volumes per hour) for the development and inclusion of an adsorption process so that phenolic compounds and flavonoids contained in low concentrations in the clarified and concentrated product and circulated as influent in the columns, are adsorbed and retained in the adsorbent material; while other secondary compounds, such as aliphatic polyhydroxycarboxylic acids, carbohydrates, alcohols and aldehydes, are eliminated in the effluent;in the e) purification phase, the columns containing the phenolic compounds and flavonoids adsorbed on the activated carbon or high specific adsorption capacity resins are washed in counterflow with 5 to 50 L, preferably with 15 to 30 L of acidified water (pH < 4), to eliminate other residual compounds, such as aliphatic polyhydroxycarboxylic acids, carbohydrates, alcohols and aldehydes, absorbed between the pores of the activated carbon or macroporous resins that could act as contaminants during the desorption or elution process. Additionally, and to facilitate the subsequent desorption or elution process, the columns are drained of the washing water and dried by circulating air in counterflow for 5 to 30 minutes, preferably for 10 to 20 minutes, and in the f) desorption phase, 10 to 40 L, preferably 15 to 25 L, are circulated in counterflow; of 96% (v / v) ethanol or an alkaline aqueous solution of 0.05 to 0.2 M potassium hydroxide, preferably 0.1 to 0.2 M, through the columns packed with the adsorbed polyphenolic compounds, maintaining the pH in a range of 7.0 to 10, preferably between 7.5 to 9.0, to favor the solubility of the adsorbed compounds and facilitate the desorption or elution process to obtain a concentrated active acid extract with polyphenols in a concentration range between 3 to 10 g equivalent of gallic acid / 100 mL 2. The active acid extract, according to claim 1, characterized in that in one of the preferred embodiments of the present invention, it is formulated at a concentration of 3 to 10 g equivalent of gallic acid / 100 mL together with glycine betaine until reaching a final concentration between 0.5 to 2.5% g equivalent of gallic acid / 100 g of sample, preferably between 1.0 to 2.0%, while the GB is at a concentration between 1 to 5%, preferably between a concentration between 2 to 3%, keeping a polyphenols (PF) / glycine betaine (GB) ratio, between 1:1 to 1:

3.

3. The active acid extract, in accordance with reinstatement 2, characterized in that it is applied for the treatment of plants, when diluted in water until reaching final concentrations of effective field doses of 0.0014 to 0.0105% (w / v) of polyphenols (PF) and 0.0030 to 0.0225% of glycine betaine (GB), applying between 400 to 1000 L / Ha of the diluted solution.

4. The active acid extract according to claim 2 and 3, characterized in that it is used to promote the growth and development of plants under normal conditions or under abiotic stress, particularly under salinity stress, and comprising the application of effective doses of the solution to monocotyledonous plants, such as a cereal or grass, preferably corn or to an amahlidae, preferably onion; and dicotyledons of the Solanaceae family, preferably to tomato, chili, pepper and potato or of the Rosaceae family, preferably to apple, or of the Malvaceae family, preferably to cotton, or of the Cucurbitaceae family, preferably to watermelon, melon or cucumber, or of the Asparagaceae family, preferably to asparagus.

5. The active acid extract according to claim 2 and 3, characterized in that it is used to promote the overexpression of genes involved in the response to oxidative stress, such as APX, ascorbate peroxidase; GST1, glutathione S-transferase-1; ion transport, such as ATPse4 and ATPase4; auxin transport, such as PIN1 and PINFORMED; and response to auxins, such as IAA5 and Inducible Indole-3-acetic acid 5 under normal conditions or under abiotic stress, particularly under salinity stress, and comprising the application of effective doses of the solution to monocotyledonous plants, such as a cereal or grass, preferably corn or an amahlidae, preferably onion; and dicotyledons of the Solanaceae family, preferably tomato, chili, pepper and potato or from the Rosaceae family, preferably apple, or from the Malvaceae family, preferably cotton, or from the Cucurbitaceae family, preferably watermelon, melon or cucumber, or from the Asparagaceae family, preferably asparagus.

6. The active acid extract according to claim 2 and 3, characterized in that in the case of cereals, such as corn, it is applied during the vegetative stage of growth, starting at V2 and ending at VT, at a concentration of 1 to 3 L / Ha, divided into 2 to 3 applications; in the case of apple crops, between 1 to 5 L per hectare, preferably 2 to 4 L per hectare, are applied, divided into 3 applications, starting after the sprouting period and at intervals of 20 to 30 days; for the cultivation of vegetables, such as asparagus, chili, tomato and onion, the same concentration is applied divided into 3 applications, starting after the sprouting period and at intervals of 15 to 20 days, while in cotton crops, in a single application, of the same concentration after the sprouting period.

7. The active acid extract according to claims 2 and 3, characterized in that it is applied with a herbicide, such as glyphosate, halosulfuron, or others; a fungicide, such as strobirulins, tetraconazole, or others; with a nematicide, such as strobirulins, tetraconazole, or others; and with nitrogen fertilizers, such as ammonium nitrate, potassium nitrate, urea, or micronutrients.

8. The active acid extract according to claim 2 and 3, characterized in that it is applied directly to the soil by any convenient means, including sprinkling, drip irrigation systems, tape irrigation systems, or others that are obvious to anyone skilled in the art. The following examples are intended to illustrate the present invention and to teach one skilled in the art how to use the formulations and methods of the present invention, but they are not limiting in any sense.

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