METHOD FOR EFFICIENT SEED DISINFECTION

Abstract

The present invention falls within the technical field of agriculture, particularly methods for treating seeds before sowing or planting. This invention aims to solve the problem of seed contamination. The purpose of seed disinfection is to eradicate contaminating microorganisms present in the seed coat and embryo, so that the resulting plants are free of contaminating bacteria and fungi. In addition to preventing contamination and diseases, the goal is for seed disinfection to improve germination rates. To achieve this, a method and a kit for efficient seed disinfection are provided, comprising contacting the seeds with a treatment system that includes at least one treatment composition with at least one disinfectant agent and / or an agronomically acceptable carrier.

Description

METHOD FOR EFFICIENT SEED DISINFECTION TECHNICAL FIELD The present invention is located in the technical field of agriculture, particularly within the area of ​​sowing and specifically is located in the field of methods for treating seeds before sowing or planting. BACKGROUND Plant reproduction specialists have focused their research on the sexual or asexual reproduction of endangered or commercially valuable plants, as they are used to obtain various products such as food, beverages, and fiber, among others. Agave is an example of a plant of interest, as it has enjoyed growing social and economic importance due to its use as a raw material for the production of fermented and distilled alcoholic beverages such as pulque, mezcal, and tequila. In seed reproduction processes, particularly in vitro cultures, the success of propagation systems depends largely on the control and prevention of contamination. Contaminating microorganisms cause significant losses of plant material in production or research processes. The purpose of seed disinfection is to eradicate contaminating microorganisms present in the seed coat and embryo, so that the resulting plants are free of contaminating bacteria and fungi. In addition to preventing contamination and disease, seed disinfection can also improve seed germination rates. Improving seed germination rates is essential for the successful in vitro cultivation of plants of interest, as a low germination rate can affect the efficiency and productivity of the in vitro cultivation process. / ant? ι η / Ρ7Π7 / Β / γι Therefore, it is important to follow appropriate disinfection procedures to ensure crop success and improve the quality, health, yield, and conservation of germplasm. Currently, various seed disinfection protocols have been reported in species such as A. victoria-reginae (Domínguez et al., 2008; Ramírez et al., 2008), A. fourcroydes (Monja, 2013), A. angustifolia (Arzate et al., 2016), A. marmorata (Álvarez et aL, 2020) and A. tequilana (Delgado et al., 2021), in which most authors agree on the use of Ethanol and Sodium and Calcium Hypochlorite. However, the use of Mercury Chloride (HgCh), Hydrogen Peroxide (H2O2) and fungicides, as well as the contaminating microorganisms found, have not been reported. On the other hand, few studies report contamination rates, which significantly increase production costs. Therefore, having an efficient disinfection protocol can reduce production costs. OBJECT OF THE INVENTION An object of the present invention is to provide an efficient method for disinfecting seeds that addresses, at least in part, one or more of the aforementioned problems. It has surprisingly been discovered that this objective can be achieved by contacting the seed with various disinfectant compositions in a single treatment. Accordingly, the invention relates in a first aspect to a method for efficient seed disinfection, which comprises contacting the seeds with at least one treatment composition comprising at least one disinfectant agent and / or an agronomically acceptable vehicle. In this sense, the main object of protection refers to a method for efficient seed disinfection, which comprises contacting the seeds with a treatment train with at least one treatment composition comprising at least one disinfectant agent and / or an agronomically acceptable vehicle, characterized in that the treatment train comprises: a) put the seeds in contact with Mercury II Chloride; b) subsequently put the seeds in contact with Copper Sulfate; c) finally, put the seeds in contact with Hydrogen Peroxide. Yet, a second object of protection relates to a kit for efficient seed disinfection, characterized in that it comprises at least one treatment composition comprising at least one disinfectant agent and / or an agronomically acceptable vehicle, wherein said compositions are selected from: a) Mercury II Chloride; b) Copper Sulfate; c) Hydrogen Peroxide. The method advantageously provides improvements in seed disinfection against contaminating microorganisms, especially seed-borne fungi. Furthermore, the method allows for the use of smaller quantities and / or less harmful substances, such as biocides. Furthermore, the method can provide a disinfecting effect on seed-borne bacteria. Furthermore, the method allows seed disinfection without significantly affecting seed quality or germination properties. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Germination of A. salmiana seeds. A) Root emergence and elongation. (14 das) B) Plumule elongation. (21 das) C) Cotyledon differentiation and root development. (30 das) D) Germination and complete development. (14-30 das) E) Fully developed normal A. salmiana seedlings. (50 das); 21 days after sowing. / RnfrLn / eznz / e / Yi Figure 2. Strains of pathogens found in the seeds of Agave spp. A) Schizosaccharomyces sp. B) Bacillus sp. C) Penicillium sp. D) Alternaría alternata. E) Aspergillus sp. F) Monilinia sp. DESCRIPTION OF THE INVENTION The present invention relates to a method for effectively disinfecting seeds. This is a crucial step in in vitro cultivation to prevent contamination, which can be caused by contaminating microorganisms such as bacteria, fungi, and viruses that can affect seedling growth and reduce seed germination rates. Therefore, proper seed disinfection is essential to ensure vigorous and healthy plant growth. The advantages conferred to the method of protection are derived from various efforts aimed at finding the most efficient treatment to achieve 100% in vitro disinfection in seeds without affecting germination. In order to clarify the description of the present invention and to enhance its understanding, it should be understood that the terminology used herein is not intended to be limiting. A glossary of technical terms used in this specification is listed below. The term seed as used herein includes, but is not limited to, a mature ovule of gymnosperms and angiosperms, which contains an embryo surrounded by a protective covering, the seed coat (testa). Some seeds comprise a pericarp or fruit cover around the seed coat. In practical terms, the term “seed” is used to include anything that can be planted (sown) in agriculture to produce plants, such as true seeds, rootstocks, plant cuttings, and plant parts such as a tuber or bulb. Preferably, however, the term seed refers to its normal meaning in the art, i.e., the mature, fertilized ovule of a flowering plant that / RnfrLn / eznz / e / Yi contains an embryo and is normally capable of germinating to produce a new plant. The term "disinfecting" plant seeds includes, for example, eliminating, killing, or reducing the impact of a pathogen (potential inoculum), particularly contaminating bacteria and fungi. Pathogens can also include nematodes and seed-borne insects, including their eggs. Pathogens are preferably bacteria, as they are more numerous and versatile. Rendering a pathogen harmless includes, for example, rendering the pathogen unable to infect the plant. Some examples of methods for preparing disinfected seeds that provide a reduction in the percentage of infected seeds and / or in the number of colony-forming units. The term "disinfection" is not used to imply a restriction on the objects that are disinfected; in particular, the seed of the plant may be considered living or non-living. The term "disinfected seed" includes, but is not limited to, seeds for which a reduction in the number of infected seedlings and / or colony-forming units for one or more pathogens is observed compared to untreated seeds. Therefore, a disinfected seed may still contain several live pathogens. An example of disinfected seed is seed in which the pathogenic inoculum for at least one type of pathogen is rendered at least partially harmless or less harmful, for example, by its removal or inactivation. The term disinfectant agent refers to a compound or composition that exhibits a disinfectant effect. The term "treatment atmosphere" as used herein includes a gas phase having a temperature and relative humidity to which the seeds are exposed. The term "relative humidity" as used herein refers to the ratio of the partial pressure of water vapor in the gas phase / RnfrLn / cznz / e / Yi to the saturated water vapor pressure at the temperature of the treatment atmosphere. The term "exposure time" as used herein refers to the duration of exposure of the seed to the treatment atmosphere. Alternatively, or in addition, contact with the disinfectant compositions comprises a liquid component that can result in wetting of the seed surface. This can help mitigate any insulating effects of roughness on some seeds. A treatment composition comprising a surfactant can provide enhanced wetting. A surfactant can also help effectively deliver the disinfectant agent to the seed endosperm. Wetting the seed can further increase process control by providing heat at a more constant temperature and also cooling the seed after exposure to a treatment atmosphere. Rapid and sufficient cooling is important for seed quality and germination properties. Notwithstanding the above-mentioned effects, which do not limit the invention by way of theory, the examples demonstrate a synergistic effect of several disinfectant compositions resulting in more effective disinfection. Preferably, the disinfectant compositions comprise water and / or are a liquid. To improve wetting, they preferably comprise a surfactant. This can also provide better penetration of the disinfectant compositions into the seed, into any air pockets and / or pores of the seed. Surfactants can also aid in the detachment of bacteria from the seed, particularly where the bacteria adhere to the seed surface through hydrophobic interaction. This is especially advantageous for crops that have seeds with a hydrophobic seed surface. For example, some seeds have a surface comprising epicutular waxes. Such waxes may contain alkanes, long-chain alcohols, ketones, and / or long-chain fatty acid esters. The seeds of some crops contain hydrophobic proteins in their seed coats. Examples include soybean and rapeseed. / AnfrLn / eznz / e / YiThe seed to be disinfected is typically capable of germination. Suitable seeds include non-germinated seeds, preferably unprepared. Optionally, the seed may be dehulled (hulled seed or dehulled seed). Preferably, immediately before contact with a treatment composition, the seeds have a temperature of -10°C to +30°C, more preferably 0-25°C, for example 10-25°C, and optionally are maintained above 5°C, 10°C, or 15°C during the method until the optional cooling step. Optionally, the method begins with opening a package containing seeds or providing a batch of seeds. The method may also comprise a step of subjecting the seeds to a treatment unit prior to said contact, wherein the unit comprises, for example, a seed chamber with at least one inlet for the disinfectant compositions.The method optionally comprises recycling the disinfectant compositions. The seed as supplied may contain pathogens and / or seed-borne contaminating microorganisms, such as bacteria, particularly dormant bacteria, or may be susceptible to or at risk of containing such bacteria. The method involves contacting the seed with the disinfectant compositions. Preferably, the contact with the disinfectant compositions is carried out at least in part at a temperature of less than 50°C or less than 40°C, such as 5-50°C. Preferably, the contact with the seed and / or the disinfectant compositions is at a temperature of less than 50°C or less than 40°C, such as 5-50°C, more preferably 15-35°C. Preferably, the disinfectant compositions are applied at a controlled temperature. This advantageously provides high efficiency and / or reliability. Preferably, the disinfectant compositions are applied at at least 15°C or at least 20°C.Preferably, the seed is contacted with the disinfectant compositions for a contact time of at least 10 seconds, such as at least 1 minute, or at least 5 minutes or at least 10 minutes, and for example less than 24 hours, such as less than 60 minutes, for example 10-30 minutes or 60-300 seconds. / «nfrLn / eznz / e / Yi The seed can be contacted with the disinfectant compositions, for example, by soaking or immersing the seed in the disinfectant compositions, or by spraying, dripping, or flowing the disinfectant compositions onto the seed, or by controlled humidity increase, condensation, soaking, or washing the seed with the seed disinfectant compositions. For example, a gaseous treatment composition can be blown onto the seed. For example, a powdered treatment composition can be mixed with the seed, or by controlled humidity increase, condensation, soaking, or washing the seed with the seed disinfectant compositions. Optionally, the seeds may be placed in a bag or basket, on a plate, or in another form of container during contact, as well as on a moving belt. Optionally, the method may result in absorption of the disinfectant compositions by the seeds. Absorption of the disinfectant compositions into the seed may provide enhanced antimicrobial efficacy, especially against internal bacteria. Preferably, the weight of the seed increases during contact with the disinfectant compositions by at least 1.0%, based on the difference in seed weight before and after such contact, with removal of excess treatment composition, divided by the weight before contact. Removal of excess treatment composition may involve, for example, wiping the seed dry.The weight gain can be, for example, from 0.10 to 25%, such as at least 1.0%, at least 2.0%, for example, less than 20%, for example 2-15% and / or 15-25%. Thus, the seeds can absorb the disinfectant compositions in an amount of, for example, at least 5% or at least 10% of their weight, such as 15-25% or 20-25%. Contact may also result in some swelling of the seed, for example, a volume increase of at least 0.5% or at least 2.0%. In principle, the method may comprise two or more steps of contacting the seed with a treatment composition, where the disinfectant compositions are different. The method may also comprise any additional step of contacting the seed with compositions that do not comprise a disinfectant agent or liquid component. Disinfectant compositions comprise at least one disinfectant agent and / or a liquid component and may comprise additional components. The liquid component is preferably water. For example, the disinfectant compositions may consist essentially of water (such as more than 99% by weight), e.g., tap water. Thus, the method may comprise contacting the seed with water. Disinfectant compositions can be applied, for example, in liquid form, e.g., as a solution, suspension, dispersion, emulsion, or foam, and also in the form of a spray, mist, or aerosol, preferably aqueous. Disinfectant compositions can also be gaseous or solid. For example, disinfectant compositions can comprise ozone or a powder. Generally, disinfectant compositions are fluid, including a fluid (liquid or gaseous), or granular, preferably the composition is liquid. Preferably, disinfectant compositions are liquid, comprise water, and / or comprise a surfactant. Preferably, the disinfectant compositions are in liquid form and comprise water and, more preferably, a surfactant. For treatment compositions comprising hydrophobic components, particularly as disinfectants, such as certain carboxylic acids, it is preferred to add a surfactant. The surfactant can enhance the effectiveness of the hydrophobic disinfectant by improving contact between said agents and the bacteria. For example, anionic, cationic, zwitterionic, and nonionic surfactants can be used. Preferably, the surfactant is an organic compound comprising a hydrophilic group. Examples include linear alkylbenzenesulfonates, lignin sulfonates, fatty alcohol ethoxylates, and alkylphenol ethoxylates. Some possible surfactants have anionic functional groups such as sulfate, sulfonate, phosphate, and carboxylates, particularly alkyl carboxylates, for example, dodecyl sulfate salts and alkyl ethers thereof. Cationic surfactants include primary, secondary, or tertiary amines and quaternary ammonium salts, particularly aromatic quaternary ammonium salts. Other surfactants are zwitterionic compounds having a phosphate anion with an amine or ammonium.Alkyl ethers of polyoxyethylene and polyoxypropylene glycol can be used, as well as, for example, alkyl glucoside ethers, alkyl glycerol ethers, polysorbate, block copolymers of polyethylene glycol and polypropylene glycol, and polyethoxylated tallow amine. The skilled person may apply suitable amounts of surfactant, such as at least 1 ppb or at least 10 ppb (parts per billion by weight) or at least 100 ppb or at least 1.0 ppm, at least 10 ppm, at least 20 ppm (parts per million by weight), at least 0.010 wt%, at least 0.10 wt%, at least 1.0 wt%, based on the total weight of the disinfectant compositions, or for example concentrations providing at least 1 µg or at least 1 mg or at least 10 mg, at least 0.10 g, at least 1.0 g, at least 5.0 g or at least 10 g per kg of seed 0.010 wt%, at least 0.10 wt%.%, at least 1.0 wt. %, based on the total weight of the disinfectant compositions, or for example concentrations providing at least 1 µg or at least 1 mg or at least 10 mg, at least 0.10 g, at least 1.0 g, at least 5.0 g or at least 10 g per kg of seed, such as at least 1 ppb or at least 10 ppb (parts per billion by weight) or at least 100 ppb or at least 1.0 ppm, at least 10 ppm, at least 20 ppm (parts per million by weight), at least 0.010 wt. %, at least 0.10 wt. %, at least 1.0 wt. %, based on the total weight of the disinfectant compositions, or for example concentrations providing at least 1 µg or at least 1 mg or at least 10 mg, at least 0.10 g, at least 1.0 g, at least 5.0 g or at least 10 g per kg of seed. Preferably, the disinfectant compositions comprise a disinfectant agent, preferably selected from the group consisting of organic acids, carboxylic acids, alcohols, aldehydes, oxidizing agents such as peroxides and peroxyacids, phenolic compounds, quaternary ammonium compounds, silver and silver compounds, including silver, ions and colloidal silver, chlorine and chlorine compounds, and iodophors. Even more preferably, a treatment of various disinfectant compositions selected from 3% Hydrogen Peroxide for between 24 to 48 h, 30% (v / v) Copper Sulfate for between 10 to 15 min and 0.1% (w / v) Mercury II Chloride for between 10 to 15 min. Preferably, the compositions further comprise a vehicle or diluting agent, preferably water. The disinfectant compositions may comprise, for example, a peroxy compound such as hydrogen peroxide, for example, at least 0.10%, at least 0.50%, or at least 1.0% by total weight of the disinfectant compositions. The composition typically contains less than 20% by weight of peroxide and preferably less than 5.0% by weight in view of the effect on germination properties. Suitable commercially available peroxy solutions are available. The peroxy solution may contain, as appropriate, the usual stabilizers for peroxy. / RnfrLn / eznz / e / Yi Seed is, for example, the seed of an agricultural crop, including vegetables. Some examples of suitable seeds also include vegetable seeds, herb seeds, wildflower seeds, ornamental seeds, grass seeds, and tree and shrub seeds. Seed can be from the order Monocotyledoneae or the order Dicotyledoneae. Some examples from the order Monocotyledoneae are rice seeds and wheat (Triticum aestivum). Some examples of suitable seeds include soybean, cotton, corn, peanut, maize, wheat, barley, oat, rye, triticale, mustard, sunflower, sugar beet, safflower, millet, chicory, flax, rapeseed, buckwheat, tobacco, and hemp seeds. Seeds include alfalfa, signal grass, clover, sorghum, chickpeas, beans, peas, and vetch.Some examples of suitable vegetable seeds include asparagus, chives, celery, leek, garlic, beetroot, spinach, beetroot, kale, cauliflower, sprouting broccoli, collard greens, white cabbage, red cabbage, swede, Chinese cabbage, turnip, escarole and chicory, watermelon, melon, cucumber, gherkin, marrow, parsley, fennel, pea, bean, radish, black salsify, eggplant, sweetcorn, popcorn, carrot, onion, tomato, pepper, lettuce, snap bean, cucurbits, shallot, broccoli, Brassica, and Brussels sprouts. Some examples of rice include Oryza sativa japonica, Oryza glaberrima javanica, Oryza sativa indica, Zizania palustris, and their hybrids. The method is not limited to these example crops. The method, for example, may comprise an additional step of removing the cooled and optionally dried seeds from the processing chamber or a cooling and / or drying chamber. The method optionally further comprises packaging the seed, for example, in a bag or container. The method may further comprise storing the treated seed, optionally at 2-10°C. Other possible steps are sowing or planting the disinfected seed, germinating the seed, and growing plants from said seed, as a way to benefit from the disinfection effect. The method preferably comprises cooling and optionally drying the seed. The drying may be carried out, for example, over a period of 2 to 8 hours, such as 3 to 6 hours, preferably 4 to 6 hours. This cooling and drying comprises, for example, exposing the seed to a cooling and drying atmosphere. The cooling atmosphere and the drying atmosphere may be the same or different. The drying and cooling may be carried out successively or partially or completely simultaneously with each other. Preferably, the method comprises a combined cooling and drying step and a subsequent cooling step. The combined cooling and drying, for example, comprises exposing the seed to a drying atmosphere, typically with a relative humidity of 50% or less, more preferably 20% or less, and a temperature lower than the treatment temperature, but higher than ambient, typically 30-45°C.The drying atmosphere is typically air. The final cooling step normally comprises exposing the seed to an atmosphere with a temperature of 30°C or less, preferably no more than 5°C above room temperature, or around room temperature, but more preferably lower, for example 10°C lower than room temperature. The cooling step preferably comprises exposing the seed to ambient air or to air cooled typically to 10-25°C. Such a final cooling step may also be applied without a combined cooling / drying step. The seed is cooled, for example, to room temperature, or to less than 5°C above room temperature, or to storage temperature. The cooling phase is preferably carried out directly after exposure of the seed to the treatment atmosphere.Preferably, the cooling is carried out at least partially with a cooling rate of at least 5°C / min, such as at least 10°C / min; for example, in an initial cooling step of 5-20°C in 1 minute or less. All references cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The use of the terms "a" and "an" and "the" and similar referents in the context of the description of the invention (especially in the context of the claims) should be construed to cover both the singular and plural forms unless otherwise indicated or clearly contradicted by the context. The terms "comprising," "having," "included," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise indicated. The enumeration of ranges of values ​​herein is merely intended to serve as a shorthand method for individually referring to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if individually enumerated herein.The use of any and all examples or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and is not intended to limit the scope of the invention unless otherwise claimed. No language in the specification should be construed to indicate that any unclaimed element is essential to the practice of the invention. For the purposes of the description and the appended claims, unless otherwise indicated, all numbers expressing amounts, quantities, percentages, etc., are to be deemed modified in all instances by the term “about.” Furthermore, all ranges include any combination of the disclosed maximum and minimum points and include intermediate ranges therein, which may or may not be specifically recited herein. Preferred embodiments of this invention are described herein. Variations from these preferred embodiments may be apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to employ such variations as appropriate, and the inventors intend that the invention be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. Furthermore, any combination of the elements described above in all possible variations is encompassed by the invention unless otherwise indicated herein or clearly contradicted by the context. The invention will now be illustrated in more detail by the following non-limiting examples. Examples The following is an example of a series of steps well known to those skilled in the art. One of these steps includes, in particular, the step comprising the essential feature discussed extensively in this specification. However, these examples are illustrative and not limiting, as those skilled in the art will understand that there are variants that fall within the scope of protection of the present invention. Mature seeds of seven Agave species were used, collected in different states of the Mexican Republic over a period of 1 to 7 years, preserved in a germplasm bank during storage (Table 1). / AnfrLn / eznz / e / Yi IVIA / a / ZUZÓ / U 1 Table 1. Agave species and seed collection data. Species Collection location Date Agave horrida Lem. ex Jacq. Ocuilan, Mexico 2015 Agave salmiana Otto ex Salm-Dyck. Acambay, Mexico 2015 Agave karwinskii Zuce. Oaxaca, Mexico 2017 Agave potatorum Zuce. Oaxaca, Mexico 2017 Agave marmorata Roezl. Oaxaca, Mexico 2017 Agave angustifolia Haw. Zacualpan, Mexico 2019 Agave cupreata Trelease. Puebla, Mexico 2021 Disinfection of biological material Twelve treatments with different disinfectant solutions and combinations were evaluated. The disinfectants used were: Hydrogen Peroxide (H2O2) at 3% for 24 h, Commercial Sodium Hypochlorite (Cloralex® 4%) (NaCIO) final concentration of 5% v / v) Calcium Hypochlorite Ca(CIO)2 at 8% (w / v) Copper Sulfate trade name PENTAMAX® 30% (v / v) Mercury II Chloride (HgCb) at 0.1% (w / v) The 12 treatments were evaluated with 10 seeds per treatment (each seed was considered a single experimental unit), and three replicates were considered for each treatment, evaluating a total of 390 seeds per species (considered a control). The experiment was monitored weekly for a period of 30 days. Initially, the seeds were placed in 50 mL Falcon tubes and washed with 15 mL of sterile osmosis water plus 1 mL of commercial soap (Axión®) and two drops of Tween 20® for 15 minutes, keeping them under agitation; after this time, they were rinsed three times with sterile distilled water. Next, they were immersed in 15 mL of 70% (v / v) ethanol for one minute and rinsed twice with sterile distilled water. Subsequently, the disinfection treatments were applied. The seeds were kept under constant agitation in all treatments and rinsed three times with sterile distilled water after the application time of each disinfectant to eliminate disinfectant residues, except for the treatments with H2O2. Everything was carried out under aseptic conditions, inside a laminar flow hood. / RnfrLn / eznz / e / Yi Table 2. Disinfection treatments evaluated in seven Agave species. Disinfectant agents Treatment Copper sulfate 30% v / v HgCb 0.1% w / v Ca(CIO)2 8% w / v NaCIO 5% v / v H2O2 3% 0 'Control 1 15 min 2 5 min 3 15 min 5 min 4 24 h 5 10 min 15 min 6 10 min 5 min 7 10 min 15 min 5 min 8 10 min 24 h 9 10 min 10 min 15 min 10 10 min 10 min 5 min 11 10 min 10 min 15 min 5 min 12 10 min 10 min 24 h Copper Sulfate (PENTAMAX®); HgCh= Mercury II Chloride; Ca(CIO)2= Calcium Hypochlorite; NaCIO= Sodium Hypochlorite; Η2θ2= Hydrogen Peroxide. 'Witness (Without any disinfectant). Seed germination After disinfection, the seeds were sown in 100 mL culture flasks, placing 10 seeds per flask. 20 mL of culture medium with MS salts (Murashige and Skoog, 1962), supplemented with 30 g L-1 sucrose, 0.5 g L-1 activated carbon, without plant growth regulators and gelled with 8 g L-1 agar, were added to each flask. The pH of the medium was adjusted to 5.7 and it was sterilized in an autoclave at 1.5 kg / cm2 pressure and a temperature of 121.5 °C for 15 minutes. The cultures were incubated for 30 days in an incubation room with a photoperiod of 16 h light and eight hours of darkness, with a light intensity of 18.83 μM·ιτΓ2·5·1 at a temperature of 25 ± 2 °C. A seed was considered germinated when the radicle was visible. When processing the data regarding seed germination, the mean tests showed significant statistical differences (p<0.05) in the different treatments, the germination response was very variable in the different treatments and independent for all species, since each species responded differently and its germination time was also variable, for the most part this variable was low (0 - 20%) (Table 3). However, the results are consistent with those obtained for the contamination variable. The only treatment that stimulated germination for all seven Agave species, in addition to eliminating pathogens, was treatment 12, where the germination percentage for most species was higher than the other treatments, such as A. marmorata (90%), A. angustifolia Haw (85%), A. salmiana (50%), A. hórrida (30%), A. potatorum Zuce (30%), A. cupreata (30%), and A. karwinskii Zuce (20%). On the other hand, A. marmorata was the one that responded best, reaching an average of 90% germination and was also the earliest species, germinating 8 days after planting, thus being the species that stood out statistically from the rest (Table 3). However, despite the low percentage of germination for the other treatments (0 - 20%), none of the seedlings obtained presented abnormalities in their germination and development, regardless of the treatment used and the species (Figure 1). The results of this study suggest that it is possible to store seeds in a germplasm bank for longer periods, achieving viability (RnfrLn / cznz / e / Yi) above 50%. Furthermore, analyzing the days of germination confirmed that the seeds did not show dormancy, a characteristic characteristic of other Agave species. ,κηπ in / pznz / B / vi In the treatments where germination was not recorded for some species, highlighting the control treatment (TO) in which no species germinated (Table 3) it can be attributed to the presence of fungi and bacteria (contamination), since the seeds affected by phytopathogenic fungi can present sclerotization, stromatization, discoloration, necrosis, radicle rot, size reduction, abortion and germination incapacity. Table 3. Comparison of means for the variables contamination and germination in seven Agave species, according to the Tukey test (p<0.05) Treatments A.salmiana A.hórrida A.marmorata A.potatorum A.karwiskii A.angustifolia A.cu preata DG (21 d) (14d) (8d) (27d) (20d) (20d) (14d) - 100 D 0 - 100 c 0 - 100 D 0 - 100 c 0 - 100 D QC 1 NaOCI 100 D 0 ~ 10AB 10 BC 0 a 80 Ae 0 A 10 BC 100 D 0 - 0 a 80 Ae 0 A 10 BC 100 D 0 - 0 a 80 AB 0 a CaO 20 - 10 CIO 0 to 85 to 100 D 40 ABC 100 c 0 - 0 to 0 - 0 to 0 - 0 to 20 AE= 3 Ca(CIO)2 / NaOCI 0 to 60 ABC 0 to 85 A 100D H2O2 0 to 10BC 0 to 0 0 0 to 70 ABC 0 to 85 A 0 to 0 - 40 ABC 20 ABC 0 to 10 BC 5 PM / Ca(CIO)2 100 D 0 3 0 to 10 BC 0 to 50 ABC 0 to 50 AB: 6 PM / NaOCI 0 A 0 - 0 to 0 ~ 0 to 80 Ae 100c 0 - 0 to 0 ~ 50 BC 0 3 0 to 85 to 7 PM / Ca(CIO)2 / NaOCI 100D 0 “ 0 to 20 ABC 100D 0 - 100c 03 0 a 80 AB 8 PM / H2O2 0 a 20 ABC 100 c 10 BC 0 a 10 SC 0 a 0 3 0 a 10 EC 00 a 10 ABC 0 a 0“ 9 PM / HgCh / CaO 30 aAB(CIO) 10 BC 0 to 40 ABC 0 to 0 3 0 to 20 ABC 30 AB 0“ 0 to 80 AB10 PM / HgCI2 / NaOCI 100 D 0 3 0A 30 AEC 0 to 10 bc 0 A 0 - 0 to 20 ABC 100 D 0 3 0 to 80 AE 11 PM / HgCI / Ca(CIO)2 / NaOCI 100 D 20 ABC 0 to 30 ABC 0 to 20 ABC 0 to 10 BC 40 ABC 10 EC 100 c 00 3 0 to 80 AB 12 PM / HgCI2 / H2O2 0 to 50 ABC 0 to 30 ABC 0 to 90 a 0 to 30 ABC 0A 20 ABC 00 to 85 a 0 to 80 AB T: Treatment; TO: Control; DG= Days to germination from in vitro sowing (^Contamination); G=Gemination. Equal letters indicate no statistically significant differences (p<0.05) between treatments. Identification of pathogens found in Agave seeds Microorganisms found in the treatments that presented contamination were isolated and sown in Petri dishes with PDA medium (Potato-Dextrose Agar) for isolation and purification. They were subsequently sent to the Microbiology Laboratory of the Center for Research and Advanced Studies in Animal Health (CIESA) of the Autonomous University of the State of Mexico, for taxonomic identification, where the samples were reseeded in PDA medium and Sabouraud Agar at 25 ± 2 ° C for 26 days, constantly monitored for subsequent morphological identification, for which lactophenol stains were performed. For the identification of bacteria, the samples were sown on blood agar and TSA agar (Tryptone-Soy Agar) incubated at 36 ° C and constantly monitored for 5 days. The Tukey test (p < 0.05) determined that there were highly significant differences between all the applied treatments, since the majority of treatments differentially decreased the contamination in the seeds. However, statistically only one treatment was better than the rest (Treatment 12), completely decreasing the contamination (100%) for all the studied species, which consisted of applying Hydrogen Peroxide (H2O2) at 3% for 24 h, Copper Sulfate at 30% (v / v) for 10 min and Mercury II Chloride (HgCl2) at 0.1% (w / v) for 10 min. In contrast, the highest levels of contamination were observed in treatments with Calcium Hypochlorite and Sodium Hypochlorite. It is important to clarify that the contamination of the Agave spp. seeds in the different treatments was mainly by fungi and less frequently by bacteria (Table 4). Overall, and for the first time, six genera of phytopathogens were detected associated with Agave seeds. The results of the taxonomic study for the six samples analyzed revealed four genera of fungi: Monilinia sp., Aspergillus sp., Penicillium sp., and Alternaría alternata, a bacterium; Bacillus sp., and a yeast, Schizosaccharomyces sp. (Figure 2). It is noteworthy that the most frequent phytopathogen isolations were Aspergillus and Penicillium, which were present in all species, while others were exclusive to some species. / AnfrLn / cznz / e / Yi Schizosaccharomyces sp. was present in A. marmorata, Alternaría alternata in A. angustifolia, and Monilinia sp. in A. hórrida. When evaluating the efficiency of the treatments on the control of pathogens, it was observed that most of the treatments had an effective control on bacteria, but poor for fungi, since the latter were the most difficult microorganisms to control, however, H2O2 presented favorable results above the other treatments even in the one where the disinfectant was used alone (treatment 5) (Table 4). / AnfrLn / eznz / e / Yi Table 4. Efficiency of Treatment j treatments on the control of pathogens A. salmiana A. angustifolia A. hórrida A. marmorata A. potatorum A. karwiski A. cupreata Η BL Η BLHBL Η BL Η BL Η BL Η BL 1 Without any disinfectant XXXXXX 2 NaOCI XXXX 3 Ca(CIO); XXXXX 4 Ca(CIO)z / NaOCI X 5 H:C>2 XXXX 6 PM / Ca(CIO); XX 7 PM / NaOCI XXXX 8 PM / Ca(CIO)2 / NaOCI X 9 ΡΜ / Ή2Ο2 XX 10 l'M llgCI. Ca(CIO): XX 11 PM / HgCk / NaOCI XXX 12 PM / HgCI / Ca(CIO); / NaOCI H: Fungi; E3: Bacteria; L: Yeasts. X: Microorganisms not controlled by the treatment No specific data have been reported on disinfection in agave. In the present study, the lowest average number of contaminated seeds occurred in treatments where H2O2 was applied; however, when 3% H2O2 was applied for 24 h in combination with 30% copper sulfate for 10 min and 0.1% mercury chloride II for 10 min (treatment 12), total seed disinfestation was achieved. Similar results have not been previously reported with these disinfectants in this genus. This may be due to the oxidizing activity with the production of free radicals, which cause oxidative damage to proteins and lipids in the cell membrane of pathogens, confirming their bactericidal, virucidal, and fungicidal properties. While the highest levels of contamination were observed in seeds free of chemical solutions, as in the control treatment (To), which showed complete contamination of the seeds, it can be deduced that simply washing the seeds with running water or soap is not enough; chemical disinfectants must be applied to reduce the presence of pathogens. Finally, when comparing different species and solutions used, the results demonstrate the effectiveness of each one for Agave spp. As mentioned above, the treatment train of the disinfectant compositions that make up the claimed method, treatment 12 was the best in controlling contaminating microorganisms in the seeds of the 7 Agave species used as biological material (6 mezcal and 1 pulquera), achieving 100% disinfection, without altering their germination. Although the foregoing description was made taking into account the preferred embodiments of the invention, those skilled in the art should bear in mind that any modification in form and detail will be within the spirit and scope of the present invention. The terms in which this specification is written should always be taken in a broad and non-limiting sense. The materials, shape, and description of the elements are subject to variation as long as this does not imply an alteration of the essential characteristics of the model. LITERATURE CITED Alvarez A, Arzate F, Martínez M, and Martínez V. 2020. Regeneration of Agave marmorata roezl plants by somatic embryogenesis. Tropical and Subtropical Agroecosystems 23(2). DOI: 10.56369 / tsaes.3117 / RnfrLn / eznz / e / Yi Arzate F, Pina E, Norman M, Reyes D, Guevara S and Vázquez G. 2016. Regeneration of mezcal agave (Agave angustifolia Haw.) from encapsulated somatic embryos. Journal of Mexican Plant Sciences 39(4): 359-366. https: / / www.scielo.org.mx / scielo.php?script=sc¡_arttext&pid=S018773802016000 400359 Delgado A, González A, Santacruz R, Folgado R and Portillo L. 2021. Indirect somatic embryogenesis and cryopreservation of the Agave tequilana cultivar Weber 'Chato'. Plants, 10(2): 249. Domínguez R, González J, Rosales G, Quiñones V, Delgadillo D, Míreles O, Pérez M. 2008. In vitro cultivation as a tool for the use, improvement, and conservation of species of the genus Agave. Research and Science 41:53–62. https: / / www.redalyc.org / articulo.oa?id=67404109 Nun M and Robert M. 2013. Direct somatic embryogenesis of Agave fourcroydes Lem. through thin cell layer culture. In Vitro Cell Dev Biol—Plant 49:541549. DOI:10.1007 / s11627-013-9535-7 Murashige T and Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Plantarum Physiology 15: 473-497. 2008. In vitro propagation of three Agave species used for liquor distillation and three for landscape. Plant Cell Tiss. Org. Cult 94:201-207. DQI:10.1007 / s11240-008-9405x

Claims

1. A method for efficient seed disinfection, comprising contacting the seeds with a treatment train with at least one treatment composition comprising at least one disinfectant agent and / or an agronomically acceptable vehicle, characterized in that the treatment train comprises: a) contacting the seeds with Mercury II Chloride; b) subsequently contacting the seeds with Copper Sulfate; c) finally, contacting the seeds with Hydrogen Peroxide.

2. The method for efficient seed disinfection according to claim 1, characterized in that the Mercury II Chloride is preferably present at least at 0.1% (w / v).

3. The method for efficient seed disinfection according to claim 1, characterized in that the Copper Sulfate is preferably present at least at 30% (v / v).

4. The method for efficient seed disinfection according to claim 1, characterized in that the Hydrogen Peroxide is preferably present at least at 3% (v / v).

5. The method for efficient seed disinfection according to claim 1, characterized in that the seeds are left to rest in Mercury II Chloride for a time between 10 to 15 min.

6. The method for efficient seed disinfection according to claim 1, characterized in that the seeds are left to stand in the Copper Sulfate for a time of between 10 and 15 minutes. / «nfrLn / eznz / e / Yi 7. The method for efficient seed disinfection according to claim 1, characterized in that the seeds are left to rest in the Hydrogen Peroxide for a time between 24 to 48 hours.

8. The method for efficient seed disinfection according to claim 1, characterized in that between each step of contact with the treatment compositions, the seeds may optionally be rinsed.

9. A kit for efficient seed disinfection, characterized in that it comprises at least one treatment composition comprising at least one disinfectant agent and / or an agronomically acceptable vehicle, wherein said compositions are selected from: d) Mercury II Chloride; e) Copper Sulfate; f) Hydrogen Peroxide.