Composition for increasing the stability and viability of agriculturally important microorganisms in the coating of seeds for storage; method of preparation and use thereof

Abstract

The present invention provides a composition for coating agricultural seeds with agriculturally important microorganisms. A composition according to the present invention provides stabilization and viability of agriculturally important microorganisms in the coating of stored seeds, resulting in long shelf life. The present invention further provides a method for preparing a composition for coating seeds with viable microorganisms, as well as a method for coating seeds with a composition according to the present invention. The agriculturally important microorganisms according to the present invention possess multiple biological functions, including but not limited to plant growth promotion, nutrient mobilization, and control of phytonematodes and phytopathogens.

Description

[0001] TITLE: COMPOSITION FOR INCREASING THE STABILITY AND VIABILITY OF MICROORGANISMS OF AGRICULTURAL IMPORTANCE IN SEED COATING FOR STORAGE; METHOD OF OBTAINING AND USING THE SAME

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to the field of agriculture, more specifically to the coating of agricultural seeds, aiming to improve the characteristics of plant crops grown from coated seeds. The present invention provides a composition for coating seeds of agronomically important plants, combining functions desirable for the seed and microorganism treatment sector, as well as maintaining their viability until planting. A composition according to the present invention provides the stabilization and viability of microorganisms of agricultural interest in the coating of stored seeds, resulting in a long shelf life. Furthermore, the present invention also provides a method for preparing a composition for coating seeds with viable microorganisms.The microorganisms, according to the present invention, have multiple biological functions including, but not limited to, the promotion of plant growth, mobilization of nutrients and control of phytonematodes and phytopathologies.

[0004] BACKGROUND OF THE INVENTION

[0005] It is widely recognized that a large proportion of food crops face the risk of pest and disease damage. To meet the ever-increasing demand for food, farmers have widely resorted to the excessive use of synthetic fertilizers and pesticides to mitigate these problems. Although chemical pesticides have contributed to increased crop production in many countries, they have also created problems, including decreased soil fertility and the degradation of local ecosystems due to soil, water, and air pollution (CHATTERJEE et al., 2017). Therefore, to make agriculture more sustainable, it is essential to expand the adoption of biological alternatives, which are known to promote benefits in both productivity and plant health (FERREIRA et al., 2023).

[0006] The vast majority of disease-causing agents can be transmitted through seeds, which are highly efficient in disseminating pathogens due to their intrinsic characteristics (PARISI and MEDINA, 2013). For this reason, seed treatment is a technique frequently employed by Brazilian farmers and plays a crucial role in protecting them from the harmful effects of pests and pathogens, especially during the early stages of seedling development (LUDWIG et al., 2015).

[0007] Seed treatment, in its broadest sense, is the application of processes and substances that preserve or improve seed performance, allowing the maximum expression of a crop's genetic potential (MACHADO, 2000). It includes the application of pesticides (fungicides, insecticides, and nematicides), biological products, stimulants, micronutrients, or physical treatments such as thermotherapy (HENNING, 2012). In Brazil, in 2020, the seed treatment market reached USD 654 million, including insecticides, fungicides, and nematicides. Approximately 90% of Brazilian soybean seeds are treated with fungicides and 80% with insecticides, and in hybrid corn, 100% of seeds are treated with fungicides and 85% with insecticides (GOULART and NUNES, 2021).

[0008] Currently, soybean seed treatment with fungicides is carried out in two ways: on-farm, performed by the farmer himself on his farm, and industrial seed treatment (IST), which is performed in seed treatment centers. The advantages of IST include superior quality due to the precision of the dose, coverage, and adhesion of the applied products, as well as the possibility of applying synthetic polymer-based film coatings that protect the seeds and improve field performance (MENTEN and MORAES, 2010).

[0009] In this context, incorporating biological control into industrial seed treatment is highly desirable, as it would provide a sustainable alternative to a management system already routinely used by farmers. The main advantage of biological products is the ability to explore different modes of action, such as antagonism and parasitism, compared to chemical pesticides (ZUCCHI et al., 2008).

[0010] However, there are no technologies that allow the use of biological products associated with industrial seed coatings due to inherent adversities in the biology of microorganisms that often make the use of some techniques unfeasible (BIRD, 2003). Therefore, the development of formulation strategies opens up possibilities for improving the quality and preservation of strains, in addition to proposing new ways of applying biological products in the field (FLORENCIO et al., 2022). The formulation stage and the association of microorganisms with a suitable vehicle with additives emerge as strategies and alternatives for improving the shelf life and efficiency of biocontrol agents, among other characteristics (SAMMAURIA et al., 2020). Formulations must be designed to ensure the long-term viability of microorganisms during production, storage, transportation, application, and at the inoculation site, providing a stable environment under various environmental conditions (BASHAN et al., 2022)., 2014). For these reasons, formulation is a key factor in the success of a bioproduct and can influence successful commercialization. The effectiveness of a formulation can be demonstrated by the significant increase in parameters such as fresh and dry biomass, changes in root and shoot morphology, increased plant nutrition, yield, and productivity (SOUMARE et al., 2020).

[0011] The main challenges in developing an efficient formulation are: increasing the shelf life of the microbial agent, finding a suitable carrier material and additive, and overcoming problems in transportation, storage, and application (HERRMANN et al., 2013).

[0012] Therefore, there is a persistent demand for a biological formulation that presents (i) long survival of microorganisms of agricultural interest in the formulation stored at room temperature (ii) long survival of agriculturally useful microorganisms on seeds treated in conjunction with chemical treatments, and (iii) efficient in economically viable doses for application in large crops of agricultural importance. BRIEF DESCRIPTION OF THE INVENTION

[0013] The present invention provides a seed coating composition comprising microorganisms with functions important to agriculture that provides stabilization and viability of the microorganisms in the coating of stored seeds, resulting in a long shelf life. The present invention also provides a method for preparing a seed coating composition with viable microorganisms. The microorganisms according to the present invention have multiple biological functions, including, but not limited to, promoting plant growth, nutrient mobilization, and controlling phytonematodes and plant diseases. Therefore, the compositions according to the present invention enable the preparation of seeds coated with microorganisms that provide desirable functions in agriculture while maintaining their viability for long periods.

[0014] The present invention further provides methods for preparing a seed coating composition according to the present invention and for producing coated seeds as well as seed coated with a composition according to the present invention.

[0015] Additional embodiments and aspects of the present invention may be derived from the following detailed description, the drawings, and also the appended set of claims.

[0016] BRIEF DESCRIPTION OF THE FIGURES Figure 1 (Fig. 1) shows the visual appearance of the formulation stored at different temperatures. A = Newly formulated, B = 14 days at 5 °C, C = 14 days at 28 °C and D = 14 days at 54 °C.

[0017] Figure 2 (Fig. 2) presents a count of colony-forming units present in the formulation stored at different temperatures. Storage for 14 days at 54 °C mimics two years of storage at room temperature.

[0018] Figure 3 (Fig. 3) shows the visual appearance / coating of soybean seeds treated with the formulation of the present invention. The filter paper shows the high adhesion of the pigment after agitation of the seeds in water.

[0019] Figure 4 (Fig. 4) shows bacterial cell recovery counts on soybean seeds treated under different conditions. The seeds were treated and stored at room temperature. Counts were performed every 30 days over 360 days.

[0020] Figure 5 (Fig. 5) shows the visual appearance of soybean seeds treated with different pigments at a dose of 2.5 ml of formulation per kilogram of seed.

[0021] Figure 6 (Fig. 6) shows the development of plants 21 days after emergence. A = control, B = Chemical treatment, C = Biological formulation, D = Chemical treatment associated with biological formulation.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides a seed coating composition comprising viable microorganisms with functions of agronomic importance that provides stabilization and viability of microorganisms in the coating of stored seeds, resulting in a long shelf life.

[0024] A composition according to the present invention comprises: at least one antifreeze in the proportion of about 1% to about 20% (w / w); at least one thickener in the proportion of about 0.1% to about 5% (w / w); water in the proportion of about 5 to about 20% (w / w); at least one defoamer in the proportion of about 0.1 to about 10% (w / w); at least one dispersant in the proportion of about 5 to about 20% (w / w); at least one biocide in the proportion of 0.1 to 10% (w / w); at least one microorganism of agricultural importance in the proportion of about 10 to about 60% (w / w); and at least one adhesive in the proportion of about 5 to about 20% (w / w).

[0025] Preferably, a composition according to the present invention further comprises a colorant in an amount of about 5 to about 30% (w / w).

[0026] In preferred embodiments of the present invention, the antifreeze can be, but not limited to, propylene glycol, glycerol, hexylene glycol, monoethylene glycol, or diethylene glycol; the thickener can be, but not limited to, xanthan gum, guar gum, gum arabic, or carboxymethyl cellulose; the defoamer is emulsified silicone; the dispersant can be, but not limited to, tristyrylphenol phosphate ether, sodium β-methyl oleyl taurate, styrene acrylic polymer, sulfosuccinate, tristyrylphenol ethoxylate, and tristyrylphenol ethoxylate sulfate, polysorbate; the biocide can be, but not limited to, 1,2-benzisothiazole-3(2H)-one, potassium sorbate, methyl paraben, sodium benzoate, benzyl alcohol; the adhesive can be, but not limited to, urethane methacrylate, ester methacrylate, epoxy acrylic, acrylate.

[0027] Still as preferred embodiments of the present invention, the microorganism of agricultural importance may be of different genera selected from the group of species but not restricted to Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus pumilus, Bacillus thuringiensis, Bacillus circulans, Bacillus haynesii, Bacillus aryabhattai, Bacillus licheniformis, Paenibacillus durus, Brevibacillus laterosporus, Pseudomonas fluorences, Pseudomoas chlororaphis, Pseudomonas protegens, Azospirillum brasilense, Bradyrhizobium japonicum, Beauveria bassiana, Metarhizium anisopliae, Metarhizium robertsii, Trichoderma asperelloides, Trichoderma harzianum, Trichoderma asperellum, Trichoderma vinde.

[0028] The present invention further provides a method for preparing a seed coating composition comprising:

[0029] (i) preparing a first premix comprising: at least one antifreeze in a proportion of about 1% to about 20% (w / w), and at least one thickener in a proportion of about 0.1% to about 5% (w / w);

[0030] (ii) preparing a second premix comprising: water in a proportion of about 5 to about 20% (w / w); at least one defoamer in a proportion of about 0.1 to about 10% (w / w); at least one dispersant in a proportion of about 5 to about 20% (w / w); at least one biocide in a proportion of about 0.1 to about 10% (w / w); at least one microorganism of agricultural importance obtained by means of solid or liquid fermentation in a proportion of about 10 to about 60% (w / w); at least one adhesive ingredient in a proportion of about 5 to about 20% (w / w); and optionally at least one colorant in a proportion of about 5 to about 30% (w / w); and mixing the first premix with the second premix to obtain the final formulation.

[0031] Still as preferred embodiments of the present invention, the mixing of the first premix with the second premix comprises is carried out in stainless steel containers, under constant stirring of approximately 40 Hz to approximately 60 Hz, until the suspension is homogeneous.

[0032] In a preferred embodiment, mixing of the first premix with the second premix comprises is carried out for between about 30 and about 180 minutes.

[0033] The compositions, methods and coated seeds according to the present invention can be applied to seeds of any cultivated plant species. Preferably, the seed is a seed of a plant selected from the group consisting of Brachiaria spp., Panicum spp., Sorghum spp., Pennisetum americanum, Glycine max, Zea mays, Gossypium hirsutum, Triticum aestivum, Solanum lycopersicum, Allium cepa, Lactuca sativa, Daucus carota, Capsicum annuum, Eucalyptus spp., Tectona grandis, Hevea brasiliensis, Pinus sp., Nicotiana tabacum, Crotalana spp., Helianthus annuus, Arachis hypogaea.

[0034] Optionally, a composition according to the present invention includes a combination of microorganisms of agricultural importance in combination with one or more chemical agents such as chemical pesticides, fungicides, insecticides and / or nematicides.

[0035] Furthermore, its composition allows the application of the formulation in low doses and the adoption of pigments of different colors, making the technique economically viable for use in conjunction with packages

[0036] The composition should be used as a concentrated suspension formulation for seed treatment (FS). However, other formulations containing these microorganisms, such as emulsions and dispersions, can also be used.

[0037] Any colorant commonly used in the art can be used in a composition according to the present invention, such as, but not limited to, red, green, blue, purple, black, pink, orange, and others. This versatility allows the formulation to be used in conjunction with different chemical treatments, depending on its original color. Additional embodiments and aspects of the present invention can be understood by those skilled in the art based on their knowledge of the prior art.

[0038] The present invention describes that the composition of an industrial seed treatment formulation is capable of increasing the product's shelf life, as well as the survival of microorganisms on chemically treated seeds. Furthermore, the agronomic effects of seeds treated with the industrial seed treatment (IST) formulation are superior to those of the formulation treated without this formula.

[0039] Phytonematodes targeted for control by a composition according to the present invention include, but are not limited to, Pratylenchus brachyurus, Pratylenchus zea, Pratylenchus coffea, Meloidogyne incognita, Meloidogyne javanica, Meloidogyne exígua, Heterodera glycines, Rotylenchulus reniformis, and Helicotilechus dihystera.

[0040] Phytopathologies targeted for control by a composition according to the present invention include, but are not limited to, Rhizoctonia solani, Fusarium solani, Fusarium oxysporum, Fusarium graminearum, Colletotrichum gloeosporioides, Colletotrichum truncatum, Colletotrichum lindemuthianum, Macrophomina phaseolina, Sclerotinia sclerotiorum, Botrytis cinerea and Penicillium digitatum.

[0041] The present invention may be used for the treatment of seeds of, but not limited to, Brachiaria spp. (Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola, Brachiaria mutica, Brachiaria ruziziensis, Brachiaria arrecta, Brachiaria dictyneura), Panicum spp. (Panicum maximum), Sorghum spp., Pennisetum americanum, Glycine max, Zea mays, Gossypium hirsutum, Triticum aestivum, vegetables (Solanum lycopersicum, Allium cepa, Lactuca sativa, Daucus carota, Capsicum annuum), forestry (Eucalyptus spp., Tectona grandis, Hevea brasiliensis, Pinus sp.), Nicotiana tabacum, ornamentals, Crotalaria spp. (Crotalaria juncea, Crotalaria spectabilis, Crotalaria ochroleuca, Crotalaria paulina, Crotalaria breviflora.

[0042] The compositions obtained by the present invention have a liquid physical nature characterized as a concentrated suspension for seed treatment (FS).

[0043] The present invention teaches that the application of the product in reduced volume solutions (e.g., approximately 2.5 mL / kg of seed) efficiently promotes growth in plants of agricultural interest, such as corn and soybeans, as will be understood by those skilled in the art.

[0044] EXAMPLES

[0045] Example 1 - Physical-chemical stability of the formulation.

[0046] Objective - To obtain ideal proportions between active and inert ingredients that provide adequate stability to the formulation.

[0047] Material and methods - A composition containing 40% Bacillus licheniformis, 20% adhesive compound, 13% dye, 8% dispersants, 5% antifreeze, 0.6% antifoams, biocides and thickeners, in addition to 13.4% water, was formulated to evaluate its physical-chemical stability.

[0048] Experimental design - The prepared formulation was stored for 14 days under different temperature conditions (5, 28, and 54°C) and evaluated for physical aspects such as appearance, color, odor, uniformity, among others; and chemical aspects, which must be maintained within the specified limits of integrity of the chemical structure, ingredient content, and other parameters. It is worth noting that this storage condition (54°C for 14 days) mimics the equivalent of two years of storage at room temperature.

[0049] Results - After the storage time, it was observed that all physical and chemical parameters evaluated maintained good stability of the formulation at all temperatures tested, when compared to the freshly prepared formulation, as observed in Figure 1.

[0050] Example 2 - Biological stability of the formulation.

[0051] Objective - To evaluate the survival of the microorganism (active ingredient) within the formulation.

[0052] Material and methods - A composition containing 40% Bacillus licheniformis, 20% adhesive compound, 13% dye, 8% dispersants, 5% antifreeze, 0.6% antifoams, biocides and thickeners, in addition to 13.4% water, was formulated and the survival of the bacteria in this composition was evaluated.

[0053] Experimental design - The prepared formulation was stored for 14 days under different temperature conditions (5, 28, and 54 °C) and evaluated for microorganism survival. It is worth noting that this storage condition (54 °C for 14 days) mimics the equivalent of two years of storage at room temperature.

[0054] Results - After the storage time, it was observed that the number of colony-forming units remained stable at all temperatures tested, when compared to the freshly prepared formulation, with a bacterial concentration of around 10 A 8 CFU / mL, as seen in figure 2.

[0055] Example 3 - Coating and adhesion on seeds.

[0056] Objective - To evaluate the coating and adhesion capacity of the present invention on soybean and corn seeds.

[0057] Material and methods - A composition containing 40% Bacillus licheniformis, 20% adhesive compound, 13% dye, 8% dispersants, 5% antifreeze, 0.6% antifoams, biocides and thickeners, in addition to 13.4% water, was formulated and evaluated for seed coating at different application doses and for the adhesion of this coating to the seeds.

[0058] The coating was evaluated according to the visual appearance of the treated seeds and adhesion was measured based on the release of the pigment on filter paper, after shaking the treated seeds in water for 10 minutes.

[0059] Experimental design - Doses of 1.0 to 3.0 ml of formulation per kilogram of seed were tested, combined with the doses recommended by the manufacturers of each chemical package evaluated. The seeds were treated in a machine that simulates the same process as industrial machines.

[0060] Results - It was observed that the dose of 2.5 ml of formulation per kilogram of seed is ideal to obtain a satisfactory visual appearance of the treated seed, without observing a large release of pigment in the adhesion test, as observed in figure 3. Example 4 - Recovery of the microorganism on the treated seeds.

[0061] Objective - To evaluate the survival of Bacillus licheniformis on seeds treated with the formulation of the present invention, over time and stored at room temperature.

[0062] Material and methods - A composition containing 40% Bacillus licheniformis, 20% adhesive compound, 13% dye, 8% dispersants, 5% antifreeze, 0.6% antifoaming agents, biocides and thickeners, in addition to 13.4% water, was used to treat soybean seeds according to the dose of the formulation described in example 3 of this document and the survival of the microorganisms was evaluated every 30 days by the solid plating method.

[0063] Experimental design - The treated seeds were stored at room temperature, and colony-forming unit counts on the seeds were performed in triplicate by plating on solid medium every 30 days. Survival differences were compared between seeds treated only with the active ingredient of the formulation (without the presence of inerts), seeds treated only with the formulation of the present invention, and seeds treated in combination with the formulation of the present invention and a chemical seed treatment package.

[0064] Results - It was observed that in the treatments in which the bacteria were present in the formulation of the present invention, there was survival of the microorganism during the evaluated storage period (360 days), as shown in Figure 4. This survival occurred regardless of the presence of the associated chemical treatment, evidencing that even when chemical and biological treatments are combined, a positive protective effect of the formulation on the bacterial cells is observed. In the case in which the seeds were treated only with the bacterial ferment, total mortality of the microorganism was observed on the seeds 90 days after treatment.

[0065] Example 5 - Diversity of pigments for coating.

[0066] Objective - To evaluate the coating quality and adhesion of other pigments to the formulation.

[0067] Material and methods - A composition containing 40% Bacillus licheniformis, 20% adhesive compound, 13% different dyes, 8% dispersants, 5% antifreeze, 0.6% antifoams, biocides, and thickeners, and 13.4% water, was formulated and evaluated for seed coating at different application rates. The coating was evaluated based on the visual appearance of the treated seeds.

[0068] Results - It was observed that the 2.5 ml dose of formulation used in example 3 of this document was also ideal for good coating of the seeds with pigments of different colors such as: blue, green, purple and black, as shown in figure 5.

[0069] Example 6 - Formulation effectiveness in field trial.

[0070] Objective - To evaluate the effect, under field conditions, of corn seeds treated with the biological formulation on plant growth promotion. Experimental design - Completely randomized blocks, with four treatments and four replicates. The plants were arranged in four rows, each 4 m long and 50 cm spacing between rows, totaling 16 m. 2 of useful portion.

[0071] Treatments and method of application - The corn seeds used to carry out the test were previously treated in the Momesso Arktos L-2K seed treater. The treatments consisted of: (1) untreated control, (2) the formulation of the present invention, (3) the chemical insecticides and fungicides, Cruiser 350 FS and Maxim XL and (4) the combination of the chemical treatment with the biological treatment of the formulation of the present invention.

[0072] The seeds were sown in the planting furrow, and in the period after sowing relevant to each evaluation, the plants were evaluated for: emergence, vigor, leaf area, root length and weight, shoot length and weight, and productivity.

[0073] Results - Figure 6 and Table 1 show the treatment results. Seed coating with a composition of the present invention containing microorganisms of agricultural impotence showed superior performance to the other treatments, from the crop establishment phase to productivity. The biological formulation combined with the chemical package (Cruiser-Maxim) showed excellent synergism, increasing productivity by 9.5%, vigor by 13.2%, and leaf area by 9.2%, compared to the chemical package used alone.

[0074] TABLE 1. Biometric evaluations carried out in a field trial with corn. The seeds were coated according to treatments (1)-(4) and the different responses of the association between the biological formulation of the present invention and a standard market chemical treatment were evaluated.

[0075] Based on the results obtained, it can be demonstrated that seed treatment with the biological formulation of the present invention can be recommended in the integrated management of various crops, for various control and plant growth promotion purposes. The use of Bacillus spp. added to the coating components of industrially treated seeds proves to be a viable alternative for agricultural management. Furthermore, in addition to the diverse mechanisms of action of these specific microorganisms, this biological formulation can be expanded to other microorganisms with numerous control pathways.

[0076] NON-PATENTARY REFERENCES CITED

[0077] BASHAN, Y., BASHAN, L.E., PRABHU, S.R. Advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998- 2013). Plant Soil 378, 1-33, 2014. Doi: 10.1007 / s11104-013-1956-x

[0078] BIRD, D.M., OPPERMAN, C.H., DAVIES, K.G. Interactions between bactéria and plant parasitic nematodes: now and then. Int. J. Parasitol. 33:1269-127, 2003.

[0079] CHATTERJEE A., SINGH S„ AGRAWAL C„ YADAV S„ RAI R„ RAI, L. C. Papel das algas como biofertilizante, Algal Green Chemistry: Recent Progress in Biotechnology . Ed. Rastogi RP, PandeyA., Madamwar D. Amesterdã, Holanda: Elsevier, 189-200, 2017.

[0080] FERREIRA, A., BASTOS, R.V., MARQUES, S.C., ACIÉN-FERNANDEZ, F.G., GOUVEIA, L. Algaeculture for agriculture: from past to future. Frontiers in Agronomy, v5, 2023. Doi: 10.3389 / fagro.2023.1064041.

[0081] FLORENCIO, C„ BORTOLETTO-SANTOS, R„ FAVARO, C„ GOVONI, MB, VELLOSO, C., KLAIC, R, RIBEIRO, C., FARINAS, C., MATTTOSO, L. Advances in the production and formulation of microbial inoculants aiming at a more sustainable agriculture. Química Nova. 45. 2022. Doi: 10.21577 / 0100-4042.20170909.

[0082] HENNING, AA More practical and efficient industrial seed treatment. Campo & Negócio Magazine, no. 115, 2012.

[0083] HERMANN, L., LESUEUR, D. Biofertilizer formulation and quality challenges for successful inoculation. Appl Microbiol Biotechnol 97 , 8859-8873, 2013. doi: 10.1007 / s00253-013-5228-8 LUDWIG, M.P., OLIVEIRA, S., AVELAR, SAG, ROSA, M.P., FILHO, O.A. L, CRIZEL, R.L. Storage of Treated Soybeans and Their Effect on Seedling Performance. Agricultural Science and Technology, v9, n.1, p.51-56, 2015.

[0084] MACHADO, JC Seed treatment in disease control. Lavras, LAPS / FAEPE, 138 p„ 2000.

[0085] MENTEN, JO, MORAES, MHD Advances in Seed Treatment and Coating. Seed Treatment: History, Types, Characteristics and Benefits. Abrates Newsletter, v.20, n3, 2010.

[0086] PARISI, JJD, MEDINA, PF Seed treatment. Agronomic Institute of Campinas (IAC) - Center for Research and Development in Plant Health, 2013.

[0087] SAMMAURIA, R., KUMAWAT, S., KUMAWAT, P. Microbial inoculants: potential tool for sustainability of agricultural production systems. Arch Microbiol 202, 677- 693, 2020. Doi: 10.1007 / S00203-019-01795-w

[0088] SOUMARE, A., BOUBEKRI, K„ LYAMLOULI, K„ HAFIDI, M„ OUHDOUCH, Y, KOUISNI, L. From Isolation of Phosphate Solubilizing Microbes to Their Formulation and use as Biofertilizers: Status and Needs. Front. Bioeng. Biotechnol. 7:425, 2020. Doi: 10.3389 / fbioe.2019.00425

[0089] ZUCCHI T.D., MORAES L.A.B., MELO I.S. Streptomyces sp. ASBV-1 reduces aflatoxin accumulation by Aspergillus parasiticus in peanut grain. J. Appl. Microbiol. 105:2153 2160, 2008.

Claims

CLAIMS 1. A seed coating composition comprising: at least one antifreeze in a proportion of from about 1% to about 20% (w / w), at least one thickener in a proportion of from about 0.1% to about 5% (w / w); water in a proportion of from about 5 to about 20% (w / w), at least one defoamer in a proportion of from about 0.1 to about 10% (w / w); at least one dispersant in a proportion of from about 5 to about 20% (w / w); at least one biocide in a proportion of from 0.1 to 10% (w / w), at least one agriculturally useful microorganism in a proportion of from about 10 to about 60% (w / w); and at least one adhesive in a proportion of from about 5 to about 20% (w / w).

2. The composition of claim 1, wherein the composition further comprises a colorant in an amount of about 5 to about 30% (w / w).

3. Composition according to claim 1, wherein the antifreeze is propylene glycol, glycerol, hexylene glycol, monoethylene glycol or diethylene glycol.

4. Composition according to claim 1, wherein the thickener is xanthan gum, guar gum, gum arabic or carboxymethyl cellulose.

5. Composition according to claim 1, wherein the antifoam is emulsified silicone.

6. The composition of claim 1, wherein the dispersant is tristyrylphenol phosphate ether, sodium N-methyl oleyl taurate, styrene acrylic polymer, sulfosuccinate, tristyrylphenol ethoxylate and tristyrylphenol ethoxylate sulfate or polysorbate.

7. Composition according to claim 1, wherein the biocide is 1,2-benzisothiazol-3(2H)-one, potassium sorbate, methyl paraben, sodium benzoate or benzyl alcohol.

8. The composition of claim 1, wherein the adhesive is an acrylic polymer, methacrylate urethane, methacrylate ester, acrylic epoxy or acrylate.

9. Composition according to claim 1, wherein the microorganism of agricultural importance is from a genus selected from the group consisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus pumilus, Bacillus thuringiensis, Bacillus circulans, Bacillus haynesii, Bacillus aryabhattai, Bacillus licheniformis, Paenibacillus durus, Brevibacillus laterosporus, Pseudomonas fluorences, Pseudomoas chlororaphis, Pseudomonas protegens, Azospiríllum brasilense, Bradyrhizobium japonicum, Beauveria bassiana, Metarhizium anisopliae, Metarhizium robertsii, Trichoderma asperelloides, Trichoderma harzianum, Trichoderma asperellum, Trichoderma viride.

10. Method for preparing a seed coating composition comprising: (i) preparing a first premix comprising: at least one antifreeze in a proportion of about 1% to about 20% (w / w), and at least one thickener in a proportion of about 0.1% to about 5% (m / m); (ii) preparing a second premix comprising: water in a proportion of about 5 to about 20% (w / w); at least one defoamer in a proportion of about 0.1 to about 10% (w / w); at least one dispersant in a proportion of about 5 to about 20% (w / w); at least one biocide in a proportion of about 0.1 to about 10% (w / w); at least one agriculturally useful microorganism in a proportion of about 10 to about 60% (w / w); at least one adhesive ingredient in a proportion of about 5 to about 20% (w / w); and optionally at least one colorant in a proportion of about 5 to about 30% (w / w); and mixing the first premix with the second premix to obtain the final formulation.

11. Method according to claim 10, wherein the mixing of the first premix with the second premix comprises is carried out in stainless steel containers, under constant agitation of about 40 Hz to about 60 Hz, until the suspension is homogeneous.

12. The method of claim 11, wherein mixing the first premix with the second premix comprises is carried out for between about 30 and about 180 minutes.

13. Method for producing coated seeds comprises: mixing seeds with a composition as defined in claim 1; and homogenizing the mixture 14. Seed coated with a composition as defined in claim 1.

15. The method of claim 13 or the seed of claim 14, wherein the seed is a seed of a plant selected from the group consisting of Brachiaria spp., Panicum spp., Sorghum spp., Pennisetum americanum, Glycine max, Zea mays, Gossypium hirsutum, Triticum aestivum, Solanum lycopersicum, Allium cepa, Lactuca sativa, Daucus carota, Capsicum annuum, Eucalyptus spp., Tectona grandis, Hevea brasiliensis, Pinus sp., Nicotiana tabacum, Crotalaria spp., Helianthus annuus, Arachis hypogaea.

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